Brain-targeted polymeric nanoparticles: in vivo

PreliminaryRCesearch ommunication Article Brain-targeted polymeric nanoparticles: in vivo evidences of different routes of administration in rodents

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Aims, materials & methods: The capacity of polymeric nanoparticles (NPs) to reach the target regardless of the administration route is a neglected field of investigation in pharmaceutical nanotechnology. Therefore, after having demonstrated in previous studies that glycopeptide-engineered NPs (g7-NPs) were able to reach the brain after intravenous administrations in rodents, this article aims to evaluate whether they can reach the CNS when administered by different routes. Results & conclusions: Results and Conclusion: The confocal microphotographs on murine brain sections showed the capability of g7-NPs to reach the target also after intraperitoneal, intranasal and oral administrations. This could open new vistas for the future application of g7-NPs in the therapeutic treatment of CNS diseases. Original submitted 19 April 2012; Revised submitted 3 September 2012

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KEYWORDS: CNS n drug targeting n intranasal administration n intraperitoneal administration n intravenous administration n nanoparticle n route of administration

with ligands was successfully exploited (‘active targeting’), emphasizing the efficacy of the approach in several applications [16–25] . In particular, several colloidal systems were designed for CNS targeting by exploiting specific surface modifications with targeting ligands capable of taking advantage of specific receptor or nonspecific pathways [26–40] . The authors emphasized the efficacy of the surface modification [41–48] with less attention given to the comparison between the results obtained by administering the colloidal systems by different routes (intranasal [49–53] , oral [54–56] , intracarotid [57] and iv. administration [58,59]). The efforts of researchers for the analysis of the factors able to improve NP targeting by modifying the biodistribution of the colloidal systems are evident. However, the analysis shows that the influence of the administration route on targeting is a neglected field of study [60–64] . Therefore, this work aims to investigate, by using confocal and fluorescent microscopy, the CNS targeting capacity of one colloidal system administered by different administration routes (intraperitoneal, intranasal and oral) in murine models. To reach this purpose, we have employed poly-lactide-co-glycolide (PLGA) NPs specifically engineered by our group for CNS targeting (g7-NPs) [57–59,65–69] . Until now, we have restricted our research to administration in rodents via carotid artery, or femoral or tail vein (iv. administration) demonstrating that g7-NPs were able to reach the brain after

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Nanomedicine (Epub ahead of print)

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Drug delivery colloidal systems, such as nanoparticles (NPs), liposomes, solid lipid NPs and dendrimers, can overcome the problems related to the instability of drugs in the bloodstream and their widespread biodistribution [1–4] . In the investigation devoted to improve the accumulation of the drug in specific organs, the researchers have demonstrated that several factors (shape, size and surface properties) can affect the biodistribution of the colloidal systems [5,6] . The influence of shape on nanosized carriers’ biodistribution was confirmed; it strongly affects biomolecular signaling and biological kinetics during the NPs’ journey inside the body [7,8] as well as the dynamics of cell entry [9] . After intravenous (iv.) administration in rodents, the biodistribution of gold NPs was strongly modified by NP size. NPs smaller than 50 nm are extravasated in many organs, while the systems larger than 100–200 nm were accumulated in the liver [10,11] . The surface properties of NPs, such as the surface charge and hydrophilic/ hydrophobic characteristics, hardly impacts their biodistribution, accumulation in organs [11,12] or the pattern of proteins absorbed onto their surface [13–15] . However, this ‘passive targeting’ is not very efficient in the control of the accumulation of the colloidal systems in target organs. Therefore, in order to improve the targeting properties of the colloidal systems, surface modification

Giovanni Tosi*1, Barbara Ruozi1, Daniela Belletti1, Antonietta Vilella2, Michele Zoli2, Maria Angela Vandelli1 & Flavio Forni1 Department of Life Sciences, University of Modena & Reggio Emilia, Via Campi 183, 41125, Modena, Italy 2 Department of Biomedical, Metabolic & Neural Sciences, University of Modena & Reggio Emilia, Via Campi 287, 41125, Modena, Italy *Author for correspondence: Tel.: +39 059 205 5128 Fax: +39 059 205 5131 [email protected] 1

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ISSN 1743-5889

Tosi, Ruozi, Belletti et al.

having crossed the blood–brain barrier (BBB) [57–59,65–69] . This study, instead, aims to provide useful information on the administration of NPs by more appreciated and acceptable to patients administrations, that is the oral and intranasal routes. According to the aim of the present work – the evaluation of the capability of g7-NPs to reach the brain regardless of the administration route – the experiments were carried out with unloaded g7-NPs.

