lipidic solip np

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Advanced Drug Delivery Reviews 56 (2004) 1257–1272

Solid lipid nanoparticles for parenteral drug delivery

S.A. Wissinga, O. Kayserb, R.H. Mullerb,*

aDDS, Drug Delivery Services, GmbH, Kronskamp 11, 24119 Kronshagen, GermanybDepartment of Pharmaceutics, Biopharmaceutics and Biotechnology, Free University of Berlin, Kelchstrasse 31, 12169 Berlin, Germany

Received 13 August 2003; accepted 20 December 2003

Abstract

This review describes the use of nanoparticles based on solid lipids for the parenteral application of drugs. Firstly, different

types of nanoparticles based on solid lipids such as ‘‘solid lipid nanoparticles’’ (SLN), ‘‘nanostructured lipid carriers’’ (NLC)

and ‘‘lipid drug conjugate’’ (LDC) nanoparticles are introduced and structural differences are pointed out. Different production

methods including the suitability for large scale production are described. Stability issues and drug incorporation mechanisms

into the particles are discussed. In the second part, the biological activity of parenterally applied SLN and biopharmaceutical

aspects such as pharmacokinetic profiles as well as toxicity aspects are reviewed.

D 2004 Elsevier B.V. All rights reserved.

Keywords: SLN; Solid lipid nanoparticles; NLC; LDC; Toxicity; Pharmacokinetics; Degradation; Parenterals

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1258

2. Nanoparticles based on solid lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1259

2.1. Definitions and structural features. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1259

2.1.1. SLN. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1259

2.1.2. NLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1259

2.1.3. LDC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1260

2.2. Production of lipid nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1260

2.2.1. High pressure homogenisation (HPH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1260

2.2.2. Production of SLN via microemulsions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1260

2.2.3. Preparation by solvent emulsification-evaporation or -diffusion . . . . . . . . . . . . . . . . . . . . . . 1261

2.2.4. Preparation by w/o/w double emulsion method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1261

2.2.5. Preparation by high speed stirring and/or ultra sonication . . . . . . . . . . . . . . . . . . . . . . . . 1261

2.2.6. Scale up feasibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1262

2.3. Stability of SLN dispersions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1262

2.4. Incorporation of drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1263

2.5. Release of incorporated drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1264

0169-409X/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.addr.2003.12.002

* Corresponding author. Tel.: +49-30-838-50678; fax: +49-30-838-50616.

E-mail address: [email protected] (R.H. Muller).

S.A. Wissing et al. / Advanced Drug Delivery Reviews 56 (2004) 1257–12721258

2.5.1. Drug release models. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1264

2.5.2. In vitro release of drugs from SLN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265

3. Biological activity and biopharmaceutical aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265

3.1. General aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1265

3.2. Administration and drug liberation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1266

3.2.1. Pharmacokinetic profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1266

3.3. Safety aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1266

3.3.1. Toxicity and status of excipients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1266

3.3.2. Blood clotting and interaction with serum proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . 1266

3.3.3. Allergic reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1267

3.4. Tissue distribution and drug targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1267

4. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1268

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1268

1. Introduction trolled release of the incorporated drugs [9]. However,

In the 1960s, the first safe parenteral fat emulsion

(Intralipid) was developed by Wretlind for parenteral

nutrition [1]. This was the beginning of a new delivery

system for lipophilic drugs, which can be incorporated

easily into the oil droplets. Successful market products

are, e.g. Diazemuls (1970s) and Diprivan (1980s). The

main advantage of this carrier system is the reduction

of side effects caused at the injection side [2]. A major

disadvantage however is the critical physical stability

of the drug containing emulsions due to a reduction of

the zeta potential (ZP) which can lead to agglomera-

tion, drug expulsion and eventually breaking of the

emulsion [3].

Another interesting parenteral carrier systems are

the liposomes. They have been described for the first

time by Bangham et al. in the 1960s and were

introduced as drug delivery vehicles in the 1970s

[4,5]. Trade products are, e.g. Ambisome (1990,

Europe, 1997, USA), DaunoXome (1996, Europe

and USA) and Doxil (1995, USA, 1996, Europe).

These products have been developed in order to

reduce toxic side effects of the incorporated highly

potent drugs and increase the efficacy of the treatment

[6,7]. Major obstacles for the development of liposo-

mal formulations were—and partly still are—limited

physical stability of the dispersions, drug leakage, low

activity due to no specific tumour targeting, non

specific clearance by the mononuclear phagocytic

system (MPS) and difficulties in upscaling [6,8].

Polymeric nanoparticles made from non-biode-

gradable and biodegradable polymers are yet another

innovative parenteral carrier system. Advantages of

these particles are site-specific targeting and con-

the cytotoxicity of the polymers after internalisation

into cells is a crucial and often discussed aspect [10].

Also, large scale production of polymeric nanopar-

ticles is problematic. Therefore, this carrier system has

so far not been relevant for the pharmaceutic market.

In the middle of the 1990s, the attention of

different research groups has focussed on alternative

nanoparticles made from solid lipids, the so-called

solid lipid nanoparticles (SLN or lipospheres or

nanospheres) [11–26]. The SLN combine the advan-

tages of other innovative carrier systems (e.g. phys-

ical stability, protection of incorporated labile drugs

from degradation, controlled release, excellent toler-

ability) while at the same time minimising the

associated problems. SLN formulations for various

application routes (parenteral, oral, dermal, ocular,

pulmonar, rectal) have been developed and thorough-

ly characterised in vitro and in vivo [27–41]. A first

product has recently been introduced to the Polish

market (Nanobase, Yamanouchi) as a topically ap-

plied moisturiser.

