Nanocomposites for neurodegenerative diseases ...

DOI: 10.5301/ijao.2011.8915

Int J Artif Organs 2011; 34 (12): 1115-1127

REVIEW

Nanocomposites for neurodegenerative diseases: hydrogel-nanoparticle combinations for a challenging drug delivery Carmen Giordano1, Diego Albani2, Antonio Gloria3, Marta Tunesi1, Serena Rodilossi2, Teresa Russo3, Gianluigi Forloni2, Luigi Ambrosio3, Alberto Cigada1 Department of Chemistry, Materials and Chemical Engineering, “G. Natta”, Politecnico di Milano, Milan - Italy Department of Neuroscience, “Mario Negri” Institute for Pharmacological Research, Milan - Italy 3 Institute of Composite and Biomedical Materials, National Research Council, Naples - Italy 1 2

ABSTRACT Neurodegenerative disorders are expected to strike social and health care systems of developed countries heavily in the coming decades. Alzheimer’s and Parkinson’s diseases (AD/PD) are the most prevalent neurodegenerative pathologies, and currently their available therapy is only symptomatic. However, innovative potential drugs are actively under development, though their efficacy is sometimes limited by poor brain bioavailability and/or sustained peripheral degradation. To partly overcome these constraints, the development of drug delivery devices made by biocompatible and easily administrable materials might be a great adjuvant. In particular, materials science can provide a powerful tool to design hydrogels and nanoparticles as basic components of more complex nanocomposites that might ameliorate drug or cell delivery in AD/PD. This kind of approach is particularly promising for intranasal delivery, which might increase brain targeting of neuroprotective molecules or proteins. Here we review these issues, with a focus on nanoparticles as nanocomponents able to carry and tune drug release in the central nervous system, without ignoring warnings concerning their potential toxicity. KEY WORDS: Alzheimer’s disease, Hydrogels, Intranasal delivery, Nanocomposites, Nanoparticles, Nanotoxicity, Parkinson’s disease Accepted: November 30, 2011

INTRODUCTION Population aging is a positive achievement but also a challenge because of the increased prevalence of age-related disorders. Many efforts are being undertaken to address these, and current treatments aim at reducing the social and economical burden of dementias and invalidating disorders such as Alzheimer’s and Parkinson’s diseases (AD/ PD), but as research goes on, it is more and more evident that to efficiently fight these pathologies, a multidisci-

plinary approach is mandatory. This is the case of materials science and basic pharmacology, which should cross-talk to develop novel tools and strategies against AD/PD. In this context, controlled drug release is an emerging field, whose challenges are made more difficult by the complexity of both neurodegeneration mechanisms and accessibility to the target tissues. Hydrogels are versatile and tunable materials which might meet many requirements for drug therapy in AD/PD. They can be prepared from biocompatible and biodegradable polymers, they can be injected, and

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Nanocomposites for drug delivery in neurodegeneration

they are suitable for drug delivery; furthermore, they can be nanostructured using nanoparticles (NPs) or other nanodevices to improve their viscoelastic properties upon injection, drug delivery, and release profile. Coupled to innovative drugs (such as neuroprotective peptides or chaperone proteins such as Hsp70 and Hsp27) or used as better delivery devices for the available drugs (acetylcholinesterase inhibitors and levo-dopa), they might allow a step forward in AD/PD therapeutic management. This review highlights the state-of-the-art for advanced drug release systems in AD/PD, underlining the available materials for hydrogel and polymer NP preparation and the experimental approaches merging drug delivery and materials science. In particular, we will focus on how nanocomposites or nanocarriers might help in nonconventional administration routes, describing the exciting field of intranasal delivery. Finally, the current concerns about nanotoxicity will be clearly pointed out.

Parkinson’s and Alzheimer’s diseases: market data and therapies The market size for neurodegenerative diseases was US $14.5 billion in 2005 and over US $18.5 billion in 2009, and it is expected to increase 62% to US $29.7 billion by 2012 (1). Parkinson’s disease (PD), one of the most common neurodegenerative disorders, affects both sexes and about 1%-2% individuals over the age of 65 (2). Its prevalence is second only to Alzheimer’s disease (AD), which has a reported annual incidence that varies from 16 to 19 individuals per 100,000 worldwide and rises with age. Its prevalence is second only to Alzheimer’s disease (AD), which has a reported prevalence after 65 years ranging from 1.6% to 40%, depending on age (3). AD is the main cerebral cortex neurodegenerative disorder. It is rare before the age of 60 but increasingly common thereafter, and it affects approximately 25 million people worldwide; in particular, in the United States more than 4 million people are suffering from AD, and this number is expected to triple by 2050 (4). In Italy, AD patients number around 500,000. Almost all AD cases are sporadic, and only less than 5% have a monogenic basis. Unfortunately, current therapies are symptomatic rather than restorative. Drugs may give temporary improvements, but at present no treatments able to stop or reverse the neurodegeneration are available, and this greatly impacts not only the medical burden on society, but especially the quality of life for patients 1116

and their relatives (5, 6). Recently, the growth of knowledge about AD/PD pathogenetic mechanisms has led to the discovery of new molecular targets which might reduce neurodegeneration. In particular, novel therapies based on neuroprotective protein delivery have been proposed to improve cognitive impairments and counteract oxidative stress, pathologic protein misfolding, and relative aggregation. For instance, prosurvival chaperone proteins such as Hsp70 or Hsp27 or proteases able to degrade pathological protein aggregates are possible candidates for these protein-based therapies (6-8). Unfortunately, systemic administration strongly limits protein bioavailability at brain level due to poor blood–brain barrier (BBB) permeability, forcing the use of high doses and/or repeated treatments, thus increasing side effects and decreasing patients’ compliance (9). In this situation, the use of polymer-based composites, in particular injectable resorbable hydrogel-based nanocomposites, represents an appealing scenario for the development of novel and minimally invasive drug delivery systems. In fact, their remarkable potential therapeutic applications lie in bioactive agent in situ release and direct action against AD/PD triggers.