Materials & methods

NP preparation NPs were obtained in accordance with the nanoprecipitation procedure [70] . Briefly, to obtain modified TMR-labeled NPs (g7-NPs) the nonconjugate polymer (PLGA 503H; 70% w/w) and the polymeric conjugates (g7-PLGA; 20% w/w, and TMR-PLGA; 10% w/w) were solubilized in organic solvent (acetone, 8 ml). Previous nuclear magnetic resonance studies clearly showed the stability of the covalent bond in the conjugates (g7-PLGA and TMR-PLGA) after solubilization in the organic solvent, and the absence of any denaturation effect on the glycopeptide sequence [57,59,69] . The organic solution was then added dropwise into deionized water (25 ml) containing poloxamer 188 (polyoxyethylene–polyoxypropylene block copolymer, Pluronic F68 ®, Sigma Aldrich, Milan, Italy). The solution was then stirred at room temperature for 10 min and the organic solvent was removed at 30°C under reduced pressure (10 mmHg). The final volume of the suspension was adjusted to 10 ml with water. All the NP preparations were then purified by gelfiltration chromatography (Sepharose® CL 4B gel, 160 ml, column 50 × 2 cm, Sigma Aldrich), using water as the mobile phase and reaching almost 80% (w/w) recovery. The colloidal suspension was then freeze-dried (Lyovac ® GT-2; Leybold-Heraeus, Hanau, Germany) without any cryoprotector and the g7-NPs were stored in the dark at 4°C.

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Animals Male albino Wistar–Hannover rats, weighing 200 ± 30 g (Charles River, Lecco, Italy), and wild-type C57Bl6 mice, weighing 23 ± 5 g (Charles River), were used for the in vivo experiments. The animals were maintained at 25°C for an average period of 15 days before the experiments, on a standard diet and water ad libitum. The experiments were carried out in accordance with the European Communities Council Directives of 24 November 1986 (86/609/EEC) for experimental animal care.

high-purity water (molecular weight of 18). All the other chemicals and solvents were obtained from commercial sources and used without further purification.

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Preliminary Communication

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Drugs & chemicals Poly(d,l-lactide-co-glycolide) (PLGA RG503H; molecular weight near to 40,000 and PLGA 502H, molecular weight near to 11,000) was used as received from the manufacturer (Boehringer-Ingelheim, Ingelheim am Rhein, Germany). Gly-l-Phe-d-Thr-Gly-l-Phe-lLeu-l-Ser(O-b-d-glucose)-CONH 2 (g7) was synthesized and characterized as previously described and conjugated with PLGA RG503H to obtain g7-PLGA [57–59] . Briefly, the covalent bond between the carboxyl terminus of the polymer PLGA RG503H and the amine terminal of the peptide was obtained by standard methods (i.e., the activation of the carboxy group of PLGA by means of an ester with N-hydroxysuccinimide, in the presence of dicyclohexylcarbodiimide and the subsequent formation of an amidic linkage with the N-terminus of the unprotected peptide), as described in [57,59] . Similarly, PLGA 502H was conjugated with tetramethylrhodamine (TMR) to obtain TMRPLGA as previously described [57,59] . As stated for g7-PLGA, the N-hydroxysuccinimide-/ dicyclohexylcarbodiimide-based technology allowed activation the PLGA-502H and the covalent formation the amidic bond. A Milli-Q® water system (Millipore, MA, USA) supplied with distilled water provided

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Morphology & particle size The morphology of the NPs was analyzed by exploiting a scanning electron microscope (XL-40 Philips, Eindhoven, The Netherlands) after coating the NPs under argon atmosphere with 10-nm gold palladium (Emitech K550 Supper Coated, Emitech LTD, Ashford, UK). The NPs were analyzed for particle size (in distilled water) and z-potential in simil–plasma f luid (NaCl 128 mM, NaH 2PO 4 2.4 mM, NaHCO3 29.0 mM, KCl 4.2 mM, CaCl 2 1.5 mM, MgCl2 0.9 mM and d-glucose 9 mM; generally used for in vivo studies in order to produce an isotonic and iso-osmolar aqueous solution; pH 7.4 ± 0.1), by photon correlation spectroscopy and laser Doppler anemometry using a Malvern Zetasizer Nano ZS (Malvern, UK; laser 4 mW He–Ne, 633 nm, laser attenuator future science group