At the turn of the millenium, modifications of

SLN, the so-called nanostructured lipid carriers

(NLC) and the lipid drug conjugate (LDC) nano-

particles have been introduced to the literature [42–

45]. These carrier systems overcome observed limi-

tations of conventional SLN.

This paper intends to describe briefly the different

lipid based carrier systems SLN, NLC and LDC,

structure and associated features, stability, applied

production methods, drug incorporation and drug

release mechanisms. The bioactivity of SLN after

parenteral application, i.e. tolerability, toxicology,

cellular uptake, albumin adsorption, pharmacokinet-

S.A. Wissing et al. / Advanced Drug Delivery Reviews 56 (2004) 1257–1272 1259

ics, tissue distribution and drug targeting is reviewed

in detail.

2. Nanoparticles based on solid lipids

2.1. Definitions and structural features

SLN, NLC and LDC are particles with a solid lipid

matrix with an average diameter in the nanometer

range. In addition to lipid and drug, the particle

dispersions contain surfactants as stabilisers. All exci-

pients are GRAS substances or have an accepted

GRAS status, therefore, a wide variety of substances

can be used for formulating purposes [46].

2.1.1. SLN

SLN are particles made from solid lipids (i.e. lipids

solid at room temperature and also at body tempera-

ture) and stabilised by surfactant(s). By definition, the

lipids can be highly purified triglycerides, complex

glyceride mixtures or even waxes [14]. Recently, SLN

based on para-acyl-calix[4]arenes have been prepared

and studied [47,48]. Through the work of various

research groups, the carrier system SLN has been

characterised intensively. For detailed information

on production, characterisation and application, the

reader is referred to the main review papers up to date

[17,49,50]. The first patents have been granted in

1993 and 1996 and contain claims on different pro-

duction methods of SLN [12,14].

The main features of SLN with regard to parenteral

application are the excellent physical stability, protec-

tion of incorporated labile drugs from degradation,

controlled drug release (fast or sustained) depending

on the incorporation model, good tolerability and site-

specific targeting. Potential disadvantages such as

insufficient loading capacity, drug expulsion after

polymorphic transition during storage and releatively

high water content of the dispersions (70–99.9%)

have been observed.

The drug loading capacity of conventional SLN is

limited (generally up to approximately 25% with

regard to the lipid matrix, up to 50% for special

actives such as Ubidecarenone) by the solubility of

drug in the lipid melt, the structure of the lipid matrix

and the polymorphic state of the lipid matrix

[13,15,18,19,21,49,51–53]. If the lipid matrix con-

sists of specially similar molecules (i.e. tristearin or

tripalmitin), a perfect crystal with few imperfections is

formed. Since incorporated drugs are located between

fatty acid chains, between the lipid layers and also in

crystal imperfections, a highly ordered crystal lattice

cannot accommodate large amounts of drug [19].

Therefore, the use of more complex lipids (mono-,

di-, triglycerides, different chain lenghths) is more

sensible for higher drug loading.

The transition to highly ordered lipid particles is

also the reason for drug expulsion. Directly after

production, lipids crystallise—partially—in higher

energy modifications (a, hV) with more imperfections

in the crystal lattice [54–57]. The preservation of the

a-modification during storage and transformation af-

ter administration (e.g. by temperature changes) could

lead to a triggered and controlled release and has

recently been investigated for topical formulations

[58]. If however a polymorphic transition to h takes

place during storage, the drug will be expelled from

the lipid matrix and it can then neither be protected

from degradation nor released in a controlled way.

2.1.2. NLC

NLC have been introduced at the end of the 1990s

in order to overcome the potential difficulties of SLN

described above [43,44,57]. The goal was the devel-

opment of a nanoparticulate lipid carrier with a certain

nanostructure in order to increase the payload and

prevent drug expulsion.

This could be realised in three ways. In the first

model, spatially different lipids, e.g. glycerides com-

posed of different fatty acids are mixed. Using spa-

tially different lipids leads to larger distances between

the fatty acid chains of the glycerides and general

imperfections in the crystal and thus to more room for

the accommodation of guest molecules. The highest

drug load could be achieved by mixing solid lipids

with small amounts of liquid lipids (oils). This model

is called ‘‘imperfect type NLC’’.

If higher amounts of oil are mixed with the solid

lipid, a different type of nanostructure is present.

Here, the solubility of the oil molecules in the solid

lipid is exceeded; this leads to phase separation and

the formation of oily nanocompartments within the

solid lipid matrix [59–61]. Many drugs show a higher

solubility in oils than in solid lipids so that they can be

dissolved in the oil and still be protected from


degradation by the surrounding solid lipids. This type

of NLC is called ‘‘multiple type NLC’’ and can be

regarded as an analogue to w/o/w emulsions since it is

an oil-in-solid lipid-in-water dispersion.

Since drug expulsion is caused by ongoing crys-

tallisation or transformation of the solid lipid, this can

be prevented by the formation of a third type, the

‘‘amorphous type NLC’’. Here, the particles are solid

but crystallisation upon cooling is avoided by mixing

special lipids (e.g. hydroxyoctacosanylhydroxystea-

rate and isopropylmyristate) [60,61].

So far, NLC have only been exploited for the

topical delivery, however their advantages over con-

ventional SLN are also of interest for parenteral

application routes.

