drug delivery to the cns and polymeric nanoparticulate carriers

1681ISSN 1756-8919Future Med. Chem. (2010) 2(11), 1681–170110.4155/FMC.10.249 © 2010 Future Science Ltd

Review

Drug discovery for the treatment of pathologies that affect CNS is one of the most challenging undertakings of the pharmaceutical industry. However, development of drugs for CNS disorders is hampered by the existence of the blood–brain barrier (BBB), which is a formidable obstacle to the entry of drugs to the CNS. This barrier is formed by specialized endothelial cells and is supported by other cell types, such as astrocytes and pericytes. Paracellular diffusion of molecules is highly restricted owing to the lack of fenestrations and the presence of tight junctions between endothelial cells; moreover, active transport, both uptake and efflux, is also prevalent, as are metabolizing enzymes [1,2]. While some pathologies involve a disruption of the BBB [3], other pathologies, and even brain tumors at first stages, do not modify BBB permeability [4]. Thus, there is a great interest in the development of systems able to deliver drugs to the CNS in the presence of an intact BBB.

Nowadays, the main characteristics needed for low molecular weight compounds to cross the BBB are known, and the research has evolved towards systems able to deliver drugs that are unable to cross the BBB to the CNS alone. To this end, peptides able to target the brain and to deliver a cargo (compounds with a molecular weight less than 500 Da, peptides or proteins) to the CNS, or nanoparticulate systems, able to carry multiple drug molecules, are actively

studied. These delivery systems should possess an increased selectivity for CNS, increased quantities of drugs could be delivered to the brain, and, in the case of nanoparticulate systems, drug degradation could be prevented and a sustained drug release obtained. This evolution has lead to an increase in complexity. This article highlights the parameters that are needed in order to fully characterize previously untreated nanoparticles (Nps) targeting CNS [5].

In the field of low molecular weight compounds, medicinal chemists use structure modifications in order to influence BBB penetration, by modulating either passive diffusion or active transport [6,7]. Various physicochemical properties influence BBB permeability. It has been shown that in order to be able to cross the BBB, drugs shoud have lipophilicity (cLogP and cLogD [pH 7.4]) in the range of 2–5, polar surface area below 90 Å2, Hbond donors below three and a molecular weight of less than 500 Da. Moreover, replacement of carboxylic acid groups, addition of an intramolecular hydrogen bond, reduction of Pglycoprotein efflux and modification or selection of structures for affinity to uptake transporters are able to increase brain drug delivery [6,8].

The characterization of brain penetration by a drug molecule is a very complex feature; there is a clear differentiation between rate and extent of brain penetration, and between total

Drug delivery to the CNS and polymeric nanoparticulate carriers

The delivery of drugs to the CNS is hampered by the existence of the blood–brain barrier (BBB). Nowadays, medicinal chemists follow defined rules for the development of drugs able to cross the BBB. At the same time, the parameters needed in order to gain valuable estimates of brain drug delivery are well defined. Despite the limits in molecular weight that allow drugs to cross the BBB, it was shown that nanotech products, in particular properly functionalized nanoparticles, spherical particles of approximately 200 nm in diameter, are able to cross the BBB after intravenous administration and act as drug carriers for CNS. Moreover, peptides as ligands for receptors present on the brain endothelium, or able to cross the BBB and to act as carriers for CNS drug delivery in the form of conjugates with drugs, have been discovered and started to be studied as targeting moieties for nanoparticulate systems. This article will discuss the results obtained so far in the field of nanoparticle drug carriers for CNS and highlight the parameters needed in order to fully characterize these hitherto largely unknown delivery systems. Even if promising results have been obtained, more studies are needed in order to fully evaluate the clinical potential of this drug-delivery system.

Luca CostantinoUniversity of Modena and Reggio Emilia, Dipartimento di Scienze Farmaceutiche, Via Campi 183, 41100 Modena, Italy Tel.: +39 059 205 5749 Fax: +39 059 205 5131 E-mail: [email protected]

For reprint orders, please contact [email protected]

Review | Costantino

Future Med. Chem. (2010) 2(11)1682 future science group

and unbound drug levels as parameters for the characterization of drug distribution within the brain [9]. Methods that measure the three parameters K

pu,u (unbound brain/unbound

plasma concentration ratio at steady state; measured by brain microdialysis), CL

in or K

in (influx

clearance into the brain; which describes the net influx clearance across the BBB, measured for example by in situ brain perfusion technique) and V

u,brain (apparent unbound volume of dis

tribution in brain; which describes the nonspecific binding to brain tissue components in relation to unbound drug in brain interstitial fluid, measured by the brainslice technique) can give clinically valuable estimates of brain drug delivery [9,10].

Peptides as drug-delivery agents for the CNSBesides chemical modifications of drugs, peptides as drug vectors that target the BBB have been considered for targeted drug delivery to the CNS. Some of these peptides have been used for brain targeting of drugs (some peptide–drug conjugates are in preclinical or in clinical trials) and organic macromolecules (dendrimers), and started to be used for the delivery of nanoparticulate systems; however, these systems have not been widely studied and data obtained until now does not allow a full assessment of the c linical usefulness of this approach.

The peptide–drug conjugates should be physiologically stable and able to cross the BBB while maintaining the integrity of the barrier. Furthermore drug release must occur at the relavant targets in the appropriate intracellular location [11,12]. Thus, there is an increase in complexity and, in addition to the parameters described above, other parameters such as serum stability, immunogenicity and the kinetics of drug release inside the CNS should be determined.

In order to deliver drugs to the CNS using peptides as drug carriers, one approach uses polycationic molecules such as cationic bovine serum albumin (CBSA) [13,14], able to cross the BBB by an adsorptivemediated transcytosis mechanism that relies on nonspecific interactions with negative charges present on the plasma membrane. However, this system lacks specific targeting and may lead to widespread absorption [15]. Cationic protein transduction domains such as HIV TAT peptide [16] and cellpenetrating peptides such as SynB [17] and other vectors [16,18] also lack targeting specificity. Some peptide–drug conjugates (e.g., doxorubicin conjugated to Dpenetratin or

to SynB1 polycationic vectors) have been successfully evaluated in an in situ rat brain perfusion/capillary depletion technique, thus taking into account the drug remaining in the endothelial cells. In fact, capillary depletion separates brain into brain parenchyma and vascular fractions and is a simple method to estimate the extent to which a drug has crossed the BBB and is not simply internalized or bound to the vascular endothelium [14]. Doxorubicin vectorialization (in situ rat brain perfusion/capillary depletion technique) either with Dpenetratin or SynB1 led to a 20fold increase in the amount of doxorubicin transported in brain parenchyma. According to the importance of the serum proteins–unbound drug fraction of the drug for BBB crossing, when the perfusion buffer was replaced by rat plasma, a dramatic decrease in doxorubicin cerebral uptake was observed [19]. SynB peptides were also conjugated with the opioid peptide dalargin; their ability to translocate across BBB were evaluated by in situ rat brain perfusion technique, and the BBB integrity was determined using [3H]sucrose as a marker of brain vascular volume, since it does not measurably penetrate the BBB during brief periods (capillary depletion experiments were not performed). While the volume of distribution for dalargin is 16.7+/1.2 µl/g, that of dalargin–SynB1 is 309+/82.7 µl/g; in vivo analgesic studies (antinociceptive assays) confirmed brain penetration [20]. Proofs were provided that the these conjugates enter the brain by adsorptivemediated endocytosis, but it has not been assessed whether the dalargin is released from the carrier or if the conjugate interacts with the opioid receptors (the affinity of dalargin for the receptor does not change in the presence of the conjugate) [20].

Another peptide family containing N-methylated amino acids was recently discovered. These peptides were studied in vitro for permeability and phospholipophilicity by means of parallel artificial membrane permeability assay and immobilized artificial membrane chromatography. The best ones have been studied in vitro on bovine brain microvessel endothelial cells for their ability to ensure a passivediffusion transport for levodopa, GABA, nipecotic acid and 5aminolevulinic acid, with positive results [21,22].

Drug vector systems (for small molecules or proteins) that target endogenous transporters at the BBB have been considered for drug delivery to the CNS, in order to exploit receptormediated

Key terms

Blood–brain barrier: Unique barrier that tightly segregates the brain from the circulating blood. This barrier, formed by special endothelial cells sealed by tight junctions and characterized by very low pinocytic activity, restricts the molecular exchange to transcellular transport.

Nanoparticle: Solid colloidal polydisperse particle ranging in size from 10 to 1000 nm. Consist of macromolecular materials in which the active principle is dissolved, entrapped, encapsulated and/or to which the active principle is adsorbed or attached. Nanoparticles are actively studied for brain targeted drug delivery.

Drug delivery: Method or process of administering a pharmaceutical compound to achieve a therapeutic effect in humans or animals.

Targeted drug delivery: Administered drug is addressed to the specific site of action.

Dendrimer: 3D organic macromolecule that possesses a highly symmetrical and branched architecture; in theory, they are perfectly monodisperse. These macromolecules are studied as drug-delivery agents.

Drug delivery to the CNS & polymeric nanoparticulate carriers | Review

www.future-science.com 1683future science group

transcytosis, by several companies: transferrin (Tf) receptor and insulin receptor (ArmaGen Technologies Inc., CA, USA), low density lipoprotein receptor related protein (LRP)1 and LRP2 (Roche and Raptor Pharmaceuticals Corp., CA, USA; Angiochem Inc. Quebec, Canada), diptheria toxin receptor (able to interact with CRM197, a nontoxic mutant of diphtheria toxin) (toBBB Inc.), gluta thione (toBBB Inc.), nicotinic acetylcholine receptor on neuronal cells (able to specifically bind the rabies virus glycoprotein, to enable viral entry into neuronal cells; RVG29, a peptide derived from rabies virus, targets this receptor) [18,23]. Other peptides (ligands specific for brain endothelium) that could facilitate brain delivery of drugs have been recently patented or published elsewhere [24,

201], but their ability to act as drug carriers and perform transcytosis across BBB with a cargo (pharmacologically active agents) has not yet been reported.

Among drugvector systems, those that target Tf or insulin receptors are of limited interest owing to the widespread expression of these receptors and the low doses (less than 4% injected dose (ID)/g tissue) that reach the brain [11,25]; RVG29 was used as a braintargeted ligand to deliver siRNA into the brain by directly conjugation with siRNA [26].

Peptides that target the receptors LRP1 and LRP2, and those for melanotransferrin and RAP are widely studied. Melanotransferrin transcytoses (0.25% ID/g tissue, mouse brain) using LRP1; RAP is a protein that participates in the folding and trafficking of LRP1 and LRP2; it was studied in mice by the in situ brain perfusion/capillary depletion technique. It was shown to to be more BBB permeable than either Tf or melano transferrin both in vitro and in vivo. In mice, between 0.5 and 1% ID/g of tissue was delivered to the brain in 30 min. The vascular marker [99mTc]albumin did not have significant entry [11,27], confirming that no damage was caused to the BBB. RAP peptide has been considered for the delivery of proteins to the CNS (NeuroTrans™, system developed by Roche and Raptor Pharmaceuticals Corp.) [28].

The most promising results for brain drug delivery have been obtained for Angiopeps, peptides designed on the basis of the binding domains of LRP [29]. Among these, Angiopep2 (19 amino acids) showed a higher brain penetration capability than other proteins of the LRPrelated family and most proteins that target the BBB, such as aprotinin and Tf [29].

The brain uptake of the conjugate between paclitaxel (three molecules) and Angiopep2, Ang1005, exceeds paclitaxel brain uptake by 4 to 5fold. In situ V

d (brain perfusion) is more

than twice that of melanotransferrin. Capillary depletion experiments showed that more than 65% of Ang1005 was found to be associated with brain parenchyma after 1–5 min perfusion [30]. However, it was shown that brain uptake (K

in) of Ang1005 is tenfold greater

than that of Angiopep2, the carrier alone, suggesting that the drug exerts a great impact in the drug delivery of the conjugate [30]. This experimental result remained unexplained. Brain [14C]sucrose space, which evaluates the BBB integrity since sucrose is a slightly permeated molecule across the normal BBB and is not metabolized by the rat, was not altered from control when determined in the presence of Ang1005 or Angiopep2, confirming that BBB was not affected by this compound. According to the free drug hypothesis, which postulates that the concentration of unbound drug is the driving force for all distribution processes, addition of 2.7% of serum albumin to the in situ brain perfusion fluid (under nonequilibrium conditions) sharply reduced the free fraction of Ang1005 in the perfusion buffer by 90% and similarly decreased [125I]Ang1005 uptake into brain [30]. However, despite this effect, clinical results obtained by the use of this conjugate are very encouraging (see later discussion). In fact, it is believed that a high level of drug–plasma protein binding is in itself no limitation for CNS action, and this association could r epresent a depot of drug in plasma [10].