Polymer-based composites: key aspects A composite material is the combination of 2 or more materials differing in composition or morphology. It is composed of a matrix and a reinforcing phase, which may be 1-dimensional (tubes and fibers), 2-dimensional (layered minerals such as clay), or 3-dimensional (spherical particles). Composites are usually classified according to several criteria, including the following: (a) fibrous composite materials, which are made up of fibers embedded in a matrix; (b) laminated composite materials, consisting of thin layers of fully bonded materials; (c) particulate composite materials, which are composed of particles within a matrix; and combinations of some of (a), (b), and (c). When the matrix is a polymer, the resulting composites are defined as “polymer-based composites”. Recently, many biodegradable and bioresorbable materials, as well as several technologies to design biomedical devices, have been experimentally and/or clinically investigated (10), and the importance of using polymers or polymer-based composites to make multifunctional materials has also been properly stressed in literature for biomedical applications. In fact, polymer-based composites have emerged as suitable can-


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didates for applications such as load bearing, tissue engineering, and drug delivery (10-12), but the concept might be rapidly extended to the development of advanced injectable materials for drug delivery in AD/PD. By designing a composite material, specific chemical, physical, and mechanical properties may be obtained; in fact, the resulting material may show a combination of the best properties of its constituents, as well as interesting features that often the single constituents do not possess (10, 13-18). One of the main factors controlling the performances of polymerbased composites is the matrix and its interaction with the reinforcing phase, represented by continuous or discontinuous fibers or micro/nanoparticles. With regards to the mechanical behavior, several theoretical and semiempirical models are available for its prediction in particulate composites consisting of spherical particles within a polymer matrix (16). As an example, the composite Young’s modulus (EC) may be predicted using the Halpin-Tsai equations or the following Kerner equation (16, 19, 20):

where

vm is the matrix Poisson’s ratio, Vf represents the filler volume fraction in the composite, and Em and Ef are the matrix and filler Young’s moduli, respectively. In a composite material, the matrix must transfer stresses to the reinforcement, providing a contribution to the composite strength. For particulate composites where the particle/matrix adhesion is poor, this may be calculated by the Nicolais-Narkis equation (16, 21):

with σc and σm representing the composite and polymer matrix strength, respectively. Weakness in the structure may be ascribed to discontinuities in the stress transfer and generation of stress concentration at the particle/matrix interface, which in the presence of purely inorganic

fillers is often due to the different ductility between the polymer matrix and the particles. Designing composite substrates consisting of a polymer matrix reinforced with sol-gel synthesized organic–inorganic hybrid microfillers is an interesting approach to overcome this limitation (2224); furthermore, the possibility of tuning the effects of microparticle composition on the mechanical and biological performances of the resulting composites has also been demonstrated (22). In the last few years, the number of published papers, patents, and conferences related to the “nano” scale has increased extraordinarily, and the beginning of the “nanotechnology era” has led to the development of nanotechnology-derived materials (25-29). Among them, polymer-based nanocomposites, which are commonly defined as the combination of a polymer matrix and fillers showing at least 1 dimension in the nanometer range (16, 30), have emerged as intriguing tools for many applications, with a potentially remarkable impact on medicine and materials science (10, 17, 28, 29, 31). Researches span the range from the synthesis of basic structures (such as colloidal NPs functionalized with molecules, simple biomolecules, or polymers) to more complex nanostructures (32). The main approach has initially focused on the control of NP shape, size, and surface functionalization and then on the possibility of modulating the topology of their chemical composition, also designing suitable single nano-objects made up of groups consisting of several types of complex molecules and NPs (33-37). Basically, the introduction of NPs improves the performance of the polymer matrix, allowing a wide range of applications involving both materials and life sciences (38, 39). For example, within the same multicomponent nanostructure, 1 or more subunits might have functionalities for detection (i.e., via interaction with a magnetic field, or light absorption, or fluorescent emission, or light scattering), while other subunits might be exploited for biochemical/biological recognition as well as selective binding of specific ligands (37) or local application of perturbations. With regard to applications related to cancer therapy, if the nanostructure has a magnetic/metallic domain, cell death may be triggered by locally producing heat in response to a precise stimulus (e.g., near-infrared light, alternating magnetic fields, or radiofrequency); furthermore, in the case of a magnetic domain, drugs attached to, or embedded in, the nanostructure may be released and directed to a specific location by applying an external field (37). However, apart from the application, the optimization of both mechanical and biological performances of


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polymer-based nanocomposites is challenging, depending upon factors such as the material and processing method (29). For example, the amount of NPs embedded in a polymer matrix may not be increased above some limitations, primarily because it may lead to a clustering effect. In fact, it has been shown that NP amounts much lower than 10% are enough to produce clusters, and while they may be effective for specific applications, such as those based on biological or magnetic features, they negatively affect mechanical properties (40).

concentration, G’ will dramatically decrease even though NP concentration is further increased. In fact, especially during injection, NPs may act as “weak points” instead of a reinforcement. It is thus clear that the optimization of injectable nanocomposites and the prediction of their mechanical behavior represent a great challenge, which may be faced by suitably integrating experimental rheological tests with mathematical models (16).

Polymer nanoparticles in composites: a challenge for drug delivery

Injectable nanocomposite gels: “reason why” Nanocomposites may be designed in the form of “solids” or gels, depending upon their specific applications. Over the past few years, there has been an increasing interest in the development and applications of hydrogel-based nanocomposites as a new class of biomaterials (41). In this context, as previously highlighted, several kinds of nanofillers (e.g., carbon nanotubes and magnetic and gold NPs) able to respond to precise stimuli by generating heat have been properly considered as remotely controlled biomaterials for designing drug delivery systems, actuators, and devices for cancer treatments (42). With regard to central nervous system (CNS) neurodegenerative disorders, the design of suitable injectable composite hydrogels for in situ drug or cell release represents an interesting and minimally invasive solution and might play a key role in the development of successful treatments (6). As a main concern, it is well known that the injection of a gel can strongly affect its rheological behavior and viscoelastic properties (storage and loss moduli). In fact, the injection through clinical catheters may lead to a partial or total disruption of the polymer network, causing a decrease of the storage modulus and a marked alteration of the gel-like behavior. The inclusion of NPs might act as a reinforcement improving drug- or cell-loaded hydrogel viscoelastic properties without altering the gel-like behavior. After the injection, viscoelastic properties may decrease, but thanks to the NP reinforcement the values of both dynamic moduli may still be suitable for the specific application (10, 13-16, 43, 44). In nanocomposite design, the concentration of NPs is a critical feature: since NPs provide a resistance of the resulting material to flow under shear, the storage modulus (G’) and the viscosity will increase with NP concentration, but beyond a threshold 1118

Apart from viscoelastic property improvement, a great advantage of embedding polymer NPs in an injectable gel is that they may be successfully exploited as drug carriers, allowing for a finely controlled release of specific biomolecules across the BBB (45-47). Since drug biodistribution and brain targeting depend on the physical-chemical properties of particles, especially size, a high degree of precision in their size selection and a reliable production process are required. To obtain the aimed-for drug release kinetics, it is also important to consider that once in situ, nanocomposites will be affected by the surrounding environment, with consequences for the rate of drug diffusion/ polymer dissolution. Examples of polymer NP applications for drug delivery in AD/PD models are quite numerous, and they take advantage of a relatively large number of materials that are biodegradable and suitable for NP synthesis, including polylactide-co-glycolide (PLGA), polylactic acid (PLA), chitosan (CS), gelatin, polycaprolactone, and polyalkyl-cyanoacrylates. At first, efforts to set up NPs specifically devoted to AD focused on the nanoencapsulation of the available drugs, and routes of administration such as intranasal or intravenous were sometimes attempted to increase brain concentration and control the release profile. Mittal et al proposed Tween 80 (T-80)–coated PLGA NPs to deliver estradiol to rat brain. They were prepared by a single-emulsion technique, and the T-80 coating was achieved by incubating the reconstituted NPs at various T-80 concentrations. They were orally administered and gave appreciably higher brain estradiol levels after 24 hours as compared with uncoated ones, being able to prevent the expression of amyloid beta-42 (Aβ42) in the hippocampus (48). A different in vitro system was tested by Carroll and coworkers, who developed Tempol-loaded PLGA NPs con-


