A review on phospholipids and their main applications in drug delivery systems

Review

A review on phospholipids and their main applications in drug delivery systems

Jing Li,Xuling Wang,Ting Zhang,Chunling Wang,Zhenjun Huang, Xiang Luo,Yihui Deng＊
School of Pharmacy,Shenyang Pharmaceutical University,No.103,Wenhua Road,Shenyang 110016,China

ARTICLEINFO

Article history:

Received 27 June 2014

Received in revised form

29 August 2014

Accepted 10 September 2014

Available online 28 September 2014

Phospholipids

A

Phospholipids have the characteristics of excellent biocompatibility and a especial amphiphilicity.These unique properties make phospholipids most appropriate to be employed as important pharmaceutical excipients and they have a very wide range of applications in drug delivery systems.The aim of this review is to summarize phospholipids and some of their related applications in drug delivery systems,and highlight the relationship between the properties and applications,and the effect of the species of phospholipids on the ef fi ciency of drug delivery.We refer to some relevant literatures, starting from the structures,main sources and properties of phospholipids to introduce their applications in drug delivery systems.The present article focuses on introducing fi ve types of carriers based on phospholipids,including liposomes,intravenous lipid emulsions,micelles,drug-phospholipids complexes and cochleates.

©2015 Shenyang Pharmaceutical University.Production and hosting by Elsevier B.V.All rights reserved.

1. Introduction

Therapeutic agents such as proteins/peptides,nucleic acids, anticarcinogens,and other drugs have the drawbacks of low bioavailability,rapid clearance,and high toxicity.Therefore, there is a great demand to develop delivery methods and carriers,which will bring a more ef fi cient delivery for therapeutics.

Drug delivery systems(DDS)are capable of designing to increase the bioavailability of drugs,control drug delivery and maintain the drug intact transport to the site of action while avoiding the non-diseased host tissues.Brie fl y,in a suitable dosage and mode of administration,using the smallest dose to achieve the best therapeutic effect is the research objective of DDS.

As main components of cellular membrane,phospholipids have excellent biocompatibility.In addition,phospholipids are renowned for their amphiphilic structures.The amphiphilicity confers phospholipids with self-assembly,emulsifying and wetting characteristics.When introduced into aqueous milieu,phospholipids self-assembly generates different supermolecular structures which are dependent on theirspeci fi c properties and conditions.Forexample,phospholipids have a propensity to form liposomes,which can be employed as the drug carriers[1].Phospholipids have good emulsifying property which can stabilize the emulsions [2].In addition,phospholipids as surface-active wetting agents which can coat on the surface of crystals to enhance the hydrophility of hydrophobic drugs[3].The above properties are successfully employed in the DDS design.

Phospholipids based DDS have been found promising for better and effective delivery of drugs and providing much appropriate systematic drugdelivery.In recentyears,a variety of phospholipid-related formulations,such as Doxil®[4], Cleviprex®[5],Valium®[6]and Silybin Phytosome™[7],have been used in clinic,and achieving good results.

Phospholipids are molecules in which hydrophilic head group and hydrophobic acyl chains are linked to the alcohol. The variation in head groups,aliphatic chains and alcohols leads to the existence of a wide variety of phospholipids.In addition,the different sources of phospholipids also enhance the species of phospholipids.Various phospholipids,such as soybean phosphatidylcholine,egg phosphatidylcholine,or syntheticphosphatidylcholine,aswellashydrogenated phosphatidylcholine,are commonly used in different types of formulations.Phospholipids become intriguing as they can offer various options.However,the species diversity of phospholipids make how to select an appropriate phospholipid to achieve the therapeutic purpose become a crucial problem in the design of DDS,so we summarized the structures,main sources,properties of phospholipids which can give a guideline in the design of DDS.In addition,we set liposomes, intravenous lipid emulsions,PC/bile salt mixed micelles, phospholipid micelles,drug-phospholipid complexes,cochleates as examples to introduce the main applications associated with phospholipids and further explain how to make a choice among the phospholipids in drug delivery.

2. Phospholipids

Phospholipids are lipids containing phosphorus,a polar potion and non-polar potion in their structures.

2.1. The structures of phospholipids

According to the alcohols contained in the phospholipids, they can be divided into glycerophospholipids and sphingomyelins.

2.1.1. Glycerophospholipids

Glycerophospholipids which are the main phospholipids in eukaryotic cells,refer to the phospholipids in whichglycerol is the backbone.All naturally occurring glycerophospholipids possess α-structure and L-con fi guration[8].

The chemical structures of glycerophospholipids can be classi fi ed by the head group,the length and the saturation of hydrophobic side chains,the type of bonding between the aliphatic moieties and glycerol backbone,and the number of aliphatic chains.Variation in the head group leads to different glycerophospholipids,such as phosphatidylcholine (PC),phosphatidylethanolamine(PE),phosphatidylserine(PS), phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylglycerol(PG)cardiolipin(CL)(Table 1).The length of the apolar moieties leads to different glycerophospholipids, e.g.dipalmitoyl,dimyristoyl,distearoyl PC.The saturation of aliphatic groups characterizes different glycerophospholipids, such as dioleoyl,distearoyl PC.The type of bonding(ester or ether)between aliphatic chains and glycerol determines different glycerophospholipids,such as plasmalogen[9].The number of aliphatic chains is different,for example,lysophospholipids have only one acyl group at the glycerol backbone[10](Fig.1).

2.1.2. Sphingomyelins

In 1884,Thudicum fi rst described sphingomyelins(SMs),but it was not until 1927 that Pick and Bielschowsky proved their structures to be N-acylsphingosine-1-phosphatidylcholine(Fig.1).In 1962,Shapiro and Flowers con fi rmed that all SMs of biological sources are D-erythro con fi guration[12].