2.1.3. LDC

SLN are useful for the incorporation of lipophilic

drugs. Due to partitioning effects during the production

process, only highly potent hydrophilic drugs which

are effective in low concentrations (e.g. LHRH or EPO)

can be firmly incorporated in the solid lipid matrix

[32,62]. In order to overcome this limitation, the so-

called LDC nanoparticles with drug loading capacities

of up to 33% have been developed at the turn of the

millenium [45,63]. Here, an insoluble drug–lipid con-

jugate bulk is prepared either by salt formation (e.g.

with a fatty acid) or by covalent linking (e.g. to esters or

ethers). In the salt formation process, the free drug base

and fatty acid are dissolved in a suitable solvent. The

solvent is then consequently evaporated under reduced

pressure. For the covalent linking, the drug (salt) and a

fatty alcohol react in presence of a catalyst and the LDC

bulk is then purified by recrystallisation. The obtained

LDC bulk is then processed with an aqueous surfactant

solution to a nanoparticle formulation using high

pressure homogenisation (HPH).

In the literature, studies on the cytotoxicity of

surface-modified diminazenestearate and -oleate

LDC, plasma protein absorption (2-DE) and activity

against trypanosomiasis via brain targeting have been

performed [45,64,65]. The results will be presented in

the context of this review.

2.2. Production of lipid nanoparticles

Different approaches exist for the production of

finely dispersed lipid nanoparticle dispersions. In this

section, the various methods are described briefly,

also with regard to scaling up possibility, a pre-

requisite for the introduction of a product to the

market.

2.2.1. High pressure homogenisation (HPH)

HPH is a suitable method for the preparation of

SLN, NLC and LDC and can be performed at elevated

temperature (hot HPH technique) or at or below room

temperature (cold HPH technique) [13,14,18,66–68].

The particle size is decreased by cavitation and

turbulences.

Briefly, for the hot HPH, the lipid and drug are

melted (approximately 5 jC above the melting point

of the lipid) and combined with an aqueous surfactant

solution having the same temperature. A hot pre-

emulsion is formed by high speed stirring. The hot

pre-emulsion is then processed in a temperature con-

trolled high pressure homogeniser, generally a maxi-

mum of three cycles at 500 bar are sufficient. The

obtained nanoemulsion recrystallises upon cooling

down to room temperature forming SLN, NLC or

LDC.

The cold HPH is a suitable technique for process-

ing temperature labile drugs or hydrophilic drugs.

Here, lipid and drug are melted together and then

rapidly ground under liquid nitrogen forming solid

lipid microparticles. A pre-suspension is formed by

high speed stirring of the particles in a cold aqueous

surfactant solution. This pre-suspension is then homo-

genised at or below room temperature forming SLN,

NLC or LDC, the homogenising conditions are gen-

erally five cycles at 500 bar.

The influence of homogeniser type, applied pres-

sure, homogenisation cycles and temperature on par-

ticle size distribution has been studied extensively

[13,18,69,70]. Both HPH techniques are suitable for

processing lipid concentrations of up to 40% and

generally yield very narrow particle size distributions

(polydispersity index < 0.2) [71,72].

2.2.2. Production of SLN via microemulsions

The group of Gasco has developed and optimised a

suitable method for the preparation of SLN via micro-

emulsions which has been adapted and/or modified by

different labs [12,16,22,66,73,74]. Firstly, a warm

microemulsion is prepared by stirring, containing

typically c 10% molten solid lipid, 15% surfactant


and up to 10% cosurfactant. This warm microemul-

sion is then dispersed under stirring in excess cold

water (typical ratio c 1:50) using an especially

developed thermostated syringe. The excess water is

removed either by ultra-filtration or by lyophilisation

in order to increase the particle concentration.

Experimental factors such as microemulsion com-

position, dispersing device, temperature and lyophili-

sation on size and structure of the obtained SLN have

been studied intensively. It has to be remarked criti-

cally, that the removal of excess water from the

prepared SLN dispersion is a difficult task with regard

to the particle size. Also, high concentrations of

surfactants and cosurfactants (e.g. butanol) are neces-

sary for formulating purposes, however less desirable

with respect to regulatory purposes and application.

2.2.3. Preparation by solvent emulsification-evapora-

tion or -diffusion

Different academic groups have attempted the

production of SLN via precipitation. In the solvent

emulsification-evaporation [25,47,48], the lipid is

dissolved in a water-immiscible organic solvent (e.g.

toluene, chloroform) which is then emulsified in an

aqueous phase before evaporation of the solvent under

reduced pressure. Upon evaporation of the solvent, the

lipid precipitates forming SLN. An important advan-

tage of this method is the avoidance of heat during the

preparation, which makes it suitable for the incorpo-

ration of highly thermolabile drugs. Problems might

arise due to solvent residues in the final dispersion;

Sjostrom et al. have calculated the amount of toluene

residues as 20–100 ppm in final dispersions. Also,

these dispersions are generally quite dilute, because of

the limited solubility of lipid in the organic material.

Typically, lipid concentrations in the final SLN dis-

persion range around 0.1 g/l, therefore, the particle

concentration has to be increased by means of, e.g.

ultra-filtration or evaporation.

In the solvent-diffusion technique, partially water-

miscible solvents (e.g. benzyl alcohol, ethyl formate)

are used [75,76]. Initially, they are mutually saturated

with water to ensure initial thermodynamic equilibri-

um of both liquids. Then, the lipid is dissolved in the

water-saturated solvent and subsequently emulsified

with solvent-saturated aqueous surfactant solution at

elevated temperatures. The SLN precipitate after the

addition of excess water (typical ratio: 1:5–1:10) due

to diffusion of the organic solvent from the emulsion

droplets to the continuous phase. Similar to the

production of SLN via microemulsions, the dispersion

is fairly dilute and needs to be concentrated by means

of ultra-filtration or lyophilisation.

