Lipid nano-systems were prepared and characterized in a series of well-established in vitro tests that could assess their interactions with the hCMEC/D3 and SH-SY5Y cell lines as a model for the blood–brain barrier and neuronal function, accordingly. The prepared formulations of nanoliposomes and nanostructured lipid carriers were characterized by z-average diameters of ~120 nm and ~105 nm, respectively, following a unimodal particle size distribution (PDI < 0.3) and negative Z-potential (−24.30 mV to −31.20 mV). Stability studies implied that the nano-systems were stable in a physiologically relevant medium as well as human plasma, except nanoliposomes containing poloxamer on their surface, where there was an increase in particle size of ~26%. The presence of stealth polymer tends to decrease the amount of adsorbed proteins onto a particle’s surface, according to protein adsorption studies. Both formulations of nanoliposomes were characterized by a low cytotoxicity, while their cell viability was reduced when incubated with the highest concentration (100 μg/mL) of nanostructured lipid formulations, which could have been associated with the consumption of cellular energy, thus resulting in a reduction in metabolic active cells. The uptake of all the nano-systems in the hCMEC/D3 and SH-SY5Y cell lines was successful, most likely following ATP-dependent internalization, as well as transport via passive diffusion.

In the present study, temozolomide (TMZ), a drug used for the treatment of anaplastic astrocytoma and glioblastoma multiforme (GBM), was incorporated into multiwalled carbon nanotubes (MWCNTs) and a MWCNTs–graphene (MWCNTs-G) hybrid compound, covalently functionalized with polyethylene glycol (PEG) 6000 and folic acid (FA), with an aim to prepare nanocarriers with the potential to prolong the drug circulation time, cross the blood–brain–tumor barrier (BBTB), and provide targeted and controlled drug release in the brain tumor cells. Cytotoxicity and effects on cell membrane integrity of the blank and TMZ-loaded dual-functionalized carbon nanostructures (CNs) were evaluated in vitro on a GBM cell line (U87MG), as well as their radiosensitizing properties after exposure of the pre-treated GBM cells to gamma radiation with a standard clinical dose for patients with GBM. All prepared formulations underwent biopharmaceutical and physicochemical characterization, including the formulations exposed to irradiation under the same conditions. For physicochemical characterization of the formulations, different techniques were used by which successful functionalization of the CNs and TMZ loading were confirmed and visualized; no significant changes in the structure of the CNs and TMZ after irradiation were observed. With single and dual functionalization, formulations with relatively high TMZ loading efficiency and drug content were prepared. They exhibited homogeneous particle size distributions and mean particle sizes and surface charges suitable for crossing the BBTB and targeting brain cancer cells. A biphasic drug release profile was observed for all functionalized TMZ-loaded formulations in simulated in vivo conditions, with a sustained release pointing to the potential for controlled release of TMZ in brain tumor cells. The formulations of the hybrid CN MWCNTs-G compared to the corresponding MWCNTs were characterized by a similar or slightly higher TMZ content, larger particle size, similar surface charge, and slightly faster TMZ release, which can be attributed to the planar structure of graphene that promotes TMZ binding to the surface on a larger scale. For the irradiated CNs, lower values for particle size, more positive values for surface charge, and accelerated TMZ release were observed, which could be explained by changes in the physicochemical characteristics of the prepared formulations upon irradiation. Significant concentration-dependent toxicity was observed for blank dual-functionalized CNs, being higher for MWCNTs-G-PEG6000-FA compared to MWCNTs-PEG6000-FA at the same formulation concentrations. With incorporation of TMZ into the functionalized CNs, the cell viability additionally decreased, maintaining the trend for higher cytotoxicity of the hybrid CN. Additional decrease in the viability of cells was observed when GBM cells pre-treated with the corresponding CNs were exposed to irradiation, which could be ascribed to changes in size, surface charge, and release kinetics of TMZ and to irradiation-induced changes in the microenvironment and cell membranes that promote uptake of a larger volume of carriers in the GBM cells. The higher cytotoxicity observed in the hybrid carrier formulations could most likely be attributed to the length of the hybrid carrier and the higher proportion of planar surface, which promotes more intense contact with the cells and rupture of cell membranes. Overall, the findings demonstrate the radiosensitizing properties of not only TMZ but also of CNs and point to a clinical benefit from combined treatment with carbon nanocarriers of TMZ and radiotherapy in GBM.

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.

Rapid development of nanomedicines necessitates advancement in internalization techniques which can accurately distinguish between the complex environments of cells and nanocarriers. Internalization (or endocytosis) studies of oil red O labeled and l...