Brain-targeted enzyme-loaded nanoparticles: A breach through the blood-brain barrier for enzyme replacement therapy in Krabbe disease

Abstract

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.

Fig. 1. Targeted GALC CLEA NP synthesis and characterization.

(A) Graphical summary of the experiment. Peptide-modified PLGA was produced by covalent linking of each peptide to a previously activated form of PLGA. GALC CLEAs were obtained by precipitation of GALC in acetone in the presence of glutaraldehyde, resulting in Schiff base formation between enzyme molecules. Last, targeted GALC CLEA NPs were obtained by nanoprecipitation. (B) Hydrodynamic Diameter of GALC CLEA NPs. Error bars represent the SEM of four independently synthesized NP batches. (C) Zeta potential of GALC CLEA NPs. Error bars represent the SEM of four independently synthesized NP batches. (D) Encapsulation efficiency of GALC CLEA NPs. Encapsulation efficiency is expressed as percentage of encapsulated GALC with respect to the amount of enzyme used in each synthesis. Error bars represent the SEM of four independently synthesized NP batches. (E) Activity yield of GALC CLEA NPs. Activity yield is expressed as percentage of specific activity (nanomole substrate hydrolyzed in 1 hour by 1 μg of enzyme) with respect to the free unaltered enzyme. Error bars represent the SEM of four independently synthesized NP batches.

Fig. 2. Intracellular localization of targeted GALC CLEA NPs.

(A) Graphical summary of the experiment. TWI or WT primary fibroblasts were incubated with fluorescently labeled ATTO 488 GALC CLEA-loaded ATTO 633 NPs for 4 hours and then washed and added with fresh medium. After 24 hours, cell lysosomes were stained, and cells were fixed and imaged with a confocal microscope. (B) Confocal imaging. Representative confocal images of TWI fibroblasts treated with fluorescently labeled GALC Ang2 NPs, GALC g7 NPs, GALC Tf2 NPs, or GALC NPs. From the left to the right column: GALC (green, stained with ATTO 488), NPs (red, stained with ATTO 633), lysosomes (blue, stained with LysoTracker Red DND-99), nuclei (yellow, stained with DAPI), superimposition of GALC, NPs, lysosomes, and nuclei fluorescence and superimposition of all channels with bright-field (BF) images. Scale bars, 10 μm. (C and D) Colocalization analysis. Manders’ coefficient of GALC/lysosomes and NPs/lysosomes overlap in TWI cells treated with GALC CLEA NPs. (E and F) Manders’ coefficient of GALC/lysosomes and NPs/lysosomes overlap in WT cells treated with GALC CLEA NPs.

Fig. 3. In vitro ERT: Dose-response experiment.

(A) Graphical summary of the experiment. GALC-deficient primary fibroblasts (derived from TWI mice) were treated with GALC CLEA NPs. After 4 hours, GALC activity was measured on cellular lysates by 6-hexadecanoylamino-4-methylumbelliferyl-β-d-galactopyranoside (HMU-βGal) assay. (B) Dose-response experiment results. Cells were treated with targeted GALC CLEA NPs (GALC Ang2 NPs, GALC g7 NPs, or GALC Tf2 NPs), with nontargeted NPs (GALC CLEA NPs), or with the free rm-GALC (GALC). For every treatment, four doses were tested: 0.75, 1.50, 3.0, and 6.0 U [unit (U) = amount of enzyme that catalyzes 1 nmol of substrate per hour]. Results are expressed in unit per microgram and reported in percentage of the activity of the WT cells [(U/μg) = unit of enzyme per microgram of cell lysate]. *P < 0.05 specific treatment versus WT, one-way analysis of variance (ANOVA) (Dunnett’s multiple-comparisons test), means ± SEM (n = 3). (C) Controls. For control, NPs nonloaded with GALC CLEAs (empty NPs) were also administered to the cells. WT- and TWI-untreated cells activity is also showed. Means ± SEM (n = 3).

Fig. 4. In vitro: ERT time-response experiment.