SMs are an important component of animal cell membranes.Although PC and SM are very similar in molecular structure,they still have some differences.1)The backbone of SM is a sphingosine,while the backbone of PC is a glycerol.2) Each SM molecule averagely contains 0.1-0.35 cis-double bonds in amide-linked acyl chains,and PC contains 1.1-1.5 cis-double bonds.It is obvious that the saturation of hydrophobic regions of SMs is higher than that of PCs.3)The typical acyl lengths of the naturally occurring SMs are usually more than 20,while the paraf fi n residues of sphingosine are relatively shorter,so the SMs are asymmetric molecules[13];in contrast,PCs typically contain moderate lengths(16-18)of the acyl chains,and the lengths of two chains are approximately equal,so the PCs are symmetric molecules[14].4)SMs are capable of forming intermolecular and intramolecular hydrogen bonds,so the SM and PC bilayer have a signi fi cant difference in the macroscopic properties[13].5)The range of phase transition temperature(Tc)of all naturally occurring SMs is 30-45°C which is above the natural PCs[13].6) Numerous observations have shown that SM and cholesterol have a very strong interaction,for example,compared with thenon-saturated PC/cholesterolbilayer,SM/cholesterol bilayer has higher compressibility,and lower permeability to water.The reason for this phenomenon is that higher saturation of the acyl chain of SM leads to stronger interaction with steroid nucleus[13].

2.2. The main sources of phospholipids

According to the sources,phospholipids can be divided into natural phospholipids and synthetic phospholipids.

2.2.1. Natural phospholipids

In 1793,Fourcroy was likely to be the fi rst to fi nd the existence evidence of complex aliphatic compounds.In 1812,Uauquelin found phospholipids in human brain.In 1846,Gobley separated phospholipids from egg yolk.The term“lecithin”which is derived from the Greek lekithos was fi rst used to describe a sticky orange material isolated from egg yolk.After 20 years, choline component in lecithin was determined[15].Currently,thereare threede fi nitionsof“lecithin”appeared in literatures, including:1)from a business perspective,“lecithin”mainly includes PC,PE,PS,PI,other phospholipids,triglycerides,fatty acids and carbohydrates;2)from a historical point of view,“lecithin”refers to the lipids containing phosphorus isolated from eggs and brains;3)from a scienti fi c point of view,“lecithin”refers to PCs[16].

Phospholipids are widely distributed in animals and plants, and the main sources include vegetable oils(e.g.soybean,cottonseed,corn,sun fl owerandrapeseed)andanimaltissues(e.g. egg yolkand bovine brain).In terms of production,egg yolk and soybean are the most important sources for phospholipids[17]. However,soybeanandeggyolkhavedifferencesinthecontents and species of phospholipids,mainly including:1)egg yolk lecithin contains a higher amounts of PC;2)phospholipids in egg yolk exist long chain polyunsaturated fatty acids of n-6 and n-3 series,primarily arachidonic acid(AA)and docosahexaenoic acid(DHA),which are absent in soybean lecithins;3)animal lecithins have characteristic of the presence of SM[18];4) the saturation level of egg yolk lecithins is higher than that of soybean lecithins[19],so their oxidative stability is better than that of soybean lecithins;5)for egg yolk phospholipids,saturatedfattyacidisusuallyatsn-1position,andunsaturatedfatty acid is at sn-2 position[9],while for soybean lecithin,sn-1 and sn-2 position can be both unsaturated fatty acids.For example, dilinoleoylphosphatidylcholine(DLPC)is the main component of soybean phosphatidylcholine(SPC)[20].

The cost of phospholipids isolated from natural sources is always lower than that obtained by synthetic or semisynthetic methods.For natural phospholipids,the more pure they are,the higher the price is[9].

Phospholipids isolated from plants and animals can be puri fi ed into different levels,including food and pharmaceutical grade.For example,lipoid E80 can contain PC,PE,lysophosphatidylcholine (LPC),lysophosphatidylethanolamine (LPE),SM and trace amounts of triglycerides,cholesterol,fatty acid,d,L-α-vitamin E and water[21].

2.2.2. Synthetic phospholipids

Since chromatographic puri fi cation techniques still cannot get single component of naturally occurring phospholipids, researchers focus on chemical synthesis which can obtain single component with de fi ned structure and con fi guration [22].The synthesis of phospholipids can be divided into semisynthesis and total synthesis.

Semi-synthesisofglycerophospholipidsreferstothe changing of head,tail groups or both on the basis of natural phospholipids.Therefore,compared with the total synthesis, it requires less reaction steps.The semi-synthetic methods of glycerophospholipids mainly include:1)the double bonds of natural phospholipids are hydrogenated to obtain the saturated phospholipids which have a higher melting point and oxidation stability; 2) Acylation of sn-glycero-3-phosphocholine(GPC)obtained by deacylation of natural PC with activated acyl derivative can get the desired PC;3) phospholipase D catalyzes glycerophospholipids to generate the phosphatidic acid(PA),and the hydroxy-containing acceptors such as glycerol and serine can attach to PA,which can converse the choline head group into various phosphorylated alcohol head groups[9].

The total synthesis of glycerophospholipids involves the formation of ester or ether bonds linking apolar moieties to glycerol backbone,and the attachement of polar head group. Ispropylidene glycerols or sugar alcohols,such as D-mannitol, are often served as the precursors of glycerol part of phospholipids,which are available from many sources.The synthetic glycerophospholipids have the advantages of single component and stable property[9].

The procedure of semi-synthesis of SM involves the deacylation of the natural SM extracts(primarily from bovine brains)to obtain the sphingosylphospocholine which is subsequently acylated to SM using the fatty acid of choice.It has been found that during the deacylation-reacylation procedure,a signi fi cant amount of the L-threo stereoisomer is formed.Thus,the fi nal product represents a mixture of D-erythro and L-threo stereoiomers[23].

Because the total synthesis of SM is considerably more complex than that of the glycerophospholipids,there is few studies on the fully synthetic and stereochemically pure SM [23].The access to synthetic SM is limited by the availability of syntheticsphingosine.Followingthetotalsynthesisof sphingosine[24],the fi rst synthetic SM was synthesized by Shapiro et al[25].The complete synthesis of SM includes the following steps:1)the synthesis of sphingosine,2)the synthesis of ceramide(N-acylsphingosine),3)the synthesis of SM.

