Targeted Polymeric Nanoparticles for Brain Delivery of High Molecular Weight Molecules in Lysosomal Storage Disorders

Abstract

Lysosomal Storage Disorders (LSDs) are a group of metabolic syndromes, each one due to the deficit of one lysosomal enzyme. Many LSDs affect most of the organ systems and overall about 75% of the patients present neurological impairment. Enzyme Replacement Therapy, although determining some systemic clinical improvements, is ineffective on the CNS disease, due to enzymes' inability to cross the blood-brain barrier (BBB). With the aim to deliver the therapeutic enzymes across the BBB, we here assayed biodegradable and biocompatible PLGA-nanoparticles (NPs) in two murine models for LSDs, Mucopolysaccharidosis type I and II (MPS I and MPS II). PLGA-NPs were modified with a 7-aminoacid glycopeptide (g7), yet demonstrated to be able to deliver low molecular weight (MW) molecules across the BBB in rodents. We specifically investigated, for the first time, the g7-NPs ability to transfer a model drug (FITC-albumin) with a high MW, comparable to the enzymes to be delivered for LSDs brain therapy. In vivo experiments, conducted on wild-type mice and knockout mouse models for MPS I and II, also included a whole series of control injections to obtain a broad preliminary view of the procedure efficiency. Results clearly showed efficient BBB crossing of albumin in all injected mice, underlying the ability of NPs to deliver high MW molecules to the brain. These results encourage successful experiments with enzyme-loaded g7-NPs to deliver sufficient amounts of the drug to the brain district on LSDs, where exerting a corrective effect on the pathological phenotype.

Fig 1. Preparation scheme of NP samples.

(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.

Fig 2. Morphologic characterization of nanoparticles.

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.

Fig 3. Number of NPs in the brain of Idua-ko and wt mice.

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.

Fig 4. Number of g7-NPs/Alb and representative confocal images of Idua-ko and wt mice liver.

(a) Idua-ko and (b) 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. (c) Number of g7-NPs/Alb per optical field. **p-value <0.01.

Fig 5. g7-NPs/Alb and u-NPs/Alb representative confocal images of Idua-ko and wt mice brain.

(a), (c) Idua-ko mice; (b), (d) wt mice; (a), (b) g7-NPs/Alb; (c), (d) u-NPs/Alb treatments. Nuclei are shown in blue (DAPI staining), NPs in red (rhodamine labeling), Albumin in green (FITC labeling); last column represents the merged images. g7-NPs/Alb: targeted nanoparticles loaded with albumin; u-NPs/Alb: untargeted nanoparticles loaded with albumin.

Fig 6. Localization of g7-NPs/Alb in Idua-ko mouse brain.

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.

Fig 7. g7-NPs/Alb representative confocal images of Ids-ko and wt mice brain.

(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.

Fig 8. Number of g7-NPs/Alb in the brain of Ids-ko and wt mice.

*p-value <0.001.