solid lipid nanoparticles for targeted brain drug...

ews 59 (2007) 454–477www.elsevier.com/locate/addr

Advanced Drug Delivery Revi

Solid lipid nanoparticles for targeted brain drug delivery☆

Paolo Blasi ⁎, Stefano Giovagnoli, Aurélie Schoubben, Maurizio Ricci, Carlo Rossi

Department of Chemistry and Technology of Drugs, School of Pharmacy, University of Perugia, Via del Liceo 1, 06123 Perugia, Italy

Received 21 November 2006; accepted 24 April 2007Available online 22 May 2007

Abstract

The present review discusses the potential use of solid lipid nanoparticles for brain drug targeting purposes. The state of the art on surfactant-coated poly(alkylcyanoacrylate) nanoparticles specifically designed for brain targeting is given by emphasizing the transfer of this technology tosolid lipid matrices. The available literature on solid lipid nanoparticles and related carriers for brain drug targeting is revised as well. The potentialadvantages of the use of solid lipid nanoparticles over polymeric nanoparticles are accounted on the bases of a lower cytotoxicity, higher drugloading capacity, and best production scalability. Solid lipid nanoparticles physicochemical characteristics are also particularly regarded in order toaddress the critical issues related to the development of suitable brain targeting formulations. A critical consideration on the potential applicationof such technology as related to the current status of brain drug development is also given.© 2007 Elsevier B.V. All rights reserved.

Keywords: CNS drug delivery; Brain targeting; Blood-brain barrier (BBB); Solid lipid nanoparticles (SLN); NLC; LDC; Polymeric nanoparticles; Polysorbate 80(Tween® 80)

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4542. Blood-brain barrier biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4553. Brain drug delivery using surfactant coated polymeric nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4574. Advantages of SLN over PACA NPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460

4.1. Preparation and sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4604.2. Toxicity, stability and biocompatibility issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461

5. SLN physicochemical features with respect to brain drug delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4645.1. SLN structure, drug loading, and stability issues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4645.2. Surfactants and brain targeting features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467

6. SLN for brain drug targeting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4687. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4718. Legend for lipid substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471

☆ This review is part of the Advanced Drug Delivery Reviews theme issue on“Lipid Nanoparticles: Recent Advances”.⁎ Corresponding author. Tel.: +39 075 5855133; fax: +39 075 5855163.E-mail address: [email protected] (P. Blasi).

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1. Introduction

In the last decade, an emerging interest has been growingtowards brain drug targeting where issues have been widelydiscussed [1–8]. The increasing awareness of the lack of rational

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455P. Blasi et al. / Advanced Drug Delivery Reviews 59 (2007) 454–477

and common efforts among different and complementary re-search areas has pointed out the need for a deeper understandingand a closer collaboration among diverse research experts [7,9]of the field. These issues along with poor knowledge regardingthe physiology of the central nervous system (CNS) have beenthe main limiting factors in the development of effective drugsand appropriate drug delivery systems (DDS) for brain targeting[1,2,6,7]. In fact, delivering drugs to the CNS is impaired by thepresence of the blood-brain barrier (BBB) that represents themain obstacle for CNS drug development [2,10].

Aworrying updated picture of the worldwide CNS pathologyincidence displayed that, in Europe alone, about 35% of the totalburden of all diseases is caused by brain diseases [1] and about1.5 billion people worldwide suffer from CNS disorders. More-over, due to an average lifespan increase to almost 70 years of ageat which about 50% of the population starts revealing evidence ofAlzheimer's disease (AD) [5], an increase of CNS disorder globalburden from the current 11% to 14%, is expected by 2020 [11].For genetic, age and contingent reasons, most of the degenerativeCNS pathologies, such as Creutzfeldt-Jakob's, Parkinson's (PD),Alzheimer's, Huntington's disease, multiple sclerosis andamyotrophic lateral sclerosis, are developing as a consequenceof wrong lifestyle and increased risk factors, such as alcohol,drugs or dietary abuse as well as stress [12–14]. In addition, CNSinvolvement may occur because of primary effects of humanimmunodeficiency virus (HIV) infection or secondary effects ofimmune suppression. About 75% of patients with acquiredimmune deficiency syndrome eventually develop some featuresof subacute encephalitis and other secondary effects, such asprimary CNS lymphoma and progressive multifocal leukoence-phalopathy [12,15–20]. This emergency is becoming particularlyserious in developing countries, as a result of a wide anduncontrolled spread of HIV infection.

In spite of this worrying picture, in the last number of yearsthe CNS drug market has become the largest of all therapeuticareas, recording worldwide an amazing +13% growth in 2003,reaching 77.2 billion USD [21]. This is also due to the advancesreported in the treatment and understanding of some seriousCNS pathologies [22–27]. In this context, the pharmaceuticaltechnology contribution to this area has been found of para-mount importance [5,6,28–30]. Several DDS and strategieshave been developed for the sole purpose of an effective drugdeposition to the CNS [31–36], while other carriers, fabricatedfor different aims, have been turned to a possible brain targetingapplication [27,37]. Among others, nanoparticles (NPs) havebeen considered as carriers of election to overcome the BBBissue [36,38,39].

Several systems have been developed and among these, poly(ethylene glycol) (PEG) stabilized poly(lactide) (PLA) [40] andchitosan [41] NPs, conjugated with the peptidomimeticmonoclonal antibody OX26, gave good results. NPs obtainedby conjugating poly(lactide-co-glycolide) PLGA to five shortsynthetic peptides, to mimic the synthetic opioid peptide MMP-2200 [42], and a Lectin–PEG–PLA conjugated system [43]showed to be equally effective in brain targeting. Moreover, asignificant brain uptake was achieved by using polysorbate 80(P80) as a coating system [44–46].

Among other NP formulations, solid lipid nanoparticles(SLN) have recently found reappraisal as potential DDS forbrain targeting [36,38,44,47–49]. The present review will reportthe state of the art on surfactant-coated NPs designed for braintargeting, and the transfer of this technology to SLN and relatedcarriers will be emphasized on the basis of their cytotoxicity,drug loading capacity, and production scalability. Besides, SLNphysicochemical characteristics will be discussed in order toaddress the issues related to the development of suitable braintargeting formulations.

2. Blood-brain barrier biology

The brain is one of the most important organs of the humanbody, if not the most, and its homeostasis is of primaryimportance. In fact, specific interfaces, also referred to asbarriers, tightly regulate the exchange between the peripheralblood circulation and the cerebrospinal fluid (CSF) circulatorysystem. These barriers are represented by the choroid plexus(CP) epithelium (blood-ventricular CSF), the arachnoid epithe-lium (blood-subarachnoid CSF), and the BBB (blood-braininterstitial fluid) [50]. The concentration and clearance ofendogenous and exogenous molecules, essential for the normalbrain functions or dangerous because of their toxicity, are strictlyregulated by the anatomic and physiologic features of suchbarriers [51,52].

The presence of the BBB is certainly the most critical issueencountered in brain drug delivery. Indeed, among the possiblestrategies to deliver therapeutic molecules into the brain, namelyintracerebral, intraventricular, and intravascular delivery, thelatest represents the most reliable one because of its potentialefficacy, safety, and compliance. The brain microvasculature, ifnot for the BBB, would represent an extraordinary way to accessthe brain, with the possibility of virtually distributing therapeuticmolecules to all brain areas [7]. In fact, even though the volumeoccupied by capillaries and endothelial cells is about 1% and0.1% of the brain volume respectively, the brain microvascu-lature develops a total surface area of ∼20 m2 and a total lengthof∼400 miles [6]. This high vascularization confers to the braina very intricate network of capillaries with an average distancebetween capillaries of ∼40 μm. This means that every singlebrain cell is placed virtually at no more than 20 μm from acapillary, thus creating a space that can be permeated (simplediffusion) by small molecules in a half a second [6]. Unfor-tunately, the physiological features of the brain microvasculaturerestrict enormously the number of drugs that can enter the brainupon systemic administration. It becomes hence easier tounderstand the difficulties in developing new CNS drugs byconsidering that more than 98% of the new potentialneurotherapeutic molecules are unable to cross the BBB [1].Generally, but not always, a molecule has to possess peculiarphysicochemical characteristics to overcome the BBB, such ashigh lipophilicity and a molecular weight b500 Da [6]. This ruleis invalidated by the presence of the transport systems (generallydivided in 3 classes: carrier-mediated transport, active effluxtransport, and receptor-mediated transport) to different regionsof the BBB, conferring to this barrier dynamic permeability

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characteristics, barely predictable only by considering itslipophilicity and molecular weight [53,54].

Brain capillaries, differently from the peripheral capillaries,present no fenestrae, a low amount of pinocytosis vesicles andparticular tight junctions (TJs) also known as zonula occludens[55–57]. TJs are structures that form a narrow and continuousseal surrounding each endothelial and epithelial cell at the apicalborder (Fig. 1) and are aimed at strictly regulating the movementof molecules through the paracellular pathway [58]. The BBBendothelial TJs show some differences in morphology, compo-sition, and complexity features from those of both the epitheliaand peripheral endothelia [59]. These structures, together withthe brain endothelial cells, make an almost impermeable barrierfor drugs administered through the peripheral circulation.

A further contribution to the peculiar BBB functions is givenby the periendothelial accessory structures represented byastrocytes [51,60], pericytes [61,62], and the basal membrane.(Fig. 1) Astrocytes, generally classified into fibrous andprotoplasmic, represent the major component (∼90%) of thebrain mass. Fibrous astrocytes have a star-like morphology andoften present many long processes, known as “end-feet”, that endon the basal membrane of the BBB (Fig. 1). These cells have amultitude of functions important for the brain homeostasis(maintenance of K+ levels, inactivation of neurotransmitters,regulation and production of growth factors and cytokines), manyof which are related to the production of apolipoprotein E (ApoE)[60]. Pericytes are spherical cells holding a prominent nucleus andmany primary and secondary lysosomes in the cytoplasm and

Fig. 1. A representative cross-section of a cerebral capillary of the BBB (adapted frohttp://www.expertreviews.org/).

seem to be virtually involved in the BBB formation, differenti-ation processes, and integrity [62].