2.3. The physiological properties of phospholipids

Phospholipids are basic substances to maintain life activity. They are widely distributed in humans,animals,plants,and so on.Phospholipids are indispensable components of all cellular and sub-cellular membranes,they can arrange as bilayer membranes.In addition to assembling the membrane, phospholipids are also used to assemble the circulating lipoproteins,the main task of which is to transport lipophilic triglycerides and cholesterols through the hydrophilic blood. The human body uses phospholipids as emulsi fi ers.Together with cholesterols and bile acids,they form mixed micelles in the gallbladder to promote the absorption of fat-soluble substances.The human body also uses phospholipids as the surface-active wetting agents in the pleura and alveoli of lung, pericardium,joints,etc[26].

Different kinds of phospholipids have some general properties,but they also own their unique physiological functions (Table 2).

2.4. The physical properties of phospholipids

2.4.1. Phospholipid polymorphisms in water

Phospholipids in water can form many kinds of assemblies, such as micelles,liposomes and hexagonal(HII)phase,which is attributed to the molecular shapes of the phospholipids. The assemblies and molecular shapes of different phospholipids in water are summarized in Table 3[1].Understanding the lipid polymorphisms is necessary for establishing a stable carrier and triggered release lipid-based delivery system.

2.4.2. Factors modulating lipid polymorphisms

Non-bilayer lipids such as unsaturated PE can be stabilized in a bilayer structure by the presence of bilayer preferring lipids such as PS.It is usually found that between 20 and 50 mol%of the bilayer preferring lipids are required to maintain a net bilayer organization when mixed with HIIpreferring lipids such as PE.The structural preferences of these pure and mixed lipid systems can be modulated by a wide variety of factors,such as head group size temperature,hydrocarbon unsaturation,ionic strength,pH,the incorporation of inverted cone molecules and the presence of divalent cations such as Ca2+[35]. (1)The smaller head group of PE(as compared to PC)is consistent with HIIorganization.In addition,in the case of PE,increasing acyl chain unsaturation,temperature and acyl chain length lead to increased cone shape and possible HIIphase formation[35].The lamellar to HIItransition temperature (Tbh)decreases with the increasing chain length and the increasing unsaturation of acyl chain of PE.Many species of naturally occurringPE preferentially adoptaHIIphaseatphysiological temperatures.In terms of Tbhfor various synthetic and naturally derived PE,the readers can refer to Tilcock's review[36].

(2)The hydration of the head group can modulate the polymorphisms.For PE,decreasing the water content or increasing the ionic strength leadsto increased proclivity for HIIstructure.In addition,under the condition of high salt concentration,unsaturated PG,CL and PA can adopt HIIphase[36].

(3)In the case of pure lipid systems,for example,protonation of the PS carboxyl and PA phosphate at low pH valuesleadsto HIIstructure[37,38].Similarobservations extend to mixed lipid systems,where bilayer-HIItransitions are observed for PS-PE and PA-PE systems as the pH is reduced below 5 as such pH values convert the PA and PS to HIIpreferring lipid species[39].

(4)Conventional liposomes as drug carriers are avidly taken up by the mononuclear phagocyte system(MPS), so in order to increase the circulation time of liposomes in vivo,researchersfocuson studyingthelongcirculating liposomes.The study for a synthetic lipid that can extend circulation times fi nally gives rise to the development of polymer grafted bilayers.Several polymer lipids lead to prolonged circulation times,but the mostwellstudied arethoseconsistingofpolyethyleneglycol(PEG)grafted onto PE(Fig.2)[40].For instance,DSPE-PEG2000is a PEGylated phospholipid which is widely applied in various preparations,and PEG layer usually serves as a steric barrier to stabilize the molecule assemblies among them.Nanostructures based on DSPE-PEG2000play an important role in DDS. For instance,DSPE-PEG2000is used in the FDA approved drug product Doxil®[4].

However,the concentration of PE-PEG derivatives can affectthestructuresand propertiesofliposomes.For phospholipid-PEG conjugates,the larger the size of the attached PEG is,the more wedge-shaped the phospholipid-PEG is.Thus,the cone shaped phospholipid-PEG2000and phospholipid-PEG5000molecules form micelles in solution and they can convertDSPC bilayerto micelles athigh phospholipid-PEG concentrations.In contrast,phospholipid-PEG350is able to separate PC head groups enough to cause interdigitation at high concentrations,but not enough to cause micelles formation even in dispersions containing pure phospholipid-PEG350.Phospholipid-PEG750has an intermediate shape,so that when added to DSPC,it induces interdigitated bilayers at moderate concentrations and mixed micelles at higher concentrations[41].

Theexperimental resultsofJohnssonetal.showedthatthe structural evolution of aggregates formed in this system with increasing phospholipid-PEG(PEG molecular weight 2000 or 5000) concentration can be summarized as: liposomes→ discoidal micelles→ spherical micelles[42]. Unlike DPPC and DSPC,the mixtures of EPC and phospholipid-PEG form cylindrical micelles rather than discoidal micelles [43].Moreover,the addition of phospholipid-PEG to DOPE is able to facilitate liposomes formation at physiological conditions(Fig.3).The observations of Johnsson et al.showed that in order to obtain a dispersed phase consisting of only liposomes(predominantly unilamellar),8 mol%phospholipid-PEG2000and greaterthan 10mol%ofphospholipid-PEG750must be added to the DOPE system at physiological conditions. When the concentration of phospholipid-PEG beyond bilayer saturating concentration,small disks and spherical micelles formed[44].

When the concentration of PE-PEG derivatives exceeds 10 mol%,a large portion of discoidal or cylindrical micelles structures formed which have no effect on the delivery for water-soluble drugs.To reach the target site,liposomes must not only possess a long circulation time,but also maintain the encapsulated drugs.Therefore,these two factors must be taken into consideration to choose the most appropriate concentration of PE-PEG derivatives[40].