Experiments suggest not only the involvement of the LRP receptor in transcytosis, but also other hitherto unknown mechanisms [31]. Ang1005 is in Phase I–II trials for brain cancers. Biological data show that Ang1005 does not elicit an immune response even in patients who received multiple treatments or experienced infusion reactions and/or rashes. Tumor stabilization and, in some cases, significant reduction in tumor size and reversal of neurological deficits were observed in patients with highgrade gliomas [32]. Other Angiopepderivatized compounds (conjugates with doxorubicin, etoposide, therapeutic peptides, monoclonal antibodies and siRNA) also showed promising results [33].

Another peptide, CRM197, a nontoxic mutant of diptheria toxin, was successfully used for targeted delivery of conjugated proteins and liposomes across the BBB [17]. A remarkable

Review | Costantino


feature of this delivery moiety is its intrinsic endosomal escape mechanism, which allows the protein to enter the cytosol of the cell, bypassing the lysosomal degradation system [17].

DendrimersRecently discovered peptides that are able to cross the BBB have started to be used as targeting moieties for dendrimers, 3D organic macromolecules with a welldefined molecular weight that possess a highly symmetrical and branched architecture, and are actively studied as drugdelivery agents [34,35]. Progress in the synthesis led to the development of structures with a large number of surface groups that can be utilized to display a range of biological motifs including peptides, proteins, sugars and targeting agents, while carrying a large therapeutic payload either within the dendrimer void or on their surface [35]. These compounds are actively studied for drug and gene delivery [36,37]; some dendrimers are in preclinical development for various indications (treatment of various cancers, malaria and HIV infections) [38] but only three reports deals with CNS delivery by these carriers. Thus, poly(amidoamine) (PAMAM) dendrimers of generation (G)5, (diameter of 5.3 nm) have been surface functionalized with Angiopep [39], lactoferrin [40] and RVG29 [41], using poly(ethyleneglycol) (PEG) as a linker, in order to target the brain and deliver genetic material to CNS. Angiopepconjugated PEGmodified PAMAM dendrimers showed a brain uptake of 0.25% ID/g of tissue (intra venous administration; capillary depletion experiments were not conducted). In vitro transport studies on the bovine brain capillary endothelial cell monolayer showed no increased permeability of [14C]sucrose, demonstrating the integrity of cell monolayer. Competition assays with different ligands of LRP (Angiopep2, RAP, lactoferrin) were successfully performed as a proof of the involvement of the receptor for BBB crossing [39]. Surface PAMAM modification with PEGlactoferrin (a singlechain ironbinding glycoprotein that belongs to the Tf family) has been reported to enhance transfection efficiency of PAMAM [40].

NanoparticlesTo date, there are over two dozen nanotechnologybased therapeutic products (nano-medicines) that have been approved for clinical use; among the firstgeneration products, liposomal drugs and polymer–drug conjugates are the two dominant classes [42]. At the same

time, Nps for intravenous administrationn are the object of intense research in the field of CNS drug delivery. These Nps are polymeric particles to which drugs can be adsorbed or incorporated. This device can protect drugs against enzyme inactivation and allow access across the BBB by masking their physicochemical characteristics. Moreover, Nps that are able to deliver multiple molecules into the CNS could ensure a sustained release of the drug and, most importantly, could increase drug tissue selectivity, if properly functionalized.

Other nanotechnology products have been shown to be able to deliver drugs to the CNS: solid lipid Nps (SLNs), which are Nps constituted of biodegradable lipids, both underivatized or surface decorated with thiamine (see later discussion), transferrin or PEG, seems to be promising brain drugdelivery agents [43,44]. Literature examples that showed an improved biodistribution and targeting effect of anticancer agents to the brain using SLNs [45] and liposomes [46] have been reviewed recently. However, these nanoparticulate systems have not been studied at the same level as polymeric Nps (quantitative data about brain drug delivery are lacking), and they are not in clinical trials for brain delivery [38]. Thus, a comparison of the brain drugdelivery ability among the various nanotechnology products available is impossible at this time. From a clinical point of view, it would be of great interest to perform studies such as that performed by Minko [47], which compared the therapeutic efficacy of drugloaded receptortargeted polymers, dendrimers and liposomes as antitumor agents. Magnetic Nps, which are able to concentrate at a specific target site within the body using a magnetic field, have recently been reviewed [48].

After intravenous administration of drugloaded Nps, the pharmacokinetic profiles of the parent drug and the drug encapsulated in Np could be different. Immune cells in the bloodstream, (e.g., monocytes, platelets, leukocytes and dendritic cells) and in tissues (e.g., resident phagocytes) can engulf and eliminate Nps. The interaction with plasma proteins (opsonins) [49–51] and blood components (via hemolysis, thrombogenicity and complement activation) may influence uptake and clearance and, hence, potentially affect distribution and delivery to the target sites [52]. Then, in the case of braintargeted Nps, they have to cross the BBB and release their cargo (drugs) into CNS; the components other than the drug should be cleared without any toxic effect.

Key term

Nanomedicine: Field of research in which the integration of nanotechnology into medicine led to nanometric systems for vectorialization and functionalization of drugs.



Several mechanisms for Npmediated drug uptake by the brain have been outlined:

nEnhanced retention in the brain capillaries, which results in a high drug concentration gradient across the BBB;

nOpening of tight junctions due to the presence of Np components;

nTranscytosis of Np through the endothelium;

nEndocytosis of Np by endothelial cells; drugs can then be released and delivered to the brain;

nInhibition of Pglycoprotein efflux system by Np components (FiguRe 1) [53].

The parameters that we propose to be considered in order to characterize this brain delivery system are:

nThose that describe Np characteristics;

nThose that describe CNS influx clearance of the Np; the assessment of a transcytosis mechanism of BBB crossing, among other possible CNS mechanisms of drug delivery [53], should be performed, as well as the intracellular localization of the Nps into neurons;

nThose that describe the drug release from the carrier once the Nps localized into brain parenchyma, after BBB crossing (FiguRe 1).

The parameters considered to be relevant for a full characterization of polymeric Np, which influence nanoparticle blood residence time and organspecific accumulation, appear to be composition, size, core properties, surface modification, targeting ligand functionalization and others reported below [54,55].

n Size & polydispersity index Size is strictly related to the pharmacokinetics of the Np [54–57] and care should be taken in the assessment of this parameter. Many tools

BBB

Brain parenchyma

Blood

Intracellular traffickingof Nps

Neuron

Intracellular trafficking of Nps

+P

-P

+P

-P

E

ET

Np

+P

-P

+P

-P

Drug

P-gp

A B

BBB

Neurons

Future Med. Chem. © Future Science Group (2010)

Figure 1. Delivery of low molecular weight drugs and drug-loaded nanoparticles to the brain parenchyma. (A) Low molecular weight drug delivery to CNS. (B) Np-mediated drug delivery to CNS (the passage through the opened tight junctions was omitted for clarity). Large molecules such as antibodies, lipoproteins, proteins and peptides can also be transferred to the central compartment by receptor-mediated transcytosis or nonspecific adsorptive-mediated transcytosis. The receptors for insulin, low-density lipoprotein, iron transferrin and lactoferrin and leptin are all involved in transcytosis. Purple squares: free (protein-unbound) drug; pink circles: drug bound to proteins; blue Np: Np containing a drug; red Np: Np containing a drug onto which proteins have been adsorbed. BBB: Blood–brain barrier; E: Endocytosis; Np: Nanoparticle; T: Transcytosis; P: proteins; P-gp: P-glycoprotein.

Review | Costantino


based on different physical principles are currently available to measure particle sizes smaller than 1 µm [58]. Techniques routinely used are light scattering and scanning electron microscopy. LS is based on the interaction of a particle viewed as a sphere with light and scanning electron microscopy is a microscopic method that allows the observation of a sample after drying and coating with a thin layer of gold or platinum. This technique provides the most direct picture of the Np, giving information about size and shape [58]. It has been reported that the use of a combination of two methods, one of which should be a microscopic method, is highly recommended for size determination. Moreover, since the size distribution is not unimodal, polydispersity should be discussed carefully, in order to validate the data from biodistribution studies [58]. Size should also be determined in the presence of plasma, owing to the opsonization process. Examples are reported that show the difference in diameters of Nps in the presence and absence of plasma [59–61]. In vitro studies showed that both size and charge of colloidal drug carriers are important in determining net brain permeation [62]. The Nps that have been shown to be able to act in vivo as CNS drug carriers are approximately 200nm diameter. A systematic study, relating diameter to BBB crossing, similar to that performed in an intestinal cell model for the intestinal absorption of Nps [63], has recently been performed [64]. It was shown that, among poly(butylcyanoacrylate)/polysorbate 80 Np, one of the best studied Nps targeting the brain, with diameters of 70, 170, 220 and 345 nm, those of 70nm diameter deliver more drug into brain, while no significant differences were observed among the other three size ranges (intravenous Np administration; quantification of drug delivered to the brain by means of HPLC on brain homogenate). Capillary depletion experiments and histological localization of Nps, in order to assess if transcytosis occurred, have not been performed [64]. However, since it has been recently shown that polyester Nps are able to transfer their cargo (fluorescence labels) rather than penetrate cells, it has been suggested that the observed increased efficiency of poly(d,llactidecoglycolide) (PLGA) Np uptake in in vitro studies, as the particle size decreased, could result from the higher surface areatovolume ratio of the smaller particles, making this fluorescence transfer easier [65]. Thus, care has to be taken in order to assess whether a true transcytosis or endocytosis process occurred.

n ShapeTo date, all the Nps studied for brain drug delivery have been spherical, owing to the ease of preparation. On the other hand, it is known that oblate Np will adhere more efficiently to the vascular endothelium than spherical particles of the same volume [66,67]. Moreover, using polystyrene particles of various sizes and shapes, it was shown that particle shape, not size, plays a dominant role in phagocytosis by alveolar macro phages [68]. Thus, it has to be expected that Nps with different shapes will be studied in the near future.

n DeformabilityThis parameter influences biodistribution (an example of its determination can be found elsewhere [69]), and it has never been determined for Nps that target the brain. This property depends on core composition, which influences flexibility and shape adaptability [70].

n Atomic composition of Np surfaceThis parameter is very important because Nps interact with body components by means of their surface. In addition, surface properties also depend on the core composition (see later discussion). Very often, the Np surface is covered by a hydrophilic polymer to stabilizing Np suspension as targeting agent; its conformation and coverage density are important parameters for Np biodistribution [49,50,71]. XPS, also known as electron spectroscopy for chemical analysis (ESCA) is now routinely used to obtain the surface composition of the polymers, since this determines the elemental and average chemical composition of the material at its surface in 5–10 nm depth. The detection limit is 0.1%, by measuring the binding energy of electrons associated with atoms. To date, this technique has only been used in the field of braintargeted Np for the surface atomic composition of unloaded polyester similopioid peptide [72], CBSA Np [73] and lactoferrinderivatized Np [74], which target the brain, and for another kind of Np, with a composition similar to those that target the brain [75].

n Toxicity of the polymer, its degradation products & other components present in the NpUsually, Np drug contents are around 10% (wt/wt) of the Np; thus, the main part of this delivery agent is composed of the Np itself and care should be taken in order to carefully check the toxicity of the components other



than the drug. The polymers mainly used for the preparation of Nps that target the CNS, (poly(butylcyanoacrylate) [PBCA] [76,77] and polyesters such as PLGA [78,79]) have been studied in this respect and have shown little toxicity. In particular, PLGA is a wellstudied, US FDAapproved, biodegradable and biocompatible polymer that has been used for decades in clinical applications [79]. However, size can be related to toxicity, since it was found that smaller particle sizes cause higher cytotoxicity (this effect was studied in the presence of poly[alkylcyanoacrylate] particles). This experimental result can be explained by the large surface area, which leads to faster and higher degradation product concentrations [80]. Thus, toxicity studies should be conducted on Nps that will be used as drugdelivery agents. Several reports showed that the Nps produced by nontoxic degradable polymers could still induce the cytotoxicity or immune toxicity [81]. Moreover, experiments conducted on cytotoxic cationic polystyrene Nps of different sizes (60 and 200 nm) showed cellspecific differences in Np processing and toxicity, which is also dependent on particle size [82]. Thus, in the case of braintargeted Np, neuronal toxicity should be routinely assessed.