Brain-targeted polymeric nanoparticles

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Oral

An exact amount of g7-NPs suspended in saline buffer (0.5–2.0 ml) was administered by oral gavage to rats (10 mg/animal; n = 3) and mice (3 mg/animal; n = 3). The rodents were maintained alive for 180 min in order to allow g7-NPs to be absorbed through the GI tract. The 180-min period was selected as it is reasonable to take into account that the presence of the gastrointestinal wall slows down the entry of g7-NPs to the blood stream compared with iv. administration.

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Animal administrations Mice and rats were treated by administering a fixed amount of g7-NPs (3 mg/mouse and 10 mg/rat) by intraperitoneal injection, intranasal and oral routes. As the different administration routes require g7-NPs to cross different barriers (peritoneal membrane, gastrointestinal wall and nasal epithelium) to reach the brain, the animals were sacrificed at different time periods after the g7-NP administration. The amount of g7-NPs administered to mice (~130 mg/kg) and rats (~40 mg/kg) is too high to be proposed for therapeutic use in humans. However, the aim of this work is to acquire, by confocal microscopy, the incontrovertible evidence of the presence of g7-NPs in the brain sections.

An exact amount of g7-NPs, suspended in saline solution (0.05 ml), was administered to mice (3 mg/animal; n = 3). Briefly, the animals were anesthetized with an ip. injection of a saline solution of xylazine (4.0 mg/kg) and ketamine (100 mg/kg). The animals were then positioned on their backs, with a rolled up 4 × 4 cm gauze under the dorsal neck. Drops of 5–6 µl of the g7-NPs suspensions were administered using a Hamilton microsyringe in each naris after having closed the opposite naris and the mouth [51] . A total volume of approximately 0.05 ml was given in approximately 2 min alternating the nares. The survival time of the animals was fixed 60 min after administration as for the iv. administration tests [68] since the g7-NPs should only cross the nasal epithelium before reaching the brain via an intra-axonal route [50,72] .

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Surface analysis In order to determine the composition of the surface of the prepared NPs, x‑ray photoelectron spectroscopy analysis was performed on an analysis system 04–153 x-ray source (PHI, Uvalca-PHI, Tokyo, Japan) and an hemispherical electron analyzer EA11 (Leybold Optics, Germany) using MgKa1,2 radiations. The spectra were recorded in fixed retardation ratio mode with 190 eV pass energy. The pressure in the sample analysis chamber was approximately 10–9 mbar. Data acquisition and processing was performed with the RBD AugerScan 2 (RBD Instruments, Inc., OR, USA).

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automatic, transmission 100 – 0.0003% , Detector Avalanche photodiode, quantum efficiency >50% at 633 nm, at 25°C). All of the results were normalized with respect to a polystyrene standard solution. The analysis of z-potential was performed in simil–plasma fluid in order to simulate conditions g7-NPs could meet in vivo and, thus, evaluate their tendency to aggregate in plasma.


Intraperitoneal

An exact amount of g7-NPs (3 mg), suspended in saline solution (0.05 ml), was injected intraperitoneally (ip.) in mice (n = 3). The animals were sacrificed 90 min after the administration. This time was selected because the transport of g7-NPs from the peritoneum to the brain is hampered by the peritoneal barrier [71] , delaying the appearance of a large amount of the g7-NPs in the CNS compared with the time (60 min) usually used after iv. administration [68] . future science group

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Figure 1. Scanning electron microscope analysis of g7-nanoparticles. The g7-nanoparticles are well formed, with a regular shape and homogeneous dimensions.

