N-acyl-O-phosphocholineserines: structures of a novel class of lipids that are biomarkers for Niemann-Pick C1 disease.

Niemann-Pick disease type C1 (NPC1) is a fatal, neurodegenerative, cholesterol storage disorder. With new therapeutics in clinical trials, there is an urgency to improve diagnostics and monitor therapeutic efficacy with biomarkers. In this study, we sought to define the structure of an unknown lipid biomarker for NPC1 with [M + H]+ ion at m/z 509.3351, previously designated as lysoSM-509. The structure of N-palmitoyl-O-phosphocholineserine (PPCS) was proposed for the lipid biomarker based on the results from mass spectrometric analyses and chemical derivatizations. As no commercial standard is available, authentic PPCS was chemically synthesized, and the structure was confirmed by comparison of endogenous and synthetic compounds as well as their derivatives using liquid chromatography-tandem mass spectrometry (LC-MS/MS). PPCS is the most abundant species among N-acyl-O-phosphocholineserines (APCS), a class of lipids that have not been previously detected in biological samples. Further analysis demonstrated that all APCS species with acyl groups ranging from C14 to C24 were elevated in NPC1 plasma. PPCS is also elevated in both central and peripheral tissues of the NPC1 cat model. Identification of APCS structures provide an opportunity for broader exploration of the roles of these novel lipids in NPC1 disease pathology and diagnosis.

Gd3+:DOTA-Modified 2-Hydroxypropyl-ß-Cyclodextrin/4-Sulfobutyl Ether-ß-Cyclodextrin-Based Polyrotaxanes as Long Circulating High Relaxivity MRI Contrast Agents.

A family of five water-soluble Gd3+:1,4,7,10-tetraazacyclododecane-1,4,7-tetraacetic acid-modified polyrotaxane (PR) magnetic resonance contrast agents bearing mixtures of 2-hydroxypropyl-ß-cyclodextrin and 4-sulfobutylether-ß-cyclodextrin macrocycles threaded onto Pluronic cores were developed as long circulating magnetic resonance contrast agents. Short diethylene glycol diamine spacers were utilized for linking the macrocyclic chelator to the PR scaffold prior to Gd3+ chelation. The PR products were characterized by 1H NMR, gel permeation chromatography/multiangle light scattering, dynamic light scattering, and analytical ultracentrifugation. Nuclear magnetic relaxation dispersion and molar relaxivity measurements at 23 °C revealed that all the PR contrast agents displayed high spin-spin T1 relaxation and spin-lattice T2 relaxation rates relative to a DOTAREM control. When injected at 0.05 mmol Gd/kg body weight in BALB/c mice, the PR contrast agents increased the T1-weighted MR image intensities with longer circulation times in the blood pool than DOTAREM. Excretion of the agents occurred predominantly via the renal or biliary routes depending on the polyrotaxane structure, with the longest circulating L81 Pluronic-based agent showing the highest liver uptake. Proteomic analysis of PR bearing different ß-cyclodextrin moieties indicated that lipoproteins were the predominant component associated with these materials after serum exposure, comprising as much as 40% of the total protein corona. We infer from these findings that Gd(III)-modified PR contrast agents are promising long-circulating candidates for blood pool analysis by MRI.

Pluronic based ß-cyclodextrin polyrotaxanes for treatment of Niemann-Pick Type C disease.

