Nano Res.2016 Jul;9(7):2190-2201

Pulmonary administration of functionalized nanoparticles significantly reduces beta-amyloid in the brain of an Alzheimer’s disease murine model.

Giulio Sancini1, Roberta Dal Magro1, Francesca Ornaghi1, Claudia Balducci2, Gianluigi Forloni2, Marco Gobbi3, Mario Salmona3, Francesca Re1

  1. Nanomedicine Center, School of Medicine and SurgeryUniversity of Milano-BicoccaMonza (MB)Italy
  2. Department of NeuroscienceIRCCS-Istituto di Ricerche Farmacologiche Mario NegriMilanoItaly
  3. Department of Biochemistry and Molecular PharmacologyIRCCS-Istituto di Ricerche Farmacologiche Mario NegriMilanoItaly
  4. San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), Division of Regenerative Medicine, Stem Cells and Gene TherapyIRCCS San Raffaele Scientific InstituteMilanoItaly

 

Abstract

Treatment options for Alzheimer’s disease (AD) are limited because of the inability of drugs to cross the blood–brain barrier (BBB). A promising strategy to overcome this obstacle is the use of nanoparticles (NPs). Previously, we showed that intraperitoneal administration of liposomes functionalized with phosphatidic acid and an ApoE-derived peptide (mApoE-PA-LIP) reduces brain beta-amyloid (Aβ) burden and ameliorates impaired memory in AD mice. Here, we investigated lung administration as an alternative, non-invasive NP delivery route for reaching the brain. Our results show that mApoE-PA-LIP were able to cross the pulmonary epithelium ([14C]-PA permeability = 6.5 ± 2.0 × 10–6 cm/min) in vitro and reach the brain (up to 0.6 μg PA/g brain) following in vivo intratracheal instillations. Lung administration of mApoE-PA-LIP to AD mice significantly decreased total brain Aβ (–60%; p < 0.05) compared to untreated mice. These results suggest that pulmonary administration could be exploited for brain delivery of NPs designed for AD therapy.

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SUPPLEMENTARY 

The pharmacological treatment of brain diseases is still a difficult task due the peculiar anatomical and physiological features that characterize the brain.

The main obstacle is represented by the blood-brain barrier (BBB), which regulates brain influx and efflux from and to the bloodstream of endogenous and exogenous compounds. Because of the peculiar properties of endothelial cells lining brain capillaries, most of potential CNS drugs do not cross the BBB or do not reach the brain parenchyma in therapeutic concentration (1). In the last decades, nanotechnologies have earned increasing interest in the field of drug delivery. The possibility to decorate the surface of nanoparticles (NPs) with multiple copies of ligands in order to achieve a site specific targeting and to control the drug dosage and release makes them attractive tools for the treatment of Alzheimer’s disease (AD).

In this work we used liposomes (LIP) functionalized with a modified synthetic peptide derived from the receptor binding region of human apolipoprotein E (mApoE), in order to achieve a BBB specific targeting, and with phosphatidic acid (PA) to bind with high affinity beta-amyloid (Aβ) aggregates, one of the main hallmarks of AD. Previous studies performed by our group demonstrated that mApoE-PA-LIP are able to cross the BBB both in vitro and after intraperitoneal (IP) administration in mice, to disaggregate Aβ assemblies in vitro, and to ameliorate memory impairment in AD-like mouse models (2-4).

Starting from these findings, we aimed to exploit the pulmonary administration, by intratracheal (IT) instillation, as an alternative route to increase mApoE-PA-LIP bioavailability in the brain. Knowing that the translocation of NPs from the lungs to extrapulmonary organs has already been described (5-7) and thanks to the properties of the lungs (i.e. extensive vasculature, easily permeable epithelium), we hypothesized that this site of administration could act as a reservoir of mApoE-PA-LIP, thus allowing a prolonged release in the systemic circulation and a sustained delivery to the brain.