(5)In 1974,Verkleij et al.[45]observed cylindrical lipid structures using dilauryl phosphatidylglycerol with the additionofCa2+byfreeze-etchelectronmicroscopy.The structurewerealsoobservedinthepresenceofMg2+[46]. In 1975,Papahadjopoulos et al.[47]observed the fusion ofsmallunilamellarPSliposomesinthepresenceofCa2+by negative staining electron microscopy and they observedthefusionofsonicated PSvesicleswithCa2+to formrolled-upcylindricalstructureswhichwerenamed as cochleate cylinders by freeze-fracture electron microscopy[48].Negativelychargedphospholipids,suchas PA,PS,PGandPI,canserveasthemainbuildingblocksof cochleates.In addition,mixture the negatively phospholipids with other phospholipids can also form cochleates,for example,vesicles prepared from PS/PC (PS＞50%)in the presence of Ca2+can form cochleates, which needs higher threshold concentration of Ca2+[47,49].Forcochleates,themolarratioofdivalentcations andphospholipidsis1:2.Ca2+maintainsthecochleatein its rolled form,and bridges each successive layer through ionic interaction [48](Fig.4).Moreover, removing the calcium by chelation with EDTA leads to the cochleates unrolling and spontaneously forming large single-bilayer vesicles[48](Fig.5).

In the case of pure lipid systems,for PS,Ca2+induces crystalline cochleate structure,whereas CL can adopt HIIphase in the presence of Ca2+[50].And for other acidic phospholipids such as PA,PG and PI,their structures on hydration may be sensitive to the fatty acid composition,for example,“cochleate”structure can be observed for saturated species in the presence of Ca2+,however,unsaturated PA adopt HIIphase[51],and unsaturated PG and PI still prefer a bilayer organization by the addition of Ca2+[52,53].

In the case of mixed systems,it has been proved that mixtures of acidic phospholipids(such as PS,CL,PA and PG) with unsaturated(HIIphase)PEaresensitiveto the presenceof Ca2+(and in some cases Mg2+)which can trigger bilayer to HIIphase transitions.In systems stabilized by CL and PA,Ca2+can convert the CL and PA to HIIpreferring species,thus allowing the entire mixture to adopt the HIIphase.This contrasts with systems stabilized by PS,where Ca2+segregates the PS into“cochleate”domains,allowing the PE to revert to the HIIphase it prefers in isolation.Alternatively,in PE systems stabilized by up to 30 mol%(unsaturated)PG,Ca2+appears to reduce the bilayer stabilizing capacity of PG and both PG and PE enter the HIIcon fi guration.However,the behavior of systems stabilized by PI suggests that none of these mechanisms apply.Nayar et al.suggested that to a limited extent Ca2+is able to segregate PI in these mixed systems, where the PI-Ca2+aggregates remain in a hydrated lamellar structure[53].An inability of Ca2+to induce a cochleate structure for PG[52],PI[53]and PA[51]may arise in part from the relatively unsaturated nature of the acyl chains.

2.4.3. The phase transition temperature of phospholipids

The Tcof phospholipids is a temperature at which phospholipids transit from gel to liquid crystalline state.Many factors affect the Tc.1)The nature of the polar head group.PC and PG with the same hydrocarbon chains have similar Tc,but the corresponding PE has a higher Tc.For example,the Tcof DPPC and DPPG are both 41°C,whereas DPPE is 63°C,which is attributed to a stronger head group interaction.2)The length of the hydrocarbon chains.Phospholipids with longer hydrocarbon chains have a higher Tcthan those with shorter ones. For example,the Tcof DSPC is 55°C,and yet DPPC is 41°C.3) The degree of saturation of the hydrocarbon chains.For phospholipids with the same head group and length of aliphatic chain,high saturation in the hydrocarbon chains increases the Tc.For example,the Tcof DSPC is 55°C,whereas DOPC is-20°C.4)Purity.The low purity of phospholipids widen the range of Tc.Naturally occurring phospholipids are usually mixtures of components having different length hydrocarbon chains.Such mixtures would usually be expected to produce broad ill de fi ned transitions,but the syntheticphospholipids usually have de fi nite Tc.For example,the Tcof SPC is-20 to-30°C,whereas DLPC is-20°C[54].Table 4 summarizes the Tcof commonly used phospholipids.

When chosing the phospholipids as carrier materials, many factors must be taken into comprehensive consideration to select a kind of phospholipid with appropriate Tc.

3. The applications of phospholipids in drug delivery systems

3.1. Liposomes

3.1.1. The development of liposomal drug delivery system

Liposomes are vesicles prepared with phospholipids as the main substance,the structure of which is similar to cellular membrane.Liposomes origined from multilaminar vesicles which were clearly taken on morphosis by electron microscope images observed by Bangham and Horne in 1964[58].In 1965,Bangham et al.reported biomembrane model system which was established based on vesicle structures and had ion gradient[59].In 1971,Gregoriadis et al. fi rst used liposomes to deliver bioactive substances[60].After that,liposomes in drug delivery fi eld fl ourish.In 1990,the fi rst injectable liposomal drug-amphotericin B liposome(AmBisome®)was available in Europe[61].In 1995,the fi rst liposomal anticancer drug(Doxil®)which is also the fi rst FDA approved nanomedicine was on the market in the USA[4].

Liposomes as carriers of therapeutic drugs have attracted attention more than 40 years.As a DDS,liposomes have many advantages as follows:delivering both hydrophilic and lipophilic drugs(Fig.6),possessing targeting,controlled release properties,cell af fi nity,tissue compatibility,reducing drug toxicity and improving drug stability.During the researches, the conventional structures of the liposomes have some changes,which have brought out a series of new type liposomes,such as long-circulating liposomes,stimuli-responsive liposomes,cationic liposomes and ligand-targeted liposomes. Liposomes can serve as the carriers of antitumor drugs,antifungal drugs,analgesic drugs,gene therapeutics and vaccines, and there have been some liposomal formulations industrialized which are listed in Table 5.