As previously mentioned, the presence of BBB transportsystems further complicates this scenario. In fact, from a drugdelivery point of view, these transporters may assist or hinder thedelivery of drugs to the brain. The carrier-mediated transport(e.g. transporter proteins for glucose, essential amino acids,monocarboxylic acids, and nucleosides) may be able to shuttledrugs or pro-drugs into the brain in therapeutic concentrations,mimicking nutrients or endogenous compounds [63–65]. Theuse of the receptor-mediated transport to gain access to the brainis another very attractive strategy. Receptors for endogenouslarge molecules (e.g. insulin, transferrin, leptin, Apo, and manyothers) highly expressed on the BBB luminal side, areresponsible for their shuttling to the brain by transcytosis. Thebinding of drugs or drug containers (e.g. liposomes and NPs) tospecific ligands (peptides) or peptidomimetic monoclonalantibodies makes it possible to ship the construct into the brainby mimicking an endogenous molecule [6,9,66,67]. Unfortu-nately, the presence of active efflux transporters to the BBB alsolimits the therapeutic efficacy of drugs virtually able to accessthe brain.

The P-glycoprotein (P-gp) is an ATP-dependent drugtransport protein present at the apical membranes of differentepithelial cell types including those forming the BBB [68–70]. Itwas demonstrated, both in vitro and in vivo, that BBB P-gp canprevent the accumulation of many molecules including a varietyof drugs (e.g. vincristine, anthracyclines, taxanes, cyclosporine

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A, digoxin, dexamethasone, and others) in the brain [70]. P-gpinhibition has been proposed as a possible strategy for enhancingthe brain drug penetration [71,72]. Some of the surfactantsgenerally employed in pharmaceutical formulations have shownto inhibit P-gp and some of them are currently undergoingclinical screening for the treatment of multidrug resistant tumors[71].

3. Brain drug delivery using surfactant coated polymericnanoparticles

Out of the several different approaches previously mentioned,the use of NPs has been regarded as having great potential for thedelivery of drugs into the CNS. The term NPs refers to well-defined particles ranging in size approximatively from 10 to1000 nm (1 μm) [73] with a core-shell structure (nanocapsules) ora continuous matrix structure (nanospheres). The active com-pounds may be entrapped within NPs and/or adsorbed or boundto their surface. Different kinds of polymers such as poly(alkylcyanoacrylate) (PACA), chitosan, PLA, poly(glycolide)(PGA) and their copolymers (PLGA) have been used to prepareNPs [40,74]. To date, poly(butylcyanoacrylate) (PBCA) has been,perhaps, the most employed for NP fabrication [75].

The major limiting factor for the systemic use of NPs is theirrapid clearance from the blood circulation by the reticuloendo-thelial system (RES). The extent of this uptake dependsmainly onparticle size, surface charge, and surface properties [76,77]. Infact, colloidal particles, characterized by variable hydrophobicsurface properties, are efficiently coated by specific plasmacomponents (opsonins), such as immunoglobulins (IgG), albu-min, the elements of the complement system, fibronectin, andothers, and then cleared from the blood stream by the phagocyticcells within minutes [78–80]. Intravenously (i.v.) administeredNPs mainly distribute into liver (60–90% of the injected dose),spleen (2–10%), lungs (3–20% and more), and bone marrow(N1%) [73]. Additionally, extravasation affects consistently NPbody distribution. In fact, the structure of the peripheral capillarywalls in different organs and tissues displays interendothelial gapsas large as 150 nm. This passive targeting to the liver stronglylimits the use of NPs in delivering drugs to sites in the body otherthan RES, such as the CNS, that, on the contrary, is characterizedby a very low permeable continuous endothelium [58].

Based on the above considerations, a proper modification ofNP surface characteristics and size, can represent a highly

Fig. 2. Schematic representation of P80-coated P

effective strategy to alter NP biodistribution, enhancing thus theirblood circulation time and deposition in non-RES organs [81].

In this respect, as early as in the '80s, different approacheswere proposed for the modification of the hydrophobic particlesurface; most of them were based on the physical adsorption ofPEG-containing surfactants [82–90].More recently, surface cova-lent modification with PEG derivatives has been explored as analternative strategy [91–95]. All authors showed that this coatingreduced NP liver uptake increasing simultaneously their persis-tance in the pheripheral circulation as well as their deposition intoother organs, including non-RES organs, for several hours.

Tröster et al. first hypothesized that the surfactant contactangle may be indicative in predicting the body distribution ofcoated NPs [96]. Based on this assumption, in a later work [88],the authors studied the in vivo body distribution of surfactant-coated and non-coated poly(methyl methacrylate) (PMMA)NPs of 131±30 nm in size. A series of poloxamers (also knownwith the proprietary name Pluronic®) (poloxamer 188, 407,184, 338, and poloxamine 908), polysorbates (also known withthe proprietary name Tween®) (polysorbates 20, 60, and 80),and Brij® 35, were used as coating material to investigate thepossible correlation between the NP surface properties and theirin vivo biodistribution [88]. The results indicated that: i) nocorrelation exists between the body distribution pattern and thecontact angle of the NP coating materials; ii) although all usedsurfactants decreased the liver uptake and increased the uptakein other organs and tissues, poloxamer 338 and poloxamine 908were the most powerful surfactants in enhancing NP bloodlevels and in increasing the spleen uptake while reducing theliver uptake; iii) P80 and poloxamer 184 yielded the highestaccumulation in the heart, muscles, kidneys, and the brain.Therefore, it was thought that polysorbates and poloxamerscould be useful for specific targeting to non-RES organs. Inparticular, the total NP brain amount was increased with allsurfactants up to 24 h, especially by polysorbates 60, and 80,poloxamers 407, 184, and 338, and poloxamine 908. However,as time increased the levels decreased approaching those of theuncoated PMMA NPs.

In light of these findings, NP surfactant coatingwas consideredas a new potential approach to improve brain drug uptake.

P80 coated PBCA NPs (Fig. 2) were first investigated in vivoto enhance the transport across the BBB of the hexapeptidedalargin, a Leu-enkephalin analogue having an opioid activity[97–99]. The peptide is not effective when administered i.v. but

ACA NPs (1) and P80 stabilized SLN (2).

Fig. 3. Molecular structure of the MePEGCA-HDCA copolymer.


when it was bound to PBCA NPs (250 nm) coated with P80,a strong analgesic effect was observed. In fact, all controls,consisting of a solution of dalargin, a solution of P80 (1% w/v,200 mg/kg), a suspension of PBCA NPs, a mixture of dalarginwith P80, a mixture of dalargin with NPs, a mixture of dalargin,P80 and NPs (mixed extemporaneously before injection) aswell as dalargin adsorbed to NPs without P80 coating, resultedineffective.

The investigation of the in vivo fate of NPs coated withdifferent surfactants further confirmed the P80 effectiveness atenhancing brain drug uptake [100]. The results showed that,after i.v. injection, only polysorbates 20, 40, 60 and 80 wereable to induce analgesia in mice. Other surfactants, such aspoloxamers 184, 188, 338, 407, poloxamine 908, Cremophor®EZ, Cremophor® RH40, and Brij® 35, were totally or almostinactive. P80 enabled the highest induction of analgesia at both7.5 and 10 mg/kg dalargin dosages.

Accordingly, other drugs that normally do not penetrate theBBB, such as tubocurarine [101], loperamide [102], 8-chloro-4-hydroxy-1-oxo1, 2-dihydropyridazino[4,5]quinoline-5-oxidecholine salt (MRZ 2/576) [103], and doxorubicin [104] showedhigher brain concentrations when associated with P80-coatedNPs. The transport across the BBB of tubocurarine loaded NPs[101] has been evaluated by in situ perfused rat brain techniquetogether with simultaneous electroencephalogram recording.Thirteen to fifteen minutes after the introduction of tubocurarineloaded P80-coated PBCANPs into the perfusate, frequent severespikes typical of epileptic seizures were observed in theelectroencephalogram. As for dalargin, neither tubocurarinesolution or NP suspension nor NP suspension in tubocurarinesolution or a simple mixture of P80 and tubocurarine influencedthe electroencephalogram in the control experiments.

The results obtained with loperamide [102], a poorly water-soluble opioid agonist unable to cross the BBB, were inexcellent agreement with the earlier results obtained withdalargin and, similarly, it was concluded that the transport ofP80-coated drug loaded NPs across the BBB may occur throughan endocytosis process by the brain capillary endothelial cells.

Similar results were observed for MRZ 2/576, a novel N-methyl-D-aspartate receptor antagonist characterized by a potentbut rather short anticonvulsant effect (5–15 min). This shortactivity was assumed to be due to its rapid elimination by effluxpump-mediated transport processes. In fact,MRZ2/576 showed aprolonged anticonvulsive activity up to 150 min after probenecidpre-treatment (an efflux process inhibitor). Better results wereachieved by i.v. administration of the drugwhen adsorbed to P80-coated PBCANPs and after previous probenecid treatment [103].In these cases, the duration of the anticonvulsive activity in micerose up to 210 and 270 min respectively. These results demon-strated that P80-coated PBCA NPs can be useful both fordelivering drugs to the brain and for prolonging CNS drug meanretention time (MRT).

Accordingly, 5 mg/kg of doxorubicin loaded PBCA NPscoated with P80 administered to healthy rats [104] were able toelicit a brain drug concentration as high as 6 mg/g by i.v.administration, versus b0.1 mg/g of the control injection.Furthermore, doxorubicin bound to P80-coated NPs produced,

in 20% of the treated rats with intracranial transplanted 101/8 glioblastomas, long term remission if compared to controlanimals or animals treated with other doxorubicin formulations.Additionally, binding to NPs may reduce doxorubicin systemictoxicity [105].

Recently, PBCA NPs coated with P80 have been proposed assuitable carriers for nerve growth factor (NGF) brain delivery[106]. NGF is essential for the survival of peripheral ganglioncells as well as central cholinergic neurons of the basal forebrain.Although its inability to cross the BBB, the NGF action on theseneuronal population and its efficacy in preventing neurodegen-eration suggested its potential use in the treatment ofneurological pathologies such as AD, diabetic neuropathiesand Huntington's disease. Systemic administration of NGFadsorbed on P80-coated PBCA NPs successfully reversedscopolamine-induced amnesia and improved recognition andmemory in acute amnesia rat model.

