Polymer-based drug delivery systems under investigation for enzyme replacement and other therapies of lysosomal storage disorders

Abstract

Lysosomes play a central role in cellular homeostasis and alterations in this compartment associate with many diseases. The most studied example is that of lysosomal storage disorders (LSDs), a group of 60 + maladies due to genetic mutations affecting lysosomal components, mostly enzymes. This leads to aberrant intracellular storage of macromolecules, altering normal cell function and causing multiorgan syndromes, often fatal within the first years of life. Several treatment modalities are available for a dozen LSDs, mostly consisting of enzyme replacement therapy (ERT) strategies. Yet, poor biodistribution to main targets such as the central nervous system, musculoskeletal tissue, and others, as well as generation of blocking antibodies and adverse effects hinder effective LSD treatment. Drug delivery systems are being studied to surmount these obstacles, including polymeric constructs and nanoparticles that constitute the focus of this article. We provide an overview of the formulations being tested, the diseases they aim to treat, and the results observed from respective in vitro and in vivo studies. We also discuss the advantages and disadvantages of these strategies, the remaining gaps of knowledge regarding their performance, and important items to consider for their clinical translation. Overall, polymeric nanoconstructs hold considerable promise to advance treatment for LSDs.

Keywords: Cellular and animal models; Enzyme replacement therapy; Lysosomal storage disorders; Nanoemulsions; Nanoparticles; Polymer-based drug delivery systems.

Figure 1.. Synthesis, endocytosis, and function of lysosomal components.

(A) Biosynthesis route for lysosomal enzymes, encompassing nuclear transcription, endoplasmic reticulum glycosylation, Golgi apparatus maturation, and transport to endosomes and lysosomes via intracellular mannose-6-phosphate receptors. (B) Secretory route for lysosomal enzymes. (C) Endocytic uptake for extracellular lysosomal enzymes, mediated by cell surface mannose-6-phosphate receptor, for delivery to lysosomes. (D) Lysosomal components, including structural membrane proteins, H⁺-ATPase pump, membrane enzymes, channels, and transporters, as well as luminal lysosomal enzymes.

Figure 2.. Properties and functions of drug delivery systems.

Drug nanocarriers (NCs) have unique bio-physicochemical properties that can be tuned to modulate drug delivery functions and improve therapeutic outcomes. Tailoring NC composition, dose, drug loading and release mechanisms, size, shape, charge, and affinity molecules can help modulate drug carriage, targeting, protection, solubility, responsiveness to physiological stimuli, and other properties.

Figure 3.. Anti-ICAM NPs for lung and brain enzyme delivery in acid sphingomyelinase deficiency.

(A) Polymer-based nanoparticles (NPs) targeted to intercellular adhesion molecule 1 (ICAM-1) used for acid sphingomyelinase (ASM) delivery, including model polystyrene (PS) NPs and poly(lactic-co-glycolide acid) (PLGA) NPs. (B) Storage level for BODIPY-FL_C12-sphingomyelin in fibroblasts from ASM-deficient patients, 3 h after treatment with naked ASM or ASM targeted by anti-ICAM-1 PS NCs. Adapted and reproduced with permission from S. Muro et al. Lysosomal enzyme delivery by ICAM-1-targeted nanocarriers bypassing glycosylation- and clathrin-dependent endocytosis, Mol. Ther. 13 (2006) 135–141 [104]. (C) Specific localization of FITC-labeled anti-ICAM-1 PS NPs in mouse lungs, 30 min after intravenous (i.v.) injection vs. control IgG NPs. Small airways = asterisks; vessels = arrowheads. Adapted and reproduced from C. Garnacho et al., Delivery of acid sphingomyelinase in normal and niemann-pick disease mice using intercellular adhesion molecule-1-targeted polymer nanocarriers, J. Pharmacol. Exp. Ther. 325 (2008) 400–408 [234]. (D) Transmission electron microscopy of wild-type mouse lungs showing anti ICAM-1/ASM PS NPs (green) interacting with endothelial cells (EC) 3 h after i.v. injection. NPs are engulfed by cells (black arrows), within endosomes (white arrowheads) and lysosomes (black arrowheads), and transcytosed into epithelial cells (white arrow). VL, vessel lumen; Cv, caveolar vesicles; Cl, clathrin vesicles; Cj, cell junction. Scale bars, 300 nm. Adapted and reproduced with permission from C. Garnacho et al., Enhanced Delivery and Effects of Acid Sphingomyelinase by ICAM-1-Targeted Nanocarriers in Type B Niemann-Pick Disease Mice, Mol. Ther. 25 (2017) 1686–1696 [235]. (E) Transmission electron microscopy of anti-ICAM-1/ASM PLGA NPs in wild-type mouse brains 3 h after i.v. injection. EC = endothelial cell; BL = basal lamina; MA = myelinated axon; VL = vessel lumen. Open arrowheads = NPs close to the abluminal side of an endothelial cell (EC); closed arrowheads = NP located passed the endothelium and basal lamina; arrow = NP within the myelinated axon of a neuron. * = clathrin-coated pits. # = caveolae-like vesicles. Scale bar = 500 μm. (F) Biodistribution of ¹²⁵I-ASM after i.v. injection in wild-type mice as naked enzyme or in anti-ICAM-1 PLGA NPs (surface-loaded or encapsulated). The tissue-over-blood localization ratio is shown. (E,F) Adapted and reproduced with permission from E. Muntimadugu et al., Comparison between Nanoparticle Encapsulation and Surface Loading for Lysosomal Enzyme Replacement Therapy. Int J Mol Sci. 2022 Apr 6;23(7):4034 [80]. (G) Biodistribution of anti-ICAM/¹²⁵I-ASM PS NPs with intermediate targeting valency, 30 min after i.v. injection in wild-type mice, as a percentage of the biodistribution found for high valency NCs. Adapted and reproduced with permission from R.L. Manthe et al., Intertwined mechanisms define transport of anti-ICAM nanocarriers across the endothelium and brain delivery of a therapeutic enzyme, J. Control. Release. 324 (2020) 181–193 [56].