Average particle sizes around 100 nm and very

narrow particle size distributions can be achieved by

both solvent evaporation methods.

2.2.4. Preparation by w/o/w double emulsion method

Recently, a novel method based on solvent emul-

sification–evaporation for the preparation of SLN

loaded with hydrophilic drugs has been introduced

to the scientific community [66]. Here, the hydrophil-

ic drug is encapsulated—along with a stabiliser to

prevent drug partitioning to the external water phase

during solvent evaporation—in the internal water

phase of a w/o/w double emulsion. This technique

has been used for the preparation of sodium cromo-

glycate-containing SLN, however, the average size

was in the micrometer range so that the term ‘‘lipo-

spheres’’ in the sense as a term for nanoparticles is not

used correctly for these particles.

2.2.5. Preparation by high speed stirring and/or ultra

sonication

The SLN were developed from lipid micropar-

ticles produced by spray congealing followed by

lipid nanopellets produced by high speed stirring or

sonication [77,78]. A great advantage of this meth-

od is the fact that the equipment is common in

every lab and the production can easily be done.

The problem of high speed stirring was a broader

particle size distribution ranging into the microme-

ter range. This lead to physical instabilities such as

particle growth upon storage. This could be im-

proved by higher surfactant concentrations, which

in order might be correlated with toxicological

problems after parenteral administration. A further

disadvantage is potential metal contamination due

to ultra sonication.

Therefore, studies have been performed by various

research groups in order to improve the stability of the

obtained SLN dispersions. Generally, high speed

stirring and ultra sonication are combined and per-

formed at elevated temperatures for some time

[79,80]. Quite narrow and physically stable distribu-

tions can be achieved, however the lipid concentration


is low ( < 1%) and the surfactant concentration is

comparably high.

2.2.6. Scale up feasibility

For the introduction of a product to the market,

scaling up feasibility is of uttermost importance. For

the two primarily used methods for the preparation

of lipid nanoparticles, HPH and production via

microemulsions, scaling up possibilities have been

investigated.

For HPH, a GMP unit for clinical batch production

was developed and validated which can achieve batch

sizes between 2 and 10 kg SLN dispersion. Further, a

large scale production line was designed having a

capacity of 50–150 kg SLN dispersion per h by

placing two homogenisers in series. The production

of SLN with these lines can be performed in discon-

tinuous or continuous modes [18,81–84].

For the production via microemulsions, a system

has been developed allowing the production of 100 ml

microemulsion which is then poured in excess cold

water forming SLN. The dispersing water ration

ranged from 1:1 to 1:10, leading to batch sizes of

up to 1.1 l. Experimental factors which have been

investigated were the pressure of the pneumatic cyl-

inder, the temperature and the needle gauge of the

syringe containing the microemulsion as well as the

volume of dispersing water [85].

2.3. Stability of SLN dispersions

The physical stability of SLN dispersions has been

investigated intensively, e.g. by measurements of

particle size (photon correlation spectroscopy, PCS;

laser diffraction, LD), charge (ZP) and thermal anal-

ysis (differential scanning calorimetry, DSC).

Physical stability of optimised aqueous SLN dis-

persions is generally more than 1 year ([51,53] and

Muller et al. could show stability for SLN made from

glyceryl palmitostearate or tribehenate for up to 3

years by PCS [20]. The average diameter of the main

population remained between 160 and 220 nm for the

investigated period.

Freitas and Muller investigated the effect of light

and temperature on the physical stability of SLN

dispersions composed of 10% tribehenate and 1.2%

poloxamer 188 [86]. They found that particle growth

could be induced by an input of kinetic energy (light,

temperature) to the system. Storage under artificial

light lead to gelation of the system within 7 days of

storage, under day light within 3 months and in

darkness particle growth started after 4 months stor-

age. The gelation was accompanied by a decrease in

ZP from � 24.7 to below � 18 mV. The influence of

the storage temperature on particle size has also been

analysed. The authors found that the particle size

measured by LD increased rapidly at elevated temper-

atures and remained stable for more than 180 days

when refrigerated. Again, particle growth could be

correlated to a decrease in ZP from � 24.7 to approx-

imately � 15 mV.

Freitas and Muller have also correlated the phys-

ical stability of the aforementioned SLN formulation

with the polymorphic state of the lipid [54]. After

hot HPH, the lipid is present in a mixture of hV, aand sub a polymorphs. The input of kinetic energy

causes a transformation to hV accompanied by gel

formation. By inhibition of this transformation (re-

frigerated, dark storage), this transformation could be

avoided. These studies show that the development of

optimal storage conditions can improve the physical

stability of previously regarded unstable SLN for-

mulations tremendously.

For the recently developed SLN based on calixar-

enes, stability data of more than 1 year have been

published. Shahgaldian et al. have further investigated

the influence of the ionic strength on stability, observ-

ing strongest destabilisation by sulphate ions [47].

Apart from optimised storage conditions of labile

SLN dispersions, they can also be spray-dried or

lyophilised. For spray-drying, a melting point of the

lipid matrix of >70 jC is a pre-requisite. Typically,

protectors such as trehalose are added to the dispersion

in concentrations of about 20–25%. For best reconsti-

tution effects, SLN concentration in the spraying me-

dium should be approximately 1%. The influence of

lipid type and concentration, carbohydrate type and

concentration, redispersion medium and spraying me-

dium have been investigated by Freitas et al. [20,87].