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jugated with a transferrin antibody (OX 26) by nanoprecipitation. Tempol is a free radical scavenger with antioxidant properties, while the presence of the transferrin receptor antibody, covalently linked to PLGA NPs by the NHS-PEG3500-Maleimide crosslinker, aimed at increasing cellular uptake, as demonstrated in RG2 rat glioma cells (49). The green tea polyphenol(-)-epigallocatechin3-gallate (EGCG) belongs to the family of natural polyphenols, and it has antioxidant and nonamyloidogenic properties. By loading EGCG into orally administered nanolipidic particles, its in vivo bioavailability was improved by more than twofold in comparison with free EGCG (50). Polyethylene glycol-polylactide-co-glycolide (PEG-PLGA) NPs conjugated to lactoferrin (Lf) were tested in a rat model of PD. The in vitro and in vivo delivery properties were evaluated by a fluorescent coumarin-6 probe, and the results pointed to an accumulation of LfNPs in bEnd.3 cells in comparison with unconjugated NPs. After Lf-NP intravenous administration, a threefold increase in coumarin-6 was found in the brain, while intravenous injection of Lf-NPs loaded with the neuroprotective peptide urocortin reduced the striatum lesion caused by 6-hydroxydopamine in rats, without showing toxicity or immunoresponse (51). A more specific approach against AD/PD neurodegeneration was designed to use the NP-mediated release of nerve growth factor (NGF). NGF is essential for the survival of central cholinergic neurons in the basal forebrain (whose deficiency leads to AD), but its administration may also slow down the progression of PD. However, NGF does not penetrate the BBB from the circulation, and to overcome this limitation, it was adsorbed on poly(butyl cyanoacrylate) (PBCA) NPs coated with polysorbate 80 and tested in rats. The efficient transport of NGF across the BBB was confirmed by direct measurement of NGF concentrations in the brain (52). Finally, taking advantage of molecular imaging modalities based on optical and hybrid contrast such as fluorescent protein tomography and multispectral optoacoustic tomography, NPs are now used in AD/PD as diagnostic tools for direct in vivo imaging (53); furthermore, since AD diagnosis also relies on the quantification of amyloid peptides (Aβ40 and Aβ42) in the cerebrospinal fluid (CSF), different nanodevices have been developed to this purpose. For instance, an ultrasensitive NP-based bio-barcode able to detect AD soluble biomarkers and an electrochemical sensing protocol based on saccha-

ride-protein interactions are available. The development of ultrasensitive immunosensors based on surface plasmon resonance (SPR) for Aβ40 peptide detection has allowed a sensitivity limit down to 1 fg/mL while the current clinical testing sensitivity is down to 10 pg/mL (54). For detecting amyloid deposition by micro magnetic resonance imaging (MRI), ultrasmall superparamagnetic iron oxide (USPIO) NPs, chemically coupled with mannitol and Aβ42 peptide, were intravenously administered in mice. Amyloid plaques detected by T2*-weighted MRI were confirmed by histology, and the USPIO NPs were able to identify the differences between AD transgenic mice and wild-type controls. This kind of methodology is very important and promising toward meeting the need for less invasive techniques for in vivo amyloid plaque detection (55). Härtig and colleagues reported the use of PE154 (a fluorescent, heterodimeric inhibitor of brain acetylcholinesterase [AChE]) for the histochemical staining of cortical amyloid plaques in tripletransgenic (TTG) mice, a model of AD. Carboxylated polyglycidylmethacrylate NPs loaded with PE154 were injected into TTG mice targeting hippocampal amyloid deposits; in addition, biodegradable core-shell polystyrene/poly(butylcyanoacrylate) (PS/PBCA) NPs were also shown to be suitable for the same purpose (56). Examples of NPs devoted to cell delivery in AD/PD are lacking, but some pioneer studies have demonstrated the feasibility of such an approach in the periphery. The main limitation of cell encapsulation is the reduced biocompatibility of encapsulating agents either with the enclosed cells or the surrounding biologic environment. For instance, assessed constraints are the use of biomaterials and scaffolds whose composition is very far from that of the natural physiological environment and the weak in vivo stability of calcium-alginate beads, a commonly used tool for cell-embedding purposes. Trying to solve such problems, Orive and coworkers developed biomimetic hydrogel capsules to promote the in vivo long-term functionality of the enclosed erythropoietin-secreting cells by coupling the adhesion peptide arginine-glycine-aspartic acid (RGD) to alginate polymer chains and by using an alginate mixture. The biomimetic capsules provided cell adhesion and prolonged in vivo long-term functionality and drug release; furthermore, results suggested a controlled in vitro and in vivo drug delivery may be achieved by tuning the cell dose within the capsules (57).


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Nanocomposites for nasal drug delivery in Alzheimer’s and Parkinson’s diseases Among the possible applications of nanocomposites in AD/ PD, an outstanding chance to overcome limited drug bioavailability at the brain level is nasal drug delivery. In fact, crossing the BBB is one of the hardest obstacles limiting the successful development of novel AD/PD drugs. Considering that the nasal upper cavity is covered by the olfactory mucosa that is directly innerved by the terminations of the olfactory nerves driving the smelling sensations from periphery to the brain olfactory bulb, the basic idea is to promote the cellular transport of drugs from the periphery to the brain. However, in this kind of administration route, the limiting step is the drug formulation, which has to bind to the nasal mucosa and promote drug uptake by nervous fibers. Moreover, the amount of delivered drug should be sufficiently high to guarantee a therapeutic threshold not only in the olfactory bulb (which is of limited relevance for the main clinical signs of AD/PD, even if in PD it undergoes neurodegeneration but also in deep brain regions (hippocampus, striatum, and substantia nigra) and at the cortical level. The exact mechanism that allows the transport of a drug form from the nasal epithelia to the brain is a matter of debate, but both a direct and a transporter-mediated uptake might be involved. The point is supported by Lee and colleagues, who assessed the kinetic transport of several intranasally administered drugs in rats, finding that a prevalent group (12 out of 17) were directly transported, while the other 5 involved the presence of transporters such as rOAT3 and rOCT2, which are expressed at significant levels in rat olfactory epithelia (58). The available literature has focused on the development of innovative formulations to deliver traditional AD/PD drugs. For instance, Arumugam and coworkers delivered the AChE inhibitor rivastigmine into the brain via the intranasal route by conventional liposomes composed of cholesterol and soya lecithin. They were able to achieve a significantly higher level of rivastigmine in the brain with liposomes in comparison with the intranasal and oral administration of the free drug; in addition, intranasal liposomes had a longer half-life in the brain (59). A similar approach was pursued by Jogani and colleagues for delivering tacrine - another AChE inhibitor - whose use is limited by its low oral bioavailability, extensive hepatic first-pass effect, rapid clearance from the systemic circulation, and hepatotoxicity. Tacrine was prepared in a suspen1120