3.1.2. Considering a variety of factors to select the appropriate phospholipid to prepare liposomes

(1)Synthetic and natural phospholipids have both advantages and disadvantages.The advantages of synthetic phospholipids are that the property is relatively stable, and purity is relatively high;the drawback is that the price is relatively high.However,the advantage of natural phospholipids is that the price is relatively low;the disadvantages are that the purity is dif fi cult to control, and the nature is relatively unstable which can be metabolized to lysophospholipids in the process of usage and storage.So the source should be considered when choosing natural phospholipids to prepare the liposomes.For example,the content of unsaturated fatty acids in soybean phospholipids is higher than that in egg yolk phospholipids.Therefore,it is dif fi cult to obtain quality controlled liposomes product using soybean lecithins[63].In addition,Wang et al.studied the relationship between drug carrying ability of liposomes and PCs.The results showed that the encapsulation ef fi ciency of EPC liposomes was higher than that of SPC liposomes,and the leakage rate was lower than that of SPC liposomes,so EPC liposomes had better drug carrying ability[64].

Based on the above advantages of EPC,the current listing liposomes prepared by natural phospholipids are substantially composed of EPC(Table 5).

(2)pH-sensitive liposomes.In order to make the drugs

escape from being degradated into inactive compounds by enzymes in lysosomes after endocytosis,usually pH-sensitive liposomes are prepared.DOPE is usually selected to prepare pH-sensitive liposomes,however, which is found to suffer from their dif fi culty to form liposomes by themselves.So in order to form stable liposomes,materials containing titratable acidic groups must be added to DOPE.The liposomes established by the above membrane materials have pH-sensitivity, because at neutral pH,the fatty acid carboxyl ion can provide effective electrostatic repulsion,which makes the liposomes stable at lamellar phase,and at acidic pH, fatty acid carboxyl groups are protonated,causing the formation of the HIIphase,which makes the liposomes unstable and easy to aggregate,fuse and release the contents[65].In addition,compared with other phospholipids,liposomes formed by PE contribute to the membranefusion,whichisbecauseofthelowhydration of the polar head of PE[66].Thus,the presence of DOPE can increase the hydrophobicity of liposomal membrane,and promote its interaction with the lipid bilayer.

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To increase the stability of pH-sensitive liposomes,the structures have undergone a series of changes,including: long-circulating DOPE-containing pH-sensitive liposomes and association of pH-sensitive polymers with liposomes.Adding DSPE-PEG to DOPE liposomes is not only able to promote the formation of DOPE liposomes,but also can increase the circulation time of liposomes in vivo[44].Another method for conferring the liposomes with pH-sensitivity is to anchor the pH-sensitive polymers to liposomes.The principle is beyound the scope of this review.

(3)Thermosensitive liposomes (temperature-sensitive liposomes)are very promising strategies for cancer treatment,which effectively utilize the dual advantages of liposomes and hyperthermia to improve the therapeutic effects and reduce toxicity.

In 1978,Yatvin et al.were the fi rst to realize the therapeutic potential of liposomes which are composed of phospholipids that can be achieved Tc(higher than the physiological temperature,but below 42°C)by gentle heating[67].Liposomes have a characteristic Tc,at which the liposome membranes transit from the gel phase to liquid crystalline phase,and the encapsulated drugs are released from the vesicles[67].

The early thermosensitive liposomes are mainly based on DPPC(Tc=41°C).Although pure DPPC liposomes can increase drug release,the amount and rate of release are relatively low. Adding other phospholipids(mainly DSPC and HSPC)to DPPC would have a positive impact on the amount and rate of drug release[68].However,the Tcof liposomes composed of DPPC/ DSPC are enhanced.Furthermore,DSPC added increases thespan of Tc.As a result,only 43-45°C can trigger the release of drugs,but this range can make normal tissue surrounding the tumor at the risk of necrosis.Therefore,it is necessary to fi nd a way that gentle heating(39-42°C)can promote the release of drugs,and simultaneously keep burst relase[68].

In 1999,Anyarambhatla and Needham proposed that incorporating 10 mol% lysolipid monopalmitoylphosphocholine(MPPS)into the PEGylated conventional thermosensitive liposomes composed of DPPC could make the Tcreduce from 43°C to 39-40°C,and enhance the release of drugs upon heating(approximately 50%of drugs will be released at 40°C for 20 s)(Fig.7)[69].ThermoDox®is a lysophospholipid-containing thermosensitive liposome, which is developed by Celsion company.Unfortunately,the combination of ThermoDox®and radiofrequency thermal ablation(RFA)did not signi fi cantly improve the therapeutic effects in third period clinical trials.There are many reasons for the failure,one of which is that drugs loading in ThermoDox®are easy to leak,leading to the loss of drugs before reaching the target site.Another important reason is that most drugs do not reach the target site,causing failure to generate substantial trigger release.Therefore,in order to achieve the desired effect,the lipid composition should be further changed[70].

Another method for preparing thermosensitive liposomes is to incorporate heat-sensitive polymers in liposomes.The temperature sensitivity of such liposomes are mainly dependent on the temperature-sensitive polymers.Moreover,DPPC incorporated to the thermal thermosensitive liposomes can achieve a synergistic effect with the polymers[71].

(4)The types of phospholipids can also affect the delivery ef fi ciency of drugs.

There is evidence that the composition of liposomes markedly contributes to their ability to escape recognition by the MPS.Liposomes that resemble the exterior layer of erythrocytes in composition are particularly resistant to clearance[72].In erythrocytes,PC and SM are mainly in the exterior layer,whereas the PE and PS are restricted to the interior layer[73].Moreover,the fatty acyl chains of phospholipids in the outer layer are more highly saturated than that in the interior layer[74].Evidence showed that the loss of phospholipid asymmetry of erythrocytes might be translated into a signal of recognition by macrophages.This result not only provides a guideline for understanding the behavior of liposomes in vivo,but also provides a reference for the design of liposome DDS.