Lyophilisation can be employed as an alternative

very sensitive drying method. The process has been

optimised with regard to operating conditions, lipid

concentration, type and concentration of cryoprotectant

and redispersing conditions [20,88]. Heiati et al. have

investigated the effect of cryoprotective sugars on the

size of neutral and negatively charged SLN after

Table 1

Examples of drugs relevant for parenteral application incorporated

into SLN

Drug References

AZT-P and derivatives [89,99,100]

Camptothecin [40,98]

Clobetasol propionate [76]

Cortisone [51]

Sodium cromoglycate [66]

Cyclosporin A [97]

Diazepam [41,51]

Diminazenediaceturate [45]

Doxorubicin [31,101–103]

Etomidate [93]

3V5V-Dioctanoyl-5-fluoro-2V-deoxuridine [79]

Hydrocortisone [34]

Idarubicin [31]

[D-Trp-6]LHRH [32]

Magnetite [74]

Mifepristone [80]

Paclitaxel [91,103,104]

Pilocarpine [33]

Piribedil [29]

Prednisolone [51,93,94]

Progesterone [34,66]

Retinoic acid [67]

Tetracaine [93]

Thymopentin [105]

Tobramycin [35,37,106]


lyophilisation and reconstitution [89]. The azidothymi-

dine palmitate (AZT-P) loaded SLN were composed of

trilaurin, stabilised with lecithin and they were pre-

pared by solvent emulsification–evaporation and sub-

sequent HPH. They found that trehalose was the most

effective cryoprotectant in a sugar/lipid ratio of 3:9 for

neutral SLN and 2:6 for negatively charged SLN. Also,

trehalose was most effective for preventing drug ex-

pulsion upon reconstitution. Lim et al. showed for all-

trans retinoic acid-loaded SLN excellent redispersion

characteristics [67]. Here, the PCS diameter increased

merely from 182 to 265 nm and the polydispersity

index from 0.173 to 0.200 upon redispering. No

changes in ZP and in drug loading were observed.

For SLN based on calixarenes, difficulties in redispers-

ing after lyophilisation have been observed, i.e. these

particles require up to 1 h of ultrasonic treatment,

however no cryoprotectant was added to the formula-

tions, so that these results have to be regarded as

preliminary.

Radomska et al. have developed an analytical meth-

od for the determination of the chemical stability of the

lipid matrix of SLN [90]. They extracted the lipid from

SLN dispersions and analysed it qualitatively and

quantitatively by gas chromatography. For SLN made

from cetyl palmitate and tristearate, they could show

chemical stability of the lipids of H90% after 2 years

storage at room temperature as well as physical stability

for up to 2 years when stored at 4–8 jC.Regarding the parenteral application of SLN, steril-

ity has to be ensured. Schwarz et al. have studied the

influence of autoclaving conditions on particle size and

ZP of different SLN formulations [13]. They observed

that steric stabilisation of trilaurin SLNwith poloxamer

188 was not suitable for autoclaving due to its critical

flocculation temperature. For lecithin stabilised SLN

formulations, autoclaving was possible. No increase in

particle size was observed by PCS. Alternatively, SLN

could be sterilised by gamma irradiation or (if the size is

well below 200 nm) by filtration. Various other re-

search groups have also published particle size and ZP

data of sterilised SLN dispersions revealing excellent

stability of the formulations [47,67,89,91].

2.4. Incorporation of drugs

An innovative and successful carrier system should

allow a high loading capacity for incorporated drugs

as well as long-term incorporation. Many different

drugs have been incorporated in SLN, NLC or LDC.

Table 1 lists examples relevant for the parenteral

application including the corresponding references.

The drug can be incorporated between fatty acid

chains, between lipid layers or in imperfections.

Depending on the drug/lipid ratio and solubility, the

drug is located mainly in the core of the particles, in

the shell or molecularly dispersed throughout the

matrix. This influences the drug release and will be

explained in more detail in Section 2.5. The influence

of drug incorporation on particle size and physical

stability has been described before [19,51]. By opti-

mising of the formulation, long term physical stability

can be achieved [20].

The solid lipid core of SLN should increase the

chemical stability of incorporated drugs and protect

them from degradation. Therefore, entrapment effi-

ciency (E.E.) and long term retention of the drug in

the lipid matrix have to be ensured. Heiati et al. have

studied the incorporation and retention of AZT-P in

Fig. 1. Proposed structural models for drug-containing SLN

(modified after [93]).


trilaurin SLN [89]. They found a dependence of the

E.E. on the phospholipid content of the formulation

and on the charge of the SLN. The formation of

phospholipid bilayers and the ability of amphiphilic

drugs such as AZT-P to integrate within these bilayers

is postulated as the reason for the increased E.E.