Niemann-Pick Type C disease (NPC) is a rare metabolic disorder characterized by disruption of normal cholesterol trafficking within the cells of the body. There are no FDA approved treatments available for NPC patients. Recently, the cycloheptaglucoside 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) has shown efficacy as a potential NPC therapeutic by extending lifetime in NPC mice, delaying neurodegeneration, and decreasing visceral and neurological cholesterol burden. Although promising, systemic HP-ß-CD treatment is limited by a pharmacokinetic profile characterized by rapid loss through renal filtration. To address these shortcomings, we sought to design a family of HP-ß-CD pro-drug delivery vehicles, known as polyrotaxanes (PR), capable of increasing the efficacy of a given injected dose by improving both pharmacokinetic profile and bioavailability of the HP-ß-CD agent. PR can effectively diminish the cholesterol pool within the liver, spleen, and kidney at molar concentrations 10-to-100-fold lower than monomeric HP-ß-CD. In addition to this proof-of-concept, use of PR scaffolds with differing physiochemical properties reveal structure-activity relationships in which PR characteristics, including hydrophobicity, threading efficiency and surface charge, were found to both decisively and subtly effect therapeutic efficacy. PR scaffolds exhibit absorption, pharmacokinetics, and biodistribution patterns that are significantly altered from monomeric HP-ß-CD. In all, PR scaffolds hold great promise as potential treatments for visceral disease in NPC patients.

Influence of Molecular Structure on the In Vivo Performance of Flexible Rod Polyrotaxanes.

Polyrotaxanes, a family of rod-shaped nanomaterials comprised of noncovalent polymer/macrocycle assemblies, are being used in a growing number of materials and biomedical applications. Their physiochemical properties can vary widely as a function of composition, potentially leading to different in vivo performance outcomes. We sought to characterize the pharmacokinetic profiles, toxicities, and protein corona compositions of 2-hydroxypropyl-ß-cyclodextrin polyrotaxanes as a function of variations in macrocycle threading efficiency, molecular weight, and triblock copolymer core structure. We show that polyrotaxane fate in vivo is governed by the structure and dynamics of their rodlike morphologies, such that highly threaded polyrotaxanes are long circulating and deposit in the liver, whereas lung deposition and rapid clearance is observed for species bearing lower 2-hydroxypropyl-ß-cyclodextrin threading percentages. Architecture differences also promote recruitment of different serum protein classes and proportions; however, physiochemical differences have little or no influence on their toxicity. These findings provide important structural insights for guiding the development of polyrotaxanes as scaffolds for biomedical applications.

Structure-property relationship for in vitro siRNA delivery performance of cationic 2-hydroxypropyl-ß-cyclodextrin: PEG-PPG-PEG polyrotaxane vectors.

Nanoparticle-mediated siRNA delivery is a promising therapeutic approach, however, the processes required for transport of these materials across the numerous extracellular and intracellular barriers are poorly understood. Efficient delivery of siRNA-containing nanoparticles would ultimately benefit from an improved understanding of how parameters associated with these barriers relate to the physicochemical properties of the nanoparticle vectors. We report the synthesis of three Pluronic(®)-based, cholesterol end-capped cationic polyrotaxanes (PR(+)) threaded with 2-hydroxypropyl-ß-cyclodextrin (HPßCD) for siRNA delivery. The biological data showed that PR(+):siRNA complexes were well tolerated (∼90% cell viability) and produced efficient silencing (>80%) in HeLa-GFP and NIH 3T3-GFP cell lines. We further used a multi-parametric approach to identify relationships between the PR(+) structure, PR(+):siRNA complex physical properties, and biological activity. Small angle X-ray scattering and cryoelectron microscopy studies reveal periodicity and lamellar architectures for PR(+):siRNA complexes, whereas the biological assays, ζ potential measurements, and imaging studies suggest that silencing efficiency is influenced by the effective charge ratio (ρeff), polypropylene oxide (PO) block length, and central PO block coverage (i.e., rigidity) of the PR(+) core. We infer from our findings that more compact PR(+):siRNA nanostructures arising from lower molecular weight, rigid rod-like PR(+) polymer cores produce improved silencing efficiency relative to higher molecular weight, more flexible PR(+) vectors of similar effective charge. This study demonstrates that PR(+):siRNA complex formulations can be produced having higher performance than Lipofectamine(®) 2000, while maintaining good cell viability and siRNA sequence protection in cell culture.

Efficient pDNA Delivery Using Cationic 2-Hydroxypropyl-ß-Cyclodextrin Pluronic-Based Polyrotaxanes.