It has been observed that liposomes containing negatively charged phospholipids such as PS,PG and PA,can be quickly eliminated from the circulation system and focused on the MPS[72].In contrast,liposomes containingnegativelycharged ganglioside(GM1)and PI are able to inhibit the uptake by the MPS and prolong the circulating time[75].Therefore,clearance rate can not be fully explained by negative charge.Allen [76]proposed that the hydrophilicity on the surface can play a very important role in phagocytosis of particles.

In addition,membrane fl uidity can also affect the circulation time ofthe liposomes.Increasingthe rigidity of liposomes by incorporating phospholipids with high Tc(e.g.DSPC)and more rigid phospholipids like SM can reduce their clearance by the MPS[75].Adding SM in liposomal formulation can not only decrease the distribution of liposomes to the MPS[77], but also stabilize the membrane structure[78].The fatty chain is connected to the skeleton via an amide bond in SM molecule,while in the glycerophospholipids,the aliphatic chains are connected to the skeleton of the molecule via an ester bond.In vivo,the amide bond is dif fi cult to be hydrolyzed and degradedby enzymesthan theesterbond,so anamidebondis more stable than an ester bond.Furthermore,SM can form intermolecular hydrogen bonds,so it also has the effect of solidifying the bilayer[79].The successful application of liposomes prepared by SM is Marqibo®[4].

Therefore,we can select the phospholipids to prepare liposomes according to the site of disease.When the sites of diseases are in the MPS,the majority of liposomes need to be removed from the blood circulation within minutes to exert therapeutic effects.So in order to make drugs target to the MPS,conventional liposomes(i.e.liposomes composed of various phospholipids and cholesterol and possibly other lipids,without speci fi c components conferring the property of long circulation in blood)can be prepared.However,the rapid elimination of liposomes has thus far been an obstacle for transporting drugs to the sites of diseases beyond the MPS, such as some tumors.So in order to enhance the circulation time,long circulation liposomes can be prepared[80].

(5)Accelerated blood clearace phenomenon.

When PEGylated liposomes are repeatedly injected into the same animal,the second dose of liposomes would rapidly be cleared from the bloodstream and accumulated in liver and spleen,and this phenomenon is called“accelerated blood clearance(ABC)”.There are many factors known to in fl uence ABC phenomenon,in this review,we mainly focus on the effects of types and dose of phospholipids.1)The effect of the dose of phospholipids on the ABC phenomenon is different with the variation of phospholipids types,for example,when the fi rstphospholipids dose of PEGylatedliposomes composed ofhydrogenatedegg phosphatidylcholine(HEPC)islargerthan 1 μmol/kg in rats,the second injection of PEGylated liposomes cannot induce the ABC phenomenon[81];however,whenliposomes are composed of partially hydrogenated egg phosphatidylcholine(PHEPC),intravenous injection of different phospholipids doses of PEGylated liposomes(i.e.0.05,0.5 or 5 μmol/kg)as a fi rst dose can cause the second speci fi c dose (5 μmol/kg)of injected PEGylated liposomes to induce the ABC phenomenon[82].In addition,the magnitude of ABC phenomenon is different when lipsomes composed of the same phospholipid,which could be reduced by the increasing of phospholipidsdose[81,82].2)Phospholipidscanalsoaffectthe magnitude of ABC phenomenon.Xu et al.demonstrated that ABC phenomenon of liposomes prepared by unsaturated phospholipids(SPC,EPC and ESM)was more obvious than that of saturated phospholipids(DPPC and HSPC)[83].

In addition,Depth study showed that PEG is not necessary for the generaion of ABC phenomenon for liposomes.Laverman et al.[82]demonstrated that conventional liposomes can also accelerate blood clearance of the second injection of conventional or PEGylated liposomes.Unlike PEGylated liposomes,the results of Ishida et al.showed that a high dose (5 μmol/kg)of conventional liposomes(without a PEG-coating) can induce the same phenomenon,while a low lipid dose (0.001 μmol/kg)did not[81].

3.2. Intravenous lipid emulsions

Intravenous emulsions are originated from the development of intravenous nutrition emulsions.In 1962,Intralipid was successfully developed in Sweden and proved to be safe and effective,which lay the foundation for the development of medicinal intravenous emulsions[84].Intravenous fat emulsions possess many advantages,including targeting,reducing drug toxicity,which make them attract more and more attentions recently.

Phospholipids as zwitterionic surfactants can be used as emulsifying agent of O/W type emulsions,and due to their biological and non-toxic characteristics,they can be used as emulsi fi ers for intravenous injection[2].The O/W type emulsions are mainly composed of two parts:the oil core and the emulsifying agents on the surface.Unlike liposomes,fat emulsions are suitable for large-scale industrial production and relatively stable below 25°C for long term.More importantly,a large quantity of lipophilic drugs can be dissolved in the hydrophobic core of emulsions[85].

Currently,the listed drug containing injectable emulsions using phospholipids as an emulsi fi er are summarized in Table 6[5].The main disadvantage of phospholipids as emulsi fi ers is that in the process of emulsi fi cation,sterilization and storage,natural lecithins are readily hydrolyzed to generate lysophospholipids which can lead to hemolysis after intravenous injection.Phospholipid mixtures derived from the egg yolk of birds and soybean have stronger emulsifying ability than single component.However,using puri fi ed lecithins[86]can reduce the generation of lysophospholipids.The physical nature of lecithins and the resulting emulsion stability are changing a lot,depending on the sources and the degrees of puri fi cation of the emulsi fi ers.