sion of propylene glycol radiolabeled with technetium 99m and intranasally or intravenously administered in BALB/c mice. The results showed that tacrine was able to reach the brain from the nasal cavity; furthermore, intranasal administration led to a higher bioavailability, reducing distribution into nontargeted tissues (60). The feasibility and advantages of intranasal AChE inhibitor delivery were also ascertained by Leonard and coworkers, who optimized galantamine formulation for intranasal administration using an in vitro tissue model and an in vivo bioavailability evaluation. In comparison with oral dosing, intranasal galantamine resulted in a reduced incidence of related side effects, such as retching and emesis (61). Concerning PD therapy, Kim and colleagues evaluated the intranasal route with levo-dopa, finding a good pharmacokinetic profile, and concluding it might be used as a good rescue therapy for PD patients with symptom fluctuation following oral levo-dopa administration (62). These systems are promising, even though they are based on very simplified interactions between drugs and the delivery device. More advanced approaches have been reported. Luppi and coworkers developed bovine serum albumin NPs carrying cyclodextrins for the nasal delivery of tacrine. The presence of beta cyclodextrins in the polymer network affected drug loading and might tune NP mucoadhesiveness and drug permeation behavior. NPs were obtained by a coacervation method and thermal cross-linking, loaded from solutions of tacrine hydrochloride, and lyophilized. They presented a mean size and a polydispersity index lower than 300 nm and 0.33 nm, respectively. NP-mediated tacrine release was measured in vitro and by ex vivo permeation studies across sheep nasal mucosa (63). Another group working on NPs for the delivery of olanzapine, a psychotropic drug also prescribed for AD/PD patients with psychosis, prepared PLGA NPs by the nanoprecipitation technique and performed pharmacokinetic studies examining in vitro NP release, and ex vivo diffusion and toxicity. They concluded that olanzapine-loaded NPs were transported directly to the brain after intranasal delivery, enhancing drug concentration in the brain, and no toxicity was observed for their crossing the sheep nasal mucosa. This system might be also extended to AChE inhibitors (64). PEG-PLGA NPs conjugated to odorranalectin were successfully used as drug carriers in an in vivo model of PD to improve nose-to-brain drug delivery and reduce the immunogenicity of traditional lectin (65). Xia and colleagues conjugated low-molecular-weight protamine (LMWP) (a


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cell-penetrating peptide able to promote cell translocation) to the surface of PEG-PLA NPs via a maleimide-mediated covalent binding procedure. Experiments with 16HBE14ohuman bronchial epithelial cells found that LMWP-loaded NPs enhanced cellular accumulation via lipid raft-mediated endocytosis and direct translocation without cytotoxicity. Following intranasal administration of a fluorescent derivative of LMWP (loaded within the same NPs), the fluorescent probe was detected in the rat cerebrum, cerebellum, olfactory tract, and olfactory bulb (66). Following nasal administration of peptide-loaded NPs, the extent of brain delivery was precisely quantified by microdialysis using a radiolabeling approach by Cheng and coworkers. Radiolabeled with sodium I125-iodide neurotoxin-I (NT-I) was encapsulated within PLA NPs with a homogenous size distribution (mean size of 65 nm), and their intranasal transport into the brain was evaluated by a microdialysis sampling technique. Results showed that the intranasal route led to a faster achievement of the peak level in comparison with intravenous administration and, again, NT-I–loaded NPs allowed for a better bioavailability of radiolabeled NT-I in comparison with the free molecule, thus suggesting that NPs really improve brain transport for active peptides (67). Polysaccharides represent a field of increasing interest for macromolecular nasal delivery (63, 68). The potential of N-trimethyl chitosan (TMC) NPs as a carrier system for protein nasal delivery was investigated by Amidi and colleagues. TMC NPs were prepared by ionic crosslinking of a TMC solution with tripolyphosphate at room temperature under stirring. The loading, integrity, and release of ovalbumin were studied. The NPs had an average size of about 350 nm and a positive zeta-potential. They released ovalbumin without cytotoxicity in vitro, and they allowed the transport of fluorescein isothiocyanate (FITC)-albumin across the nasal mucosa in vivo (69). Another formulation developed for intranasal protein delivery was tested by Goycoolea and coworkers. They prepared NPs by ionic gelation of CS hydrochloride with pentasodium tripolyphosphate (TPP) and concomitant complexation with sodium alginate (ALG), obtaining NPs with a size ranging from 260 to 525 nm. Insulin, taken as a model peptide, was associated to CS-TPP-ALG NPs with efficiencies in the range from 41% to 52%, and after nasal administration to conscious rabbits these NPs enhanced the systemic absorption of insulin (70). It is apparent that the above-described NPs might also be suitable for delivering other possible therapeutic proteins

in AD/PD - for instance, chaperone proteins of the heatshock family, or antibodies with intended antiaggregating features that in other ways are unable to efficiently reach the brain (71). Hydrogels are also an intensively investigated field for nasal delivery because of their advantage of film-thickness behavior and mucoadhesiveness to the nasal mucosa. Up to now, several hydrogel formulations have been proposed enhancing adhesiveness and/or drug release (insulin, antivirals, and antiinflammatory drugs) (72).

Concerns on potential nanocomposite toxicity in vivo The previous section has documented the increase of experimental results supporting the use of various kinds of materials with increasing nanocomplexity to set up devices able to ameliorate drug delivery in AD/PD, with a particular focus on intranasal administration. This very specific application is just one of several others in the field of neurorestorative or neuroprotective strategies that might benefit from material and nanotechnology exploitation. However, this growing field has been paralleled by some warnings and concerns about the potential cytotoxicity of nanostructured systems in biological or clinical settings. The main concerns are (i) the persistence in the brain of exogenously added materials due to low biodegradation and absorption, (ii) the possible, unknown effects of nanomaterials on cell physiology, and (iii) the possible overspreading of nanomaterials to other body organs, where the residual effect of the transported drug might be unwanted or even toxic. The science of NPs (and generally speaking of all nanotechnologies) is relatively recent, and attempts at rationalization are underway with a particular emphasis on the potential toxicity of NPs (73-76). The concept of nanotoxicity is also intriguing because human beings are continuously facing nanopollutants or other biological nanoagents. In fact, we make use of efficient endothelial filters to neutralize and eliminate foreign matter in the body, including viruses and chemical particles (for instance, those arising from smoke, combustion, and urban traffic). However, these NPs are overloading our natural protective systems, with a consequent increase in the risk of severe diseases, and triggering toxic effects including inflammation, oxidative stress, and unbalanced gene expression. Because of this, introducing other NPs into the body, even if with a therapeutic purpose, should be carefully considered (73).