(A) Graphical summary of the experiment. GALC-deficient primary murine (from TWI mice) and human (from patients with KD with GALC 30kbΔ) fibroblasts were treated with a single dose [3.0 U; unit (U) = amount of enzyme that catalyzes 1 nmol of substrate/hour] of targeted GALC CLEA NPs (GALC Ang2 NPs, GALC g7 NPs, or GALC Tf2 NPs), nontargeted NPs (GALC CLEA NPs), or with the free rm-GALC (GALC). Four hours later, cells were incubated with fresh medium. GALC activity was measured in the cell lysates 24 or 96 hours later by HMU-βGal assay. (B) NPs mediated ERT in GALC deficient mouse cells. Results are expressed in unit per microgram and reported as percentage of the activity of the WT cells (U/μg = unit of enzyme per microgram of cell lysate). *P < 0.05 specific treatment versus WT, one-way ANOVA (Dunnett’s multiple comparisons test). #P < 0.05 GALC 96 hours versus GALC 24 hours, Student’s t test, means ± SEM (n = 3). (C) Controls. WT and TWI untreated cells activity at 24 and 96 hours. Means ± SEM (n = 3). (D) NPs mediated ERT in GALC deficient human cells. Results are expressed in unit per microgram and reported in percentage of the activity of the healthy (HD, human donor) cells (U/μg = unit of enzyme per microgram of cell lysate). *P < 0.05 specific treatment versus WT, one-way ANOVA (Dunnett’s multiple comparisons test). #P < 0.05 GALC Tf2 NPs 96 hours versus GALC Tf2 NPs 24 hours and ##P < 0.01 GALC NPs 96 hours versus GALC NPs 24 hours, Student’s t test, means ± SEM (n = 3). (E) Controls. HD and 30kbΔ untreated cells activity at 24 and 96 hours. Means ± SEM (n = 3).

Fig. 5. In vivo NP mediated ERT in the TWI mice.

(A) Graphical summary of the experiment. TWI mice were treated with targeted GALC CLEA ATTO 633 NPs (GALC Ang2 NPs, GALC g7 NPs, or GALC Tf2 NPs), nontargeted GALC CLEA ATTO 633 NPs (GALC CLEA NPs), or with the free rm-GALC (GALC). Four hours later mice were euthanized, and GALC activity was assayed in extracted brain, sciatic nerves, spinal cord, kidneys, and liver by HMU-βGal assay. (B) Legend. Untreated: WT (WT-UT), heterozygous (HET-UT), and TWI (TWI-UT). Targeted GALC CLEA ATTO 633 NPs (TWI Targ-NPs): TWI-GALC Ang2 NPs (lot a and lot b), TWI-GALC g7 NPs (lot a and lot b), and TWI-GALC Tf2 NPs (lot a and lot b). Control treatments (TWI Controls): GALC CLEA ATTO 633 NPs (TWI-GALC NPs lot a and lot b) and free rm-GALC (GALC-lot a and lot b). (C) Brain GALC activity. ***P < 0.001 HET versus WT; ****P < 0.0001 TWI, TWI Targ-NPs, and TWI Controls versus WT; +P < 0.05 TWI versus HET; #P < 0.05 TWI Controls versus TWI; ####P < 0.0001 TWI Targ-NPs versus TWI one-way ANOVA (Tukey’s test). Means ± SEM (n = 3 to 12 per group). (D) Liver GALC activity. **P < 0.01 TWI versus WT and #P < 0.05 TWI Controls versus TWI, one-way ANOVA (Tukey’s test). ^P < 0.05 TWI Targ-NPs versus WT and ^^P < 0.01 TWI Targ-NPs versus TWI, Student’s t test. Means ± SEM (n = 3 to 6 per group). (E) Kidney GALC activity. ***P < 0.001 TWI and TWI Controls versus WT and ****P < 0.0001 TWI Targ-NPs versus WT, one-way ANOVA (Tukey’s test). Means ± SEM (n = 3 to 6 per group). (F) Sciatic nerve GALC activity. Results are expressed in unit per microgram and reported as percentage of the WT activity (U/μg = unit of enzyme per microgram of cell lysate). ****P < 0.0001 all groups versus WT, +P < 0.05 TWI versus HET, ++P < 0.01 WT, TWI Targ-NPs and TWI Controls versus HET, one-way ANOVA (Tukey’s test). Means ± SEM (n = 3 to 8 per group). (G) Spinal cord GALC activity. ****P < 0.0001 HET, TWI Targ-NPs and TWI Controls versus WT, +P < 0.05 TWI Targ-NPs versus HET, and ++P < 0.01 TWI and TWI Controls versus HET, one-way ANOVA (Tukey’s test). Means ± SEM (n = 3 to 7 per group). (C to G) Results are expressed in unit per microgram and reported in percentage of the WT activity (U/μg = unit of enzyme per microgram of cell lysate). (H) Confocal imaging. Representative confocal images of brains and livers of the treated TWI mice. NPs are shown in red in the brains of the mice administered with the functionalized targeted GALC CLEA ATTO 633 NPs (GALC Ang2 NPs, GALC g7 NPs, or GALC Tf2 NPs), while they are not present in the brain of the mice treated with the nontargeted GALC CLEA ATTO 633 NPs (GALC CLEA NPs). All livers present NPs. Scale bars, 5 μm.