Hybrid nanoparticles as a new technological approach to enhance the delivery of cholesterol into the brain

Restoration of the Chol homeostasis in the Central Nervous System (CNS) could be beneficial for the treatment of Huntington's Disease (HD), a progressive, fatal, adult-onset, neurodegenerative disorder. Unfortunately, Chol is unable to cross the blood-brain barrier (BBB), thus a novel strategy for a targeted delivery of Chol into the brain is highly desired. This article aims to investigate the production of hybrid nanoparticles composed by Chol and PLGA (MIX-NPs) modified with g7 ligand for BBB crossing. We described the impact of ratio between components (Chol and PLGA) and formulation process (nanoprecipitation or single emulsion process) on physico-chemical and structural characteristics, we tested MIX-NPs in vitro using primary hippocampal cell cultures evaluating possible toxicity, uptake, and the ability to influence excitatory synaptic receptors. Our results elucidated that both formulation processes produce MIX-NPs with a Chol content higher that 40%, meaning that Chol is a structural particle component and active compound at the same time. The formulation strategy impacted the architecture and reorganization of components leading to some differences in Chol availability between the two types of g7 MIX-NPs. Our results identified that both kinds of MIX-NPs are efficiently taken up by neurons, able to escape lysosomes and release Chol into the cells resulting in an efficient modification in expression of synaptic receptors that could be beneficial in HD

Synthesis, characterization, and In vitro studies of an reactive oxygen species (ROS)-responsive methoxy polyethylene glycol-thioketal- melphalan prodrug for glioblastoma treatment

Glioblastoma (GBM) is the most frequent and aggressive primary tumor of the brain and averages a life expectancy in diagnosed patients of only 15 months. Hence, more effective therapies against thismalignancy are urgently needed. Several diseases, including cancer, are featured by high levels of reactive oxygen species (ROS), which are possible GBM hallmarks to target or benefit from. Therefore, the covalent linkage of drugs to ROS-responsive molecules can be exploited aiming for a selective drug release within relevant pathological environments. In this work, we designed a new ROS-responsive prodrug by using Melphalan (MPH) covalently coupled with methoxy polyethylene glycol (mPEG) through a ROS-cleavable group thioketal (TK), demonstrating the capacity to self assembly into nanosized micelles. Full chemical-physical characterization was conducted on the polymeric-prodrug and proper controls, along with in vitro cytotoxicity assayed on different GBM cell lines and “healthy” astrocyte cells confirming the absence of any cytotoxicity of the prodrug on healthy cells (i.e. astrocytes). These results were compared with the non-ROS responsive counterpart, underlining the anti-tumoral activity of ROS-responsive compared to the non-ROSresponsive prodrug on GBM cells expressing high levels of ROS. On the other hand, the combination treatment with this ROS-responsive prodrug and X-ray irradiation on human GBM cells resulted in an increase of the antitumoral effect, and this might be connected to radiotherapy. Hence, these results represent a starting point for a rationale design of innovative and tailored ROS-responsive prodrugs to be used in GBM therapy and in combination with radiotherapy

g7-NPs/Alb representative confocal images of Ids-ko and wt mice brain.

<p>(a) Ids-ko and (b) syngeneic wt mice. Nuclei are shown in blue (DAPI staining), NPs in red (rhodamine labeling), Albumin in green (FITC labeling); last column represents the merged images.</p

Localization of g7-NPs/Alb in Idua-ko mouse brain.

<p>Representative confocal images of the g7-NPs/Alb perinuclear localization in the brain of Idua-ko mice injected with g7-NPs/Alb. (a) Albumin is shown in green (FITC labeling), (b) NPs in red (rhodamine labeling), (c) nuclei in blue (DAPI staining); (d) represents the merged images.</p

Morphologic characterization of nanoparticles.

<p>AFM (panels a, c, e) and SEM (panels b, d, f) images of unloaded g7-NPs, used as control, loaded u-NPs/Alb and g7-NPs/Alb, respectively. g7-NPs: unloaded targeted nanoparticles; u-NPs/Alb: untargeted nanoparticles loaded with albumin;g7-NPs/Alb: targeted nanoparticles loaded with albumin.</p

Chemico-physical characterization of nanoparticles.

<p>Chemico-physical characterization of nanoparticles.</p

Type of injection performed in the two mouse models.

<p>Type of injection performed in the two mouse models.</p

Number of g7-NPs/Alb in the brain of Ids-ko and wt mice.

<p>*p-value <0.001.</p

Number of NPs in the brain of Idua-ko and wt mice.

<p>g7-NPs: unloaded targeted nanoparticles; u-NPs: unloaded untargeted nanoparticles; g7-NPs/Alb: targeted nanoparticles loaded with albumin: u-NPs/Alb, untargeted nanoparticles loaded with albumin; MIX1 (g7-NPs+Alb): unloaded targeted nanoparticles suspended in FITC-albumin solution; MIX2 (u-NPs+Alb): unloaded untargeted nanoparticles suspended in FITC-albumin solution. *p-value <0.05.</p

Preparation scheme of NP samples.

<p>(a) Un-modified NPs were prepared both as un-loaded NPs (empty left half circle) and as albumin loaded-NPs (right half circle). b) Un-modified NPs were chemically modified on their surface (EDC/NHS activation followed by NA-maleido reaction and biotinylated-g7 conjugation) in order to produce modified NPs both un-loaded (empty left half circle) and loaded with albumin (right half circle). c) MIX samples: MIX1 = physical mixture of un-loaded g7-NPs + albumin solution; MIX2 = physical mixture of un-loaded u-NPs + albumin solution. u-NPs = un-modified NPs, Alb = albumin, EDC = 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide, NHS = N-hydroxysulfoxuccinimide, NA = neutravidin.</p