However, it should be kept in mind that phospholipids

can also form independent vesicles and integrate

drugs. These vesicles scatter only weakly and might

not be detected with PCS or LD. Ahlin et al. have

studied the location and E.E. of lipophilic spin probes

(C14- and C18-Tempo) by electron paramagnetic res-

onance (EPR) [92]. They could locate the spin probes

between the solid glyceride core and the soft phos-

pholipid layer on the particle surface or at the lipid/

water interface and found E.E.s close to 100%. Hou et

al. have investigated mifepristone-loaded SLN based

on glycerol monostearate, stabilised with Tween 80

regarding E.E. and retention after 1 month [80]. For

formulations optimised regarding physical stability,

they found an E.E. of 87.89% on day 1 and 82.05%

after 1 month storage at 4–8 jC, the decrease corre-

lated with lipid transitions. It has to be pointed out that

lipid transition during storage and subsequent drug

expulsion from the matrix can be avoided by formu-

lation as NLC. Also, for hydrophilic drugs the for-

mulation as LDC is more suitable for higher loading

capacities and retention within the particle matrix

upon storage.

2.5. Release of incorporated drugs

Next to the characterisation of the carrier system

SLN, the release characteristics have been studied by

various research groups. It could be shown that the

release profile can be influenced by modifications in

the lipid matrix, surfactant concentration and produc-

tion parameters. Mehnert et al. have performed inten-

sive in vitro release studies and developed structural

models for different release characteristics [93]. This

section of the paper presents in vitro and in vivo

release data published by different groups.

2.5.1. Drug release models

Mehnert et al. have observed variable release

profiles for SLN loaded with different drugs

[19,93–95]. They found the fastest release for tetra-

caine, followed by etomidate and extremely sustained

release for prednisolone. The proposed structural

models represented in Fig. 1 serve as an explanation.

Tetracaine SLN were prepared produced by hot

HPH at a drug concentration well below saturation

solubility in the lipid, i.e. during the production, the

drug partitioned to the water phase. Upon cooling, the

lipid precipitates first due to phase separation. Simul-

taneously, the drug re-partitions into the liquid lipid

phase and its concentration in the outer shell liquid

lipid increases continuously. Finally, the drug-

enriched shell crystallises. Atomic force microscopy

(AFM) studies on coenzyme Q10 loaded SLN have

shown a soft surface of the particles undermining this

thesis [96]. If the drug is located primarily in the shell

of the particles, a burst release is highly likely. Other

factors contributing to a fast release are large surface

area, high diffusion coefficient (small molecular size),

low matrix viscosity and short diffusion distance of

the drug.

Etomidate SLN represent the homogeneous solid

solution matrix model [93]. A homogeneous distribu-

tion of drugs in the particle matrix can be achieved by

cold HPH or by the avoidance of potentially drug-

solubilising surfactants.

SLN loaded with prednisolone released the drug in

vitro (i.e. in absence of enzymes) over a period of

more than 5 weeks [93,94]. For them, the drug-

enriched core model is proposed. The prednisolone

concentration in the matrix material was close to its

saturation solubility. Cooling of the hot nanoemulsion

prepared by HPH leads to supersaturation of the drug

in the liquid lipid and subsequently to drug precipita-

tion prior to lipid crystallisation. The drug-enriched

core is surrounded by a practically drug-free lipid

shell. Due to the increased diffusional distance and


hindering effects by the surrounding solid lipid shell,

the drug has a sustained release profile.

2.5.2. In vitro release of drugs from SLN

Apart from the in vitro release data presented in the

above section, various other studies have been pub-

lished with regard to potential parenteral application.

Cavalli et al. have prepared stealth and non-stealth

tripalmitin SLN loaded with paclitaxel in order to

provide an alternative for the parenteral administration

[91]. The commercially available product TaxolR is a

toxicologically critical micellar solution of the drug in

Cremophor EL. Cavalli et al. report sustained in vitro

release: 0.1% of the paclitaxel is released into the

receptor medium (phosphate buffer, pH 7.4) after 120

min, this is correlated to first pseudo zero oder

kinetics. The same academic group has previously

shown similarly sustained in vitro release profiles for

doxorubicin and idarubicin (0.1% after 120 min) in

contrast to burst release from reference solutions [31].

For stearic acid SLN containing cyclosporin A, they

determined an in vitro release of < 4% after 2 h com-

pared to >60% from solution [97].

Yang et al. determined the in vitro release of

camptothecin from stearic acid SLN in conjunction

with potential targeting to the brain using a dialysis

bag technique at 37 jC [98]. The data revealed a

sustained release and could be fitted to a Weibull

distribution (t1/2 = 23.1 h).

Heiati et al. have studied the in vitro release of 3V-azido-3V-deoxythymidine palmitate (AZT-P) from tri-

laurin SLN using a bulk-equilibrium reverse dialysis

sac technique at 37 jC [99]. The observed initial burst

is attributed to partial AZT-P localisation in phospho-

lipid micelles. Further, a dependence of release profile

on the type of phospholipid could be shown, i.e.

phospholipids with phase transition temperatures

(PTT) below 37 jC lead to fast release, PTT >37

jC represented a stronger diffusional barrier causing

slower release.

Wang et al. have prepared an optimised SLN

formulation containing 3V,5V-dioctanoyl-5-fluoro-2V-deoxyuridine (DO-FUdR) for enhanced brain target-

ing [79]. They studied the in vitro release by a bulk-

equilibrium reverse dialysis sac technique in at 37 jCcompared to DO-FUdR and also FUdR reference

solutions. Hundred parcent drug were released from

the FUdR solution after less than 2 h. After 4 h,

approximately 80% drug were released from DO-

FUdR solution and only 60% from the SLN formula-

tion. The SLN release profile was biphasic, the initial

burst release was followed by a prolonged release

(80% drug released after 48 h). This can be correlated

to the drug-enriched shell model. The drug present in

the shell is released fast, followed by gradually release

from the lipid core.