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in different cerebral macroareas. Each frozen tissue was cut in all its thicknesses by a cryotome into 5-µm slides (~40 slides for each brain). The slides were then fixed in absolute MeOH and stored at 4°C for a maximum period of 24 h before examination on the fluorescent microscope. In order to highlight the cellular structures, some sections were treated with 4´,6-diamidino2-phenylindole (DAPI; LabVision Corporation, CA, USA), which is known to form fluorescent complexes with natural dsDNA [73] . Thus, the samples were stained for 10 min with 50 µl of a DAPI solution (125 ng/ml) and observed using a fluorescence microscope (Axiophot, Zeiss, Stuggart, Germany; Olympus Analyzer; excitation bands for DAPI and tetra-methyl rhodamine isothiocyanate using an emission filter set for f luorescence imaging) and a confocal microscope (Leica DM IRE 2, IL, USA; Leica Confocal System: scan head multiband

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Sample preparation for microscopic analysis For histological evaluation, animals were anesthetized with chloral hydrate (400 mg/ kg/10 ml, ip.). Intracardial perfusion was performed with 4% paraformaldehyde and 0.2% picric acid for 10 min (7 ml/min) and the organs (brain, liver, spleen and kidney) were dissected. The brain was postfixed in the same solution for 12 h, cryoprotected in 15% sucrose in phosphate buffered saline (PBS) pH 7.4 for approximately 12 h and then in 30% sucrose in PBS for 1 day. The brains were frozen using dry ice and coronal 50-µm thick sections series were cut on a cryotome (Cryotome CM3000; Leica Instruments GmbH, Germany), washed three times in cold 1 × PBS and stored at -20°C in a glycerol-PBS solution.

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Fluorescence study Regardless of the route of g7-NPs admini stration, the brains of the rodents were dissected

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Figure 2. Spatial distribution of g7-nanoparticles. (A & B) Brain paraventricular hypothalamic nucleus and (C & D) external pyramidal layers of the somatosensory cortex. B & D report higher magnifications of the squared areas bounded with dashed lines in A & C. (B & D) Nanoparticles (red dots) mainly decorate neuronal (large spotted) nuclei and only rarely glial (small homogeneously intense) nuclei. Confocal microscopy analysis using 4´,6-diamidino-2-phenylindole (blue) and g7-NPs (red) on brain cryosections obtained from mice sacrificed 90 min after g7-nanoparticle intraperitoneal injection (3 mg/mouse). I–IV represents the different cortical layers. 3V: Third ventricle PV: Paraventricular hypothalamic nucleus.

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3 channels Leica TCS SP2 with AOBS, laser diode blu COH [405 nm/25 mW], laser Ar [458 nm/5 mW; 476 nm/5 mW; 488 nm/20 mW; 496 nm/5 mW; 514 nm/20 mW], laser HeNe [543 nm/1.2 mW], laser HeNe [594 nm; Orange], laser HeNe [633 nm/102 mW]). The red fluorescent spots, due to fluorescent labeling with TMR, were considered as the visible markers of NPs.

Results

In vivo results

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Figure 3. Spatial distribution of g7-nanoparticles in the brain. (A & B) CA3 subfield and (C & D) dentate gyrus of the hippocampus. (B & D) Higher magnifications of the squared areas bounded with dashed lines in (A & C). (B & D) Nanoparticles (red dots) mainly decorate neuronal (large spotted) nuclei and only rarely glial (small homogeneously intense) nuclei. In particular, the neurons whose nuclei are most intensely labeled are interneurons of the (A & B) stratum lacunosum-moleculare and (C & D) hilus. Confocal microscopy analysis using 4´,6-diamidino-2-phenylindole (blue) and g7-nanoparticles (red) on brain cryosections obtained from mice sacrificed 60 min after g7-nanoparticles nasal administration (3 mg/mouse). CA3: Cornu Ammonis field 3; Gr: Granule cell layer; Hil: Hilus; LMol: Stratum lacunosum moleculare; Pyr: Pyramidal layer.

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Nanoparticle preparation & characterization PCS analysis clearly demonstrated that the size of the NPs ranged from 140 to 200 nm with a homogenous size distribution (polydispersity index ranging from 0.1 to 0.13). The g7-NPs showed a negative surface charge (-15 mV/30 mV). As the PLGA molecule does not contain nitrogen, the ratio between nitrogen and carbon (0.03), determined by x‑ray photoelectron spectroscopy analysis, in comparison with the nitrogen and carbon theoretical ratio of the g7 molecule (0.23), allowed us to hypothesize that approximately 15% of g7 is exposed on the g7-NP surface. The scanning electron microscope microphotographs emphasized the spherical shape, the surface integrity of g7-NPs and size values close to those evaluated by the PCS analysis (Figure 1) .