Lately, clioquinol (CQ) (5-chloro-7-iodo-8-hydroxyquinoline)loaded NPs have been proposed for diagnostic and therapeuticapplications in AD [28]. CQ is a highly hydrophobic moleculeable to cross the BBB and it is known to solubilize, in vitro, theamyloid beta protein (Aβ) (senile plaques or Aβ plaques)presumably by chelation of the Aβ-associated zinc and copper[107]. Moreover, after oral treatment, CQ inhibits the Aβaccumulation in AD transgenic mice model [107,108]. CQtreatment appears to inhibit successfully Aβ accumulation andhence CQ may be considered the first credible drug candidate totake care of AD. In addition, after coating with 1% P80 solution,the NPs were shown to be able to enhance the drug transportacross the BBB.

Likewise PEG-containing surfactants as surface modifiers,also PEGylated NPs have been accounted as potentially usefultool to deliver drugs into the brain. New synthetic methoxy-polyethyleneglycol cyanoacrylate-co-hexadecylcyanoacrylate(MePEGCA-HDCA) amphiphilic block-copolymer have beenfabricated for the preparation of novel PEGylated polymeric NPs(Fig. 3) [91]. Upon [14C]MePEGCA-HDCA NPs i.v. adminis-tration in mice, 30% of the radioactivity was still found in theblood circulation 6 h after injection, regardless of the PEGylationdegree employed. On the other hand, the non-PEGylated NPswere cleared from the bloodstreamwithin a fewminutes [92]. Theoverall biodistribution showed that the hepatic accumulation wasdramatically reduced whereas an increased spleen uptake wasobserved as already noticed for PEGylated PLA NPs [93,94].

PEGylated NPs have been hence proposed as a new carrierfor brain drug delivery and a comparison of the biodistributionprofiles, after i.v. administration in mice and rats, of radi-olabelled PEGylated NPs, P80- or poloxamine 908-coated poly(hexadecyl cyanoacrylate) (PHDCA) NPs and the uncoatedPHDCA NPs has been performed [95]. In this case [14C]

Fig. 4. Schematic representation of the proposed mechanism of P80-coated NPbrain uptake. 1. Apo adsorption on P80-coated NP surface. 2. NP binding to theBBBLDL receptors. 3. Drug release fromNPs upon endocytosis and transcytosisthrough the BBB endothelial cells.


PEGylated NPs (60 mg/kg) penetrated into the brain to a largerextent than P80- or poloxamine 908-coated PHDCA NPs, anduncoated PHDCA NPs, both in mice and rats. On the contrary,the injection of half of the P80-coated NP dose (30mg/kg) in ratsproduced a significant accumulation enhancement in the brainthat resulted to be the double of that recorded for PEGylatedparticles. The authors hypothesized that low amounts ofsuspended PHDCA NPs in the P80 solution (1%), increasedthe concentration of free surfactant in the blood to a levelsufficient to modify the BBB permeability. However, thisreasonable hypothesis is not supported by experimental data andis strongly contrasting with what reported in the literature, wheregood performances by P80-coated NPs are described but notheories on the effect on BBB permeability due to P80 arepostulated [95].

Following the evidences that P80-coated NPs could targettherapeutic molecules to the brain, a number of potentialmechanisms have been proposed [81,109,110]. In particular, sixplausible explanations were provided: (i) the increased NPretention in the brain blood capillaries, and/or their adsorption tothe capillary walls, can create a higher concentration gradientenhancing the transport across the BBB and leading to braindrug accumulation; (ii) the solubilization of the endothelial cellmembrane lipids by the surfactant can lead to membranefluidization and then an enhanced BBB drug permeability; (iii)the opening of the tight junctions byNPs can lead to an increaseddrug paracellular permeation in the free or NP-bound form; (iv)the NP endocytosis by the endothelial cells can permit the drugrelease within these cells and the following drug diffusion in thebrain parenchyma; (v) the transcytosis of NPs with the bounddrug can release directly into the brain parenchyma; (vi) theinhibition of the efflux system (especially P-gp) by means ofP80 can prolong the activity of drugs. These mechanisms canalso work in combination. Nevertheless, some of these possibleexplanations can be excluded. In fact, it has been demonstratedthat control groups treated with: a mixture of drug and P80,blank NP suspension, P80-uncoated drug loaded NPs, and anextemporaneously prepared mixture of the three components(P80, NPs, and drug), failed to display any pharmacologicaleffect [97,99,101–103]. By reason of these results, themechanisms (ii), (iii) and (vi) may be obviously excluded.Moreover, the fact that NP-coated with other PEG-containingsurfactants (e.g.: poloxamers 184, 188, 338, 407, poloxamine908, Cremophor® EL, Cremophor® RH 40 and Brij® 35) werenot able to significantly induce antinociceptive activity, ruled outthe possibility of an enhanced BBB drug permeability due to ageneral surfactant effect (ii).

The remaining three hypothetic mechanisms are usuallyconsidered the most likely. In fact, evidence of P80-coated NPadsorption on the brain capillary wall [111] and NP endocytosis,[97,112–114] has been reported both in vitro and in vivo.Furthermore, transcytosis was observed with different types ofNPs [115,116].

However, none of these mechanisms may be completelyconfirmed or rejected. Lately the hypothesis of NP endocytosisby the BBB endothelial cells, followed by drug release anddiffusion to the brain, has gained more credit [117–119]. The

P80-coated NP endocytosis is believed to bemediated by proteinadsorption on their surface followed by subsequent interactions(triggering the endocytosis) with specific BBB surfacereceptors.

As previously mentioned, when an exogenous particulatematerial (e.g. polymeric NPs and SLN) is exposed to serum orplasma, proteins readily adsorb (within the first few seconds) onits surface [120,121]. The type and the relative amount ofadsorbed proteins will decide the fate of particles. This conceptwas originally defined as “differential protein adsorption” in1989 [121]. Based on this concept, the hypothesis waspostulated that an adsorption of the ApoE or B on P80-coatedNPs could be responsible for the interaction with the BBB andthe subsequent endocytosis (Fig. 4) [81,109,110]. Abundantevidence of Apo adsorption on P80-coated NPs by differenttechniques have been reported [45,109,111,122–126].


It has been shown that Apo (particularly ApoJ, ApoA-I andApoC-III) were the dominant proteins adsorbed on cetylpalmitateSLN, regardless of the surfactant used (polysorbates 20, 40, 60,80) [45]. All those SLN formulations showed ApoE adsorption,while typical opsonins (e.g. IgG or complement factors) wereeither not at all detected or in very low amounts. Interestinglyenough, there seems to be a relationship between ApoEadsorption on polysorbate-stabilized SLN and the surfactanthydrophilic–lipophilic balance (HLB) number [45]. This mayexplain some of the findings regarding a significant effect ofdalargin when using PBCA NPs coated with polysorbates 20, 40,60, 80 although the highest effect was achieved using P80 [100].However, it was not possible to correlate the amount of adsorbedApoE with the observed pharmacological effect.

Naturally, the fate of colloidal drug carriers cannot bedetermined by the sole amount of a single protein adsorbed ontheir surface. More likely, the composition of the entire proteinlayer, the type, and the relative quantity of the adsorbedproteins, will determine the body distribution of the carrier. Infact, ApoC-I and ApoC-II were found to inhibit the binding ofApoE-containing lipoproteins to the low-density lipoproteinreceptors [127]. Therefore, a high ApoE/ApoC-II ratio on theNP surface should be achieved to successfully accomplish braintargeting. P80 stabilized SLN displayed the highest ApoE/ApoC-II ratio [45].

In order to attempt to elucidate the mechanism leading toP80-coated NP brain targeting, by means of systematicexperiments, it has been shown [117,119] that just dalargin-or loperamide-loaded NPs, coated with P80 and/or ApoB orApoE, were able to significantly induce the antinociceptiveeffect. The highest effects were observed when both P80 andApoB or ApoE were coating NPs. In ApoE deficient mice, areduced effect was observed [117]. Further studies using ApoElinked to human serum albumin NPs were carried out by meansof the biotin–neutravidin strategy. This system loaded withloperamide was able to induce an analgesic effect in a dose-dependent manner. In particular, the variant ApoE3 was foundresponsible for this effect [119]. Both results sustained thespecific role of ApoE in mediating the brain targeting of thistype of NPs, supporting the hypothesis of NP endocytosis [128]and transcytosis (Fig. 4) [129]. In order to further investigateP80-coated NP uptake mechanism and to establish whether themain role was played by phagocytosis or pinocytosis, additionalexperiments were performed. The preincubation of bovine brainendothelial cells with cytochalasin, a phagocytosis inhibitor ledto a significantly reduced uptake (−40%) of labeled P80-coatedNPs, while the pretreatment with colchicine, a pinocytosisinhibitor, had no effect on NP uptake [112].

Even though these results support the aforementionedmechanisms, further investigation and other evidence isrequired in order to draw an unambiguous conclusion on thismatter. In fact, up to now, none of the studies performed havedemonstrated the presence of P80-PBCA NPs in the vesicularstructures of the brain endothelial cells [130].

Besides, if this hypothesis will turn out to be true, NP sizewill be demonstrated to represent a critical parameter for braintargeting. Indeed, NPs with particle size close to low density

lipoproteins (LDL), in the range from 18 to 25 nm should bemore effectively taken up by the BBB [129]. This hypothesis isnow being supported only by indirect evidence [131].Methotrexate loaded NPs with various mean sizes (70, 170,220, 345 nm) were coated with P80, administered by i.v.injection and the methotrexate concentrations in CSF and braintissues were measured. In this experiment, among the testedNPs, those with a diameter below 100 nm led to the highest druglevel in the brain, even though no statistically significantdifferences were observed [131].

Recent preliminary results demonstrated the possibility oftargeting P80-coated NPs to the brain following oral adminis-tration as well [132,133]. This opportunity appears mostinteresting and should be further investigated as a possiblealternative to the parenteral route due to its higher compliance.

