Nanoparticle Tracking Analysis for the Enumeration and Characterization of Mineralo-Organic Nanoparticles in Feline Urine

Abstract

Urinary stone disease, particularly calcium oxalate, is common in both humans and cats. Calcifying nanoparticles (CNP) are spherical nanocrystallite material, and are composed of proteins (fetuin, albumin) and inorganic minerals. CNP are suggested to play a role in a wide array of pathologic mineralization syndromes including urolithiasis. We documented the development of a clinically relevant protocol to assess urinary CNP in 9 healthy cats consuming the same diet in a controlled environment using Nanoparticle Tracking Analysis (NTA®). NTA® is a novel method that allows for characterization of the CNP in an efficient, accurate method that can differentiate these particles from other urinary submicron particulates. The predominant nanoscale particles in feline urine are characteristic of CNP in terms of their size, their ability to spontaneously form under suitable conditions, and the presence of an outer layer that is rich in calcium and capable of binding to hydroxyapatite binders such as alendronate and osteopontin. The expansion of this particle population can be suppressed by the addition of citrate to urine samples. Further, compounds targeting exosomal surfaces do not label these particulates. As CNP have been associated with a number of significant urologic maladies, the method described herein may prove to be a useful adjunct in evaluating lithogenesis risk in mammals.

Dietary Oxalate Induces Urinary Nanocrystals in Humans

“…35,52 It has been used to visualize calcium-containing microcrystals (calcium pyrophosphate dihydrate, basic CaP, hydroxyapatite, and CaOx) in synovial fluid from patients with arthritis using microscopy and flow cytometry, 35 and to calcify nanoparticles in feline urine. 51 In our hands, crystals labelled with Fluo-4, Calcium green, or Calcein Ultragreen AM did not effectively detect nanocrystals using NTA. However, we were able to detect crystals using Fluo-4 AM, a membrane-permeable form of Fluo-4.…”

“…NTA using NanoSight technology has previously been used to detect nanoparticles in biological samples. 30,32,51 Fluo-4 is a calcium-binding probe that was developed to detect free calcium in biological systems and has high fluorescence upon binding to calcium. 35,52 It has been used to visualize calcium-containing microcrystals (calcium pyrophosphate dihydrate, basic CaP, hydroxyapatite, and CaOx) in synovial fluid from patients with arthritis using microscopy and flow cytometry, 35 and to calcify nanoparticles in feline urine.…”

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“…Calcified nanoparticles were analyzed by Nano sight LH I0HS-48814TS instrument. The nanoparticle tracking analyzer identified a population of submicron-size particles with the calcifying nanoparticle size range 21 .…”

“…The release patterns of the drug also vary as per the release conditions, i.e., Sink or non-sink conditions and release medium etc. 21,55,56 . Amar, S. K. Chandrashekar et.…”