Hu et al. have studied in vitro release kinetics of

clobetasol propionate from SLN prepared by solvent

emulsification-diffusion [76]. The lyophilised product

was dispersed in aqueous dissolution medium (PEG

400 solution containing Tween 80) without dividing

membranes. The authors observed a biphasic release

profile following Higuchi (45% release after 3 h,

followed by 5.9% release per day for 4 days). How-

ever, the chosen release model is not quite suitable for

lipophilic drugs. Therefore, these data have to be

judged critically.

For detailed information of the in vitro release of

the mentioned drugs from SLN, the reader is kindly

referred to the corresponding references. Concluding,

it can be stated that depending on the formulation,

sustained in vitro release can be achieved for various

drugs that are of interest for the parenteral application.

3. Biological activity and biopharmaceutical

aspects

3.1. General aspects

Because of their small size, SLN may be injected

intravenously and used to target drugs to particular

organs. The particles, as with all intravenously

injected and colloidal particulates, are cleared from

the circulation by the liver and spleen. To facilitate

drug targeting, in tumour tissue for example, a retic-

uloendothelial system avoidance (stealth) facility may

be incorporated using polyoxyethylene as described

well for classical polymeric nanoparticels in the past.

This may be achieved using block polyoxyethylene

polypropylene copolymers like Pluronic F188 in

which the hydrophobic portion of the molecule forms

the nanoparticle matrix while the water soluble poly-

oxyethylene block forms a hydrophilic coating on the

particle Stealth SLN increase the tumour accumula-

tion [104], antibacterial activity [106] of antiparasitic


and antifungal drugs and allow brain delivery of

anticancer drugs not capable of crossing the blood

brain barrier (BBB) [102]. Besides of new attractive

drug delivery strategies we have also addressed bio-

pharmaceutical aspects regarding safety and toxicity

of the drug delivery system relevant for patients.

3.2. Administration and drug liberation

SLN are generally injected either intravenously,

intramuscularly or subcutaneously or to the target

organ. Because of their minimum size below 1 Am,

SLN formulations can be used for systemic body

distribution with a minimised risk of blood clotting

and aggregation leading to embolism. Also SLN

provide a sustained release depot of the drug when

administered subcutaneously or accumulated in the

MPS. Incorporated drug is gradually released on

erosion (e.g. degradation by enzymes) or by diffusion

from the particles. The rate of release may be con-

trolled by the nature of the lipid material [108], particle

size [109], and choice of surfactant [107,109,110] and

also by inner structure of SLN as discussed above. The

particle size of intravenously administered drug must

be below 5 Am to avoid blocking of fine capillaries

leading to embolism. By production techniques de-

scribed before, the size is mostly below this value.

Special problems regarding blood clotting and aggre-

gation under high electrolyte conditions in vivo are

discussed in Section 3.3.2.

3.2.1. Pharmacokinetic profile

Administration of drugs incorporated in SLN ver-

sus free drugs leads to different pharmacokinetic

profiles as well described for doxorubicin [101,102]

and paclitaxel [103]. Using SLN as carriers mostly a

three to 5-fold enhancement of plasma peaks is

observed. Interestingly plasma formulations for doxo-

rubicin SLN showed a bi-exponential curve [101,102]

with high AUC, a lower rate of clearance, and a

smaller volume of distribution in comparison to the

free drug. The biphasic behaviour is explained prob-

ably due to the slow distribution of doxorubicin in

SLN. SLN formulations reduced cardiotoxic side

effects of doxorubicin in Wistar rats and prolonged

the metabolisation of doxorubicin to doxorubicinol.

Uptake of the SLN in cells of the MPS differed

from the size and composition of the particles. Uptake

of SLN can be avoided by PEG-ylation leading to

long circulating particles, so-called Stealth SLN. Sev-

eral authors have worked on Stealth SLN, but finally

only a few reports on the in vivo behaviour have been

published yet. Interestingly, stealth doxorubicin solid

lipid nanoparticels showed similar circulating time

and pharmacokinetic behaviour in comparison to

unmodified nanoparticels [102]. Main reasons might

be explained by similar low surface hydrophobicity of

both types of particles avoiding adsorption of blood

proteins mediating liver uptake. The modified tissue

distribution of doxorubicin in SLN was related to a

slower distribution and passage of particles through

biological barriers and slow drug release from the

lipid matrix into the blood [49]. This biopharmaceut-

ical behaviour may explain lower drug blood concen-

trations and reduction of severe side effects.

3.3. Safety aspects

3.3.1. Toxicity and status of excipients

Since today no SLN product for parenteral use is

on the market, but intensive studies for SLN in

different bioassays have been published recently

[49,50]. Most studies have been conducted with

glycerides composed of fatty acids, which are mostly

accepted as safe. Good tolerability depends in the first

line of the used surfactant and secondarily on the

lipid. To formulate parental SLN, surfactant of GRAS

status must be employed, e.g. lecithin, Tween 80,

Poloxamer 188, Span 85, and sodium glycocholate.

When performing bolus injections into mice good

tolerability was found for most of these surfactants

coating SLN. As described, for cetyl palmitate SLN

with different surfactants no acute toxicity, and no

increase in liver and spleen weight was observed

[111]. After autopsy and histopathology no significant

evidence was documented that SLN were acute toxic

to tested animals.