A family of cationic Pluronic-based polyrotaxanes (PR(+)), threaded with 2-hydroxypropyl-ß-cyclodextrin (HPCD), was synthesized for pDNA delivery into multiple cell lines. All PR(+) formed highly stable, positively charged pDNA complexes that were < 250 nm in diameter. The cellular uptake and pDNA transfection efficiencies of the PR(+):pDNA complexes was enhanced relative to the commercial transfection standards L2K and bPEI, while displaying similar or lower toxicity profiles. Charge density and threading efficiency of the PR(+) agent significantly influenced the colloidal stability and physical properties of the complexes, which impacted their intracellular transfection efficiencies. Taken together, our results suggest that HPCD: Pluronic PR(+) can be used as potent vectors for pDNA-based therapeutics.

Impact of Mixed ß-Cyclodextrin Ratios on Pluronic Rotaxanation Efficiency and Product Solubility.

Water-soluble polyrotaxanes have been prepared under heterogeneous conditions from mixtures of ß-cyclodextrin (ß-CD), 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), methyl-ß-cyclodextrin, or 6-monoazido-ß-cyclodextrin with 4-sulfobutyl ether-ß-cyclodextrin (SBE-ß-CD) and Pluronic L81 copolymer modified with cholesterol end caps. Threading reactions gave polyrotaxane products in modest chemical yield that were reflective of the ß-CD feed ratios in the reaction. Polyrotaxanes containing mixtures of HP-ß-CD and SBE-ß-CD were screened and found to be biologically active in an in vitro model of Niemann-Pick Type C disease where they mobilize aberrantly stored cholesterol similarly to monomeric cyclodextrin controls.

Gd3+-1,4,7,10-Tetraazacyclododecane-1,4,7-triacetic-2-hydroxypropyl-ß-cyclodextrin/Pluronic Polyrotaxane as a Long Circulating High Relaxivity MRI Contrast Agent.

A multivalent magnetic resonance imaging agent based on a 2-hydroxypropyl-ß-cyclodextrin (HPCD):Pluronic F127 polyrotaxane carrier has been synthesized, and its blood pool contrast properties have been characterized. This Gd3+-DO3A-HPCD/Pluronic polyrotaxane construct is shown to circulate for more than 30 min and provide >100-fold vascular enhancement relative to the monomeric Gd3+-DO3A-HPCD control that is rapidly cleared via the kidney. The high r1 relaxivity at 37 °C (23.83 mM(-1) s(-1) at 1.5 T; 34.08 mM(-1) s(-1) at 0.5 T), extended blood circulation, well-known pharmacology of the polyrotaxane precursors, and absence of acute toxicity make it a highly attractive blood pool contrast agent candidate.

Synthesis of 2-hydroxypropyl-ß-cyclodextrin/pluronic-based polyrotaxanes via heterogeneous reaction as potential Niemann-Pick type C therapeutics.

Five polyrotaxanes were synthesized by threading 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) onto a variety of α,ω-ditriethylenediamino-N-carbamoyl-poly-(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethylene oxide) (Pluronic) triblock copolymers using a two-pot strategy under heterogeneous, nonaqueous conditions. The threaded HP-ß-CD units were retained on the pseudopolyrotaxane precursors by end-capping the branched diamine termini with sodium 2,4,6-trinitrobenzene sulfonate. Inclusion of the Pluronic copolymers within the HP-ß-CD cavities was more favorable in nonpolar solvents, such as diethyl ether and n-hexane, both of which gave better coverage ratios than polar solvents. (1)H NMR and MALDI-TOF were used to estimate the average molecular weights of the purified polyrotaxane products. A globular morphology of aggregated polyrotaxanes was observed by tapping-mode AFM imaging of dried samples. Treatment of Niemann-Pick C (NPC) type 2-deficient fibroblasts with the polyrotaxane derivatives produced substantial reductions in sterol accumulation, as seen by diminished filipin staining in these cells, suggesting that Pluronic-based polyrotaxanes may be promising vehicles for delivery of HP-ß-CD to cells with abnormal cholesterol accumulation.