In light of the promising results obtained with P80-coatedpolymeric NPs for brain targeting, the transfer of this technologyto novel lipid nanocarriers, such as SLN, nanostructured lipidcarriers (NLC) and lipid drug conjugated (LDC) NPs (Fig. 2)will be discussed. In fact, SLN, developed almost fifteen yearsago [134–136], have been proposed as alternative carrier topolymeric NPs to overcome some of their common problems.SLN have been used for various purposes, such as drugbioavailability enhancement [137,138], controlled release [139]and drug targeting [140]. Due to the high lipid biocompatibility,all the existing administration routes are virtually possible andmany of them have been investigated, namely the parenteral[49], oral [141], transdermal [142], pulmonary [143] and ocular[144].

4. Advantages of SLN over PACA NPs

4.1. Preparation and sterilization

Many approaches for SLN preparation exist. The mostcommon methods consist in high pressure homogenization atelevated or low temperatures, microemulsion, solvent emulsi-fication–evaporation or –diffusion, w/o/w double emulsion andhigh speed stirring and/or sonication [47–49]. High pressurehomogenization at elevated temperatures [145,146] and micro-emulsion [147–149] are among the most versatile techniquesand for this reason have been largely employed for SLNpreparation. Briefly, the high pressure homogenization methodregards the production of a lipid and drugmelt, which is added toan aqueous surfactant solution at the same temperature. Then, apre-emulsion is produced by high shear stirring and subsequent-ly processed by a high pressure homogenizer at controlledtemperature for several cycles and finally cooled down. Incontrast, the microemulsion method consists in the formation ofa warm microemulsion containing about 10% melted lipid, 15%surfactant and up to 10% cosurfactant. The microemulsion isthen poured upon stirring in a large volume of cold water and theexceeding water is eliminated by ultra-filtration or lyophiliza-tion. Both these techniques, avoiding the use of organic solvents,do not lead to residue contaminations. SLN metallic particlecontamination, that may occur by using sonication, is avoided aswell. Moreover, the elevated temperatures used do not represent

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an issue because of the limited time of exposure. Anotherfundamental advantage of these two methods is the scale upfeasibility as high pressure homogenization easily allows alaboratory, pilot or large scale production. In fact, 50 to 150 kg ofSLN dispersions per hour can be obtained by placing twohomogenizers in series [145]. However, the microemulsiontechnique is less scalable, even though the production of batchesas large as 1.1 L has been achieved [49].

Differently from SLN, the preparation of PACA NPs can beperformed either by polymerization of alkylcyanoacrylate mono-mers or by using the formed polymers [150]. The first processincludes twomethods of preparation, emulsion [102] or interfacialpolymerization [151]. Emulsion polymerization is the mostwidely used method and consists in a very complex mediumwhere its initiation is carried out by the water hydroxyl ions andelongation of the polymer occurs by anionic mechanism. Theinterfacial polymerization technique has been developed to obtainnanocapsules containing awater or oil core by using awater-in-oilor oil-in-water emulsion system respectively. PACA NPsprepared with block copolymer can be obtained by nanoprecipita-tion or by emulsion-solvent evaporation [91,92,95]. In the formercase, colloidal particles are obtained adding a non-solvent to adilute solution of the polymer. Finally, the solvent is removedfrom the suspension. In the latter case, a polymer solution is addedto an aqueous phase upon high pressure homogenization to form afine emulsion. The solvent is then evaporated to induce polymerprecipitation and NP formation. The scale up of these preparationmethods is more complicated than for SLN as a result of theintensive use of solvents needed to solubilize polymers ormonomers. Unfortunately, in the literature there is a consistentlack of studies concerning the scale up issues in the production ofthese systems, although in recent years more attention has beendevoted to the industrial development aspect of batch production.

Sterilization is another fundamental step in the production ofNPs intended for parenteral administration. In the case of SLN,normal procedures consist in carrying out the SLN preparation inaseptic conditions and the possible application of commonsterilization processes, such as filtration, γ-irradiation andautoclaving [48]. However, these methods can present someimportant drawbacks. In particular, sterilization by filtrationrequires particle sizes below 0.2 μm and therefore cannot be usedin all circumstances. Sterilization by γ-irradiation generally maylead to free radical production that could provoke SLN chemicalmodification, while heat sterilization can induce modificationsrelated to their physical stability such as the increase of particlesize due to their aggregation [48]. In fact, it seems that poloxamerdid not provide enough stabilization to avoid particle size increaseupon heating [135,151]. Even though, autoclaving has beenproposed for SLN suspensions containing reduced lipid concen-trations (2%), since no important alterations were observed [139].Beyond the nature of the lipid and the presence of surfactants, themedium represents an important parameter that may allowautoclaving without particle size increase. In fact, when SLNwere suspended in a solution of Pluronic® F68 prior tosterilization, no increase in particle size was observed [152]. Ofcourse, the possible drug loss and lipid or drug degradations haveto be carefully considered as well [151,153].

Sterilization of PACA NPs is hardly accounted for in theliterature [154], although general methods, already consideredfor SLN and applied to other kind of particle systems [155–157]can be considered useful. However, for both particulatesystems, the preparation performed in sterile conditions stillremains the best choice over all the methods above reported.

4.2. Toxicity, stability and biocompatibility issues

Since these DDS are designed for human application, thematerial used for their preparation has to be non-toxic,biocompatible, and able to be excreted or biodegradable.PACA NPs are completely excreted only when the molecularweight of the used polymers is sufficiently low [150]. Hence, it isfundamental to understand the degradation mechanisms whichcan determine the possible PACA NP suitability or limitations.

Three mechanisms of degradation have been described forPACA NPs [150]. The first, which is believed to be the mostimportant, is based on the enzymatic degradation by esterases ofthe serum, lysosomes and pancreatic juice. This mechanismgives rise to alkylalcohol and poly(cyanoacrylic acid) bypro-ducts. The second is based on the unzipping polymerizationfollowed by an instantaneous repolymerization into a lowermolecular weight polymer. The third, that at the early stages ofPACA NP development was believed to be the most important,is well known under the name of Knoevenagel reaction. Thedegradation products are formaldehyde and cyanoacetic esterand the reaction yields only 5% of degradation after 24 h. Thesebyproducts are all potentially harmful to cells as they are small ormedium size non-self molecules. In this regard, the knowledgeof the degradation pathway is fundamental as it deeplyinfluences the kind and the accumulation rate of byproducts intissues and organs from which the toxicity depends. For thisreason, toxicity studies have been performed to understand theimplication of PACA biodegradability in the occurrence ofpossible side effects.

The investigation of PACA NP in vitro cytotoxicity wascarried out on several cell lines, namely macrophages [158,159],L929 fibroblasts [160,161], hepatocytes [162,163], cancer cellsDC3F [164], mesenchymal cells [165], Swiss 3T3 cells[166,167] and murine cerebral microvessels [168]. Sincemacrophages are characterized by a high phagocytic propensity,they represent good models to study the toxicity of PACA NPsintended for intracellular targeting [158,159]. The resultsrevealed that polyisohexylcyanoacrylate induced a respiratoryburst in macrophages, and thus macrophage activation [159].Another study showed that polymethylcyanoacrylate NPsprovoked perforation of the macrophage membrane. In thesame conditions, perforation was not observed with polyisobu-tylcyanoacrylate NPs. The contrasting results can be explainedby the different degradation rate that is correlated to the alkylchain length [158]. The influence of the alkyl chain length on thePACA NP cytotoxicity was investigated more accurately in aL929 fibroblast cell model analyzing cell growth inhibition[161]. Polyethyl-, polyisobutyl-, polymethyl-, and polyisohexyl-cyanoacrylates were investigated for cell growth at an amount of20 μg/ml. The first two polymers showed a considerably higher


toxicity compared to polyisohexyl-cyanoacrylate, while poly-methyl-cyanoacrylate is only slightly less toxic. The observedtoxic effects could be due to the combination of twomechanisms, PACA degradation and adhesion. In the presenceof longer alkyl chains, NPs undergo a slower degradationfavoring cell adhesion likely leading to high local degradationproduct concentrations [161]. Besides the length of the alkylchain, the particle size influences the toxicity too. In fact, it wasfound that smaller particle sizes provoke higher cytotoxicity.This has been explained by the large surface area that leads to afaster degradation and a higher degradation product concentra-tion [161].

Another very useful model to study PACA NP toxicity is theliver [162,163] since it is responsible for a rapid uptake uponNP i.v. administration. PACA NP i.v. administrations initiate aninflammation process accompanied by metabolic disorders inthe glucidic metabolism and by massive glycoprotein under-sialylation. This process, reversible 14 days after the end of thetreatment, can be correlated to the NP phagocytosis by Kupffercells. In fact, in vivo, PACA NPs are supposed to principallydistribute in the Kupffer cells initiating their activation, whilethe direct contact with hepatocytes is not likely to occur. For thisreason, PACA degradation products are not mainly responsiblefor the low level of reduced glutathione. Indeed, the decrease ofthe reduced glutathione level could be due to a phagocytosisinduced burst respiratory effect.

It can be concluded that the PACA NP cytotoxicity is likelydue to the burst release of degradation byproducts, directlyrelated to the length of the polymer alkyl chains. Even if NPsmade of longer alkyl chain polymers are less toxic, they couldadhere to cells, leading to a high local degradation productconcentration. For brain targeting, less toxic slow degradingPACA NPs should be more suitable, although their chronicadministration would lead to ‘waste disposal' in brainendothelial cells and/or other brain structures.

On the other hand, since SLN are prepared from biocompatiblematerials, better tolerability with respect to polymeric NPs can behypothesized. In addition, triglycerides, fatty acids, or waxes areknown to release natural occurring degradation byproducts. Todate, no SLN formulations for parenteral use are present on themarket. Nevertheless, lipid emulsions composed by fatty acids arelargely used in parenteral nutrition since the '60s [49].Consequently, the use of glycerides for SLN preparation couldbe conceivablewithout expecting important toxic effects resultingfrom their degradation products [47]. Although SLN forparenteral administration are of great clinical interest, little isknown about their metabolism. Moreover, GRAS (GenerallyRecognized As Safe) surfactants must be used for theirpreparation to obtain approval and a good in vivo tolerability [49].