3.3.2. Blood clotting and interaction with serum

proteins

Cellular binding of SLN formulations, with em-

phasis on erythrocytes, is important because it affects

not only blood clotting, embolic effects but also may

change pharmacokinetic behaviour. SLN have distinct

affinities to red blood cells depending on the surfac-

tant used. By Flow Cytometry, Olbrich et al. showed


that SLN formulation consisting of Compritol as

matrix material and Tween 80 and Poloxamer 188

showed no binding to erythrocytes (SLN binding

< 10.0%) [112]. In contrast, when Span 85 was used,

blood cell affinity of labelled SLN was increased

leading to significant aggregation of red blood cells

(75.3%).

The interaction of SLN with the major circulatory

protein, serum albumin, has been investigated recent-

ly. By PCS and AFM albumin adsorption on the

particle surface formed a capping layer of 17 nm

increasing the size of tested particle populations only

slightly (final particle size: 150, 183, 193 nm, respec-

tively) [113]. AFM imaging revealed that the SLNs

are protected by this layer against flattening on

surfaces. At physiological albumin concentrations

(35–50 g/l) the increased size was not important

enough to explain blood cell aggregation [113].

3.3.3. Allergic reactions

SLN has been used as drug delivery systems as

discussed here intensively, but also drug unloaded

as Vaccine adjuvants. Focussing on drug delivery

systems mainly, we want to describe SLN as

vaccine adjuvants briefly. For further detailed

reviews, we refer to contributions by Muller et al.

[114,115]. Shortly, antigens adsorbed on the surface

or entrapped in the matrix of SLN induce an

enhanced immunological response. SLN showed to

be equivalent to Freud incomplete adjuvants (FIA)

protecting against different infections in chicken

and sheep. Decreasing particle size and increasing

hydrophobicity improves the adjuvancy of these

particles [114]. The particles offer a prolonged

and controlled presentation of the antigen to the

immune system.

Immunologic and finally allergic reactions are not

desired for SLN as drug delivery systems. Immuno-

logic reactions can be affected by the production

process or by the lipids and additives used in the

formulation. Applying HPH and producing with a

LAB 40 homogeniser made of V2A steel may lead

to a final product that contains traces of nickel with a

risk of allergic reaction. Krause et al. showed by

atomic absorption spectroscopy that no iron as the

dominant metal in steel was detectable in traces in

nanosuspension formulations, e.g. Fe < 1 ppm [116].

All detectable metals were distinctly below concen-

trations of 20 ppm accepted by the German Federal

Institute for Drugs (BfArM).

Since Cremophor EL was recognised as allergic

principle in paclitaxel emulsions for parenteral use

[117], the use surfactants for i.v. administration is

under critical debate. Today tested SLN were proved

to be not toxic in in vivo and in vitro systems. Muller

et al. showed in in vivo test that test animals did not

suffer from anaphylatic shock or related symptoms

[111]. Detailed investigations regarding the immunos-

timulation and release of interleukins from activated

macrophages showed no significant interleukin con-

centrations (IL 6, IL 12, TNF, IFN) causing proin-

flammatory reactions [110,115]. Even the absence of

IL 12, that is mostly associated with severe toxic

effects in vivo, indicates an acceptable non acute toxic

profile of SLN [110,115].

3.4. Tissue distribution and drug targeting

The accumulation of SLN within the Kupffer

cells of the liver is predominantly found after

intravenously injection in case non-stealth SLN

are injected. With the exception of liver diseases

like hepatic neoplasms, liver infections like hepatitis

and visceral leishmaniasis and systemic candidasis,

and physiologic disorders (e.g. hypercholesterine-

mia), passive liver targeting should be avoided.

The systemic use of colloidal carriers is limited

by the presence of MPS, and it is consequently

necessary to avoid such recognition. SLN carriers

are mostly recognised by macrophages due to the

physicochemical characteristics in particle size, sur-

face charge and surface hydrophobicity as discussed

before [108,109]. Various attempts have been made

to achieve long circulation times by avoiding MPS

uptake as discussed for Stealth SLN before. One

outstanding example of targeting specific organs is

brain delivery. The uptake of SLN by the brain

might be explained by adsorption of blood proteins

like apolipoproteins on particle surfaces mediating

the adherence to endothelial cells of the BBB. This

effect was well described for the trypanocidal drug

diminazene formulated as LDC. Diminazen acetu-

rate does not cross the BBB because of its hydro-

philicity. Formulation as LDC increased crossing of

the BBB and reducing CNS infection of Trypano-

soma brucei infected mice [45].


4. Outlook

Most of the discussed drugs belong to classical

low molecular weight drugs. In the future we can

expect an increasing number of therapeutic mole-

cules like proteins [62], oligonucleotides [118] and

DNA as vaccine or as drug for gene therapy [119].

Because of their physical and chemical instability in

the GIT, these drugs must be administered parenter-

ally. With the exception of gene therapy, up to today

only a few publications have reported about protein

or nucleotide formulation with SLN [62,105]. But, as

SLN are a new and innovative therapeutic delivery

system, we can expect in the future an increasing

number of contributions describing delivery of re-

combinant proteins.

In contrast to recombinant proteins, first studies

have been published about gene delivery we want to

discuss here briefly. This platform technology—called

TransoplexR—has been developed as an alternative

gene delivery system for lipoplexes. Olbrich et al.

showed their efficacy in transfection of COS-1 cells

with the beta Galactosidase gene [119]. Transoplex

were produced in a mixture of a cationic lipids like

cetylpyridinium chloride, benzalkonium chloride or

cetrimide and a Compritol or cetyl palmitate as helper

lipid. Main advantages of the technology are easy

production, higher physical stability, lower cytotox-

icity and increased transfection in comparison to

commonly used liposome formulations like [119].

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