The component of PC determines the surface properties of fat emulsions,and also affects their distribution in the body. Lenzo et al.studied the in vivo behavior of emulsions using EPC,DOPC,DMPC,DPPC,and 1-palmitoyl-2-oleoyl phosphatidylcholine(POPC)as an emulsi fi er.In that study,emulsions were prepared in the size of about 150 nm,and the elimination rate in rat plasma mainly depended on the types of phospholipids.When these emulsions were injected into the blood of conscious rats,the elimination rate of formulations using DPPC as an emulsi fi er was the slowest.The fat emulsions stabilized by EPC and POPC were metabolized similarly to natural chylomicrons.The triacylglycerols were rapidly hydrolyzed by lipoprotein lipase(LPL),followed by the remnants derived from the emulsions was taken up by liver. Phospholipids from the injected emulsions were removed more slowly and became associated with the high-density lipoprotein(HDL)fractions of the plasma.Emulsions containing DPPC were metabolized differently.The triacylglycerols were eliminated very slowly from plasma, which indicated that LPL hydrolysis did not occur and phospholipids were not transferred to the HDL.For the emulsions containing DMPC,the triacylglycerols and cholesterol esters of emulsion were eliminated very fast,but phospholipids were not transferred to the HDL.For DOPC emulsions,clearances were slower than EPC and POPC emulsions,but it was effectively transferred to the HDL[87].Redgrave et al.showed that the clearances of DSPC-stabilized fat emulsions from the plasma were faster than the DPPC-stabilized fat emulsions.Although DSPC fat emulsions can hardly be hydrolyzed by LPL,it can be avidly taken up by the macrophages in liver and spleen,which is different from the liposomes prepared by DSPC[88].

SM is a very important component of lipoproteins surface, andthesurfaceconcentration ofSMplays animportantrole in metabolism and distribution of emulsions.Redgrave et al.[88] observed that adding SM to emulsions caused the prolongation of circulation time in plasma and the reduction of uptake by liver,which are attributed to the decrease of binding of apolipoprotein E and the reduction of LPL-mediated lipolysis induced by SM.

Recently,in order to increase the circulation time of intravenous emulsions(Fig.8A)and enhance the antitumor effect of drugs,PEGylated derivatives as emulsi fi ers are usually added to prepare long-circulating emulsions(Fig.8B). These effects of PEG derivatives can be attributed to the increase of hydrophilicity and steric stability of the emulsion surface[80].However,repeatedinjectionsofPEGylated emulsions can also induce the ABC phenomenon[89],and the factors related to phospholipids affecting it still need in-depth study.

3.3. Micelles

The applications of phospholipids in micelle systems mainly includePC/Bilesalt mixedmicellesand phospholipid micelles.

3.3.1. PC/bile salt mixed micelles

In classical mixed micelles(MMs),water-insoluble phospholipid molecules combine with another surfactant such as bile salt(BS)to form MMs,and the hydrophobic core of MM can encapsulate poorly soluble drugs[90].Insoluble drugs dispersed in MMs in molecular state can improve their bioavailability.Phospholipids can form MMs with multiple substances,and PC/BS MMs have entered into pharmaceutical drug market.

BS,detergent-like chemicals produced by liver and stored in the gallbladder,are able to solubilize PC to a large extent, andform a clear MMs solutionwhich promotes the adsorption of fat-soluble substances.Therefore,PC/BS MMs serve as a carrier that can greatly improve solubility of poorly soluble drugs.In addition,the results of a previous research showed that PC can protect against plasma membrane disruption by BS[91].

Valium®and Konakion®representing the classical MM system prepared by SPC and glycocholic acid are two MM preparations which can be available in currently medical market[6].Intravenous injection of these MMs exhibits good stability and compatibity[92].

There are two essentially different types of PC/BS MMs, which is dependent on the ratio of BS and PC.When BS:lecithin molar ratios are less than approximately 2:1,lamellar particles similar to a lecithin bilayer arrangement are found (Fig.9A).When molar ratios exceed 2:1,a different type of micelle structure is found.This is a highly isometrical particle of globular shape(Fig.9B),probably having a centrosymmetric arrangement of the molecular constituents[93].Therefore,a drawback of PC/BS MMs is that a change in the lecithin/BS ratio or in the total concentration leads to a change in the MM size and structure[94].

3.3.2. Phospholipid micelles

In 1994,it was observed that PE-PEG molecules are able to form micelles in an aqueous environment[95](Fig.10).When trying to prepare long-circulating liposomes,phoshpholipid-PE-PEG mixtures could form micelles rather than liposomes if PE-PEG content exceeds certain critical limit[40]. This phenomenon is considered as a nuisance until it is realized that PE-PEG molecules can aggregate to form sterically stabilized micelles(SSMs)which have potential as a particulate carrier to deliver poorly water-soluble therapeutics,especially anticancer molecules[96].In PE-PEG micelles, two fatty acyl chains of the phospholipid residues make hydrophobic interaction of micellar core stronger which leads to higher stability of micelles,and PEG residue exposed on the surface can avert the uptake by the MPS which can prolong the circulating time of micelles[97].

The saturation level of PE has an effect on the circulating half-life of the micelles.Anatoly et al.demonstrated that replacing DSPE that is lipid component of PE-PEG with DOPE to form micelles can reduce the circulating half-life[98].

Although the SSMs have many advantages,they also have drawbacks,one of which is the limited solubilization capabilityforpoorly soluble drugs.In some cases,to improvedrugsolubilization,additionalmicelle-forming compounds can be added to PE-PEG micelles to form MMs. EPC is added to the micelle composition to form sterically stabilized mixed micelles(SSMMs)[99].Insertion of a small percentage of EPC into a PE-PEG micellar system is able to increase the volume of the hydrophobic region of micelles. This would therefore provide a larger space for the hydrophobic drugs to be solubilized.The results showed that the SSMMs approximately double the paclitaxel encapsulation ef fi ciency[99].

The addition of EPC into the micelle composition is able to increase the encapsulation ef fi ciency of paclitaxel,but when EPC content exceeds certain critical limit,cylindrical micelles begin to dominate and the solubilization potential for drugs decreases[100].

Kaminskas et al.studied the ABC phenomenon of PEGylated micelles and liposomes.The results demonstrated that although PEG micelles could stimulate production of anti-PEG IgM,which led to accelerated clearance of subsequently administered PEGylated liposomes.But the second injection of PEGylated micelles were not substrates for IgM binding and did not exhibit a similar ABC phenomenon[101].