The phagocytosis of SLN can be controlled modifying theirsurface properties as has been done for liposomes andpolymeric micro- and nanoparticles [169,170]. In this way, itis possible to target molecules to the brain by limiting RESuptake. At any rate, it is essential to understand if the interactionof SLN with cells leads to cytotoxic effects.

The in vivo toxicity on the liver and the spleen of mice after i.v.injection of two SLN formulations composed of cetylpalmitate

or Compritol® has been assessed [171]. Even if cetylpalmitateis not a physiological wax, no accumulation was observedsince its degradation is very rapid. On the contrary, Compritol®provoked a reversible liver and spleen weight increase.Moreover, the liver histological analyses revealed the presenceof mononuclear cell infiltration, Kupffer cell hyperplasia andliver cell necrosis. These effects were almost totally reversible6 weeks after the injection since neither inflammation nor freshinfiltrations were observed. Besides, the Compritol® SLNinjected dose was remarkably high, in fact, if correlated to thehuman body mass, the dose was equivalent to 100 g solid lipidmass administered 6 times by i.v. injection. In light of theseconsiderations, the effects observed can be ascribed to theenormous dose administered, much larger than that theoreti-cally needed for therapy.

Several studies have investigated the influence of the lipidmatrix, surfactants, and even particle size on the SLNdegradation rate. The degradation of the lipid matrix occursmainly by lipases while only a small part is degraded by non-enzymatic hydrolytic processes [172]. Another enzyme respon-sible for SLN degradation is the endogenous alcohol dehydro-genase [173]. The influence of surfactants, namely cholic acidsodium salt, lecithin, P80 and poloxamer 407, on the degradationrate of SLN has been evaluated [174]. The last two stabilizers arePEG-containing surfactants that provide a sterically protectivelayer of varying thickness depending on the structure andnumber of PEG units. The protective layer more or less hindersthe lipid from the enzyme attack obstructing the anchorage of thelipase/colipase system. When comparing the same surfactant, ahigher degradation rate was observed for glyceride based SLNthan for wax based SLN (e.g. cetylpalmitate). In fact, waxes arenot an optimal substrate for lipase/colipase that preferentiallymetabolizes glycerides. The faster degradation occurred withcholic acid sodium salt that is recognized as promoting enzymeanchorage to surfaces [175]. Since lecithin did not promote orhinder the enzyme anchorage, SLN degradation rate is notinfluenced by the presence of this stabilizer. Poloxamer 407 wasable to reduce the SLN degradation rate to a larger extentcompared to P80 that had a limited steric effect. In fact, theadsorption layer thickness was approximately 20 Å for P80 andaround 120 Å for poloxamer 407. A faster degradation rate wasobserved for triglycerides containing short fatty acid chains,while an opposite effect for long fatty acid chain triglycerideswas noticed. Since SLN are degraded by surface erosion, size isexpected to have an influence on the degradation rate [176]. ForSLN possessing the same diameter and polydispersity index,degradation was faster with cholic acid sodium salt than withpoloxamer 407. Poloxamer 407-coated SLN degradationseemed to be more sensible to size variations. In fact, smalldifferences in particle diameter led to a significantly differentdegradation behavior. In addition, two poloxamer 407 SLNpossessing two different diameters were characterized by asimilar degradation rate. This phenomenon was explained by anincreased polydispersity of the smaller size SLN batch, due tothe presence of microparticle population fractions. However, ingeneral small size SLN were characterized by a fasterdegradation rate while cholic acid sodium salt-coated SLN


degradation was just slightly sensible to particle size. In fact,different degradation rates were observed only when sufficientlydifferent particle populations (300 nm and 800 nm mean size)were compared. Generally, the influence of particle size on SLNdegradation rate was observed when hindering surfactants wereused in SLN preparation.

Investigation on SLN in vitro cytotoxicity have been carriedout on several cell lines, such as granulocytes, HL 60 and MCF-7 cells and murine peritoneal macrophages [149,177–180]. Incomparison to Food and Drug Administration (FDA) approvedpolymers, such as PLA or PLGA, that show 50% viability at0.2% of polymeric NPs, SLN (Compritol®, cetylpalmitate)were 20 times less toxic on human granulocytes and HL 60[177]. Moreover, tripalmitin and stearic acid based SLN, in therange 0.1 to 0.25%, resulted non-toxic to HL60 or MCF-7 cells[149]. Comparing Dynasan® 114 and Compritol® SLN in therange 0.015–1.5%, no significant viability differences on HL 60cells were observed. These results mean that SLN are very welltolerated by HL60 cells even if their degradation rate issignificantly different [179]. These two SLN formulations didnot have any effect on murine peritoneal macrophage viabilitywhile an amount of 0.1% Dynasan® 118 and Imwitor® SLNprovoked a clear viability reduction. Other lipid-based SLN,such as dimethylstearoyl-ammonium bromide (0.01%), cetyl-palmitate and stearic acid (0.1%) resulted in a macrophageviability of 15.3%, 61.8% and 2.2%, respectively [178,180].These results show how the lipid nature fundamentallyinfluenced the cytotoxicity. Since Dynasan® 118 and Imwitor®are two lipids containing glycerides of stearic acid, theircytotoxicity was compared with stearic acid SLN; the latterresulted much more toxic than the former ones. This highercytotoxicity was explained by the presence of larger amounts ofstearic acid produced by SLN biodegradation [180].

SLN size did not lead to macrophage activation, and thus tocytokine production. Moreover, no cytotoxic effects wereassociated to SLN size modifications, confirming the findingspreviously reported [181]. In addition to the lipid type, thesurfactant, used to stabilize SLN, could affect cell viability. Infact, while poloxamer 407 (up to 10%) did not have any effecton HL 60 cell viability, P80 reduced it markedly when used at aconcentration ≥0.01% [179]. Moreover, P80 toxicity resultedhigher when used as a free molecule (concentration of 0.001%,viability of 50%) with respect to P80 bound or incorporated inSLN. Non-ionic surfactants (poloxamine 908, poloxamer 407and 188, Solutol® HS15 and P80) revealed a slight concentra-tion dependent cytotoxicity on murine peritoneal macrophages,while cationic surfactants, such as cetylpyridinium chloride,provoked a clear viability reduction [178,181]. Furthermore, thesame concentration (concentration presented in 0.01 or 0.1%SLN) of free cetylpyridinium chloride incubated with macro-phages led to a lower toxicity if compared with the toxicityobserved when it was incorporated in SLN. The cetylpyridi-nium chloride antimicrobial activity is based on its interactionwith microorganism membranes [182]. Indeed, the positivelycharged surfactant present at the surface of SLN can interactwith the negatively charged cellular membrane resulting in acytotoxic effect. The lipid matrix seems to play a role in cell

adherence since such toxic effects are not observed with freecetylpyridinium chloride [181].

The comparison between polyester NPs and SLN cytotoxi-city showed that SLN were ten-fold [183] or even twenty-fold[177] less toxic to human granulocytes if compared to thepolyester particles. In fact, for 0.5% PLGA and PLA NPs,important cytotoxic effects were detected, while, for 5% SLN, a70–90% granulocyte viability was still observed [184]. SLNcytotoxicity resulted much lower than that of PACA NPs[179,185], since the cytotoxic concentration of the less toxicPACA polymer (polyhexylcyanoacrylate) was 0.005% whenincubated with L929 fibroblasts and 0.05% when incubatedwith hepatocytes. Moreover, the exposure of human granulo-cytes to 0.05% hexylcyanoacrylate NPs killed all cells.

The very low cytotoxicity of SLNmakes them very attractivecandidates for brain delivery. It is important to underline thattheir toxicity is not only related to the lipid or wax used but alsoto the surfactant employed. The most common surfactantexploited for NP brain targeting is P80 and it has beendemonstrated that free P80 is more toxic than when bounded[179]. Because P80 is incorporated into the SLN matrix while itis only adsorbed on PACA NPs [46], its leakage is more likelyto occur from PACA NPs (Fig. 2). That means that even if thecytotoxicity was similar for both systems, SLN would be saferfor brain targeting since little P80 would be released in the body.High incorporation efficiencies are desirable, of course, since itcan reduce the amount of particles needed to reach pharmaco-logical levels. In the case of SLN, NLC or LDC NPs, drugs arelocated in the core, in the shell or are molecularly dispersed inthe entire matrix according to their lipophilicity and hydro-philicity. SLN are generally able to entrap lipophilic drugs,yielding high encapsulation efficiencies [49]. On the contrary,hydrophilic molecules are hardly incorporated into SLN due tothe low affinity with the lipid matrix. However, biotechnolog-ical drug loaded SLN have been successfully formulated[186,187]. Conscious of this issue, Müller et al. developedP80 stabilized NPs for brain targeting made by lipophilic pro-drug, or LDC NPs, of diminazene (diminazene covalentlylinked to oleic and stearic acids) [188]. In this fashion, a drugcontent, up to 33%, was achieved.

In the case of PACA NPs, drugs can be incorporated byusing the interfacial polymerization method that allows theentrapment of both hydrophilic and hydrophobic molecules.However, polymerization can produce a consistent amountof free radicals that may react with the loaded drug uponNP formation. Perhaps for this reason, in the preparation ofPACA NPs for brain targeting, drugs are generally adsorbed onthe surface of the carrier made with the preformed polymer[98,100,101]. It is thus evident that drug loading in PACANPs, intended for brain targeting, is much lower than in SLNand, even under the unlikely assumption of equal toxicity, thelatter are preferable over the former. In fact, larger amounts ofPACA NPs with respect to SLN are needed to carry the sameamount of drug to the brain. Moreover, considering that only1–2% of the injected dose generally reaches the brain, drugloading is an essential issue to address in order to develop aneffective therapy.


5. SLN physicochemical features with respect to brain drugdelivery

Lipids are rightly being considered as safe and usefulmaterials for drug delivery [49,135,189–193]. Conventionallipid-based systems, consisting of emulsions and microemul-sions, have been widely used to enhance bioavailability of classII molecules [194], and the absorption of class III molecules[194]. The stability of such systems is strictly related to particlesize distribution, the lipid content, and the presence of asurfactant capable of stabilizing the dispersion. Fig. 5 reports ageneral ideal diagram for the possible situations that can begenerated according to surfactant, lipid, and aqueous phaseinterplay. Of course, the molecular properties of the phasesinvolved deeply influence lipid organization and assembly.

