Influence of surface characteristics on the in vitro stability and cell uptake of nanoliposomes for brain delivery

Abstract

In contemporary research, there is a clear emphasis on the physicochemical characteristics and effectiveness of nanoliposomal (NLs) formulations. However, there has been minimal focus on elucidating nano-bio interactions and understanding the behavior of these formulations at organ and cellular levels. Specifically, it is widely recognized that when exposed to biological fluids, nanodelivery systems, including NLs, rapidly interact with various biomolecules which have a significant impact on the functionality and fate of the nanosystems but also influence cellular biological functions. Hence, the primary objective of this study was to elucidate the evolution of physicochemical characteristics and surface properties of NLs in biorelevant media. Additionally, in order to point out the influence of specific characteristics on the brain targeting potential of these formulations, we investigated interactions between NLs and blood-brain barrier (BBB, hCMEC/D3) and neuroblastoma cells (SH-SY5Y) under different conditions. The results obtained from comparative in vitro cell uptake studies on both cell culture lines after treatment with three different concentrations of fluorescently labelled NLs (5, 10, and 100 μg/mL) over a period of 1, 2, and 4 h showed a time- and concentration-dependent internalization pattern, with high impact of the surface characteristics of the different formulations. In addition, transport studies on hCMEC/D3/SH-SY5Y co-cultures confirmed the successful transport of NLs across the BBB cells and their subsequent uptake by neurons (ranging from 25.17% to 27.54%). Fluorescence and confocal microscopy micrographs revealed that, once internalized, NLs were concentrated in the perinuclear cell regions.

Keywords: blood–brain barrier; cell co-culture; cell uptake; internalization; nanoliposomes; stability; surface characteristics.

Figure 1

Fractograms from the qualitative evaluation using AF4–UV–MALS–DLS of the native formulations a) NLb0, b) NLb1, and c) NLb2.

Figure 2

Comparative representation of the UV signal and z-average diameter of NLb1 after a) 1 h incubation in serum-supplemented (S) and serum-free (M) cell culture medium, b) 4 h incubation in serum-supplemented and serum-free cell culture medium, c) 1 and 4 h of incubation in serum-free cell culture medium, d) 1 and 4 h incubation in serum-supplemented cell culture medium.

Figure 3

Comparative representation of the UV signal and z-average diameter of NLb2 after a) 1 h incubation in serum-supplemented (S) and serum-free (M) cell culture medium, b) 1 and 4 h incubation in serum-supplemented cell culture medium, c) 4 h incubation in serum-supplemented and serum-free cell culture medium, d) 1 and 4 h of incubation in serum-free cell culture medium.

Figure 4

Comparative representation of the UV signal and z-average diameter of NLb0 after a) 1 h incubation in serum-supplemented (S) and serum-free (M) cell culture medium, b) 1 and 4 h incubation in serum-supplemented cell culture medium, c) 4 h incubation in serum-supplemented and serum-free cell culture medium, d) 1 and 4 h of incubation in serum-free cell culture medium.

Figure 5

High-resolution automated electrophoresis band representation of a) NLb1 and NLb2 after 1 and 4 h incubation in serum-free cell medium (M) and serum-supplemented cell medium (S), b) NLb0 after 1 and 4 h incubation in serum-free cell medium (M) and serum-supplemented cell medium (S).

Figure 6

Cell uptake of NLs (10 μg/mL) in a) hCMEC/D3 cells and b) SH-SY5Y after a 2 h incubation at 4 °C with chlorpromazine or indomethacin.

Figure 7

Fluorescent microscopy (Nile-red channel used) showing internalization in live hCMEC/D3 cells of a) NLb0, b) NLb1, and c) NLb2.

Figure 8

Fluorescent microscopy (Nile-red channel used) showing internalization in SH-SY5Y cells of a) NLb0, b) NLb1, and c) NLb2.

Figure 9

Representation of a) NLb0, b) NLb1, and c) NLb2 in live SH-SY5Y cells (4 h) by fluorescent microscopy. Left image – phase contrast; second image – red fluorescence by NLs marked with Dil red; third image – green fluorescence by endosomes marked with pHrodo Green dextran conjugate, right image – superimposed image.

Figure 10

Confocal microscopy showing internalization and distribution of a) NLb0, b) NLb1, and c) NLb2 in hCMEC/D3 cells. Left image – blue channel – nucleus counterstained with Hoechst fluorescent stain and DAPI excited at 405 nm and detected by a band-pass filter (BP 420/480 nm). Second image – green channel – actin cytoskeleton stained with Alexa Fluor 488 Phalloidin excited at 488 nm and detected by a band-pass filter (BP 505/550 nm). Third image – red channel – Dil-labelled samples detected at a 549 nm excitation wavelength by a long-pass filter (LP 560 nm). Right image – superimposed micrograph.