Lysosomal storage disorders (LSDs) result from an enzyme deficiency within lysosomes. The systemic administration of the missing enzyme, however, is not effective in the case of LSDs with central nervous system (CNS)-involvement. Here, an enzyme delivery system based on the encapsulation of cross-linked enzyme aggregates (CLEAs) into poly-(lactide-co-glycolide) (PLGA) nanoparticles (NPs) functionalized with brain targeting peptides (Ang2, g7 or Tf2) is demonstrated for Krabbe disease, a neurodegenerative LSD caused by galactosylceramidase (GALC) deficiency. We first synthesize and characterize Ang2-, g7- and Tf2-targeted GALC CLEA NPs. We study NP cell trafficking and capability to reinstate enzymatic activity in vitro. Then, we successfully test our formulations in the Twitcher mouse. We report enzymatic activity measurements in the nervous system and in accumulation districts upon intraperitoneal injections, demonstrating activity recovery in the brain up to the unaffected mice level. Together, these results open new therapeutic perspectives for all LSDs with major CNS-involvement.
Polymeric nanoparticles (NPs) represent one of the most promising tools in nanomedicine and have been extensively studied for the delivery of water-insoluble drugs. However, the efficient loading of therapeutic enzymes and proteins in polymer-based nanostructures remains an open challenge. Here, we report a synthesis method for a new enzyme delivery system based on cross-linked enzyme aggregates (CLEAs) encapsulation into poly(lactide- co-glycolide) (PLGA) NPs. We tested the encapsulation strategy on four enzymes currently investigated for enzyme replacement therapy: palmitoyl protein thioesterase 1 (PPT1; defective in NCL1 disease), galactosylceramidase (GALC; defective in globoid cell leukodystrophy), alpha glucosidase (aGLU; defective in Pompe disease), and beta glucosidase (bGLU; defective in Gaucher's disease). We demonstrated that our system allows encapsulation of enzymes with excellent activity retention (usually around 60%), thus leading to functional and targeted nanostructures suitable for enzyme delivery. We then demonstrated that CLEA NPs efficiently deliver PPT1 in cultured cells, with almost complete enzyme release occurring in 48 h. Finally, we demonstrated that enzymatic activity is fully recovered in primary NCL1 fibroblasts upon treatment with PPT1 CLEA NPs.
Krabbe disease (KD; or globoid cell leukodystrophy) is an autosomal recessive lysosomal storage disorder caused by deficiency of the galactosylceramidase (GALC) enzyme. No cure is currently available for KD. Clinical applied treatments are supportive only. Recently, we demonstrated that two differently acting autophagy inducers (lithium and rapamycin) can improve some KD hallmarks in‐vitro, laying the foundation for their in‐vivo pre‐clinical testing. Here, we test lithium carbonate in‐vivo, in the spontaneous mouse model for KD, the Twitcher (TWI) mouse. The drug is administered ad libitum via drinking water (600 mg/L) starting from post natal day 20. We longitudinally monitor the mouse motor performance through the grip strength, the hanging wire and the rotarod tests, and a set of biochemical parameters related to the KD pathogenesis [i.e., GALC enzymatic activity, psychosine (PSY) accumulation and astrogliosis]. Additionally, we investigate the expression of some crucial markers related to the two pathways that could be altered by lithium: the autophagy and the β‐catenin‐dependent pathways. Results demonstrate that lithium has not a significant rescue effect on the TWI phenotype, although it can slightly and transiently improves muscle strength. We also show that lithium, with this administration protocol, is unable to stimulate autophagy in the TWI mice central nervous system, whereas results suggest that it can restore the β‐catenin activation status in the TWI sciatic nerve. Overall, these data provide intriguing inputs for further evaluations of lithium treatment in TWI mice.
In acute malaria, the bulk of erythrocyte loss occurs after therapy, with a nadir of hemoglobin generally observed 3–7 days after treatment. The fine mechanisms leading to this early post-treatment anemia are still elusive. We explored pathological changes in RBC subpopulations by quantifying biochemical and mechanical alterations during severe malaria treated with artemisinin derivatives, a drug family that induce “pitting” in the spleen. In this study, the hemoglobin concentration dropped by 1.93 G/dl during therapy. During the same period, iRBC accounting for 6.12% of all RBC before therapy (BT) were replaced by pitted-RBC, accounting for 5.33% of RBC after therapy (AT). RBC loss was thus of 15.9%, of which only a minor part was due to the loss of iRBC or pitted-RBC. When comparing RBC BT and AT to normal controls, lipidomics revealed an increase in the cholesterol/phosphatidylethanolamine ratio (0.17 versus 0.24, p < 0.001) and cholesterol/phosphatidylinositol ratio (0.36 versus 0.67, p = 0.001). Using ektacytometry, we observed a reduced deformability of circulating RBC, similar BT and AT, compared to health control donors. The mean Elongation Index at 1.69Pa was 0.24 BT and 0.23 AT vs. 0.28 in controls (p < 0.0001). At 30Pa EI was 0.56 BT and 0.56 AT vs. 0.60 in controls (p < 0.001). The retention rate (rr) of RBC subpopulations in spleen-mimetic microsphere layers was higher for iRBC (rr = 20% p = 0.0033) and pitted-RBC (rr = 19%, p = 0.0031) than for healthy RBC (0.12%). Somewhat surprisingly, the post-treatment anemia in malaria results from the elimination of RBC that were never infected.
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