Longterm toxicity of braintargeted Nps has never been conducted (usually Np drug content is approximately 10% wt/wt; the main part is represented by a polymer and other formulation components). However, the toxicity of Nps can be an issue, especially in light of the short duration of the pharmacological effect seen in the presence of drugloaded Nps (a few hours at best; in only one case was a sustained release on the order of weeks seen [83]), implying that daily Np administrations will be required. Moreover, in order to avoid accumulation, the Np components other than the drug should be removed from the body as quickly as the embedded drug. Thus, intense research must be performed on the development of fully biocompatible formulating agents.

n ImmunogenicityImmunological events can compromise the efficacy and safety of drugdelivery systems by changing their pharmacokinetics, biodistribution and targeting capability and, thus, inducing hypersensitivity reactions. Little attention has been paid to the potential immunogenicity (the capability to elicit an immune response) of Nps; antibodies induced by administration of

drugdelivery systems can be directed against the carrier material, the drug and/or targeting ligands associated with drugdelivery systems (reviewed in [84]) and these immunological events can compromise the efficacy and safety of these systems. Although the literature describing antidrugdelivery system antibodies is largely restricted to liposomes, there is no fundamental reason why antibodies would not be formed against other drugdelivery systems, including polymeric Nps [84]. In fact, poly(isobutyl cyanoacrylate)/dextran Nps have been reported to activate complement in vitro, with size and configuration of dextran (‘brush’ or ‘loops’) playing an important role [85]. Even PEG itself, FDA approved for use as vehicle or base in foods, cosmetics and pharmaceuticals, including injectable formulations, initially though to act as an inert steric barrier for the attachment of opsonins, was shown to be able to generate complement activation products [86]. PEGylated liposomes are also able to induce IgM production and complement activation. This leads to accelerated blood clearance (reviewed in [48]). Since braintargeted Nps contain components with a PEG substructure, the ability of these Nps to induce immunological events should be carefully checked. The effect of accelerated blood clearance observed after repeated injection of PEGylated liposomes, which are cleared much faster than after the first administration, was also seen in presence of braintargeted CBSA Nps [87]; it is not known at present if this effect is shared by other kinds of Np. Studies conducted on PBCA/dextran Nps, carrier widely studied for brain drug delivery (see later discussion), showed a variable effect on specific immune response in mice receiving high or low doses of Np [88].

The immunogenicity of polymers should be studied in the form of Nps. Even if the solution of polymers carrying surfacehydroxyl groups (poly[vinyl alcohol] [PVA]; dextran), widely used for Np preparation are not immunogenic, when they are linked to a surface, these are able to strongly activate the complement system through the alternative pathway [89].

Up to now, in the case of braintargeted Np, the assessment of hemocompatibility, with a particular focus on hemolytic activity, platelet function and blood coagulation, has been performed only in the case of E78 Np (emulsifying wax/Brij 78 and distearoylphosphatidylethanolamine (DSPE)–PEGemulsifying wax/Brij 78 Np) and demonstrated to be safe in this regard [90]. Another study tested the hemocompatibility of

Review | Costantino


PLGA, surfactantmodified (lauric acid sodium salt) PLGA and PEGylated PLGA Nps. These Nps have not been tested as CNS drugdelivery agents. Results obtained underlined the positive effect of PEGylation among the studied Nps with respect to biocompatibility [91].

n z potential of Nps The z potential plays an important role in affecting biodistribution but also the intracellular trafficking of Nps [92], a factor that is important for drug delivery to the site of action. Nps that shows a reversal in their zpotential (from anionic to cationic) in acidic pH (e.g., PLGA Nps) can escape the degradative endolysosomal compartment into the cytosol, whereas Nps that remain anionic at all pH values (e.g., polystyrene Nps) do not exhibit endolysosomal escape [93]. This parameter is also very important since it was shown that Np surface charges alter BBB integrity and permeability [94]. In fact, it was shown by in situ rat brain perfusion that neutral emulsifying wax Nps and low concentrations of anionic Nps can be utilized as colloidal drug carriers to the brain, while cationic Nps have an immediate toxic effect at the BBB. Experiments carried out on cyanoacrylate Nps loaded with doxorubicin showed the importance and the influence of the loaded drug on this parameter [60,95].

n Interaction with plasma proteins This is a very important parameter, since it relates to particle biodistribution, biocompatibility and therapeutic efficacy [51]; a systematic study showed that size and surface properties determine the composition of protein corona [96]. However, this parameter is not easily measurable [50] since the complete composition of the protein coating at any given time is a function of the concentrations of all plasma proteins and their binding kinetics (equilibrium constants, on/off rates and binding affinity). Moreover, the presence of a drug molecule modifies protein adsorption pattern [95] and, at the same time, drug is released from Nps over time, after their intravenous administration. In light of this, it appears that the true composition of the Np–protein complex changes continuously during its time within the body [49,50].

n Core compositionCore composition inf luences f lexibility and shape adaptability, which are important factors for biodistribution [69], but also influences the surface characteristics, and should always

be determined. It is known that PBCA Nps, widely studied as CNS drug delivery agents, are usually prepared in the presence of the polymer dextran (PBCA Nps contain more than 87% dextran [97]). This quantity should always be determined on the Np being tested; PLGA Nps are usually prepared in the presence of the surface ative agent pluronic F68, but the quantity of this agent loaded on braintargeted Nps has never been determined.

n Surfactants With the exception of the apolipoproteinconjugated albumin Nps, Nps are usually prepared using a polymer (in the case of braintargeted Nps, these are mainly polyesters and polycyanoacrylates) together with a polyhydroxylated polymer (PVA and dextran, respectively) and/or in presence of surfactants based on PEG substructure (polysorbate 80, pluronic F68) (FiguRe 2). PEG itself can be also covalently linked to polyalkylcyanoacrylate, the starting material for Np preparation. It is known that pluronics exert biological actions beside Np stabilization [98,99], and residual PVA on Np is able to prevent uptake by cells [100]. However, it has been recently hypothesized that this effect of PVA could be due to a reduced dye (a label of Np) transfer from the Np to cells, owing to a contact limitation between Nps and cells caused by PVA shielding, rather than a reduced Np uptake [65]. The amount of surface active agent adsorbed on braintargeted Np has been determined only in the case of model Nps composed of PLA, fluorescein isothiocyanate labeled dextran and polysorbate 80 [101]. The disso ciation equilibrium constants of the surface active agents in presence or in absence of plasma proteins has never been determined.

n Presence of the drugIt is known that the embedded drug could be able to modify Np size, zpotential, protein adsorption and biodistribution [60,95]. Thus, Np behavior could change in the presence of a loaded drug. Nps should therfore be studied in the presence of the drug that has to be delivered to the brain.

n Uptake mechanisms & intracellular trafficking of NpsIn contrast to free drugs, which can cross the plasma membrane by active or passive diffusion, drugdelivery systems are internalized by a process known as endocytosis upon binding

Key term

z potential: Represents the difference in the electrical charge between the dense layer of ions that surrounds the particle and the charge of the bulk of the suspended fluid surrounding the particle.



to cellsurface receptors. Endocytosis occurs by multiple mechanisms that fall into two broad categories: phagocytosis (in the case of phagocytes) and pinocytosis [102,103]. Endocytic pathways occur via four main mechanisms (clathrinmediated endocytosis, caveolaemediated endocytosis, macropinocytosis and caveolaeindependent endocytosis). The macropinocytosis and clathrinmediated endocytosis pathways end up in small vesicles known as endosomes; these then evolve in late endosomes. Next, these vesicles fuse together and with lysosomes, where degradation by several enzymes or by the presence of an acidic pH (pH 4.5) occurs. It appears that the intracellular drugdelivery processes depend on nanocarrierintrinsic properties [103,104] and also on endocytic pathways in a given cell type [103,105]. Endolysosomal escape is fundamental for drugs sensitive to lysosomal degradation or for those that need to reach extra endolysosomal targets [102,106,107], thus intracellular trafficking of Nps is a characteristic that should be studied indepth. While many studies dealing with Nps endocytosis have been performed [102,103,107], the precise mechanism of transcytosis across polarized endothelial cells has not been determined yet.

Experiments show that the use of polymers with buffering capacity or pHsensitive carriers prevent the drug degradation in lysosomes [102]. Interestingly, the comparison between PLGA/PVA/polysorbate 80 and PLGA/PVA Np, which share the same diameter (250 nm) and zpotential, (30 mV) showed that Nps containing polysorbate 80 (the amount was not quantified) possess an increased cellular uptake and a different intracellular distribution (among cytosol, membrane/organelle, nucleus and cytoskeleton) [108]. These results also underline the necessity of fully characterizing Nps, beside the two parameters considered, diameter and zpotential.

Few studies dealing with intracellular trafficking are available in the field of brain targeted Nps. PEG–PHDCA Nps, able to cross the BBB in vivo (as determined by fluorescence microscopy experiments) [109], were internalized in in vitro rat brain endothelial cell cultures through the clathrinmediated endo cytosis pathway after specific recognition by LDL receptors [110] (the recognition by these receptors is also shared by PBCA/polysorbate 80 Np [53]) and were shown to be localized in different cellular compartments (incubation time 20 min). A total of 48% of Nps were associated with the plasma membrane, 24% in cytoplasm, 20% in vesicular compartment

and 8% in a Tritoninsoluble fraction (it has been suggested that this fraction could be associated to the nucleus, cytoskeleton or caveolae). The closely related PHDCA Np (PEG–PHDCA Np diameter: 171 nm; PHDCA Np diameter: 166 nm; zpotential for both: 20 mV) localizes in the brain at a level threetimes lower than PEG–PHDCA Np (80% remained in the plasma membrane and 10% found both in the cytoplasm and vesicular compartments) [111].

Another set of parameters that are considered for low molecular weight compounds are those that describe brain penetration (rate, extent). In the case of Nps, it has to first be determined, whether transcytosis or endocytosis occurs; in other words, if Nps are able to translocate across the BBB or if the Nps simply enter into endothelial cells.

Usually, the assessment of transcytosis is made using Nps loaded with fluorescent probes (lipophilic compounds), and the localization of the Nps into brain parenchyma is determined by means of fluorescence microscopy. However, since it was recently shown that polyester Nps are able to increase intracellular fluorescence of cultured cells either by dye transfer or by Np uptake (in vitro studies), results obtained using loaded dyes should be considered cautiously [65].

By analogy with the parameters related to low molecular weight compounds, K

in, which

describes the net influx clearance into the brain, appears to be very important in order to allow

OHO O

O OH

O

O

OH

zy

x

w

O

Polysorbate 80

HOO

Hn

PEG

PEG–PHDCA

CN CN CN

OO

O OO

O n

w + x + y + z = 20

OO

O

HO

nm

Hn

Pluronic F68 (n = 75, m = 30)

O

Figure 2. PEG-based compounds used for the development of brain-targeted nanoparticles.PEG: Poly(ethyleneglycol).

Review | Costantino


a comparison between different kinds of Nps. However, since it is known that plasma lipoproteins play an important role in Nps crossing the BBB (see later discussion), this parameter should be determined in the presence of plasma to give a valuable estimate of in vivo brain delivery.

Previously, this value was determined only for SLN (without discrimination between endo cytosis and/or transcytosis) by means of in situ rat brain perfusion technique in the absence of plasma. Moreover, capillary depletion experiments have not been performed; thus, Np amount present in the brain endothelium vessels is unknown. These Nps were comprised of emulsifying wax/Brij78/DSPE–PEG–thiamine as the delivery agent and radiolabeled with [3H]cetyl alcohol (blank Np) or [3H]thiamine (t hiaminedecorated Np, in order to use the thiamine transporter to cross the BBB). Thiaminecoated Nps have a K

in of 9.8 ± 1.1 × 10–3 ml/s/g

compared with a Kin of 7.0 ± 0.3 × 10–3 ml/s/g

for uncoated Nps [112].WaxBrij 78/polysorbate 80 Np labeled with

[3H]cetyl alcohol showed brain uptake (endocytosis and/or transcytosis) without any effect on significant BBB baseline parameters (cerebral perfusion flow, barrier integrity and permeability E78 Np K

in = 4.1 ± 0.5 × 10–3 ml/s/g; E72:

Kin = 5.7 ± 1.1 × 10–3 ml/s/g [113,114]. Washout

studies indicate an absence of [3H]Nps loosely associated with endothelium.