3.4. Drug-phospholipid complexes

Many synthetic and herbal drugs possess the problem of poor oral bioavailability,and the reason is their very low water solubility or poor permeation through the biological membrane.Poorly soluble drugs have suffered from low bioavailability and inef fi cacy in therapy due to their low dissolution pro fi le in biological fl uid.Without a proper level of drug concentration in the gastrointestinal(GI) fl uid,the drugs cannot be effectively transported by the epithelia of the GI tract, resulting in low systemic absorption[102].However,although most bioactive molecules of plants are biologically polar or water-soluble,they are dif fi cult to pass through the lipid-rich biological membrane and be absorbed by human,the reasons of which include:1)large molecular weight,2)low lipid solubility[103].

When studied liposomes,Bombardelli et al.stumbled that natural fl avonoids have a special af fi nity for phospholipids, and they can combine to form complexes which exhibit markedly different biological properties and pharmacological activities from parent drugs[104].Thereafter,the drugphospholipid complexes(aptly called as phytosomes)gradually attracted people'sattention,and theirstudiesare increasing.Later researchers foundthat manytypesofnatural ingredients and natural extracts can be made from phospholipid complexes.The interaction had been attributed to the formation ofhydrogen bondsand/orhydrophobicinteractions between the two molecules[105].

Phytosomes are characterized by amphiphilicity,which makesthem havenot only better dissolution in the GI fl uid but also better absorption through the lipophilic membrane system or tissue.Drug-phospholipid complexes can improve the bioavailability ofparentdrugswhichhaveeither verylow lipid solubility or very poor water solubility.Therefore,both kinds of drugs can be complexed for improving biopharmaceutical properties[106].In addition to improving the drug absorption, drug-phospholipid complexes also have the following advantages:1)increasing the stability of drugs,2)prolonging the duration of action of drugs[7].

When treated with water,phytosomes form liposome-like structures(Fig.11A).However,there are many differences between phytosomes and liposomes,mainly including:1)thesize of liposomes is much bigger than that of phytosomes;2) there is the formation of new bonds in phytosomes,whereas no chemical bond is formed in liposomes;3)in liposomes, there are hundreds or thousands of PC molecules surrounding the water-soluble compounds.Conversely,the molar ratio of PC and natural ingredients is 1:1 or 2:1 for phytosomes,which depends on the chemical bond-forming material;4)the natural active ingredients are dissolved in the medium or wrapped by the membrane in liposomes,while in phytosomes the active principle is anchored through the chemical bond to the polar head of phospholipids[107](Fig.11B).

In recent years,the reports about phospholipid complexes of natural active ingredients are gradually increasing.In abroad,there have been dozens of phytosome products such as Silybin Phytosome™,Ginkgo Phytosome™,and Ginseng Phytosome™[7].

3.5. Cochleates

Cochleates are a lipid-based macromolecular assembles, having enormous potential in drug delivery.They have many characteristics including:1)Ability to deliver a variety of drugs,such as antifungal agents,polypeptides,proteins, vaccines,oligonucleotides and genes[108].Cochleates as a drug delivery platform are applicable to macromolecules as well as small molecule drugs that are hydrophobic,amphiphilic,negatively charged,positively charged which have poor bioavailability[109](Fig.12).At fi rst,cochleates are formed by ionic interaction between negatively charged liposomes and bivalent cations,subsequently,Syed et al. used cationic drugs themselves as the bridging agents and successfully prepared the cochleates without adding any bivalent cations[110](Fig.12E).2)High stability.There is evidence that drug molecules encochleated in the cochleate cylinders are present in the inner layers of a solid,stable, water and oxygen impermeable structure which is capable of increasing the stability of drugs[111].The encochleated drug molecules can remain intact,even though the outermost layer of cochleates can be exposed to the harsh environmental condition or enzyme,which contributes to the ef fi caciousness of cochleates for the oral delivery of drugs [112].Cochleates can be stored in a cation-containing buffer solution for over 2 years at 4°C,and at least a year at room temperature as a lyophilized powder[113].Cochleates can be lyophilized to a free fl owing powder that can be incorporated in capsules for oral administration or redispersed in water for injection[114].3)Ability to signi fi cantly improved oral absorption of encochleated drug molecules[110].This enhanced activity can be attributed to the membrane fusion capability of cochleates which can be envisioned as membrane fusion intermediates in many naturally occurring membrane fusion events[110,115].The high tension of edges of cochleates makes them interact with the tissue membrane[110].4)High safety.Cochleates are usually composed of PS and Ca2+,which are derived from naturally occurring substances.PS has been used as a nutrient supplement which have a role in supporting the mental functions in aging brains[116].

4. Conclusions

In the material world,applications are determined by properties,which are essentially determined by structure.Phospholipids are endogenous substances,so understanding their nature and physiological functions in vivo is very necessary for us to comprehend and design its application in drug delivery platform.For example,from the distribution of phospholipids in the cell membrane,we can understand the circulation time of liposome composed of different phospholipids in vivo.The discussed examples show that phospholipids offer manifold possibilities to be used as excipients in DDS,which can deliver both hydrophilic and lipophilic drugs.Thus,suitable carrier have to be thoughtfully selected for every therapeutic.The applications of phospholipids in drug delivery are certainly not limited to those described in the present article,and there are still other applications,such as suspensions,solid lipid nanoparticle and so on.In the future there will be a broad space for the development of DDS based on phospholipids.Although the phospholipids have wide applications in DDS,they also face challenges,for instance,liposomes and intravenous lipid emulsions have ABC phenomenon.In sum,the applications ofphospholipids in DDS are the coexistence of opportunities and challenges.

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*Corresponding author.Shenyang Pharmaceutical University,No.103,Wenhua Road,Shenyang 110016,China.Tel./fax:+86 24 23986316. E-mail address:pharmdeng@gmail.com(Y.Deng).

Peer review under responsibility of Shenyang Pharmaceutical University.

http://dx.doi.org/10.1016/j.ajps.2014.09.004

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Biocompatibility

Amphiphilicity

Drug delivery systems