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In particular, size and size distribution, shape, surface area, crystal structure, chemical composition, surface chemistry, surface charge, porosity, and eventual clustering effects have been emphasized in the assessment of NPs’ toxic effects on the CNS cellular environment (77, 78). As reported, the effective NP content used in antibacterial/antiviral compositions (i.e., 0.1%-1% w/w) should be much below the toxicity dosage limit by the time NPs reach the CNS from the point of contact. This estimation takes into consideration the fact that eventually released NPs also need to enter the body, cross the BBB, and finally reach the CNS. Most of NP release rates refer to works carried out on matrix–NP composites, and they have shown interesting results. In the case of metallic and ceramic NPs, their concentration or dosage in body fluids is considered to be well below the maximum drug toxicity test limit (10-5 g/mL), but no data on relationships among NP size, composition, and their eventual presence in the brain are available. As an example, Yang and coworkers reported that 50-nm silica-coated cobalt ferrite, and 100-nm and 1,000nm latex fluorosphere NPs were found in the brain following intravenous injection, while 20-nm latex fluorosphere NPs were not detected. Furthermore, apart from the large amount of reported data, it appears clear that results normally obtained for metals, metal oxides, and ceramics would be totally different if natural polymers were considered (78). A possible global strategy to overcome the potential dangerous side effects of exogenous biomaterials is developing smart devices made up of fully characterized and biocompatible materials, tuning up biodegradation, and increasing tissue specificity by promoting adhesion to selective cellular surface molecules. However, we have to admit that our comprehension of the exact cellular fate after exposition to exogenous materials and nanomaterials requires much more investigation. For instance, Yang and coworkers reported a study using single-wall carbon nanotubes (SWCNTs) that might represent excellent targeted delivery systems for AD/PD drugs. Lysosomes are the pharmacological target organelles of SWCNTs, and in this way they are removed from cells, but in large doses or in cases of poor lysosomal activity, mitochondria also take up SWCNTs with ensuing toxicity, which overcomes the benefit of efficient drug delivery. In the work by Yang et al, SWCNTs were successfully used to deliver acetylcholine (the neurotransmitter lacking in AD) into the brain, with a high safety range avoiding mitochondrial involvement (79). This kind of nanomaterial was also checked in vivo as a di1122

etary supplement for rainbow trouts, fed with a control diet (no SWCNT addition), or a diet supplemented with 500 mg SWCNTs/kg or 500 mg fullerenes C(60)/kg for 6 weeks. The only observed alteration was an increase in lipid peroxidation; no other significant treatment-related differences were observed (80). The concept that nanostructured materials might have direct toxic effects on nervous cells is supported by Nyitrai and colleagues who assessed the interactions between native plasma membranes and polyamidoamine (PAMAM) dendrimers (1-0.0001 mg/mL) applied to freshly prepared brain slices. They observed enhanced toxicity, electrophysiological failure, and synaptic transmission defects; however, conjugation of surface amino groups with β-D-glucopyranose units reduced functional neurotoxicity (81). Nanotoxicity was also proved in mice following copper NP (23.5 nm) intranasal administration. At 3 different doses, copper NPs caused different degrees of pathological lesions in certain tissues (including the olfactory bulb), where the secretion levels of neurotransmitters were influenced (82). In conclusion, this article reinforces the need to develop fully biocompatible systems for drug delivery in AD/ PD. In fact, several other studies assessing the nanotoxicity of gold and silver NPs have revealed they may modify brain endothelial and nervous cells at biochemical and even transcriptional levels, causing bioaccumulation that should be completely avoided in AD/PD (83).

Future perspectives Recently, we presented 2 intriguing multidisciplinary drug delivery– and cell conveying–based strategies for AD/PD, both relying on diffusible recombinant proteins and injectable biodegradable hydrogel-based devices: (i) in drug delivery–based strategies, recombinant proteins are directly loaded within a polymer matrix and then slowly released in brain tissues - for instance, also taking advantage of an alternative route of administration such as intranasal spray; (ii) in cell conveying–based strategies, patients’ cells are collected, engineered for neuroprotective recombinant protein production, encapsulated and then in situ reinjected to act as drug reservoirs for a sustained protein release (6). For both approaches, NPs might be loaded within hydrogels as a reinforcement to preserve gel rheological properties upon injection, and with regard to the first strategy, proteins might be embedded in NPs to obtain


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Fig. 1 - Neuronal populations involved in Alzheimer’s and Parkinson’s diseases (AD/PD) and possible intranasal drug delivery strategy (A, B). Microphotographs of primary cultures of mouse cortical neurons in presence of glial cells. Neurons are characterized by long branches, while glial cells have rounded cellular bodies with evident nuclei (C). Dopaminergic neurons from mouse mesencephalic cultures (dark structures) (D). Nanostructured hydrogel as intranasal delivery system: (I) adhesion to nasal epithelial cells, (II) drug crossing, (III) retrotranslocation by olfactory neurons, and (IV) hydrogel degradation.

a defined release profile (6). As thoroughly presented in this review, we have summarized the experimental results supporting different kinds of materials as potentially suitable for macromolecules delivery in vivo. This might allow a rational choice for the best material for the final application (e.g., the release of diffusible engineered proteins) according to protein size, net charge, and tertiary structure. A smart nanocomposite made up of NPs of different materials able to control the release of 2 or more neurotrophic macromolecules, whose action might be optimized thanks to a timely synergy between each factor, might also be effective. For instance, NPs might aid in reducing a pro-oxidant environment and maximizing cell survival (in the case of replacement therapies or cell-conveying strategies) or prolong the half-life of redox-sensitive prosurvival proteins (in the case of drug delivery approaches) by releasing a strong antioxidant molecule. This might be the case for the PD-related protein DJ-1, which is redox-sensitive and whose protective action might be prolonged in the target tissue by releasing other neuroprotective molecules such as resveratrol or other polyphenols (84, 85). Concerning drug delivery in brain-related applications, the most challenging feature is probably the extreme com-

plexity of the nervous tissue and the selective vulnerability of neuronal populations, which are differently affected by neurodegeneration. For instance, cortical neurons are involved in AD neurodegenerative process, while mesencephalic dopaminergic neurons are the target of PD-related pathophysiology. These different neuronal populations are depicted in Figures 1A-C. Figures 1A and B show immunostaining of mouse cortical neurons, with fluorescent antibodies staining glial cells in green and 2 different neuronal markers in yellow and red. Figure 1C presents well-developed dopaminergic neurons from mouse mesencephalic cultures (dark structures). Several potential AD/PD drugs are not efficiently delivered across the BBB, and consequently their actual utility is limited. A possible strategy to overcome this bottleneck is intranasal delivery, summarized in Figure 1D. In this scenario, nanocomposites might greatly help in ameliorating the quantity and kinetic release profile of potential and/ or existing AD/PD drugs. The schematic depicts a nanostructured hydrogel that as a first step should be able to stably adhere to outer nasal olfactory epithelium (I) to promote the release of NPs (in green) loaded with a drug or a neuroactive protein. NPs should then be able to cross the epithelium (II) and reach the terminations of nervous cells


