Lysosomal pH-inducible supramolecular dissociation of polyrotaxanes possessing acid-labile N-triphenylmethyl end groups and their therapeutic potential for Niemann-Pick type C disease

Abstract

Niemann-Pick type C (NPC) disease is characterized by the accumulation of cholesterol in lysosomes. We have previously reported that biocleavable polyrotaxanes (PRXs) composed of β-cyclodextrins (β-CDs) threaded onto a linear polymer capped with bulky stopper molecules via intracellularly cleavable linkers show remarkable cholesterol reducing effects in NPC disease patient-derived fibroblasts owing to the stimuli-responsive intracellular dissociation of PRXs and subsequent β-CD release from the PRXs. Herein, we describe a series of novel acid-labile 2-(2-hydroxyethoxy)ethyl group-modified PRXs (HEE-PRXs) bearing terminal N-triphenylmethyl (N-Trt) groups as a cleavable component for the treatment of NPC disease. The N-Trt end groups of the HEE-PRXs underwent acidic pH-induced cleavage and led to the dissociation of their supramolecular structure. A kinetic study revealed that the number of HEE groups on the PRX did not affect the cleavage kinetics of the N-Trt end groups of the HEE-PRXs. The effect of the number of HEE groups of the HEE-PRXs, which was modified to impart water solubility to the PRXs, on cellular internalization efficiency, lysosomal localization efficiency, and cholesterol reduction ability in NPC disease-derived fibroblasts (NPC1 fibroblasts) was also investigated. The cellular uptake and lysosomal localization efficiency were almost equivalent for HEE-PRXs with different numbers of HEE groups. However, the cholesterol reducing ability of the HEE-PRXs in NPC1 fibroblasts was affected by the number of HEE groups, and HEE-PRXs with a high number of HEE groups were unable to reduce lysosomal cholesterol accumulation. This deficiency is most likely due to the cholesterol-solubilizing ability of HEE-modified β-CDs released from the HEE-PRXs. We conclude that the N-Trt group acts as a cleavable component to induce the lysosomal dissociation of HEE-PRXs, and acid-labile HEE-PRXs with an optimal number of HEE groups (4.1 to 5.4 HEE groups per single β-CD threaded onto the PRX) have great therapeutic potential for treating NPC disease.

Scavenger Receptor A‐Mediated Targeting of Carboxylated Polyrotaxanes to Macrophages and the Impacts of Supramolecular Structure

“…To clarify the effects of CEE‐PRXs on cultured cells, the toxicity and inflammatory responses of CEE‐PRX ( 4 ) in RAW264.7 cells were investigated. In these experiments, HEE‐PRX was used as a control, because the toxic and inflammatory effects of HEE‐PRX is negligible . The toxicity of RAW 264.7 cells treated with CEE‐PRX ( 4 ) and HEE‐PRX for 24 h was assessed based on the release of LDH.…”

“…Carboxyethyl ester‐modified PRX (CES‐PRX, Figure A) ( M n of PEG axle: 10 000, the number of threading α‐CD: 41.6, number of carboxyethyl esters modified on PRX: 127, M n : 65 800) was synthesized according to a previous report . 2‐(2‐Hydroxyethoxy)ethyl (HEE) carbamate group‐modified PRX (HEE‐PRX, Figure A) ( M n of PEG axle: 10 000, the number of threading α‐CD: 40.4, the number of HEE groups modified on PRX: 191, M n : 74 700) was synthesized according to a previous report . Methyl 3‐bromopropionate and dextran sulfate ( M n = 5000) were obtained from Wako Pure Chemical Industries (Osaka, Japan).…”

Versatile synthesis of end-reactive polyrotaxanes applicable to fabrication of supramolecular biomaterials

“…Herein, alkynyl group-bearing fluorescent molecules were modified at the terminal azide group of the PRXs, and intracellular fluorescence imaging of the PRXs was performed to verify whether the terminally modified fluorescent molecules could be used for fluorescence imaging. First, 4a was modified with 2-(2-hydroxyethoxy)ethyl (HEE) groups to impart water solubility, which was necessary for in vitro cellular experiments (Scheme 3) [32–33]. Then, the terminal benzylazide groups of HEE-PRX-Bn-N 3 ( 6 ) were modified with dibenzylcyclooctyne (DBCO)-conjugated fluorescent molecules (DF488) via a copper-free click reaction (Scheme 3) [34–35].…”

Cyclodextrin‐Based Macromolecular Systems as Cholesterol‐Mopping Therapeutic Agents in Niemann–Pick Disease Type C

“…Such CD‐PRX was synthesized from pluronic P123 (PEG‐b‐PPG‐b‐PEG triblock copolymer) as the axle polymer, the threaded βCDs were modified with 2‐(2‐hydroxyethoxy)ethyl carbamate to improve water solubility and N ‐triphenylmethyl selected as acid‐cleavable bulky stoppers . The high molecular weight of CD‐PRX translates into prolonged blood half‐life and therefore a higher accumulation of CDs in tissues and more effective cholesterol lowering activity in comparison to CD derivatives .…”