Figure 4.. NC formulations for enzyme delivery for Fabry disease.

(A) Trimethyl chitosan (TMC) polyelctrolyte complexes (PEC), targeted by RGD peptide or not, for delivery of GLA to endothelial cells in culture. (B) In vitro activity of PEC-RGD or untargeted PEC vs. naked GLA in MAEC knockout cells, measured as Gb3 reduction (mean±SEM). Adapted and reproduced with permission from M.I. Giannotti et al., Highly Versatile Polyelectrolyte Complexes for Improving the Enzyme Replacement Therapy of Lysosomal Storage Disorders, ACS Appl. Mater. Interfaces. 8 (2016) 25741–25752 [272].

Figure 5.. Polyrotaxanes nanoformulations for Niemann-Pick type C disease.

(A) Acid-labile 2-(2-hydroxyethoxy)ethyl group-modified polytrioxanes (HEE-PRX) based for pH-mediated delivery of β-cyclodextrin (β-CD) into lysosomes of NPC-1 cells and remove stored cholesterol, to avoid toxic effects of current hydroxypropyl-β-cyclodextrin (HPβCD). (B) Reduction of total cholesterol levels in normal fibroblasts vs. fibroblasts from NPC-1 patients, after treatment for 24 h with described concentrations of HEE-PRX vs. HP-β-CD (mean±SD). Adapted and reproduced with permission from A. Tamura et al. Lysosomal pH-inducible supramolecular dissociation of polyrotaxanes possessing acid-labile N-triphenylmethyl end groups and their therapeutic potential for Niemann-Pick type C disease, Sci. Technol. Adv. Mater. 17 (2016) [340].

Figure 6.. Polymer-based nanoparticles to correct lysosomal pH in lysosomal storage disorders.

Poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) can enter cells by endocytosis and traffic to lysosomes, where their degradation into lactic acid and glycolic acid can help restore a normally acidic pH, which is less acidic in lysosomal storage disorders. This property was used by M. Bourdenx et al., [380] to ameliorate lysosomal pH alterations associated with GBA mutantions, which aare characteristic of Gauche disease and associated with Parkinson’s disease.

Figure 7.. Poly(lactic-co-glycolic acid) nanoparticles for Mucopolysaccharidosis II.

(A) Poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) modified or not with g7 glycopeptide for targeting and uptake in cells after intravenous i.v. injection in mice. NPs were loaded with iduronate-2-sulfatase (IDS) for treatment of Mucopolysaccharididosis type II (MPS2). (B) Fibroblasts from MPS2 patients were left untreated (UT) or treated with naked IDS or IDS-loaded PLGA NPs (u-NPs-IDS), and IDS activity delivered to cells was measured after 0 (T0), 7 (T7) or 14 (T14) days. IDS activity is expressed in nmols of 4-methylumbelliferyl produced in 4 h per mg of protein. (C) GAG content detected in the brain of IDS knockout mice 6 weeks after i.v. injection with g7-targeted PLGA NPs loaded with IDS, compared to non-loaded formulations, naked enzyme, untreated (UT) mice or control wild-type (wt) mice. Data are mean±SD. (B,C) Adapted and reproduced with permission from L. Rigon et al., Targeting brain disease in MPSII: Preclinical evaluation of IDS-loaded PLGA nanoparticles, Int. J. Mol. Sci. 20 (2019) 1–15 [439].

Figure 8.. ICAM-1 targeted nanoparticles for enzyme delivery in Pompe disease.

(A) Enzyme α-glucosidase (GAA), which can bind to the mannose-6-phosphate receptor (M6PR), either naked or loaded in model polystyrene (PS) nanoparticles (NPs) targeted to ICAM-1 (anti-ICAM-1 NPs) for treatment of Pompe disease, which is characterized by abnormal glycogen storage in cells. (B) Cells incubated with turanose to induce glycogen storage as in Pompe disease, were treated for 5 h with naked GAA or anti-CAM-1/GAA NPs. Excess glycogen remaining was determined compared to normal cells. (mean±SEM). (C) Biodistribution of ¹²⁵I-GAA 30 min after i.v. injection in wild-type mice as either naked enzyme or targeted by anti-ICAM-1 NPs. The tissue-over-blood localization ratio is shown. Adapted and reproduced with permission from J. Hsu et al., Enhanced delivery of α-glucosidase for Pompe disease by ICAM-1-targeted nanocarriers: comparative performance of a strategy for three distinct lysosomal storage disorders, Nanomedicine. 8 (2012) 731–739 [507].