The third series of parameters to be considered are those that describe the drug release from the carrier once the Np has crossed the BBB.

n Np degradation time This parameter is important for drug release if the drug is embedded into the Np, and not if it is simply adsorbed onto the Np surface. It appears to be related to Np size. Nps that target the brain are usually composed of polyesters and polybutylcyanoacrylate. The degradation of these polymers in bulk was recently reviewed [46]; some studies were conducted on Np stability in vitro (buffered solutions), but very few were performed into cells or in the presence of plasma. While PLGA microspheres (10–40 µm in diameter) injected into rat brain remain in the tissue for approximately 2 months [115,116], the degradation of PLGA Nps (~200 nm diameter) within 6 h was observed in nonphagocytic HeLa Cells [117], while PLGA beads (10 µm size) phagocytosed by macrophages took longer to degrade (1–2 weeks) [118]. On the other hand, it was shown that brain levels of the drug ritonavir loaded into PLA/PVA Nps

(320 nm diameter) are still present 28 days after administration, suggesting a slow Np degradation [83]. Furthermore, after intravenous administration of another polyester – poly(lactide), [14C]PLA radiolabeled Nps – to rats, 90% of the recovered 14C was eliminated within 25 days, of which 80% was CO

2. In this case, however,

both the hydrolysis of the polymer PLA that form the Nps leading to the lactic acid monomer, and its subsequent metabolism to CO

2 were

evaluated [119]. Studies conducted in the presence of plasma showed that PBCA Nps are quickly biodegradated [120] and the halflife of PBCA/polysorbate 80 Np (200–250 nm diameter) into neurons in vitro was 27 min [121]. Thus, more studies are needed in order to determine Np degradation time into cells, in order to avoid a cumulative effect of Np components other than the drug in the case of repeated administrations.

n Release kinetics of drug from the NpIn order to compare the efficacy of the Np, release studies evaluating the amount of Npunbound drug present in brain parenchyma should be performed in the CNS (e.g., by centrifugation [83]), or at least in vitro in the presence of proteins, and not in buffer. This has been shown for PLGA Nps. In contrast to that observed in buffer, PLGA Nps release a moderate amount of Nile red in the presence of serum proteins [65]. To date, the pharmacological effects observed after braintargeted drugloaded Np administration last approximately 3 h, thus, it can be hypothesized that a fast release occurs, excluding other factors that could influence drug amounts at the site of action. If the loaded drug is a protein/peptide, it has to be determined whether these compounds are released in the active form or if they are degraded, for example by the acidic pH present during the degradation of Nps made of polyesters.

The drug levels present in brain both in the free (unencapsulated) and bound (Npencapsulated or tissuesequestered) form were evaluated only in the presence of TATconjugated PLA/PVA Np by means of centrifugation of brain tissue [83].

n Uptake of Nps by neurons (in the case of Np BBB transcytosis) This property has to be considered in relation to the cellular localization of the receptor that is the target of the administered drug. It was shown that PBCA/polysorbate 80 Nps are able to deliver a loaded protein into neurons in vitro by interaction with LDL receptor [121], and, after intravenous administration, apolipoprotein Econjugated



albumin Nps were found in neurons, indicating transcytosis across the BBB, with a subsequent reuptake by neurons [122]. Among Nps that have been studied for their braintargeting ability, it was found that proteinloaded PBCA/polysorbate 80 Nps localize into the cytoplasm of cultured neurons, thus escaping the endolysosomal compartment [121]. Another kind of Np, composed of PLGA (used for braintargeted Nps) and PVA (a surfactant not used for Np that target the brain) loaded with a fluorescent dye, showed the same behavior (in human arterial smooth muscle cells in culture). This effect has been attributed to the reversal in their zpotential, from anionic, which is a characteristic of PLGA Nps suspended in water, to cationic, in the presence of an acidic pH, as is found in lysosomes [93]. Another study explored the uptake and intra cellular fate of Nps with the same composition (on three different epithelial cell lines); the results obtained confirmed the ability of Nps to escape the endolysosomal compartment; at the same time, a difference in the rate and extent of Np uptake and trafficking among cell lines was noted [123]. Studies on neurons (or on brain endothelial cells) should therfore be conducted to fully understand in vivo Np behaviour.

n Neuronal toxicityThis topic gained particular attention following the rapid developments in the field of nanotechnology. The toxic effects that polymeric Nps could exert after crossing the BBB have not been studied in detail, and investigations regarding neurotoxicity are needed [124]. Data showed that PBCA/polysorbate 80 Np are well tolerated by cultured neurons at doses at which uptake saturation occurs; however, it appears that the presence of a loaded protein can influence toxicity. TAT peptide, was shown to exert neuronal toxicity when present conjugated with the Fragile X Mental Retardation protein [125].

Moreover, pharmacokinetic studies, which involve measurements of Np concentrations in all major tissues after Np administration over a period of time until the elimination phase, useful in order to fully describe the behaviour of Nps in vivo and to assess their potentiality in therapy, are available (reviewed in [49,55,126]). However, since it was shown that loaded doxorubicin modifies Np biodistribution [60,95], it seems that these parameters must be determined in the presence of the drug that has to be delivered, in order to obtain clinically useful data. The use of model drugs should be avoided.

Nanoparticles that target the brain have not been studied in all aspects in a systematic manner; moreover, since the presence of a drug can modify Np characteristics (z potential, diameter, interaction with plasma proteins [95]), it could be hypothesized that it is very difficult to find a carrier that can be used for all the drugs to be delivered to the CNS. Further, the assessment of the toxic effects of these Nps on the BBB has to be carefully checked. If breakdown of the BBB occurs, the passage of various serum components into the brain can occur, leading to CNS pathology [127]. Thus, experiments should always be conducted in order to test BBB integrity after Np administration. The opening of tight junctions could be investigated by measurement of the inulin spaces by the Oldendorf method [128,129], or by quantification of the diffusion of [14C]sucrose into the brain [109]. Another method involves the perfusion of a lanthanium salt solution, followed by electron microscopy studies [122].

Considering all these aspects, it is evident that the characterization of this delivery system is more complex than that needed for low molecular weight compounds.

Several strategies have been applied in order to obtain Nps that are able to cross the BBB, namely:nCationization

nPEGbased surface coverage of Np

nNps surfacedecorated with receptor ligands

nNpd surfacedecorated with a similopioid peptide

This subdivision is somewhat arbitrary, since positive results depend, as will be shown, not only on the surface characteristics but also on the Np core (type of polymer), the presence of other formulating agents and the characteristics of the drug loaded onto the Nps.

n CationizationThe strategy of using cationic vectors for BBB crossing was also applied to nanosized carriers (liposomes and Nps) made of CBSA, chitosan or TAT peptide.

Liposomes coupled with PEG–CBSA have been shown to be taken up by cultured porcine brain microvessel endothelial cells and intact brain capillaries [130]. Fluorescence studies showed that CBSA–PEG–PLA Nps are able to cross the BBB in vivo by means of adsorptivemediated transcyosis; however, contrary to what is expected, the zpotential of the Nps, determined in NaCl

Review | Costantino


solution, is negative, probably owing to a low surface coverage of the PLA Np by CBSA [73,131]. CBSA–PEG–PLA Np showed a twotothreefold increase with respect to PLA Nps [87]. Nps made of trimethylated chitosan, a permanently quaternarized chitosan derivative, positively charged under physiological conditions, were shown to cross the BBB by fluorescence microscopy and deliver a drug into the CNS, on the basis of the observed pharmacological activity [132].

TATconjugated PLA/PVA Np (zpotential: +2.4 mV) were shown by f luorescence microscopy to cross the BBB without inducing damage (Evans blue permeability test). Despite this, the delivery based on TATpeptide lacks specific brain targeting and can lead to widespread adsorption [16], a great quantity of ritonavir is delivered to the brain and a sustained release is obtained. Quantitative data ([3H]ritonavir levels) after intravenous administration in mice, together with capillary depletion experiments, showed a ritonavir brain level 800fold higher with TATconjugated Np than with ritonavir in solution and approximately sevenfold higher than the same with unconjugated Np, with a large amount of drug in a free form present in brain [83].

However, there are some concerning issues in the development of cationic Nps, since it is known from in situ rat brain perfusion experiments that cationic Nps have an immediate toxic effect at the BBB, altering BBB integrity and permeability [94]. Therefore BBB intregrity, especially in the presence of cationic Nps, should be carefully examined.

n Interaction with receptorsPEG-based strategiesBraintargeted Nps mainly synthesized from two kind of polymers: those based on a cyanoacrylate and those based on a polyester, coated with surfaceactive agents containing the PEG substructure, polysorbate 80 or pluronic F68. Another strategy covalently links PEG chains to the polymer: The copolymer obtained is then used as starting material for the preparation of Nps.

Among cyanoacrylate Nps, PBCApolysorbate 80 Nps (that contain more than 80% of dextran [97]), are the most studied as drugdelivery agents for the CNS; they have been studied in a number of ways [53,133]:

nRecently, a relevant in vitro rat BBB model consisting of a coculture of rat brain endothelial cells and rat astrocytes, which show good correlation between in vitro/in vivo results in

the presence of PEG–PHDCA Np (FiguRe 2) able to cross the BBB, has been established. This method allowed the assessment of translocation of these Nps [134]; a method to study the intracellular distribution of the same kind of Nps in cultured brain endothelial cells has been set up by the same group [111].

nIn vivo; their drugdelivery ability was assessed by evaluating the pharmacological response exerted by the pharmacologically active substances loperamide, dalargin, kytorphin, tubocurarine and MRZ 2/576. The loperamide delivery ability of polysorbate 80 adsorbed on PBCA Nps is also shared by pluronic F68 [135].

nEvidence of transcytosis was provided by in vivo histological fluorescence studies in the presence of PBCA Nps containing dextran [136] or without it [137].

nThe opening of the tight junctions exerted by PBCA/polysorbate 80 Nps appears to be excluded, despite having a small effect on the inulin space [53]. Conflicting results have been obtained: Olivier et al. (in vitro studies) showed that PBCA–polysorbate 80 Nps displayed some toxic effect towards the BBB, allowing the opening of the tight junctions [138], but Kreuter (in vitro and in vivo studies) did not demonstrate any disruption of the BBB by the presence of polysorbate 80coated Nps since the permeability of the extracellular markers (sucrose and inulin) was not modified in the presence of 10 or 20 µg/ml of PBCA Np with and without polysorbate 80 [139]. In vivo studies showed that polysorbate 80 solution increased the concentration of sucrose in all brain structures, suggesting an effect of permeabilization exerted by this surfactant over the BBB [109]. This effect was not observed when the same dose of polysorbate 80 was injected as adsorbed onto another kind of cyanoacrylate Np (PHDCA Np), probably because an important part of the administered surfactant is retained on the Np surface. A correlation between Np concentration and free polysorbate was observed; in fact, decreasing the amount of Np increases the percentage of ID/g tissue, suggesting that free polysorbate, likely present in solution owing to the smaller number of Nps, has an effect on BBB permeability [109]. On the other hand, it is known that low doses of polysorbate 80 (3 mg/kg) cause BBB disruption [140]. CNS effects of systemic administration of a



neuropeptide that does not cross the BBB could only be elicited if the neuropeptide was coadministered with polysorbate 80 [141]. Thus, there are some concerns about the use of PEGbased compounds for drug delivery.

nQuantitative analysis of drugs that crossed the BBB; this was performed in the presence of doxorubicin, dalargin and NGFloaded Nps without capillary depletion experiments (doxorubicin: 0.44% of the administered dose into healty rats [40], [3H]dalargin [142] and NGF: 3 ng/g tissue (brain), after an administered dose of 5 µg/mouse [143]). Thus, the results obtained do not discriminate between drug present in brain parenchyma and drug present in brain capillaries.

nQuantification of [14C]PBCA/polysorbate 80 Np able to reach brain parenchyma; this showed that after intravenous administration in rats, the brain uptake of Np was less than 1% ID/g tissue. Capillary depletion experiments were not conducted [60].

nMechanism of Np BBB crossing, this seems to be due to the adsorption of apolipoproteins by polysorbate 80 and pluronic F68 from blood. The adsorbed apolipoproteins then interact with the apolipoprotein receptors present at the BBB, resulting in their endocytotic uptake into endothelium [133]. Nps are then transcytosed into the brain [136,137]. This hypothesis is supported by the fact that covalent attachment of apolipoproteins AI, E and B to human serum albumin Nps enables the brain uptake of loperamide adsorbed on these particles, leading to a pronounced antinociceptive (analgesic) effect, whereas free drug produces no CNS effect [144,145]. The adsorption of apolipoproteins is shared by poly(caprolactone)dextran Nps and PEG–PCL Nps [146], but these Nps have never been tested for their ability to cross the BBB. Subsequent experiments with fluorescently labeled Nps showed that albumin–ApoE Nps enter the CNS by transcytosis, without any damage to the tight junctions [122].

nThe effect of the surfactants polysorbate 80 and pluronic F68 depends on Np composition. SLN decorated with polysorbate 80 and pluronic F68 showed a similar pattern of apolipoprotein adsorption [147]. PLGA and PBCA–doxorubicinloaded Nps, to which pluronic F68 was adsorbed, appear very promising for the treatment of brain tumors, while polysorbate 80 was effective only in the case of PBCA

Nps [148]. Other experiments showed that both surfactants enabled similarly pronounced pharmacological effects with loperamide embedded in PBCA/PVA Nps, while in PLGA/PVA Nps, polysorbate 80 appeared to be less effective than pluronic F68 [149]. Pluronic F68 is not able to deliver PBCA/PVA–dalarginloaded Nps [150], and unloaded PLGA Np to the CNS [72]. On the other hand, histological localization of fluorescent Nps showed that PLA–polysorbate 80 Nps were able to cross the BBB [151] as were PBCA/polysorbate 80 Nps [136,137]. These observations suggest that differences in the core properties influence the nature of the coating behavior and possibly the structure of the coating [148]. Studies of the structure of PLGA–pluronic F68 Nps and their colloidal stability have been conducted [152].