Stable lipid aggregates form only when the right surfactantand concentration are being employed. Surfactants are usuallychosen according to their HLB and the Israelachvili's surfactantpacking parameter (P) [195] reported in the following form(Eq. (1)):

P ¼ VAL

ð1Þ

where V is the molar volume of the hydrophobic moiety of thesurfactant molecules, A the cross sectional area of thehydrophilic portion when situated at the interface, and L thecritical length of the hydrophobic chain. HLB and packingparameter describe the same basic concept, though the latter ismore suitable for microemulsions. According to the geometry ofthe surfactant molecules, P values can range from 1/3 to 1 andto values N1 for conical, cylinders or reverse phase conicalgeometries, respectively [194,195]. For lipid aggregatesdispersed in an aqueous phase a value between 1 /3 and 1 isconsidered optimal [194].

These general considerations also apply to peculiar lipidsystems such as SLN. In this context, stable SLN dispersionshave been associated with the use of surfactants possessingHLB values lower than 12 [194,195]. Furthermore, lipidmolecular characteristics, bulk, and surface properties heavily

Fig. 5. Ternary phase diagram of a generic lipid-based system showing thepossible lipid aggregates (adapted from Ref. [194]).

affect physical and chemical stability and suitability of SLNformulations such as DDS.

5.1. SLN structure, drug loading, and stability issues

Recently, some papers have highlighted the need for a widercomprehensive investigation of SLN properties, which havebeen correlated to their composition and nanoscale size[49,194]. The connection existing between SLN physicalcharacteristics and their in vitro and in vivo behavior has beenalso pointed out [196,197]. Investigations on the possible SLNinner structure should always be developed since its lack canbring to possible misinterpretation of the in vivo results [198]. Ithas been reported that materials formulated at nanoscale levelshow different characteristics compared to the bulk material[194,199]. These differences can be ascribed to changes in thephysical state and level of interaction as well as in the energiesinvolved. In fact, nanosize systems always possess peculiarcharacteristics not found in the microscale systems, such as largesurface areas, high curvature radius, and high interactionenergies. In this regard, surface properties deeply impactnanosystems and are correlated to the diameter of the particles.In fact, when their size is 10–100 nm, a large portion ofmolecules (10% or more) are located at or near the surface. Thesurface molecules will affect the mechanical (strength, flexibil-ity, etc.) and physical behaviors of particles in a way that is notyet fully understood [199].

For a volume fraction FV of randomly distributed sphericalparticles of radius r, the number of particles per unit volume NV

(number density) is given by Eq. (2).

Nv ¼ 3Fv

4kr3ð2Þ

This feature is important if one considers that the particledose administered is proportional to their loading capacity andto NV. In this regard, being NV closely related to particle size, forcolloidal systems the number of particles per volume fraction isseveral orders of magnitude higher than that for microparticlesand it decreases rapidly as size increases (Fig. 6). Thisconsideration has to be accounted for when assessing NP invivo performances. In addition, the inner structure of the lipidmatrix has been seen to vary from large to small size particles[200,201]. Since SLN are made of solid lipids, long and shortchain tri-, di-, or monoglycerides, their inner structure can resultquite different compared to the bulk material. Therefore, it isobvious that the investigation of the solid lipid structure is ofgreat importance in order to improve the understanding of SLNproperties and to handle possible stability issues.

Several techniques have been applied to outline the SLNphysical and chemical inner organization, such as differentialscanning calorimetry (DSC), nuclear magnetic resonance(NMR), and small angle and wide angle X-ray scatteringtechniques (SAXS, WAXS) [202–206]. Likewise, rheologicalcharacterization has also been found useful to assess the SLNlipid matrix structural characteristics [201,207,208]. In thisregard, Compritol®, stearic alcohol and Softisan® 138 SLN

Fig. 6. Particle size effect on the surface area (–) and on the number of particles per volume fraction (—) (Eq. (2)) of lipid-based systems.


revealed high storage modulus G′ and loss modulus G″1,suggesting the presence of a gel-like organization [209]. Inparticular, Compritol® and stearic alcohol SLN had a larger G″elastic modulus, whereas Softisan® 138 SLN resulted moreviscous (larger G′). These features were ascribed to the highercrystallinity of Compritol® and stearic alcohol compared toSoftisan® SLN that, in contrast, were depicted as supercooledmelt dispersions. In addition, cetylpalmitate SLN flow proper-ties in comparison with cetylpalmitate microparticles wereinvestigated as well. Cetylpalmitate SLN have been found topossess about ten fold increased G′ and G″ moduli compared tocetylpalmitate microparticles [201]. This effect was correlatedwith the higher viscoelasticity of the NP inner structure, whichwas assumed to resemble a gel-like structure. These results alsodemonstrated that size may have a predominant role inassessing lipid particle physical characteristics.

The formation, in the SLN, of a network-like structurehaving a semisolid consistency was correlated to the lipidmolecules assembling themselves into a platelet-like arrange-ment rather than a conventional spherical particle (Fig. 7). Infact, platelet structures are being formed mainly according tosize and when highly ordered and compact crystallinearrangements occur, whereas it is likely that a contingententropy increase followed by a size increase, forces the systemto redistribute into a more thermodynamically stable spheroid.The assumption of a platelet structural assembling of lipidlayers has been confirmed by transmission electron microscopy(TEM) analysis [207,210]. These non-spherical particles offer amuch larger surface available for particle–particle interactionsthus favoring the formation of a network-like structure. Suchstructures are naturally occurring especially for pure triglycerideSLN. This layer organization has been reported in lipid systems

1 G′=Measurement of energy stored during deformation and related to thesolid-like or elastic portion of the material. G″=Measurement of energy lost(usually lost as heat) during deformation and related to the liquid-like orviscous portion of the material.

such as Compritol® SLN characterized by field flow fraction-ation, photon correlation spectroscopy (PCS), and cryo-TEM[210]. Platelets are consistent with few lipid layers (two orthree) forming a 10–18 nm thick structure. Solid cetylpalmitateSLN tend to crystallize in a platelet-like structure with a step-like surface. SAXS studies highlighted an orthorhombiclamellar lattice arrangement for cetylpalmitate layers [211].

Since the conventional preparation methods imply the SLNformation upon fast cooling from hot lipid melts [49], the solidcolloidal dispersion is the result of lipid crystallization into wellestablished ordered structures. Crystallization of triglycerides

Fig. 7. Schematic representation of solid lipid nanoparticle shapes (adapted fromRef. [210]).


from the melt occurs in a metastable hexagonal α-form whichconverts, via an orthorombic β′-form, into a stable triclinic β-form upon reheating and storage [212–214]. The rate of suchtransition is sensitive to size, and the α-to-β transformation hasbeen observed to be much faster in colloidal dispersions than inthe bulk material and, likely, via an altered course. In general, itseems that the lower the lipid melting point, the better thechance is of α-to-β transformation during storage. Also particleshape and morphology were modified as a result of the changein the intimate physical structure of the lipid matrix. In fact, aplatelet-like shape was observed when the content of β-formwas higher (in the case of ionic surfactants), whereas a higheramount of α-form produced particles of different shapes as afunction of size. Larger particles (N200 nm) were mostlyspherical, while smaller particles (b100 nm) had a blockyisometric layered shape [215]. Another point to be accountedfor is the fact that, as a consequence of the α-to-β transition, theparticle surface area increases considerably as the platelets dueto the β polymorph are being formed (Fig. 8) [213] This is theresult of the building up of a more complex step-like plateletstructure as is reported for cetylpalmitate SLN [211].

Melting and crystallization temperatures can be verydifferent between bulk materials and SLN, according to lipidchain length [212].

The particle size dependence of the melting temperature hasbeen often described by using the Thomson equation modifiedfor crystalline materials or Gibbs–Thomson equation (Eq. (3))[216],

� T0 � TT0

clnTT0

¼ � 2gslVs

rDHfusð3Þ

where T is the melting temperature of a particle with radius r, T0is the melting temperature of the bulk material at the sameexternal pressure, γsl is the interfacial tension at the solid–liquidinterface, Vs is the specific volume of the solid, and ΔHfus is thespecific heat of fusion. It has been shown that this equation doesnot only apply to spherical (curved) particles but can be used todescribe the behavior of particles with plane surfaces [217] andit turned out to be useful to explain the melting point depressionof triglyceride NPs compared to their bulk phase [217]. Thiseffect can represent an important issue for the development ofsuitable SLN formulations. In fact, not always does crystalli-zation occur properly and the formation of supercooled meltshas been reported for several lipid systems [196,199]. Tristearinand tripalmitin SLN are crystalline at room temperature. Underthe same conditions, trimyristin and trilaurin particles remainliquid for several months upon storage. According to DSC

Fig. 8. Schematic representation of surface area increase upon step-like plateletSLN formation (adapted from Ref. [213]).

analysis trimyristin particles start to crystallize at 10°C whiletrilaurin dispersions retain the emulsion state even at 4 °C [212].As already reported, the small size itself can reduce meltingtemperatures of several degrees with respect to the bulkmaterial. The same effect may happen also for the crystalliza-tion temperature. This suggests that supercooling may oftenoccur especially for short chain lipids or lipids that are not of thepurest quality. Since the main advantages of SLN reside in theparticle solid state, low purity along with the presence ofstabilizers can indeed represent a serious issue. Moreover, thepossibility of polymorph coexistence heavily affects SLNstability. In this regard, trilaurin exists as four differentpolymorphic forms: α–β′–β1–β2 [203]. The melt of trilaurinSLN was found to crystallize straight into the metastable α-formupon fast cooling.

Another factor strongly affecting SLN characteristics is drugloading. In spite of the high efficiency achieved, especially forlipophilic drugs, the amount of entrapped drug into SLN islimited by their small size. Lipid polymorphic structures oftenundergo modification upon drug loading as a result of theintercalation of the drug between lipid layers [196]. As proof,blank Compritol® SLN showed small amounts of the unstableα-form that disappeared upon heating or by drug moleculeloading [218]. Generally, drug loading was found to decreaselipid layer organization. In this regard, several examples ofsupercooled lipid melts have been described [196,199,212].Trimyristin SLN and SLN made of a blend of triglycerides withchain lengths between 12 and 18 carbons and stabilized with amix of lecithins and glycerols were evaluated upon loading ofseveral steroidal drugs [196]. A large depression of melting andcrystallization temperatures was observed in agreement withlipid chain length, indicating a strong tendency towardssupercooling, even the recrystallization course was differentbetween bulk material and SLN as shown by X-ray patternanalysis [196].

