gH625-liposomes deliver PACAP through a dynamic in vitro model of the blood–brain barrier

Abstract

The blood–brain barrier (BBB) selectively protects the central nervous system (CNS) from external insults, but its function can represent a limit for the passage of therapeutic molecules. Numerous in vitro models of the BBB have been realized in order to study the passage of drugs for neurodegenerative diseases, but these in vitro models are not very representative of the physiological conditions because of a limited supply of oxygen and nutrients due to static conditions. To avoid this phenomenon, we used a millifluidic bioreactor model that ensures a circulation of the medium and, therefore, of the nutrients, thanks to the continuous laminar flow. This dynamic model consists of a double-culture chamber separated by a membrane on which brain endothelial cells are cultured in order to evaluate the passage of the drug. Furthermore, in the lower chamber, SH-SY5Y were seeded as 3D spheroids to evaluate the drug passage through these cells. As nanodelivery system, we used liposomes functionalized with viral fusion peptide to evaluate the passage of a neuroprotective agent, pituitary adenylate cyclase-activating polypeptide (PACAP), through the dynamic in vitro model of the BBB. We showed that our nanodelivery system, made of functionalized liposomes and loaded with specific molecules, efficiently crosses the in vitro fluid-dynamic model of the BBB. Our findings represent an important step for further experimental investigations on PACAP administration as a therapeutic agent by an enhanced drug delivery system. Our results can improve the diffusion of good practice in neuroscience laboratories, helping to spread the 3R rules.

Neuroprotective Effects of gH625-lipoPACAP in an In Vitro Fluid Dynamic Model of Parkinson’s Disease

“…Large unilamellar vesicles (LUVs) derived from PPC/Chol (70/30 mol/mol) were obtained in the protocol, as previously reported [ 18 ]. Briefly, chloroform was used to dissolve all lipid mixture (lipid concentration 1 mM), SPE-PEG2000-gH625 and PACAP-Rho.…”

“…A dynamic light scattering (DLS) (Malvern Zetasizer Nano ZS, Malven, UK) was employed to obtain the measure of the hydrodynamic diameters (DH) and polydispersity index (PDI) of gH625-lipoPACAP-Rho [ 18 ]. He–Ne laser 4 mW operating at 633 nm at scattering angle fixed at 173° and at 25° at pH 7.2 was used to perform all measurements in triplicate for each sample.…”

“…After evaluating the neuroprotective effects of PACAP on 2D SH-SY5Y/RA, we produced 3D SH-SY5Y/RA cells by hanging drop method (2 × 10 4 cells per aggregate) [ 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 ]. After 48 h from the aggregate’s formation, they were transferred in an LB1 bioreactor.…”

“…Therefore, retinoic acid (RA) has been used to induce SH-SY5Y differentiation into neuronal phenotype, since it causes the inhibition of cell growth and increased norepinephrine production by SH-SY5Y cells [ 16 ]; furthermore, SH-SY5Y cells also express dopamine transporter (DAT) that regulates DAn homeostasis [ 17 ]. In our previous work [ 18 ] we showed that SH-SY5Y 3D spheroids express βIII-tubulin neural marker. The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine is the prodrug of 1-methyl-4-phenylpyridinium (MPP + ), acting on mitochondrial complexes, causing adenosine triphosphate (ATP) depletion, indirectly stimulating reactive species of oxygen (ROS) production, causing DAn apoptosis with the spill of dopamine from synaptic vesicles, resulting in the induction of apoptosis in DAn [ 19 ].…”

“…The in vitro fluid-dynamic model is represented by two different bioreactors Livebox1 and Livebox2 (LB1, LB2, IVTech, Ospedaletto, PI, Italy) in which 3D SH-SY5Y cells differentiated with retinoic acid (RA) are treated with MPP + . In a recent piece of work [ 18 ], we have used a LB2 bioreactor to recreate a blood–brain barrier (BBB) in vitro fluid dynamic model, placing mouse endothelial brain cells (bEnd.3) on a porous membrane in the upper chamber and 3D SH-SY5Y cells enriched to the neural portion, in the lower chamber, to demonstrate the passage of gH625-liposomePACAP-Rho through the endothelial monolayer in the lower chamber. These bioreactors are made in polydimethylsiloxane (PDMS) that ensure oxygen permeability and adequate channels for cell culture thanks to the soft lithography techniques.…”