Nanoparticles synthesized from PEG covalently linked to a cyanoacrylate polymer – [14C]PEG–PHDCA and pluronic F68 [109,128] – triple Np delivery to the brain with respect to PHDCA Nps without modification of BBB permeability (brain levels: 0.006% ID dose/g tissue) [109]. It was suggested that the adsorbed apolipoproteins Apo E and Apo B100 by PEG present on the surface of PEG–PHDCA/pluronic F68 Nps are involved in the brain transport of these Nps [153]; the atomic composition of the Np surface was not determined. Evidence of transcytosis is administered by histological localization of f luorescently labeled PEG–PHDCAcoated polystyrene Nps. The integrity of the BBB was evaluated in vivo by means of quantification of the diffusion of [14C]sucrose into the brain; capillary depletion experiments have been not performed.

PEG coating is also able to deliver SLNs to the brain (capillary depletion experiments were not performed) [154].

n Nps that target transportersNanoparticles have been surface decorated with several ligands able to interact with transporters present on the brain endothelium, but these new Nps have not been studied in depth.

Transferrin Different kinds of Np were conjugated with transferrin and transferrinreceptor antibodies; proof of BBB passage was based on the pharmacological activity exerted by the loaded drug or by fluorescence studies of brain parenchyma [155–157].

Review | Costantino


LactoferrinLactoferrinconjugated PEG–PLA Nps have been evaluated in vitro and in vivo. The fluorescent dye coumarin6 embedded into Nps was found to be threefold higher in the brain for unconjugated Nps. Capillary depletion experiments have been not performed and the Np surface was studied by ESCA. Receptormediated transcytosis was assessed on the basis of histological studies of brain slices [74].

ThiamineThe consideration of thiamine as a cellspecific ligand for targeted delivery can be rationalized since all eukaryotic cells have a specified transport mechanism for thiamine. SLNs comprised of emulsifying wax and Brij 78 and DSPE–PEG–thiamine as the delivery agent, radiolabeled with [3H]cetyl alcohol (blank Np) or [3H]thiamine (thiaminedecorated Np) were evaluated in situ by the rat brain perfusion technique in the absence of plasma. BBB integrity during experiments was verified by concurrent vascular volume measurements with [14C]sucrose [112].

To date, no experiments have been conducted on recently discovered peptides (e.g., angiopeps) as targeting moieties for brain delivery of polymeric Nps, but studies of them for brain targeting of dendrimers have been undertaken.

n Nps that are able to cross the BBB by an unknown mechanismPLGA Nps of approximately 160nm diameter, surface decorated with the peptide GlyPhedThrGlyPheLeuSer(Obdglucose)CONH

2

and containing pluronic F68 (the quantity remaining on the Np surface has not been determined) have been studied for their ability to cross the BBB by means of rat brain perfusion technique and in vivo. These Nps were able to cross the BBB (by rat brain perfusion technique), as shown by histological localization of fluorescent Nps (fluorescent probe covalently linked to the polymer), without BBB damage, while unmodified PLGA/pluronic F68 Nps were unable to cross the BBB [72]. In vivo studies were performed in rats using loperamideloaded Nps (tail vein administration); loperamide is a drug that is able to interact with the opioid receptors but unable to cross the BBB. The results of the anti nociceptive assays showed that these Np are able to deliver the model drug loperamide into the CNS. Biodistribution studies of these Nps showed a localization into the CNS in a quantity approximately two orders of magnitude greater

than that found in the presence of other known Np drug carriers [158], reaching a brain level (fluorescence marker: rhodamine123 loaded into Nps) of approximately 15% ID/g of tissue. Capillary depletion experiments have not been conducted, thus this value does not take into account the quantity of Np present in the endothelium. This result is remarkable, as the other Nps that have been studied so far that target the CNS reached brain levels on the order of 0.1–0.2% ID/g of tissue [60,95,109,142,159–161]. However, biodistribution studies were based on a fluorescence marker loaded into the Np, and this experimental setting can lead to an over estimation of the results, since it is known (from in vitro studies) that PLGA Nps are able to transfer a dye into cells; thus, intracellular fluorescence increases observed with physically encapsulated probes in PLGA Nps appear to be, at least in part, due to a dye transfer from Nps to cells rather than Np uptake [65]. On the other hand, the results of the biodistribution studies that have been obtained are of the same order of magnitude as those obtained by intracerebroventricular administration of the drug (evaluation of the pharmacological effect exerted by loperamide showed that at least 13% of the injected dose is delivered to the brain by Nps), thus the effect of dye transfer appears to be negligible [158]. At present, the BBB crossing mechanism of these Nps is unknown.

ConclusionPolymeric Nps were first explored for drugdelivery purposes and vaccines in 1960, and the first commercial Np product containing a drug (Abraxane®, human serum albumin Np containing paclitaxel) appeared on the market in 2005. Another kind of Np (cyanoacrylatebased) loaded with doxorubicin was recently tested in a Phase I/II clinical trial for hepatocellular carcinoma with positive results (DoxorubicinTransdrug®, BioAlliance Pharma) [301], and irinotecan–Nps are in preclinical development for the oral delivery of irinotecan (BioAlliance Pharma) [301]. However, the first article to report the successful delivery of a drug to the CNS using an Np was published in 1995. Currently, no clinical trials are in progress for Nps as drugdelivery agents targeting the brain; this fact suggests that, even if promising results have been obtained, this technology requires further investigations and improvements.

Despite the stringent properties that low molecular weight compounds must have in order to cross the BBB, it is now recognized



that peptides, cargopeptides and even intravenously administered Nps of approximately 200nm diameter can cross the BBB and act as drugdelivery agents for the CNS. However, these drugdelivery systems have not been studied in a systematic way, and the parameters that are useful for their characterization have seldom been determined. This drug delivery approach to the CNS implies an increase in complexity with respect to low molecular weight compounds; as such, the number of parameters that have to be taken into account in order to fully characterize these systems rises markedly. Thus, more studies are needed in order to allow a comparison among the performances of these drugdelivery systems and to fully evaluate their clinical potential.

Other conclusions on polymeric Nps can be drawn. In general, proof of the Nps crossing the BBB was provided by the pharmacological effect exerted by the embedded drug and by fluorescence spectroscopy of brain parenchyma after tail vein Np administration, while quantitative experiments (the assessment of the amount of drug or Np that has been delivered to the brain) were conducted only in very few cases ([3H]dalargin and rhodamine123 as loaded markers, or by the use of radioactive polymers) without capillary depletion experiments. Results obtained so far show that, with one exception (approximately 15% ID/g tissue (brain [158]), the Nps localize into the CNS by just below 2% ID/g tissue.

Thus, Np drugdelivery systems show great promise if more efficient/selective targeting moieties can be found. Several promising peptides that target the brain and are able to carry cargo into the CNS have been discovered, and some of their drug conjugates have begun clinical trials (angiopeps). These peptides were first used as targeting moieties for dendrimers, and use of angiopeps in the field of Nps is expected in the near future.

Moreover, it appears that Nps should be optimized in the presence of a given drug, since the embedded drug can modify Np characteristics [60,95].

Attempts have been made to develop Nps in order to deliver drugs to the CNS by an oral or nasal route. A drug administered by the nasal route may enter into the blood of the general circulation, permeate the brain directly, bypassing the BBB (via the olfactory or trigeminal nerve systems), or follow both pathways [162]. Clinical pharmacokinetics of opioids, benzodiazepines and antimigraine drugs delivered intranasally show that this administration route is suitable for

rapid delivery [163]. Among the strategies that have been successfully followed in order to enhance nasal absorption of molecules, there are nano and microparticulate systems surface decorated with chitosan, PEG and lectins [164,165]. While drugloaded Nps can enhance nosetobrain delivery of drugs compared with equivalent drug solution formulations, the quantities of drug administered nasally that have been shown to be transported directly from nose to brain (bypassing the BBB) are very low, less than 0.1% [164]. No proof for the actual mechanism by which the drug improved transport was given; most studies on nasal drug delivery simply evaluated the effect of the drug when administered in a Np formulation in comparison with the drug in solution. Only a limited number of publications have evaluated the fate of Nps after their application in the nasal cavity and the subsequent transport across the mucosal membrane into the underlying tissue or into the bloodstream [164,166]. After nasal administration of polystyrene Nps, (100nm diameter) and wheat germ agglutinindecorated PEG–PLA Nps (90 nm in diameter), negligible amounts were found in whole brain homogenate [164]. Less than 3% of the administered dose of other Nps was found in the systemic circulation [166]. Thus, on the basis of the limited data available, it seems that it will be difficult to obtain clinically useful Nps that are able to translocate into the CNS following the nasal route of administration [166]. Moreover, Nps larger than 100 nm have restricted access to the brain via the intraaxonal route because their diameter exceeds that of the axons in the filia olfactoria [164]. Orally administered dalarginloaded PBCA Nps overcoated with polysorbate 85, uncoated [167] or doublecoated with polysorbate 80 and PEG

20000 [168] were shown

to be able, on the basis of the results of antinociceptive assays, to deliver the opioid hexapeptide dalargin to the brain. However, the pharmacological effect – proof of the brain delivery – was observed at doses higher than those effective in the presence of PBCA/polysorbate 80 Nps (polysorbate 80coated dalarginloaded intravenously administered Np: 50% maximum effect was obtained at 7.5 mg/kg in mice [53], while PBCA Np doublecoated with polysorbate 80 and PEG

20000 and orally administered showed

the same activity in mice at 25 mg/kg [168]). Thus, it appears that the best results for braintargeted Np as drugdelivery agents able to translocate into brain parenchyma obtained to date have been observed in the presence of Nps administered intravenously.

Review | Costantino


Another field of intense research in the field of Np deals with the delivery of proteins (biotech products), for which a medicinal chemistry approach cannot be applied. Nanoparticulate systems able to encapsulate and release proteins, without any degradation, are actively studied [169–172], and the development of proteinloaded braintargeted Nps would be clinically very useful.

Executive summary

n The transport of compounds from the circulating blood into the CNS is restricted by the blood–brain barrier (BBB).

n The characterization of brain penetration by a ‘low molecular weight’ (<500 Da) drug molecule can be based on three parameters able to give clinically valuable estimates of brain drug delivery.

n Several strategies have been developed by medicinal chemists in order to allow low molecular weight compounds to cross the BBB; however, there is a need for a drug-delivery system that is able to increase drug stability, shield physicochemical characteristics unsuitable for BBB crossing, ensure a sustained drug release and to selectively target the CNS.

n A growing number of peptides able to cross the BBB have been discovered and some of them have been considered drug carriers for the CNS. However, it is important to bear in mind that there is an increase in complexity of the system and other parameters are needed to characterize these new entities.

n Nanotechnology products, in particular polymeric nanoparticles, spherical particles of approximately 200 nm in diameter, were shown, if properly functionalized, to be able to cross the BBB. These nanoparticles can be used as drug carriers, ensuring a sustained drug release into the brain and masking the negative characteristics of the embedded drugs. However, the complexity of the system increases further at this level and many more parameters must be taken into account in order to fully characterize these systems.

n These delivery systems seem to be promising, especially for selective drug delivery to the CNS, and for the brain delivery of peptides and proteins, but the knowledge level of these systems has to be improved, in order to understand their full clinical potential.

Financial & competing interests disclosureThe author has no relevant affiliations or financial involve-ment with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, con-sultancies, honoraria, stock ownership or options, expert t estimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

BibliographyPapers of special note have been highlighted as:n of interestnn of considerable interest

1 Calabria AR, Shusta EV. Blood–brain barrier genomics and proteomics: elucidating phenotype, identifying disease targets and enabling brain drug delivery. Drug Discov. Today 11, 792–799 (2006).