Drug loading capacity was higher in the case of glycerideblends as a result of their lower crystallinity with respect to purelipids, and different drug distribution and motility could bedetected by NMR analysis [196]. Drug loading from 1% up to50% (w/w) caused drastic changes in the lipid structure anddrug expulsion was reported upon storage. The rate of drugexpulsion was different for different lipid matrices and thisfeature was correlated to the rate of polymorph transformation.Lipid annealing was also taken into consideration.

Further studies on tripalmitin SLN loaded with ubidecar-enone (coenzyme Q10, CQ10) highlighted again the destabiliz-ing effect because of the entrapped drug as shown by thedifferent DSC profiles of drug free and up to 70% (w/w) drugloaded systems [219].

Melting point depletion was observed as the CQ10 concen-tration was increased and it persisted even after long-termstorage. Such behavior was ascribed to an eutectic formationbetween the drug and tripalmitin. Above a 50% CQ10 loading,SLN remained as a supercooled melt even upon cooling belowroom temperature. However, CQ10 was found to be stronglyassociated with the NPs and consequently no separationoccurred even with 50–70% drug loading.


Characteristic platelet structures were detected by TEM. Theexcess drug was seen to remain outside of the particle as liquiddroplets adsorbed to their surface forming thus a two phaseparticle (Fig. 7). In contrast, solid state NMR studies performedonCQ10 (∼5%w/w) loaded cetylpalmitate SLN showedmainly asolid state occurrence of the particles [220]. About 60% of thedrug was found homogeneously dispersed in the lipid matrix.Other studies demonstrated that drugs, such as mifepristone,incorporated into SLN, can lead to stable formulations with a lessordered crystalline organization [221]. The advantage is that lessrigid and ordered structures provide a larger space to accommo-date drugs. In this situation drug expulsion is also less likely tooccur upon storage. Moreover, SLN showed a more regularspherical shape and this may be correlated to the lower crystallinegrade. The physical state of particles is thus of paramountimportance both from the technological and biopharmaceuticalviewpoint. Since the actual physical condition of the lipid systemis not known, misinterpretation of in vitro or in vivo behavior caneasily occur as supercooled melts behave mainly as emulsions.When solubilization of poorly soluble drugs is required whiletheir modified release does not constitute a major aim, the use ofsupercooled melts may be advantageous as they can hold largeramounts of drug and they possess fewer stability issues. However,supercooled melts are not thermodynamically stable andrecrystallization over a long period of time cannot be excluded.Such an issue enforces the possibility of product changes uponlong-term storage; and their pharmaceutical quality, especiallywhen intended for parenteral administration, cannot be ensured.In fact, even though some of these systems, such as trimyristinSLN, can be forced to recrystallize if cooled below their criticalcrystallization point, undesired irreversible phenomena, such asgelling or expulsion of the incorporated drug, can always occur inan unpredictable manner.

5.2. Surfactants and brain targeting features

The choice of a proper stabilizer is essential to ensure SLNsuitability as brain drug targeting systems. SLN inner crystalstructures have been extensively characterized because of theirimportance in determining particle behavior and stability. Parallelinvestigations have been carried out to determine the bestsurfactant characteristics impacting positively SLN stability andtheir in vitro and in vivo behavior [214,222,223]. The surfactantsuitability is being based on the mentioned HLB and packingparameter. However, the ability to stabilize lipid dispersionscannot be only related to the surfactant intrinsic properties but isalso influenced by lipid and particle characteristics. Severalstudies report the effect of stabilizers on size, polydispersity andstructure of triglyceride SLN [224–226].

The most common stabilizers employed are ionic andnonionic molecules, such as lecithins, polysorbates, poloxa-mers, derivatized fatty acids, and their combinations [96].

Surfactants are being used to prevent aggregation but theyhave been found to change SLN characteristics, such ascrystallinity [227]. In particular, the extent of SLN triglyceridecrystallinity and stability is correlated to their chain length andthe presence of stabilizers, such as lecithin [228].

Tripalmitin SLN have been observed to organize into thestable β-form when different surfactants were used for SLNpreparation. However, ionic surfactants caused the retention of ahigher amount of α-form compared to nonionic surfactantsupon cooling. This can affect the SLN behavior during storageas the kinetic pattern of transformation into the β-form isprofoundly altered [215].

The different polymorphic behavior related to the hydro-philic surfactants suggests that polymorphic transitions intriglycerides start mainly from the surface of the particle. Aspreviously mentioned, SLN degradation rate was modifiedaccording to the surfactant used for their preparation[174,176,229]. Surfactants, therefore, are fundamental fordeveloping proper SLN formulations. In fact, even uponpreparation, these molecules have been reported to influenceparticle size and aggregation of freshly produced SLN intendedfor brain drug delivery. As reported earlier, most of thepreparation methods are based on high pressure homogeniza-tion techniques to improve SLN dispersion, their effectivenessand scale up perspectives [145]. Several studies have beenconducted on homogenization pressure, cycle number, andequipment effects on SLN size [226,230,231]. Tri-, di-, andmonoglycerides show a similar sensitivity to pressure andnumber of homogenization cycles [226]; triglyceride SLN sizeincreased when using higher pressures, meanwhile di- andmonoglyceride SLN remained almost unchanged. Thesecontrasting effects of the homogenization pressure have beenelsewhere reported also for Compritol® and Softisan® 142 SLN[231]. The explanation may reside in the coupled effect ofcavitation forces and surface area increase [226]. In fact,although the expected effect at higher pressures and number ofcycles is size reduction, the high shear at which the particles arebeing processed and the larger surface area that is generated byparticle breaking down favor aggregation. These effects arebeing observed for many SLN preparations and, of course, aresusceptible to lipid and surfactant molecular characteristics. Inaddition, the same effects can be obtained by increasing theprocessing temperature, which may lead to size reduction as aconsequence of a reduced melt viscosity, but, similarly, thesurface area increase can produce higher tendency to aggrega-tion. In this regard, the formation of microparticle populations isbeing generally observed [226,231].

All these features have to be accounted for especially whendeveloping SLN intended for brain delivery. In fact, as alreadystated, size and surface properties are fundamental for particlebrain uptake [110,131]. In this context, the use of a propersurfactant is even more important because of its role of particlesurface modifier. In particular, polysorbates have been foundpotentially predominant over other surfactants for brain delivery[100]. Pressure and number of cycles upon homogenizationaffect similarly the P80-coated Compritol® SLN size [231]. P80concentration had a larger effect on particle size compared to thenumber of homogenization cycles (Fig. 9). Moreover, in thecase of Softisan® 142, it was observed that concentration valueshigher than 2%may be required in order to reduce the size downto 150 nm [231,232]. The question arising is what kind of effectP80 exerts on the lipid matrix. Hypothetically, the P80 oleic





Fig. 9. Response surface plot of the effect of P80 concentration and number of homogenization cycles on the size intended as mean volume diameter (1.) and Gaussiandistribution width (2.) of P80 stabilized Compritol® SLN (adapted from Ref. [231]).


chain may penetrate the lipid matrix and anchor the surfactantmolecule onto the SLN surface (Fig. 2). This may have a largeimpact on the lipid physical state. However, DSC investigationsshowed a much larger melting point depression for Softisan®142 with respect to Compritol® SLN (Fig. 10). This effect couldbe due to the diverse impact of the surfactant on the lipid matrixorganization as well as to the different SLN particle size [232].

How these features can affect the in vivo effectiveness ofsuch preparations is yet to be understood. Moreover, in spite ofthe long history of P80 application to NPs for brain delivery, acomprehensive investigation of the physicochemical character-istics of the coated SLN is still partially missing. Indeed, such

information could be helpful to the development of suitableSLN for improving brain therapies.

6. SLN for brain drug targeting

In the late '90s SLN were proposed for brain drug targetingapplication independently by two research groups [233,234]even though the first proof of lipid particle transport across theBBB had already been provided [235].

Studying the pharmacokinetics of two anticancer agents,namely camptothecin and doxorubicin, drug accumulation intothe brain was observed after both oral and i.v. administration

Fig. 10. DSC data of the bulk Softisan 142 and Compritol® and their respective P80 stabilized SLN formulations.


when loaded into SLN [233,234]. As previously shown withPACA NPs, better results in brain targeting were achieved whenSLN surface characteristics were modified by means of PEG-derivatives or PEG-containing surfactants [116,233,236,237].

Both stealth and non-stealth stearic acid labeled SLN werefound in rat CSF 20min after i.v. administration even though lowamount of radioactivity was found in the CSF samples. Anunderestimation due to the sampling into the cysterna magnawas then hypothesized [116]. When the same kind of SLN wereloaded with doxorubicin, significantly higher drug concentra-tion was found in the brain of the group treated with stealth SLNas compared to non-stealth SLN and doxorubicin solution.Remarkably, 30 min after administration of stealth SLN, thesame doxorubicin concentration (∼10 μg/g of tissue) was foundeither in the brain, heart, liver, lungs and spleen. The overallplasma kinetics of stealth and non-stealth SLN provided to besignificantly different from that of the doxorubicin solution[237].

Poloxamer 188 stabilized stearic acid camptothecin-loadedSLN targeted to the brain after both oral and i.v. administrationin mice [233,236]. Following SLN i.v. administration, themaximum concentration (Cmax) increased by 180% if comparedto the Cmax of the drug solution. The area under the curve(AUC)/dose and the MRTof SLN were 10.4- and 4-fold higher,respectively [236].