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making up the olfactory nerve and projecting to the olfactory bulbs. Ideally, NPs should be captured by neuronal axons and retrotranslocated up to the olfactory bulbs, where the effective molecule should be released and also be able to reach other brain regions (III). It is worth noting that the nanostructured hydrogel, after residing for a sufficient time to release its NPs, should be degraded and eliminated without discomfort for the patient (IV). At present this is a very promising situation where the individual actors (hydrogel, NPs, and selected drugs) are already available, but we still need to deepen our knowledge of their properties and the peculiar features that the resulting nanocomposites are able to gain upon their carefully arranged mixture. In fact, for instance, while designing appropriate composite devices with suitable mechanical properties for biomedical applications, several tests are needed to evaluate their static and dynamic behavior (44, 86-89). Another field of particular relevance for preventative medicine where great strides are expected is nanodiagnostics; in fact, when AD/PD are clinically suspected, the underpinning molecular neurodegeneration is already very

advanced, and an effective therapy is more difficult. NPs might improve the sensitivity of diagnostic tests and aid in early detection of subtle alterations before AD/PD symptoms. For instance, an implantable nanodevice able to monitor over time the accumulation of pathological forms of amyloid proteins involved in AD/PD might help toward early screening of patients at-risk, supporting a preventative approach.

REFERENCES

7.

1.

2.

3.

4. 5. 6.

Cutting Edge Info Reports. Neurodegenerative market forecast to 2013 (Report TA222). 2009. Available at: http://www. cuttingedgeinfo.com/research/market-intelligence/neurodegenerative-forecast/. Accessed December 5, 2011. Gasser T. Molecular pathogenesis of Parkinson disease: insights from genetic studies. Expert Rev Mol Med. 2009;11:e22. doi: 10.1017/S1462399409001148 Twelves D, Perkins KS, Counsell C. Systematic review of incidence studies of Parkinson’s disease. Mov Disord. 2003;18(1):19-31. Waldau B, Shetty AK. Behavior of neural stem cells in the Alzheimer brain. Cell Mol Life Sci. 2008;65(15):2372-2384. Mattson MP. Pathways towards and away from Alzheimer’s disease. Nature. 2004;430(7000):631-639. Giordano C, Albani D, Gloria A, et al. Multidisciplinary perspectives for Alzheimer’s and Parkinson’s diseases: hydrogels for protein delivery and cell-based drug delivery as therapeutic strategies. Int J Artif Organs. 2009;32(12):836-850.

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Conflict of Interest Statement: None of the authors has any conflicts of interest to disclose.

Address for correspondence: Carmen Giordano Department of Chemistry, Materials and Chemical Engineering, “G. Natta” Politecnico di Milano Via Luigi Mancinelli 7 IT-20131, Milan, Italy e-mail: [email protected]

8.

9.

10.

11.

12.

13.

Aridon P, Geraci F, Turturici G, D’Amelio M, Savettieri G, Sconzo G. Protective role of heat shock proteins in Parkinson’s disease. Neurodegener Dis. 2011;8(4):155-168. Laskowska E, Matuszewska E, Kuczyńska-Wiśnik D. Small heat shock proteins and protein-misfolding diseases. Curr Pharm Biotechnol. 2010;11(2):146-157. Popovic N, Brundin P. Therapeutic potential of controlled drug delivery systems in neurodegenerative diseases. Int J Pharm. 2006;314(2):120-126. Gloria A, De Santis R, Ambrosio L. Polymer-based composite scaffolds for tissue engineering. J Appl Biomater Biomech. 2010;8(2):57-67. Sionkowka A. Current research on the blends of natural and synthetic polymers as new biomaterials: review. Prog Polym Sci. 2011;36(9):1254-1276. Gloria A, Russo T, De Santis R, Ambrosio L. 3D fiber deposition technique to make multifunctional and tailor-made scaffolds for tissue engineering applications. J Appl Biomater Biomech. 2009;7(3):141-152. Schwartz MM. Composite materials handbook. New York,


Giordano et al

NY: McGraw-Hill, 1992. 14. Mallick PK. Composites engineering handbook. New York, NY: CRC Press, 1997. 15. Jones RM. Mechanics of composite materials. London: Taylor & Francis, 1999. 16. Nicolais L, Gloria A, Ambrosio L. The mechanics of biocomposites. In: Ambrosio L, ed. Biomedical composites. London: CRC Press, 2010; 411-440. 17. Gloria A, Ronca D, Russo T, et al. Technical features and criteria in designing fiber-reinforced composite materials: from the aerospace and aeronautical field to biomedical applications. J Appl Biomater Biomech. 2011;9(2):151-163. 18. Kabiri K, Zohuriaan-Mehr MJ. Porous superabsorbent hydrogel composites: synthesis, morphology and swelling rate. Macromol Mater Eng. 2004;289(7):653-661. 19. Schapery RA. Thermal expansion coefficients of composite materials based on energy principles. J Comp Mat. 1968;2(3):380-404. 20. Nicolais L. Mechanics of composites. Polym Eng Sci. 1975;15(3):137-149. 21. Nicolais L, Narkis M. Stress-strain behaviour of styrene-acrylonitrile/glass bead composites in the glassy region. Polym Eng Sci. 1971;11(3):194-199. 22. Russo T, Gloria A, D’Antò V, et al. Poly(e-caprolactone) reinforced with sol-gel synthesized organic-inorganic hybrid fillers as composite substrates for tissue engineering. J Appl Biomater Biomech. 2010;8(3):146-152. 23. Alfieri I, Lorenzi A, Montenero A, Gnappi G, Fiori F. Sol-gel silicon alkoxides-polyethylene glycol derived hybrids for drug delivery systems. J Appl Biomater Biomech. 2010;8(1):1419. 24. Catauro M, Verardi D, Melisi D, Belotti F, Mustarelli P. Novel sol-gel organic-inorganic hybrid materials for drug delivery. J Appl Biomater Biomech. 2010;8(1):42-51. 25. Lamponi S, Forbicioni M, Barbucci R. The role of fibronectin in cell adhesion to spiral patterned TiO2 nanoparticles. J Appl Biomater Biomech. 2009;7(2):104-110. 26. Sandin K, Kloo L, Odselius R, Ollsson LF. The observation of nano-crystalline calcium phosphate precipitate in a simple supersaturated inorganic blood serum model - composition and morphology. J Appl Biomater Biomech. 2009;7(1):1322. 27. Zaffe D, Traversa G, Mozzati M, Morelli F, D’Angeli G. Behavior of aqueous nanocrystalline hydroxyapatite in oral bone regeneration. J Appl Biomater Biomech. 2011;9(1):19-25. 28. Yang L, Zhang L, Webster TJ. Nanobiomaterials: state of the art and future trends. Adv Eng Mat. 2011;13(6):B197-217. 29. Camargo PH, Satyanarayana KG, Wypych F. Nanocomposites: synthesis, structure, properties and new application opportunities. Mat Res. 2009;12:1-39. 30. Liao K, Ren Y, Xiao T. Single-walled carbon nanotubes in epoxy composites. In: Mai YW, Yu ZZ, eds. Polymer nanocomposites. Cambridge: Woodhead, 2006. 31. Pietronave S, Iafisco M, Locarno D, Rimondini L, Prat M.