2 Pardridge WM. Blood–brain barrier delivery. Drug Discov. Today 12, 54–61 (2007).

3 GarciaGarcia E, Andrieux K, Gil S, Couvreur P. Colloidal carriers and blood–brain translocation: a way to deliver drugs to the brain? Int. J. Pharm. 298, 274–292 (2005).

4 Lampson LA. Targeted therapy for neurooncology: reviewing the menu. Drug Discov. Today 14, 185–191 (2009).

5 Stenehjem DD, Hartz AMS, Bauer B, Anderson GW. Novel and emerging strategies in drug delivery for overcoming the blood–brain barrier. Fut. Med. Chem. 1, 1623–1641 (2009).

6 Hitchcock SA, Pennington LD. Structurebrain exposure relationships. J. Med. Chem. 49, 7559–7583 (2006).

7 ProkaiTatrai K, Prokai L. Modifying peptide properties by prodrug design for enhanced transport into the CNS. In: Peptide Transport and Delivery Into the Central Nervous System. ProkaiTatrai K, Prokai L (Eds.). BirkhauserVerlag, Basel, Switzerland, 155–188 (2003).

8 Kerns EH, Di L. Chapter 10. In: Drug-like Properties: Concepts, Structure Design and Methods. Academic Press 122–136 (2008).

9 Reichel A. Addressing central nervous system (CNS) penetration in drug discovery: basics and implications of the evolving new concept. Chem. Biodiversity 6, 2030–2049 (2009).

10 HammarlundUdenaes M, Friden M, Syvanen S, Gupta A. On the rate and extent of drug delivery to the brain. Pharm. Res. 25, 1737–1750 (2008).

11 Jones AR, Shusta EV. Blood–brain barrier transport of therapeutics via receptor mediation. Pharm. Res. 24, 1759–1771 (2007).

12 JuilleratJeanneret L. The targeted delivery of cancer drugs across the blood–brain barrier: chemical modifications of drugs or drugnanoparticles? Drug Discov. Today 13, 1099–1106 (2008).

13 Kumagai AK, Eisenberg WM, Pardridge WM. Absorptivemediated endocytosis of cationized albumin and a bendorphincationized albumin chimeric

peptide by isolated brain capillaries. Model system of blood–brain barrier transport. J. Biol. Chem. 262, 15214–15219 (1987).

14 Trigueiro D, Buciak JB, Pardridge WM. Capillary depletion method for quantifying blood–brain barrier transcytosis of circulating peptides and plasma proteins. J. Neurochem. 54, 1882–1888 (1990).

15 Bickel U, Yoshikawa T, Pardridge WM. Delivery of peptides and proteins through the blood–brain barrier. Adv. Drug Del. Rev. 46, 247–279 (2001).

16 Lee HJ, Pardridge WM. Pharmacokinetics and delivery of Tat and Tatprotein conjugates to tissues in vivo. Bioconj. Chem. 12, 995–999 (2001).

17 Zorko M, Langel U. Cellpenetrating peptides: mechanism and kinetics of cargo delivery. Adv. Drug Del. Rev. 57, 529–545 (2005).

18 DeBoer AG, Gaillard PJ. Strategies to improve drug delivery across the blood–brain barrier. Clin. Pharmacokinet. 46, 553–576 (2007).

19 Rousselle C. New advances in the transport of doxorubicin through the blood–brain barrier by a peptide vectormediated strategy. Mol. Pharmacol. 57, 679–686 (2000).



20 Rousselle C, Clair P, Smirnova M et al. Improved brain uptake and pharmacological activity of dalargin using a peptide vector mediated strategy. J. Pharmacol. Exp. Ther. 306, 371–376 (2003).

21 Malakoutikhah M, Teixido M, Giralt E. Toward an optimal blood–brain barrier shuttle by synthesis and evaluation of peptide libraries. J. Med. Chem. 51, 4881–4889 (2008).

22 Malakoutikhah M, Prades R, Teixido M, Giralt E. Nmethyl phenylalaninerich peptides as highly versatile blood–brain barrier shuttles. J. Med. Chem. 2354–2363 (2010).

23 Gabathuler R. Approaches to transport therapeutic drugs across the blood–brain barrier to treat brain diseases. Neurobiol. Disease 37, 48–57 (2010).

24 van Rooy I, CakirTascioglu S, Couraud PO et al. Identification of peptide ligands for targeting to the blood–brain barrier. Pharm. Res. 27, 673–682 (2010).

25 Gan CW, Feng SS. Transferrinconjugated nanoparticles of poly(lactide)datocopheryl. polyethylene glycol succinate diblock copolymer for targeted drug delivery across the blood–brain barrier. Biomaterials 31(30), 7748–7757 (2010).

26 Kumar P, Wu HQ, Jodi LM et al. Transvascular delivery of small interfering RNA to the central nervous system. Nature 448, 39–43 (2007).

27 Pan W, Kastin AJ, Zankel TC, van Kerkhof P, Terasaki T, Bu G. Efficient transfer of receptorassociated protein (RAP) across the blood–brain barrier J. Cell Sci. 117, 5071–5078 (2004).

28 Prince WS, Cormick LM, Wendt DJ et al. Lipoprotein receptor binding, cellular uptake, and lysosomal delivery of fusion between the receptorassociated protein (RAP) and aiduronidase or acid aglucosidase. J. Biol. Chem. 279, 35037–35046 (2004).

29 Demeule M, Regina A, Che C et al. Identification and design of peptides as new drug delivery system for the brain. J. Pharmacol. Exp. Ther. 324, 1064–1072 (2008).

30 Thomas FC, Taskar K, Rudraraju V et al. Uptake of ANG1005, a novel paclitaxel derivative, through the blood–brain barrier into brain and experimental brain metastases of breast cancer. Pharm. Res. 26, 2486–2494 (2009).

31 Regina A, Demeule M, Che C et al. Antitumor activity of ANG1005, a conjugate between paclitaxel and the new brain delivery vector Angiopep2. Br. J. Pharmacol. 155, 185–197 (2008).

32 Drappatz J, Brenner A, Rosenfeld S et al. Ang1005: new EPiC compound for the treatment of recurrent malignant glioma. Presentated at: The EORTC-NCI-AACR Annual Meeting 2009, Boston, MA, USA, 15–19 November, 2009.

33 Che C, Yang G, Thiot C et al. New angiopepmodified doxorubicin (ANG1007) and etoposide (ANG1009) chemotherapeutics with increased brain penetration. J. Med. Chem. 53, 2814–2824 (2010).

n One of the most interesting peptide brain drug-delivery agents. The ability of Angiopep to deliver antitumor drugs into the brain does not seem to be dependent on the drug considered; however, the precise mechanism of delivery has not yet been fully characterized.

34 Esfand R, Tomalia DA. Poly(amidoamine) (PAMAM) dendrimers: from biomimicry to drug delivery and biomedical applications. Drug Discov. Today 6,427 436 (2001).

35 Medina SH, ElSayed EH. Dendrimers as carriers for delivery of chemotherapeutic agents. Chem. Rev. 109, 3141–3157 (2009).

36 Astruc D, Boisselier E, Ornelas C. Dendrimers designed for functions: from physical, photophysical, and supramolecular properties to applications in sensing, catalysis, molecular electronics, photonics, and nanomedicine. Chem. Rev. 110, 1857–1959 (2010).

37 Nanjwade BK, Bechra HM, Derkar GK, Manvi FV, Nanjwade VK. Dendrimers: emerging polymers for drugdelivery systems. Eur. J. Pharm. Sci. 38, 185–196 (2009).

38 Zhang L, Gu F, Chan JM, Wang AZ, Langer RS, Farokhzad OC. Nanoparticles in medicine: therapeutic applications and developments. Clin. Pharmacol. Ther. 83, 761–769 (2008).

39 Ke W, Shao K, Huang R et al. Gene delivery targeted to the brain using an angiopep conjugated polyethyleneglycolmodified polyamidoamine dendrimers. Biomaterials 30, 6976–6985 (2009).

40 Huang RQ, Ke WL, Liu Y, Chen J, Pei YY. The use of lactoferrin as a ligand for targeting the polyamidoaminebased gene delivery system to the brain. Biomaterials 29, 238–246 (2008).

41 Liu Y, Huang R, Han L et al. Braintargeting gene delivery and cellular internalization mechanisms for modified rabies virus glycoprotein RVG29 nanoparticles. Biomaterials 30, 4195–4202 (2009).

42 Farokhzad OC, Langer R. Impact of nanotechnology on drug delivery. ACS Nano 3, 16–20 (2009).

43 Huang G, Zhang N, Bi X, Dou M. Solid lipid nanoparticles of temozolomide: potential reduction of cardiac and nephric toxicity. Int. J. Pharm. 355, 314–320 (2008).

44 Kaur IP, Bhandari R, Bhandari S, Kakkar V, Potential of solidlipid nanoparticles in brain targeting. J. Contr. Rel. 127, 97–109 (2007).

45 Joshi MD, Muller RH. Lipid nanoparticles for parenteral delivery of actives. Eur. J. Pharm. Biopharm. 71, 161–172 (2009).

46 Costantino L, Tosi G, Ruozi B, Bondioli L, Vandelli MA, Forni F. Colloidal systems for CNS drug delivery. Progr. Brain Res. 180, 35–69 (2009).

47 Saad M, Garbuzenko OB, Ber E et al. Receptor targeted polymers, dendrimers, liposomes: which nanocarrier is the most efficient for tumorspecific treatment and imaging? J. Contr. Rel. 130, 107–114 (2008).

48 Tosi G, Costantino L, Ruozi B, Forni F, Vandelli MA. Polymeric nanoparticles for the drug delivery to the central nervous system. Exp. Opin. Drug Del. 5, 155–174 (2008).

49 Owens DE, Peppas NA. Opsonization, biodistribution and pharmacokinetics of polymeric nanoparticles. Int. J. Pharm. 307, 93–102 (2006).

50 Moghimi SM, Szebeni J. Stealth liposomes and long circulating nanoparticles: critical issues in pharmacokinetics, opsonisation and proteinbinding properties. Prog. Lipid Res. 42, 463–478 (2003).

51 Aggarwal P, Hall JB, McLeland CB, Dobrovolskaia MA, McNeil SE. Nanoparticle interaction with plasma proteins as it relates to particle biodistribution, biocompatibility and therapeutic efficacy. Adv. Drug Del. Rev. 61, 428–437 (2009).

52 Dobrovolskaia MA, Aggarwal P, Hall JB, McNeil S. Preclinical studies to understand nanoparticle interaction with the immune system and its potential effects of nanoparticle biodistribution. Mol. Pharmaceutics 5, 487–495 (2008).

53 Kreuter J. Nanoparticulate systems for brain delivery of drugs. Adv. Drug Del. Rev. 47, 65–81 (2001).

54 Alexis F, Pridgen E, Molnar LK, Farokhzad OC. Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol. Pharmaceutics 5, 505–515 (2008).

55 Li SD, Huang L. Pharmacokinetics and biodistribution of nanoparticles. Mol. Pharmaceutics 5, 496–504 (2008).

56 Cho WS, Cho M, Jeong J et al. Sizedependent kinetics of PEGcoated gold nanoparticles. Toxicol. App. Pharmacol. 245, 116–123 (2010).

Review | Costantino


57 He C, Hu Y, Tang C, Yin C. Effect of particle size and surface charge on cellular uptake and biodistribution of polymeric nanoparticles. Biomaterials 31, 3657–3666 (2010).

58 Gaumet M, Vargas A, Gurny R, Delie F. Nanoparticles for drug delivery: the need for precision in reporting particle size parameters. Eur. J. Pharm. Biopharm. 69, 1–9 (2008).

n Reports the rules that should be followed in order to obtain reliable nanoparticle (Np) size data for meaningful correlations with biodistribution data.

59 Zhang L, Chan JM, Gu FX et al. Selfassembled lipidpolymer hybrid nanoparticles: a robust drug delivery platform. ACS Nano 2, 1696–1702 (2008).

60 Ambruosi A, Khalasky AS, Yamamoto H, Gelperina SE, Begley DJ, Kreuter J. Biodistribution of polysorbate 80coated doxorubicinloaded [14C]poly(butyl cyanoacrylate) nanoparticles after intravenous administration to glioblastomabearing rats. J. Drug Targ. 14, 97–105 (2006).

61 Besheer A, Vogel J, Glanz D, Kressler J, Groth T, Mader K. Characterization of PLGA nanospheres stabilized with amphiphilic polymers: hydrophobically modified hydroxyethyl starch vs Pluronics. Mol. Pharmaceutics 6, 407–415 (2009).

62 Sahagun G, Moore SA, Hart MN. Permeability of neutral vs anionic dextrans in cultured brain microvascular endothelium. Am. J. Physiol. 259, H162–H166 (1990).