To substantiate our assumption, radiolabeled mApoE-PA-LIP were administered to healthy BALB/c mice by IT instillation and the biodistribution was assessed at different time points (from 24 to 168 h) by measuring the lipid-associated radioactivity. Our results showed that the radioactivity was detected in the lungs up to 168 h after the administration and a low but quite constant percentage of mApoE-PA-LIP was measured both in the brain and in the blood (Figure 1).

 

 

fig1
Figure 1. Biodistribution of mApoE-PA-LIP after a single intratracheal instillation. The radioactivity in the blood, liver, spleen, kidneys, lungs and brain was measured at different time points by liquid scintillation counting.

 

By performing repeated IT instillations (3 administrations, 48 h apart), we observed a progressive accumulation of mApoE-PA-LIP in the brain of BALB/c mice, while the amount of radioactivity detected in the blood remained low (less than 0.3% of the injected dose) and almost unchanged. Interestingly, 24 h after the last administration 0.8% of the instilled dose was measured in the brain. These findings suggest that a slow and sustained mApoE-PA-LIP translocation from to lungs to the blood stream can occur, thus supporting a constant biovailability of mApoE-PA-LIP at the BBB to reach the brain.

Finally, we investigated the effect of the treatment with mApoE-PA-LIP in a AD-like mouse model (APP23 mice). Mice were instilled with mApoE-PA-LIP 3 times/week for 3 weeks. The analysis on collected brains showed a significant reduction of Aβ aggregates, both extracellular plaques and intracellular deposits as well as of the total brain Aβ level (-60%), thus confirming the potential therapeutic effect of mApoE-PA-LIP (Figure 2).

 

 

fig2

Figure 2. Immunostaining of Aβ aggregates in cortical slices from AD-like mice showed a reduction of extracellular plaques after 3 weeks treatment with mApoE-PA-LIP (B) compared to PBS-treated mice (A). Repeated IT administration of mApoE-PA-LIP cleared also intracellular Aβ deposits (a, b) and significantly reduced total (Aβ 1-40 and Aβ 1-42) Aβ levels (C). * p<0.05 by Student’s t test

 

Relevance of the study

This work highlights the feasibility of pulmonary administration of brain targeted NPs as an alternative, non-invasive route to reach the brain. This strategy, which to our knowledge has never been exploited to deliver engineered NPs to the brain, reduces the first pass effect and the amount of NPs in the systemic circulation compared to IP and IV administrations. Therefore, the here obtained results open the possibility to exploit this route to improve the NPs performance in reaching the brain for the treatment of CNS diseases.

 

References

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(2)   Bana, L.; Minniti, S.; Salvati, E.; Sesana, S.; Zambelli, V.; Cagnotto, A.; Orlando, A.; Cazzaniga, E.; Zwart, R.; Scheper, W. et al. Liposomes bi-functionalized with phosphatidic acid and an ApoE-derived peptide affect Aβ aggregation features and cross the blood-brain-barrier: Implications for therapy of Alzheimer disease. Nanomedicine 2014, 10, 1583–1590.

(3)   Balducci, C.; Mancini, S.; Minniti, S.; La Vitola, P.; Zotti, M.; Sancini, G.; Mauri, M.; Cagnotto, A.; Colombo, L.; Fiordaliso, F.et al. Multifunctional liposomes reduce brain β-amyloid burden and ameliorate memory impairment in Alzheimer’s disease mouse models. J. Neurosci. 2014,34, 14022–14031.

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(5)   Videira, M. A.; Gano, L.; Santos, C.; Neves, M.; Almeida, A. J. Lymphatic uptake of lipid nanoparticles following endotracheal administration. J. Microencapsul. 2006, 23, 855–862.

(6)   Videira, M. A.; Botelho, M. F.; Santos, A. C.; Gouveia, L. F.; Pedrosode Lima, J. J.; Almeida, A. J. Lymphatic uptake of pulmonary delivered radiolabelled solid lipid nanoparticles. J. Drug Target. 2002, 10, 607–613.

(7)   Weber S, Zimmer A, Pardeike J. Solid Lipid Nanoparticles (SLN) and Nanostructured Lipid Carriers (NLC) for pulmonary application: a review of the state of the art. Eur J Pharm Biopharm. 2014; 86:7-22.

 

 

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