32.

33.

34.

35.

36.

37.

38. 39.

40.

41. 42.

43.

44.

45.

46.

47. 48.

Functionalized nanomaterials for diagnosis and therapy of cancer. J Appl Biomater Biomech. 2009;7(2):77-89. Dvir T, Timko BP, Kohane DS, Langer R. Nanotechnological strategies for engineering complex tissues. Nat Nanotechnol 2011;6(1):13-22. Fu AH, Micheel CM, Cha J, Chang H, Yang H, Alivisatos AP. Discrete nanostructures of quantum dots/Au with DNA. J Am Chem Soc. 2004;126(35):10832-10833. Yin Y. Alivisatos AP. Colloidal nanocrystal synthesis and the organic-inorganic interface. Nature. 2005;437(7059):664670. Cozzoli PD, Pellegrino T, Manna L. Synthesis, properties and perspectives of hybrid nanocrystal structures. Chem Soc Rev. 2006;35(11):1195-1208. Srivastava S, Samanta B, Arumugam P, Han G, Rotello VM. DNA-mediated assembly of iron platinum (FePt) nanoparticles. J Mater Chem. 2007;17:52-55. Quarta A, Di Corato R, Manna L, Ragusa A, Pellegrino T. Fluorescent-magnetic hybrid nanostructures: preparation, properties, and applications in biology. IEEE Trans Nanobioscience. 2007;6(4):298-308. Paul DR, Robeson LM. Polymer nanotechnology: nanocomposites. Polymer. 2008;49(15):3187-3204. Raffaini G, Ganazzoli F. Protein adsorption on biomaterial and nanomaterial surfaces: a molecular modelling approach to study non-covalent interactions. J Appl Biomater Biomech. 2010;8(3):135-145. De Santis R, Gloria A, Russo T, et al. A basic approach toward the development of nanocomposite magnetic scaffolds for advanced bone tissue engineering. J Appl Pol Sci. 2011;122(6):3599-3605. Giannoni P, Narcisi R. Nano-approaches in cartilage repair. J Appl Biomater Biomech. 2009;7(1):1-12. Satarkar NS, Biswal D, Hilt JZ. Hydrogel nanocomposites: a review of applications as remote controlled biomaterials. Soft Matter. 2010;6:2364-2371. Fischer H. Polymer nanocomposites: from fundamental research to specific applications. Mater Sci Eng. 2003;23(68):763-772. Gloria A, De Santis R, Ambrosio L, Causa F, Tanner KE. A multi-component fiber-reinforced PHEMA-based hydrogel/ HAPEX™ device for customized intervertebral disc prosthesis. J Biomat Appl. 2011;25(8):795-810. Roney C, Kulkarni P, Arora V, et al. Targeted nanoparticles for drug delivery through the blood-brain barrier for Alzheimer’s disease. J Control Release. 2005;108(2-3):193-214. Nahar M, Dutta T, Murugesan S, et al. Functional polymeric nanoparticles: an efficient and promising tool for active delivery of bioactives. Crit Rev Ther Drug Carrier Syst. 2006;23(4):259-318. Zhong Y, Bellamkonda RV. Biomaterials for the central nervous system. J R Soc Interface. 2008;5(26):957-975. Mittal G, Carswell H, Brett R, Currie S, Kumar MN. Development and evaluation of polymer nanoparticles for oral deliv-


1125


49.

50.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

ery of estradiol to rat brain in a model of Alzheimer’s pathology. J Control Release. 2011;150(2):220-228. Carroll RT, Bhatia D, Geldenhuys W, et al. Brain-targeted delivery of Tempol-loaded nanoparticles for neurological disorders. J Drug Target. 2010;18(9):665-674. Smith A, Giunta B, Bickford PC, Fountain M, Tan J, Shytle RD. Nanolipidic particles improve the bioavailability and alpha-secretase inducing ability of epigallocatechin-3-gallate (EGCG) for the treatment of Alzheimer’s disease. Int J Pharm. 2010;389(1-2):207-212. Hu K, Shi Y, Jiang W, Han J, Huang S, Jiang X. Lactoferrin conjugated PEG-PLGA nanoparticles for brain delivery: preparation, characterization and efficacy in Parkinson’s disease. Int J Pharm. 2011;415(1-2):273-283. Kurakhmaeva KB, Djindjikhashvili IA, Petrov VE, et al. Brain targeting of nerve growth factor using poly(butyl cyanoacrylate) nanoparticles. J Drug Target. 2009;17(8):564-574. Bhaskar S, Tian F, Stoeger T, et al. Multifunctional nanocarriers for diagnostics, drug delivery and targeted treatment across blood-brain barrier: perspectives on tracking and neuroimaging. Part Fibre Toxicol. 2010;7:3. Brambilla D, Le Droumaguet B, Nicolas J, et al. Nanotechnologies for Alzheimer’s disease: diagnosis, therapy, and safety issues. Nanomedicine. 2011;7(5):521-540. Yang J, Wadghiri YZ, Hoang DM, et al. Detection of amyloid plaques targeted by USPIO-Aβ1-42 in Alzheimer’s disease transgenic mice using magnetic resonance microimaging. Neuroimage. 2011;55(4):1600-1609. Härtig W, Kacza J, Paulke BR, et al. In vivo labelling of hippocampal beta-amyloid in triple-transgenic mice with a fluorescent acetylcholinesterase inhibitor released from nanoparticles. Eur J Neurosci. 2010;31(1):99-109. Orive G, De Castro M, Kong HJ, et al. Bioactive cell-hydrogel microcapsules for cell-based drug delivery. J Control Release. 2009;135(3):203-210. Lee KR, Maeng HJ, Chae JB, et al. Lack of a primary physicochemical determinant in the direct transport of drugs to the brain after nasal administration in rats: potential involvement of transporters in the pathway. Drug Metab Pharmacokinet. 2010;25(5):430-441. Arumugam K, Subramanian GS, Mallayasamy SR, Averineni RK, Reddy MS, Udupa N. A study of rivastigmine liposomes for delivery into the brain through intranasal route. Acta Pharm. 2008;58(3):287-297. Jogani VV, Shah PJ, Mishra P, Mishra AK, Misra AR. Nose-to-brain delivery of tacrine. J Pharm Pharmacol. 2007;59(9):1199-1205. Leonard AK, Sileno AP, Brandt GC, Foerder CA, Quay SC, Costantino HR. In vitro formulation optimization of intranasal galantamine leading to enhanced bioavailability and reduced emetic response in vivo. Int J Pharm. 2007;335(12):138-146. Kim TK, Kang W, Chun IK, Oh SY, Lee YH, Gwak HS. Pharmacokinetic evaluation and modeling of formulated

1126

63.