Two new SLN formulations made with biocompatiblematerials, such as emulsifying wax and Brij® 72, and stabilizedby P80 and Brij® 78 were proposed for brain drug targeting[46,140,238]. The aforementioned particles showed a signifi-cant brain uptake, measured during a short term in situ rat brainperfusion experiment [46]. Brij® 78 stabilized wax NPscontaining paclitaxel were able to significantly increase braindrug distribution as compared to paclitaxel dissolved in a 50:50v/v mixture of the surfactant Cremophor® ELR and dehydrated

ethanol (as the commercially available Taxol® formulation). Inthis case it was speculated that the employed carrier, maskingpaclitaxel characteristics, limited its binding to P-gp leading tohigher brain drug concentration due to the avoided efflux [140].A very interesting series of experiments, regarding the effect ofthese NPs on the baseline BBB parameters were carried out aswell [238]. Both in vitro and in vivo data showed no significantchanges on cerebral perfusion flow, integrity, permeability, andthe facilitated transport of choline [238]. The tight junctionmolecular integrity was also confirmed by occludin andclaudin-1 western blot analyses, showing no changes in proteinexpression. The authors concluded that the two investigated NPformulations turned out to have minimal effects on the primaryBBB parameters [238].

Surface charged SLN were also proposed in order to achievebrain targeting. Positively (+5 mV; 391 nm) and negatively(−47 mV; 362 nm) charged tripalmitin SLN were loaded withlabeled etoposide and their organ biodistribution was comparedto that of free labeled drug in mice after i.v. administration.Positively charged SLN showed a higher brain accumulationcompared with both negatively charged SLN and free labeleddrug. The etoposide concentration, achieved using positivelycharged SLN, was 10- and 14-fold higher with respect to thenegatively charged SLN at 1 and 4 hours after administration,respectively. Even though the overall concentration was not ashigh, some preferential BBB uptake could be hypothesized[239].

Clozapine loaded tripalmitin SLN, with (+23.2±0.9 mV;163 nm) and without stearylamine (+0.2±0.1 mV; 233 nm),were able to significantly increase drug brain concentration aftermice i.v. administration when compared to clozapine suspension[240]. Particularly, if compared to the clozapine suspension, theCmax increased by 199 to 243%, the AUC by 5.3- and 4.1-fold,and the MRT by 3.9- and 4.3-fold, for charged and non-charged

http://www.aapsj.org


particles, respectively. It is noteworthy that, for the positivelycharged SLN preparation, over the investigated organs (liver,spleen, heart, kidney, and brain), the highest relative bioavail-ability was seen in the brain [240]. Significantly higher brainetoposide concentrations, after mice intraperitoneal administra-tion of negatively charged tripalmitin SLN (−47 mV; 387 nm),with respect to etoposide alone were observed [241]. Unfortu-nately, the strategy of using surface charged SLN, or NPs ingeneral, to cross the BBB has shown some important drawbackslimiting its usefulness [242]. Neutral (−14 mV; 75±53 nm),negatively (−60 mV; 127±71 nm) and positively (+45 mV; 97±69 nm) charged SLN were prepared and their rat BBBpermeability was studied using the in situ brain perfusionmethod. Neutral SLN or low amounts (10 μg/mL) of negativelycharged SLN showed no effect on the cortical cerebrovascularvolume, indicating a good BBB integrity. Higher amounts ofnegatively charged SLN (20 μg/mL) or positively charged SLNsignificantly increased the cortical cerebrovascular volumepointing out a BBB disruption. Significant increases in vascularvolume were noted in all brain regions when perfused with highdoses (20 mg/mL) of either anionic or cationic NPs. Comparingorgan distribution of tobramycin-loaded SLN and tobramycinsolution, it was found that a statistically higher brainconcentration was achieved with i.v. administered SLN ifcompared to duodenal administration. Nevertheless, SLN couldreach the brain after duodenal administration in rats [243]. Inother works, biodistribution studies showed that idarubicin-loaded SLN were able to cross the BBB after duodenaladministration [244,245].

Superparamagnetic iron oxide loaded SLN (159 nm) wereproposed as a new type of nuclear magnetic resonance contrastagent [246]. After i.v. injection in rats, SLN were able to crossthe BBB and to accumulate in the brain parenchyma. A highsignal suppression was found in the hypothalamus, even thoughthe strongest suppression was in the liquoral space, where SLN

Fig. 11. Situation of the CNS drug research pr

were present in high amounts [116]. Different results wereobtained with charged NPs in vitro. This discrepancy wasprobably due to the non-adequate BBB in vitro model, havinglow permeability to sucrose and inulin but an electricalresistance of only 500–800 Ω cm2 [115].

A lipophilic pro-drug of 5-fluoro-2′-deoxyuridine (FUdR),the 3′,5′-dioctanoyl-5-fluoro-2′-deoxyuridine (DO-FUdR), wasefficiently incorporated into SLN (76 nm) [247]. Drug brainconcentration (intended as FUdR or DO-FUdR) was higher ateach time point for the group treated with the SLN formulationswith respect to FUdR solution. In the same study, DO-FUdRpharmacokinetics and body distribution were investigated aswell. Unfortunately, statistical data analysis and informationconcerning DO-FUdR formulation (solution, suspension, par-ticle size, stabilizer, etc.) are missing [247], impeding furthercomparisons between the brain distribution of DO-FUdR aloneand that loaded into SLN. However, the pro-drug approach canbe very useful in the development of SLN for brain drugtargeting. In fact, not all the therapeutic molecules possesssuitable characteristics to be efficiently incorporated into a lipidmatrix. Moreover, as a result of the low particle uptake availableat the BBB surface, SLN drug content may become a criticalissue for the clinical success of this strategy. For this reasonLDC NPs have been developed [188]. Even though preliminaryin vivo studies showed just an accumulation of NPs on the BBBsurface in place of a real particle uptake [111], it is our opinionthat this elegant approach should be more deeply investigatedbecause of its enormous potentiality. Another possibility tomaximize NP drug loading may be represented, when possible,by the formulation of the sole drug into nanocrystals,successively coated with a proper surfactant (e.g. P80)[126,248]. This technology would permit the delivery of thetherapeutic molecules to the target site, maximizing the amountdelivered and avoiding all possible toxic effects from the carriermatrix.

ogress updated through September 2005.


7. Concluding remarks

The amazing growth in recent years of CNS drugs on themarket has generated enormous research efforts in an attempt todevelop new drugs for brain diseases. However, the main interesthas been focused on the discovery of new therapeutic moleculesrather than developing new approaches and systems to targetactives to the brain. This is a general trend in pharmaceuticalscience. Since the importance of drug delivery to improvetherapies was understood more than 30 years ago, it is deeplydisappointing that most efforts are still oriented to the soledevelopment of drug discovery programs. Such a picture isexacerbated in the case of CNS drugs, for which virtually no BBBprograms have ever been accounted [1,2]. In fact, between the 40and 50% of the compounds under development remained at theearly stages of the discovery process while between the 23 and30% are those divided within the clinical phase I–phase III(Fig. 11). Less than 5% of the total molecules under screeninghave reached the final step beforemarketing and less than 1%havebeen registered over the past three years. In addition, about 15–20% of molecules have reported no development over a period of18 months [249–251]. Of the whole drug development program,more than 43% is represented by AD and PD therapeutics,whereas just about 14% are related to epilepsy, Huntington'sdisease, and multiple sclerosis. This disproportion on behalf ofAD and PD is likely a consequence of the large incidence of thesediseases in the population as a result of lifespan increase.

The low number of drugs entering the market reflects thecomplexity of the CNS drug development, certainly in part dueto the presence of the BBB. It is clear that this failure isascribable to a wrong design in the drug discovery and devel-opment programs that overlook the fundamental contribution ofa well sustained drug delivery program. Only the integration ofthese two complementary steps from the early stages of thedevelopment pathway can lead to more successful achievementsin brain disease therapy. This is even more evident in light of thefact that most of the potentially available drugs for AD and PDtherapies are large hydrophilic molecules, e.g. peptides, pro-teins, and oligonucleotides that do not cross the BBB. Amongthe several strategies attempted in order to overcome this prob-lem, properly tailored NPs may have a great potential.

The large amount of evidence regarding brain drug deliveryby means of P80-coated NPs cannot be ignored or considered assingle evidence even though its action mechanism is notcompletely understood. Lipid NPs, e.g. SLN, NLC, LDC NPs,may represent, in fact, promising carriers since their prevalenceover other formulations in terms of toxicity, productionfeasibility, and scalability is widely documented in the literature.

The use of surfactants is the main controversial point of thisapproach [252], but, at the same time, data on P80 toxicity, whenformulated together with NPs, are less conspicuous [95,253]than those reported by other authors [97–100,102,104,106,109]and not very convincing [254]. Moreover, such a controversialpoint is less applicable to lipid NPs by the fact that the use of alipid matrix strongly reduces the possibility of P80 desorptionupon administration [46]. Therefore, the use of these carriersshould be more deeply investigated as brain DDS for newer and

older neurotherapeutics. Furthermore, an optimal CNS drugdevelopment strategy should consider the design of suitablecarriers for these drugs as soon as they are demonstrated to beactive but unable to reach their molecular target. This would allowone to find the proper carrier, according to molecule char-acteristics, for an early preclinical and clinical evaluation of thefinal formulation. Such an approachwould certainly improve drugdeveloping strategies by increasing the number of therapeuticsthus far not exploited but that could effectively reach the market.

8. Legend for lipid substances

Brij® 35: Polyoxyethylene (35) lauryl etherBrij® 72: Polyoxyethylene 2 stearyl etherBrij® 78: Polyoxyethylene (20) stearyl etherCompritol® 888ATO:Mixture ofmono-, di-, and triglycerides

of behenic acid (referred in the text as Compritol®)Cremophor® ELR: Polyoxyethylated castor oilCremophor® RH40: Polyoxyl 40 hydrogenated castor oilCremophor® EL: Polyoxyl 35 castor oilDynasan® 114: TrimyristinDynasan® 118: TristearinImwitor®: Glycerol monostearatesPluronic® F68: Ethylene oxide/propylene oxide block

copolymerSoftisan® 138: Hydrogenated coco glyceridesSoftisan® 142: Hydrogenated coco glyceridesSolutol® HS15: Polyethyleneglycol 660 12-hydroxystearate

Acknowledgment

Thanks are due to Miss Maria Vigilante for kindly revisingthe English version of the manuscript.

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