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Intraperitoneal administration of g7-NPs

Red spots of the TMR labeling of g7-NPs were heterogeneously distributed in the entire CNS, demonstrating the ability of g7-NPs to cross the BBB 90 min after ip. administration. As an example, representative images of the hypothalamus (Figure 2A & B) and cerebral cortex (F igur e 2C & D) sections show that red spots preferentially decorate the nuclei of neurons and only rarely those of glial cells. Intranasal administration of g7-NPs

The localization of g7-NPs within the brain areas was widespread and heterogeneous. We measured the distribution of the TMR signal in the hippocampal CA3 field (Figure 3A & B) and dentate gyrus (F igur e 3C & D) , cingulate cortex (Figure 4A & B) and striatum (Figure 4C & D) . The images highlighted, at a regional level, a remarkable accumulation of g7-NPs without specific tropism to any particular brain area, and at a cellular level, a preferential accumulation of g7-NP signal around neuronal nuclei (see also the high-magnification images of Figure 5). future science group

Oral administration of g7-NPs

The images of CNS parenchyma of rats (Figure 6) and mice (Figure 7) 180 min after oral administration clearly show the localization of g7-NPs inside the brain compartment, showing the ability of g7-NPs to cross the BBB by this administration route. Indeed, as with ip. and intranasal administration, the NP signal accumulation appears to be preferential around neuronal nuclei.

Discussion After iv. administration in rodents via carotid artery [57] , femoral [57] or tail vein [58,59,66–68] , a remarkable amount of g7-NP was able to reach the brain, demonstrating the capability of g7-NPs to cross the BBB both in rats and mice. Electron microscopy studies allowed us to hypothesize that g7-NPs are able to promote the membrane curvature at the BBB, stimulating the www.futuremedicine.com

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Figure 4. Spatial distribution of g7-nanoparticles in the brain. (A & B) Cingulate cortex and (C & D) striatum. (B & D) Higher magnifications of the squared areas bounded with dashed lines in (A & C). (B & D) Nanoparticles (red dots) mainly decorate neuronal (large spotted) nuclei and only rarely glial (small homogeneously intense) nuclei. Confocal microscopy analysis using 4´,6-diamidino2-phenylindole (blue) and g7-nanoparticles (red) on brain cryosections obtained from mice sacrificed 60 min after g7‑nanoparticles nasal administration (3 mg/mouse). CC: Corpus callosum; Cg: Cingulate cortex; LV: Lateral ventricle; Str: Striatum.

endocytotic pathways for crossing the BBB [58] . An endocytosis-mediated uptake mechanism was also responsible for the g7-NP uptake by neurons in vitro [65] . At present, no studies have been carried out to elucidated the mechanism through which the g7-NPs are able cross the peritoneal and gastrointestinal barriers and to pass the intranasal epithelium. However, it can be reasonable to hypothesize the involvement of an endocytotic process in these cases. The iv. route of administration is clearly one of the most successful, since it assures immediate and fast circulation within the bloodstream and, therefore, a short time to reach brain capillaries. Oral and ip. administrations allow the g7-NPs to interact with the BBB and reach the CNS after a longer period, owing to the time necessary to cross other barriers (intestinal and peritoneal walls) [71] .

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Thus, the possibility of g7-NPs to reach the brain after oral administration is an important finding as, to our best knowledge, until now only few papers have dealt with CNS targeting after this administration route [54–56] . However, an important drawback must be clarif ied before proposing the oral administration of g7-NPs for brain targeting. Notwithstanding the capability of g7-NPs to reach the CNS, an attempt to transport loperamide, an opioid-receptor agonist, without any analgesic effect across the BBB via the oral route by means of g7-NPs failed. As previous papers [57–59,66,67] demonstrated the analgesic activity of loperamide targeted to the brain via iv. administration of g7-NPs, it can be reasonable to confirm that the failure in loperamide targeting to the CNS was due to the release of loperamide (low molecular weight, future science group



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this finding is not surprising as the glycopeptide (MMP-2200 – the Tyr in MMP2200 was substituted in g7 by Phe in order to avoid a potential opioid effect [57–59]) demonstrated a higher stability in the bloodstream with respect to native peptidic opiods. The photomicrographs emphasized the presence of g7-NPs in the CNS after intranasal administration (Figur es 3–5) . It is well known that NPs have to cross the mucosal barrier and be taken up by neurons and supporting cells through a number of endocytic mechanisms before reaching the brain via an intra-axonal route [72,79,80] . Since a endocytotic pathway was observed in vivo for the BBB crossing [58] and in vitro for the neuron uptake [65] , a similar endocytotic mechanism can, therefore, [74–78]