63 Gaumet M, Gurny R, Delie F. Localization and quantification of biodegradable particles in an intestinal cell model: the influence of particle size. Eur. J. Pharm. Sci. 36, 465–473 (2009).

64 Gao K, Jiang X, Influence of particle size on transport of methotrexate across blood–brain barrier by polysorbate 80coated polybutylcyanoacrylate nanoparticles. Int. J. Pharm. 310, 213–219 (2006).

n Systematic study that relates Np diameter with brain delivery ability.

65 Xu P, Gullotti E, Tong L et al. Intracellular drug delivery by poly(lacticcoglycolic acid) nanoparticles, revisited. Mol. Pharmaceutics 6, 190–201 (2009).

nn Discovery of the ability of polymeric Nps to transfer their loaded contents into cultured cells without Np internalization.

66 Mitragotri S, Lahann J. Physical approaches to biomaterial design. Nat. Mater. 8, 15–23 (2009).

67 Doshi N, Mitragotri S. Designer biomaterials for nanomedicine. Adv. Funct. Mater. 19, 3843–3854 (2009).

68 Champion JA, Mitragotri S. Role of target geometry in phagocytosis. Proc. Natl Acad. Sci. USA 103, 4930–4934 (2006).

69 Hwang HY, Kim IS, Kwon IC, Kim YH. Tumor targetability and antitumor effect of Docetaxelloaded hydrophobically modified glycol chitosan nanoparticles. J. Contr. Rel. 128, 23–31 (2008).

70 Sun X, Rossin R, Turner JL et al. An assessment of the effects of shell crosslinked nanoparticle size, core composition, and surface PEGylation on in vivo biodistribution. Biomacromolecules 6, 2541–2554 (2005).

71 Bertholon I, Vauthier C, Labarre D. Complement activation by coreshell poly(isobutylcyanoacrylate)polysaccharide nanoparticles: influences of surface morphology, length, and type of polysaccharide. Pharm. Res. 23, 1313–1323 (2006).

72 Costantino L, Gandolfi F, Tosi G, Rivasi F, Vandelli MA, Forni F. Peptide derivatized biodegradable nanoparticles able to cross the blood–brain barrier. J. Contr. Rel. 108, 84–96 (2005).

73 Lu W, Zhang Y, Tan YZ, Hu KL, Jiang XG, Fu SK. Cationic albuminconjugated pegylated nanoparticles as novel drug carrier for brain delivery. J. Contr. Rel. 107, 428–448 (2005).

74 Hu K, Li J, Shen Y et al. Lactoferrinconjugated PEGPLA nanoparticles with improved brain delivery: in vitro and in vivo evaluations. J. Contr. Rel. 134, 55–61 (2009).

75 Zhang Z, Feng SS. In vitro investigation of poly(lactide)Tween 80 copolymer nanoparticles fabricated by dialisys method for chemotherapy. Biomacromolecules 7, 1139–1146 (2006).

76 Huang CY, Lee YD. Coreshell type of nanoparticles composed of poly[(nbutyl cyanoacrylate)co(2octyl cyanoacrylate)] copolymers for drug delivery application: synthesis, characterization and in vivo degradation. Int. J. Pharm. 325, 132–139 (2006).

77 Vauthier C, Dubernet C, Fattal E, PintoAlphandary H, Couvreur P. Poly(alkylcyanoacrylates) as biodegradable materials for biomedical applications. Adv. Drug Del. Rev. 55, 519–548 (2003).

78 Yoon SJ, Kim SH, Ha HJ et al. Reduction of inflammatory reaction of poly(d,llacticcoglycolic acid) using demineralised bone particles. Tissue Eng. Part A, 14, 539–547 (2008).

79 Anderson JM, Shive MS. Biodegradation and biocompatibility of PLA and PLGA microspheres. Adv. Drug Del. Rev. 28, 5–24 (1997).

80 Lherm C, Muller RH, Puisieux F, Couvreur P. Alkylcyanoacrylate drug carriers II. Cytotoxicity of cyanoacrylate nanoparticles with different alkyl chain length. Int. J. Pharm. 84, 13–22 (1992).

81 MaurerJones MA, Bantz KC, Love SA, Marquis BJ, Haynes CL. Toxicity of therapeutic nanoparticles. Nanomedicine 4, 219–241 (2009).

82 Xia T, Kovochich M, Liong M, Zink JI, Nel AE. Cationic polystyrene nanosphere toxicity depends on cellspecific endocytic and mitochondrial injury pathways. ACS Nano 2, 85–96 (2008).

83 Rao KS, Reddy MK, Horning JL, Labhasetwar V. TATconjugated nanoparticles for the CNS delivery of antiHIV drugs. Biomaterials 29, 4429–4438 (2008).

nn Determined Np-delivered brain drug levels in a Np-free form for the first time.

84 Jiskoot W, van Schie RMF, Carstens MG, Schellekens H. Immunological risk of injectable drug delivery systems. Pharm. Res. 26, 1303–1314 (2009).

85 Bertholon I, Vauthier C, Labarre C. Complement activation by coreshell poly(isobutylcyanoacrylate)olysaccharide nanoparticles: influences of surface morphology, length, and type of polysaccharide. Pharm. Res. 23, 1313–1323 (2006).

86 Hamada I, Hunter AC, Szebeni J, Moghimi SM. Poly(ethylene glycol) generate complement activation products in human serum through increased alternative pathway turnover and a MASP2dependent process. Molecular Immunol. 46, 225–232 (2008).

87 Lu W, Wan J, She Z, Jiang X, Brain delivery property and accelerated blood clearance of cationic albumin conjugated pegylated nanoparticle. J. Contr. Rel. 118, 38–53 (2007).

88 Simeonova M, Chorbadjiev K, Antcheva M. Study of the effect of polybutylcyanoacrylate nanoparticles and their metabolites on the primary immune response in mice to sheep red blood cells. Biomaterials 19, 2187–2193 (1998).

89 Arima Y, Kawagoe M, Toda M, Iwata H. Complement activation by polymers carrying dhydroxyl groups. ACS Appl. Mat. Interfaces 1, 2400–2407 (2009).

90 Koziara JM, Oh JJ, Akers WS, Ferraris SP, Mumper RJ. Blood compatibility of cetyl alcohol/polysorbatebased nanoparticles. Pharm. Res. 22, 1821–1828 (2005).

nn Reports an assessment of the blood compatibility of Nps.



91 Kim D, ElShall H, Dennis D, Morey T. Interaction of PLGA nanoparticles with human blood constituents. Colloids and Surf. B Biointerfaces 40, 83–91 (2005).

nn Reports an assessment of the blood compatibility of Np.

92 Rajendran L, Knolker HJ, Simons K. Subcellular targeting strategies for drug design and delivery. Nature Rev. Drug Disc. 9, 29–42 (2010).

93 Panyam J, Zhou WZ, Prabha S, Sahoo SK, Labhasetwar V. Rapid endolysosomal escape of poly(d,llactidecoglycolide) nanoparticles: implications for drug and gene delivery. FASEB J. 16, 1217–1226 (2002).

94 Lockman PR, Koziara JM, Mumper RJ, Allen DD. Nanoparticle surface charges alter blood–brain barrier integrity and permeability. J. Drug Target. 12, 635–641 (2004).

95 Brigger I, Morizet J, Laudani L et al. Negative preclinical results with stealth nasnospheresencapsulated Doxorubicin in an orthotopic murine brain tumor model. J. Contr. Rel. 100, 29–40 (2004).

96 Lundqvist M, Stigler J, Elia G, Lynch I, Cedervall T, Dawson KA. Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proc. Natl Acad. Sci. USA 105, 14265–14270 (2008).

nn Systematic study of Np characteristics with respect to their ability to adsorb selected proteins from plasma.

97 Langer K, Seegmuller E, Zimmer A, Kreuter J. Characterization of polybutylcyanoacrylate nanoparticles: I. Quatification of PBCA polymer and dextrans. Int. J. Pharm. 110, 21–27 (1994).

98 Batrakova EV, Kabanov AV. Pluronic block copolymers: evolution of drug delivery concept from inert nanocarriers to biological response modifiers. J. Contr. Rel. 130, 98–106 (2008).

99 Huang JG, Si LQ, Jiang LL, Fan ZZ, Qiu J, Li G. Effect of Pluronic F68 block copolymer on Pglycoprotein transport and CYP3A4 metabolism. Int. J. Pharm. 351–353 (2008)

100 Sahoo SK, Panyam J, Prabha S, Labhasetwar V. Residual polyvinyl alcohol associated with poly(d,llactidecoglycolide) nanoparticles affects their physical properties and cellular uptake. J. Contr. Rel. 82, 105–114 (2002).

101 Sun W, Xie C, Wang H, Hu Y. Specific role of polysorbate 80 coating on the targeting of nanoparticles to the brain. Biomaterials 25, 3065–3071 (2004).

102 Breunig M, Bauer S, Goepferich A. Polymers and nanoparticles: intelligent tools for intracellular targeting? Eur J. Pharm. Biopharm. 68, 112–128 (2008).

nn Review dealing with the progress that has been accomplished in recent years in the field of carrier design for efficient delivery to target cellular structures.

103 Sahay G, Alakhova DY, Kabanov AV. Endocytosis of nanomedicines. J. Contr. Rel. 145, 182–195 (2010).

104 Prabha S, Zhou WZ, Panyam J, Labhasetwar V. Sizedependency of nanoparticlemediated gene transfection: studies with fractionated nanoparticles. Int. J. Pharm. 244, 105–115 (2002).

105 Zauner W, Farrow NA, Haines AM. In vitro uptake of polystyrene microspheres: effect of particle size, cell line and cell density. J. Contr. Rel. 71, 39–51 (2001).

106 Torchilin VP. Rrecent approaches to intracellular delivery of drugs and DNA and organelle targeting. Annu. Rev. Biomed. Eng. 8, 343–375 (2006).

107 Khalil IA, Kogure K, Akita H, Harashima H. Uptake pathways and subsequent intracellular trafficking in nonviral gene delivery. Pharmacol. Rev. 58, 32–45 (2006).

108 Tahara K, Yamamoto H, Kawashima Y. Cellular uptake mechanism and intracellular distribution of polysorbate 80modified poly(d,llactidecoglycolide) nanospheres for gene delivery. Eur. J. Pharm. Biopharm. 75, 218–224 (2010).

109 Calvo P, Gouritin B, Chacun H et al. Longcirculating PEGylated polycyanoacrylate nanoparticles as new drug carrier for brain delivery. Pharm. Res. 18, 1157–1166 (2001).

110 Kim HR, Gil S, Andrieux K et al. Low density lipoprotein receptormediated endocytosis of PEGylated nanoparticles in rat brain endothelial cells. Cell. Mol. Life Sci. 64, 356–364 (2007).

111 GarciaGarcia E, Andrieux K, Gil S et al. A methodology to study intracellular distribution of nanoparticles in brain endothelial cells. Int. J. Pharm. 298, 310–314 (2005).

112 Lockman PR, Oyewumi MO, Koziara JM, Roder KE, Mumper RJ, Allen DD. Brain uptake of thiaminecoated nanoparticles. J. Contr. Rel. 93, 271–282 (2003).

113 Koziara J, Lockman PR, Allen DD, Mumper RJ, In situ blood–brain barrier transport of nanoparticles. Pharm. Res. 20, 1772–1778 (2003).

114 Lockman PR, Koziara J, Roder KE, Paulson J, Abbruscato TJ, Mumper RJ, Allen DD. In vivo and in vitro assessment of baseline blood–brain barrier parameters in the presence of novel nanoparticles. Pharm. Res. 20, 705–713 (2003).

115 Menei P, Daniel V, MonteroMenei C, Brouillard M, PouplardBarthelaix A, Benoit JP. Biodegradation and brain tissue reaction of poly(d,llactidecoglycolide) microspheres. Biomaterials 14, 470–478 (1993).

116 Nicholas AP, McInnis C, Gupta KB et al. The fate of biodegradable microspheres injected into rat brain. Neurosci. Lett. 323, 85–88 (2002).

117 Chernenko T, Matthaus C, Milane L, Quintero L, Amiji M, Diem M. Labelfree raman spectral imaging of intracellular delivery and degradation of polymeric nanoparticle system. ACS Nano 3, 3552–3558 (2009).

118 van Apeldoorn A, van Manen H, Bezemer J, de Bruijn, J, van Blitterswijk C, Otto C. Raman imaging of PLGA microspheres degradation inside macrophages. J. Am. Chem. Soc. 126, 13226–13227 (2004).