64.

65.

66.

67.

68.

69.

70.

71. 72.

73.

74.

75.

76.

77.

levodopa intranasal delivery systems. Eur J Pharm Sci. 2009;38(5):525-532. Luppi B, Bigucci F, Corace G, et al. Albumin nanoparticles carrying cyclodextrins for nasal delivery of the anti-Alzheimer drug tacrine. Eur J Pharm Sci. 2011;44(4):559-565. Seju U, Kumar A, Sawant KK. Development and evaluation of olanzapine-loaded PLGA nanoparticles for nose-tobrain delivery: in vitro and in vivo studies. Acta Biomater. 2011;7(12):4169-4176. Wen Z, Yan Z, Hu K, et al. Odorranalectin-conjugated nanoparticles: preparation, brain delivery and pharmacodynamic study on Parkinson’s disease following intranasal administration. J Control Release. 2011;151(2):131-138. Xia H, Gao X, Gu G, et al. Low molecular weight protamine-functionalized nanoparticles for drug delivery to the brain after intranasal administration. Biomaterials. 2011;32(36):9888-9898. Cheng Q, Feng J, Chen J, Zhu X, Li F. Brain transport of neurotoxin-I with PLA nanoparticles through intranasal administration in rats: a microdialysis study. Biopharm Drug Dispos. 2008;29(8):431-439. Wang JJ, Zeng ZW, Xiao RZ, et al. Recent advances of chitosan nanoparticles as drug carriers. Int J Nanomedicine. 2011;6:765-774. Amidi M, Romeijn SG, Borchard G, Junginger HE, Hennink WE, Jiskoot W. Preparation and characterization of proteinloaded N-trimethyl chitosan nanoparticles as nasal delivery system. J Control Release. 2006;111(1-2):107-116. Goycoolea FM, Lollo G, Remuñán-López C, Quaglia F, Alonso MJ. Chitosan-alginate blended nanoparticles as carriers for the transmucosal delivery of macromolecules. Biomacromolecules. 2009;10(7):1736-1743. Bednar MM. Anti-amyloid antibody drugs in clinical testing for Alzheimer’s disease. Drugs. 2009;12(9):566-575. Alsarra IA, Hamed AY, Alanazi FK, Neau SH. Rheological and mucoadhesive characterization of poly(vinylpyrrolidone) hydrogels designed for nasal mucosal drug delivery. Arch Pharm Res. 2011;34(4):573-582. Buzea C, Pacheco II, Robbie K. Nanomaterials and nanoparticles: sources and toxicity. Biointerphases. 2007;2(4):MR17MR71. Suh WH, Suslick KS, Stucky DG, Suh YH. Nanotechnology, nanotoxicology, and neuroscience. Prog Neurobiol. 2009;87(3):133-170. Billi F, Campbell P. Nanotoxicology of metal wear particles in total joint arthroplasty: a review of current concepts. J Appl Biomater Biomech. 2010;8(1):1-6. Kunzmann A, Andersson B, Thurnherr T, Krug H, Scheynius A, Fadeel B. Toxicology of engineered nanomaterials: focus on biocompatibility, biodistribution and biodegradation. Biochim Biophys Acta. 2011;1810(3):361-373. 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. 2008;69(1):1-9.


Giordano et al

78. Yang Z, Liu Z.W, Allaker RP, et al. A review of nanoparticle functionality and toxicity on the central nervous system. J R Soc Interface. 2010;7:S411-S422. 79. Yang Z, Zhang Y, Yang Y, et al. Pharmacological and toxicological target organelles and safe use of single-walled carbon nanotubes as drug carriers in treating Alzheimer disease. Nanomedicine. 2010;6(3):427-441. 80. Fraser TW, Reinardy HC, Shaw BJ, Henry TB, Handy RD. Dietary toxicity of single-walled carbon nanotubes and fullerenes (C60) in rainbow trout (Oncorhynchus mykiss). Nanotoxicology. 2011;5(1):98-108. 81. Nyitrai G, Kékesi O, Pál I, et al. Assessing toxicity of polyamidoamine dendrimers by neuronal signaling functions. Nanotoxicology 2011. [Epub ahead of print]. 82. Zhang L, Bai R, Liu Y, et al. The dose-dependent toxicological effects and potential perturbation on the neurotransmitter secretion in brain following intranasal instillation of copper nanoparticles. Nanotoxicology 2011. [Epub ahead of print]. 83. Johnston HJ, Hutchison G, Christensen FM, Peters S, Hankin S, Stone V. A review of the in vivo and in vitro toxicity of silver and gold particulates: particle attributes and biological mechanisms responsible for the observed toxicity. Crit Rev Toxicol. 2010;40(4):328-346.

84. Batelli S, Albani D, Rametta R, et al. DJ-1 modulates alphasynuclein aggregation state in a cellular model of oxidative stress: relevance for Parkinson’s disease and involvement of HSP70. PLoS One. 2008;3(4):e1884. 85. Albani D, Polito L, Batelli S, et al. The SIRT1 activator resveratrol protects SK-N-BE cells from oxidative stress and against toxicity caused by alpha-synuclein or amyloid-beta (1-42) peptide. J Neurochem. 2009;110(5):1445-1456. 86. Kyriakidou K, Lucarini G, Zizzi A, et al. Dynamic co-seeding of osteoblast and endothelial cells on 3D polycaprolactone scaffolds for enhanced bone tissue engineering. J Bioact Compat Pol. 2008;23(3):227-243. 87. Gloria A, Causa F, De Santis R, Netti PA, Ambrosio L. Dynamic-mechanical properties of a novel composite intervertebral disc prosthesis. J Mater Sci Mater Med. 2007;18(11):21592165. 88. Gloria A, Manto L, De Santis R, Ambrosio L. Biomechanical behavior of a novel composite intervertebral body fusion device. J Appl Biomat Biomech. 2008;6(3):163-169. 89. De Santis R, Gloria A, Sano H, et al. Effect of light curing and dark reaction phases on the thermomechanical properties of a Bis-GMA based dental restorative material. J Appl Biomat Biomech. 2009;7(2):132-140.


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