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close to 500 Da) into the gastric environment [54] . Thus, the g7-NPs cannot be used to target the CNS with low-molecular-weight drugs, but this administration route could be reserved for the administration of high-molecular-weight macromolecules (macropeptides, proteins and gene materials) that are unstable after an oral administration. Notwithstanding the failure to target low-molecular-weight drug to the brain, the observation that g7-NPs reach the brain after oral administration is essential evidence of the stability of g7 in the acid medium of the stomach and against proteolytic enzymes. The g7 stability is due to the presence of d-Thr in the glycopeptide molecule, which is more resistant to proteases than l-amino acids [67,68] . Really,

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Figure 5. g7-nanoparticles in the brain. (A) Hippocampus and (B) somatosensory cortex. High magnification images showing that a high density of nanoparticles (red dots) decorates some neuronal (large spotted) nuclei. Confocal microscopy analysis using 4´,6-diamidino-2-phenylindole (blue) and g7-nanoparticles (red ) on brain cryosections obtained from mice sacrificed sacrificed 60 min after g7-nanoparticles nasal administration (3 mg/mouse).

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Figure 6. g7-nanoparticles in the brain. (A) Somatosensory cortex and (B) hippocampus. Confocal microscopy analysis using 4´,6-diamidino-2-phenylindole (blue) and g7-nanoparticles (red) on brain cryosections obtained from mice sacrificed 180 min after g7-nanoparticles oral administration (10 mg/rat).



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Figure 7. g7-nanoparticles in the brain. (A) Somatosensory cortex and (B) hippocampus. High-magnification images showing that high density of nanoparticles (red dots) decorates some neuronal (large spotted) nuclei. Confocal microscopy analysis using 4´,6-diamidino-2-phenylindole (blue) and g7-nanoparticles (red) on brain cryosections obtained from mice sacrificed 180 min after g7-nanoparticles oral administration (3 mg/mouse).

confirmed by the impressive number of papers, patents, clinical trials and pharmaceutical products [34,81,82] , clearly indicating the worldwide effort in the nanomedicine applications field. The improvement in the knowledge of colloidal systems has allowed the targeting of drugs to specific cell populations or organs playing, at first, on the morphology, size and surface properties of the systems (passive targeting) and then on the surface modifications (active targeting). In-depth attention to the influence of the administration route could demonstrate the feasibility of the oral route, extending the use of the colloidal systems for the self-treatment of patient affected by chronic CNS diseases such as Alzheimer’s and Parkinson’s diseases. The inroduction of the nanomedicine to these pathologies, which have a high total number of cases in the population, will facilitate the industrial development of the colloidal systems and, thus, reducing the production costs.

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be hypothesized for the CNS access of the g7-NPs. To date, our experiments cannot evaluate whether the conjugation of g7 on the NPs surface improves brain targeting via the nasal route of the g7-NPs compared with the unmodified NPs. However, we are confident of the possibility of taking advantage of the g-7 modification for targeting the CNS via the nasal route, as the surface modification approach was already been successfully used to improve the targeting of NPs to the brain [81,82] . These data represent a starting point for promoting a deeper examination of the influence of the administration route on the brain targeting and on distribution of g7-NPs in different brain areas.

Conclusion The achievement of noninvasive innovative drug delivery systems for CNS targeting is one of the topics at the cutting edge of current pharmaceutical nanotechnology as the noninvasive intranasal and oral administration routes could result in added value for patient compliance. These preliminary and merely qualitative data, suggest the possibility to reach the brain regardless of the route of administration. Future perspective The drug delivery colloidal systems represent the most innovative approaches for improving the treatment of difficult-to-treat diseases. This field of research is growing year-by-year, as

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Financial & competing interests disclosure The author has no relevant affiliations or financial involvement 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, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript. future science group


Ethical conduct of research The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all


human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Executive summary In order to improve the targeting properties of the colloidal systems, their surface modification with ligands was successfully exploited. The influence of the administration route on nanoparticle (NP) targeting is a neglected field of investigation. This study aimed to evaluate the influence of the administration route on brain targeting by means of NPs modified on the surface with glycopeptides, g7-NPs, able to reach the brain by intravenous injection. The g7-NPs were administered in rodents (rats and mice) by intraperitoneal, oral and intranasal routes, and their brain sections were evaluated by means of confocal microscopy.

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