119 Bazole DV, Ropert C, Huve P et al. Body distribution of fully biodegradable [14C]poly(lactic acid) nanoparticles coated with albumin after parenteral administration to rats. Biomaterials 13, 1093–1102 (1992).

120 Muller RH, Lherm C, Herbert J, Couvreur P. In vitro model for the degradation of alkylcyanoacrylate nanoparticles. Biomaterials 11, 590–595 (1990).

121 Hasadsri L, Kreuter J, Hattori H, Iwasaki T, George JM. Functional protein delivery into neurons using polymeric nanoparticles. J. Biol. Chem. 284, 6972–6981 (2009).

nn Very important article that describes the fate of protein-loaded PBCA Np cultured neurons.

122 Zensi A, Begley D, Pontikis C et al. Albumin nanoparticles targeted with Apo E enter the CNS by transcytosis and are delivered to neurones. J. Contr. Rel. 137, 78–86 (2009).

123 Cartiera M, Johnson KM, Rajendran V, Caplan MJ, Saltzman WM. The uptake and intracellular fate of PLGA nanoparticles in epithelial cells. Biomaterials 30, 2790–2798 (2009).

nn Demonstrates the dependence of PLGA Np uptake (rate and extent, intracellular trafficking) on the cell lines considered; moreover, PLGA intracellular trafficking has been studied in detail.

124 Hu YL, Gao JQ. Potential neurotoxicity of nanoparticles. Int. J. Pharm. 394, 115–121 (2010).

Review | Costantino


125 Reis SA, Willemsen R, van Unen L, Hoogeveen AT, Oostra BA. Prospect of TATmediated protein therapy for fragile X syndrome. J. Mol. Histol. 35, 389–395 (2004).

126 Olivier JC. Drug transport to brain with targeted nanoparticles. NeuroRx 2, 108–119 (2005).

127 Sharma HS. Nanoneuroscience: emerging concepts on nanoneurotoxicity and nanoneuroprotection. Nanomedicine 2, 753–758 (2007).

128 Sisson WB, Oldendorf WH. Brain distribution spaces of mannitol3H, inulin14C, and Dextran14C in the rat. Am. J. Physiol. 221, 214–217 (1971).

129 Alyautdin RN, Reichel A, Lobenberg R, Ramge P, Kreuter J, Begley DJ. Interaction of poly(butyl cyanoacrylate) nanoparticles with the blood–brain barrier in vivo and in vitro. J. Drug Targ. 19, 209–221 (2001).

130 Thole M, Nobmann S, Huwyler J, Bartmann A, Fricker G. Uptake of cationized albumin coupled liposomes by cultured porcine brain microvessel endothelial cells and intact brain capillaries. J. Drug Targ. 10, 337–344 (2002).

131 Parikh T, Bommana MM, Squillante E. Efficacy of surface charge in targeting pegylated nanoparticles of sulpiride to the brain. Eur. J. Pharm. Biopharm. 74, 442–450 (2010).

132 Wang ZH, Wang ZY, Sun CS, Wang CY, Jiang TY, Wang SL. Trimethylated chitosanconjugated PLGA nanoparticles fort the delivery of drugs to the brain. Biomaterials 31, 908–915 (2010).

133 Kreuter J. Influence of the surface properties on nanoparticlemediated transport of drugs to the brain. J. Nanosci. Nanotech. 4, 484–488 (2004).

134 GarciaGarcia E, Gil S, Andrieux K et al. A relevant in vitro rat model for the evaluation of blood–brain barrier translocation of nanoparticles. CMLS Cell. Mol. Life Sci. 62, 1400–1408 (2005).

nn A relevant in vitro BBB model for the study of brain-targeted Nps, showing a correlation with the results obtained in in vivo studies.

135 Hekmatara T, Gelperina S, Vitali V, Yang, SR, Kreuter J. Encapsulation of waterinsoluble drugs in poly(butyl cyanoacrylate) nanoparticles J. Nanosci. Nanotech. 9, 5091–5098 (2009).

136 Reimold I, Domke D, Bender J, Seyfried CA, Radunz HE, Fricker G. Delivery of nanoparticles to the brain detected by fluorescence microscopy. Eur. J. Pharm. Biopharm. 70, 627–632 (2008).

137 Weiss CK, Kohnle MV, Landfester K et al. The first step into the brain: uptake of NIO PBCA nanoparticles by endothelial cells in vitro and in vivo, and direct evidence for their blood–brain barrier permeation. ChemMedChem. 3, 1395–1403 (2008).

138 Olivier JC, Fenart L, Chauvet R, Pariat C, Cecchelli R, Couet W. Indirect evidence that drug brain targeting using polysorbate 80coated polybutylcyanoacrylate nanoparticles is related to toxicity. Pharm. Res. 16, 1836–1842 (1999).

139 Kreuter J, Ramge P, Petrov V et al. Direct evidence that polysorbate80coated poly(butylcyanoacrylate) nanoparticles deliver drugs to the CNS via specific mechanisms requiring prior binding of drug to the nanoparticles. Pharm. Res. 20, 409–416 (2003).

140 Azmin MN, Stuart JFB, Florence AT. The distribution and elimination of metotrexate in mouse blood and brain after concurrent administration of polysorbate 80. Cancer Chemother. Pharmacol. 14, 238–242 (1985).

141 Sakane T, Tanaka C, Yamamoto A et al. The effect of polysorbate 80 on brain uptake and analgesic effect of Dkyothorphin. Int. J. Pharm. 57, 77–83 (1989).

142 Schroeder U, Schroeder H, Sabel BA. Body distribution of 3Hlabelled dalargin bound to poly(butyl cyanoacrylate) nanoparticles after i.v. injections to mice. Life Sci. 66, 495–502 (2000)

143 Kurakhmaeva KB, Djindjikhashvili IA, Petrov VE et al. Brain targeting of Nerve Growth Factor using poly(butyl cyanoacrylate) nanoparticles. J. Drug Targ. 17, 564–574 (2009).

144 Michaelis K, Hoffmann MM, Dreis S et al. Covalent linkage of apolipoprotein E to albumin nanoparticles strongly enhances drug transport into the brain. J. Pharmacol. Exp. Ther. 317, 1246–1253 (2006 ).

145 Kreuter J, Hekmatara T, Dreis S, Vogel T, Gelperina S, Langer K, Covalent attachment of apolipoprotein AI and apolipoprotein B100 to albumin nanoparticles enables drug transport into the brain. J. Contr. Rel. 118, 54–58 (2007).

146 Lemarchand C, Gref R, Passirani C et al. Influence of polysaccharide coating on the interactions of nanoparticles with biological systems. Biomaterials 27, 108–118 (2006).

147 Goppert TM, Muller RH. Plasma protein adsorption on Tween 80 and Poloxamer 188stabilized solid lipid nanoparticles. J. Drug Targ. 11, 225–231 (2003).

148 Kreuter J. Use of nanoparticles for cerebral cancer. Tumor. 94, 271–277 (2008).

149 Gelperina S, Maksimenko O, Khalansky A et al. Drug delivery to the brain using surfactantcoated poly(lactidecoglycolide) nanoparticles: influence of the formulation parameters. Eur. J. Pharm. Biopharm. 74, 157–163 (2010).

nn First article in which a comparison between the brain delivery ability of a model drug (loperamide) and two kinds of polymeric Np is made.

150 Petri B, Bootz A., Khalansky A et al. Chemotherapy of brain tumor using doxorubicin bound to surfactantcoated poly(butyl cyanoacrylate) nanoparticles: revisiting the role of surfactants. J. Contr. Rel. 117, 51–58 (2007).

151 Sun W, Wang H, Xie C, Hu Y, Yang X, Xu H. An attempt to directly trace polymeric nanoparticles in vivo with electron microscopy. J. Contr. Rel. 115, 259–265 (2006).

152 SantanderOrtega MJ, JodarReyes AB, Csaba N, BastosGonzalez D, OrtegaVinuesa JL. Colloidal stability of Pluronic F68coated PLGA nanoparticles: A variety of stabilisation mechanisms. J. Colloid Interface Sci. 302, 522–529 (2006).

153 Kim HR, Andrieux K, Gil S et al. Translocation of poly(ethylene glycolcohexadecyl)cyanoacrylate nanoparticles into rat brain endothelial cells: role of apolipoproteins in receptormediated endocytosis. Biomacromolecules 8, 793–799 (2007).

154 Zara GP, Cavalli R, Bargoni A, Fundaro A, Vighetto D, Gasco MR. Intravenous administration to rabbits of nonstealth and stealth Doxorubicinloaded solid lipid nanoparticles at increasing concentrations of stealth agent: pharmacokinetics and distribution of doxorubicin in brain and other tissues. J. Drug Targ. 10, 327–335 (2002).

155 Ulbrich K, Hekmatara E, Kreuter J. Transferrin and transferrinreceptorantibodymodified nanoparticles enable drug delivery across the blood–brain barrier (BBB). Eur. J. Pharm. Biopharm. 71, 251–256 (2009).

156 Aktas Y, Yemisci M, Andrieux K.et al. Development and brain delivery of chitosanPEG nanoparticles functionalized with the monoclonal antibody OX26. Bioconj. Chem. 16, 1503–1511 (2005).

157 Karatas H, Aktas Y, GursoyOzdemir Y et al. A nanomedicine transports a peptide Caspase3 inhibitor across the blood–brain barrier and provides neuroprotection. J. Neurosci. 29, 13761–13769 (2009).



158 Vergoni AV, Tosi G, Tacchi R, Vandelli MA, Berolini A, Costantino L. Nanoparticles as drug delivery agents specific for CNS: in vivo biodistribution. Nanomedicine: NBM 5, 369–377 (2009).

159 Brigger I, Morizet J, Aubert G et al. Poly(ethylene glycol)coated hexadecylcyanoacrylate nanospheres display a combined effect for brain tumor targeting. J. Pharmacol. Exp. Ther. 303, 928–936 (2002).

160 LeRay AM, Vert M, Gautier JC, Benoit JP. Fate of [14C]poly(d,llactidecoglycolide) nanoparticles after intravenous and oral administration to mice. Int. J. Pharm. 106, 201–211 (1994).

161 Lode J, Fichtner I, Kreuter J, Berndt A, Diederichs JE, Reszka R. Influence of surfacemodifying surfactants on the pharmacokinetic behaviour of 14Cpoly(methylmethacrylate) nanoparticles in experimental tumor models. Pharm. Res. 18, 1613–1619 (2001).

162 Dhuria SV, Hanson LR, Frey WH. Intranasal delivery to the central nervous system: mechanisms and experimental considerations. J. Pharm. Sci. 99, 1654–1673 (2010).

163 VeldhorstJanssen NML, Fiddelers AAA, van der Ruy PHM, Neef C, Marcus MAE. A review of the clinical pharmacokinetics of opioids, benzodiazepines, and antimigraine drugs delivered intranasally. Clin. Ther. 31, 2954–2987 (2009).

164 Mistry A, Stolnik S, Illum L. Nanoparticles for direct nose to brain delivery of drugs. Int. J. Pharm. 379, 146–157 (2009).

165 Ozsoy Y, Gungor S, Cevher E. Nasal delivery of high molecular weight drugs. Molecules 14, 3754–3779 (2009).

166 Illum L. Nanoparticulate systems for nasal delivery of drugs: a real improvement over simple systems? J. Pharm. Sci. 96, 473–483 (2007).

167 Schroeder U, Sommerfeld P, Sabel BA, Efficacy of oral dalarginloaded nanoparticle delivery across the blood–brain barrier. Peptides 19, 777–780 (1998).

168 Das D, Lin SS. Doublecoated poly(butylcyanoacrylate) nanoparticulate delivery systems for brain targeting of dalargin via oral administration. J. Pharm. Sci. 94, 1343–1353 (2005).

169 Putney SD, Burke PA. Improving protein therapeutics with sustainedrelease formulations. Nat. Biotech. 16, 153–157 (1998).

170 He W, Jiang X, Zhang ZR. Preparation and evaluation of polybutylcyanoacrylate nanoparticles for oral delivery of thymopentin. J. Pharm. Sci. 97, 2250–2259 (2008).

171 van de Weert M, Hennink WE, Jiskoot W. Protein instability in poly(lacticcoglycolic acid) microparticles. Pharm. Res. 17, 1159–1167 (2000).

172 Yang SX, Yuan WE, Jin T. Formulating protein therapeutics into particulate forms. Exp. Opin. Drug Del. 6, 1123–1133 (2009).

n Patent201 Khrestchatisky M, Marion D, Yves M,

Vlieghe P. 2937322 A1 20100423 (2010).

n Website301 Bioalliance Pharma.

www.bioalliancepharma.com

drug delivery to the cns and polymeric nanoparticulate carriers

Documents