Test in vitro assay to determine the material\'s potential to damage red bloodce

Tag : medical material.
Posted: July 18, 2008 Analyzing biocompatibility of medical nanotechnology applicationswith blood ( Nanowerk Spotlight ) Any drug intended for systemic administration and all medicaldevices which will contact blood (e.g. oxygenators, tubing,catheters, artificial hearts) must undergo thoroughbiocompatibility testing. These tests include an in vitro assay to determine the material's potential to damage red bloodcells (hemolysis). Hemolysis, the abnormal breakdown of red bloodcells either in the blood vessels (intravascular hemolysis) orelsewhere in the body (extravascular), can lead to anemia or otherpathological conditions. In the pharmaceutical industry,hematocompatibility testing is harmonized through the use ofinternationally recognized standard protocols. ASTM F756 –Standard Practice for Assessment of Hemolytic Properties ofMaterials – is a widely used standard for blood-damagetesting. Another standard, ISO 10993-4, recommends investigatingred blood cell (erythrocyte) damage as a way to study a material'scompatibility with blood. Nanotechnology-based medical devices and drug carriers are emergingas alternatives to conventional small-molecule drugs, and in vitro evaluation of their biocompatibility with blood components is anecessary part of early preclinical development. Many researchpapers have reported nanoparticle hemolytic properties but, so far,no in vitro hemolysis protocol has been available that is specific tonanoparticles. A new study published this month describes in vitro assays to study nanoparticle hemolytic properties, identifiesnanoparticle interferences with these in vitro tests and provides the first comprehensive insight to potentialsources of this interference, demonstrates the usefulness ofincluding nanoparticle-only controls, and illustrates theimportance of physicochemical characterization of nanoparticleformulations and visually monitoring test samples to avoidfalse-positive or false-negative results. "Since biomedical nanoparticle engineering is a rapidlygrowing field, we realized that having a protocol designedspecifically to work with nanomaterials would be useful to manyparties" Dr. Marina A. Dobrovolskaia tells Nanowerk. "We started from an existing standard (ASTM756-00) for evaluating medical devices. The first task to adaptthis to nanoparticles was to minimize material requirements,because biomedical nanoparticles are often expensive andcomplicated to produce and not available in gram-quantities. Thenext task was to test various nanoparticles representing differentclasses of materials and to identify whether or not our method wasgenerally applicable. The main finding of our study is thatnanoparticles have unique physicochemical properties that can leadto a host of interference issues with traditional in vitro tests." Dobrovolskaia is an Immunologist for the Nanotechnology Characterization Laboratory (NCL), a formal collaboration between the National CancerInstitute (NCI), the U.S. Food and Drug Administration (FDA), andthe National Institute of Standards and Technology (NIST). The NCL was established to accelerate the transition of basicnanotechnology research into clinical applications to effectivelytreat and diagnose cancer. The NCL performs preclinicalcharacterization of nanoparticles intended for cancer therapeuticsand diagnostics. It is a free resource available to investigatorsfrom academia, industry, and government laboratories. The NCL iscomprised of staff from the fields of chemistry, physics,immunology, cell biology and toxicology, and NCL staff is nowfamiliar with a wide variety of nanoparticle types, such asdendrimers, liposomes, gold colloids, fullerenes, and polymers. Oneof the NCL's objectives is to establish a standardized assaycascade which would aid researchers and regulatory agencies inunderstanding nanoparticle properties that affect biocompatibility. In their paper in Nano Letters ( "Method for Analysis of Nanoparticle Hemolytic Properties inVitro" ), Dobrovolskaia together with several NCL colleagues and Dr. Scott E. McNeil , Director of the NCL, describe validation of an in vitro method designed to analyze nanoparticle potential to damage redblood cells and the use of this method to study a variety ofnanoparticles. Specifically, the study describes approaches toidentify nanoparticle/erythrocyte interferences, when they occurand how to resolve them to get accurate results (i.e. to avoidfalse-positive or false-negative results). The NCL team's assay leverages the ASTM F-756-00 standard foranalysis of hemolytic properties of medical devices. "Wescaled this standard practice to a 96-well plate format assay andconducted a one month validation aimed at determining itsreproducibility, precision, and accuracy, as well as qualificationof negative and positive nanoparticle-relevant controls"explains Dobrovolskaia. "We subsequently used our assay toanalyze various types of nanomaterials including polymers, goldnanoshells, nanoliposomes, nanoemulsions, fullerene derivatives,gold colloids, and dendrimers. This second phase was conducted overa two year period and included identification and resolution ofnanoparticle interference with the assay, in addition to evaluationof reproducibility, precision, accuracy, and controlqualification." "Several previous studies evaluating nanoparticle hemolyticproperties in vitro have appeared in the literature" says Dobrovolskaia."However, all these tests were conduced using differentmethods, and even when similar approaches were used, factors suchas plasma anti-coagulant, blood incubation times, centrifugalforces, assay detection wavelength varied from study to study. Thismade it difficult to compare results." She points out that, most importantly, none of these earlierstudies included special controls to identify nanoparticleinterference, and therefore it was really impossible to concludewhat nanoparticle properties were responsible for the hemolysis– i.e. did it depend on size, charge, surface groups or wasdue to the particle absorbance? The problem therefore was to develop a hemolysis assay that isapplicable to a wide variety of nanoparticles so that data can becompared between the many labs testing nanoparticlebiocompatibility. Consequently, the NCL scientists specifically designed their assayto be applicable to various nanoparticles. That way, it that itcould be used by different investigators working with differentnanoparticles, and allow comparison of the results. The protocol described in this paper is one of a set of assaysdeveloped specifically for use with nanoparticles and available tothe public through the NCL web site ( Assay Cascade Protocols ). When used in early preclinical drug development, this particularmethod may help differentiate strong candidates from ones withproperties that need further tuning before in vivo applications. If studies from numerous laboratories on a widevariety of nanoparticles are conducted using the same method, itwill increase confidence in the quality of data and conclusionsdrawn from that data regarding nanoparticle biocompatibility. Hemolysis is not the only traditional biocompatibility test thatnanoparticles interfere with. Nanoparticles are intricate, oftendelicate systems with unique properties, and their characterizationis challenging. Dobrovolskaia mentions that, for instance, many nanoparticles havecatalytic properties and can enhance assays that rely on enzymaticreactions, generating false-positive results. "Assays routineto the preclinical characterization of conventionalpharmaceuticals, such as the Limulus amebocyte lysate (LAL) testfor endotoxin contamination detection may yield spurious resultswhen applied to nanoparticle samples. Multifunctional nanoparticleshave to be characterized quite rigorously, as there are multiplecomponents that must work in concert to achievefunctionality." She adds that a thorough characterization of a nanoparticle-basedtherapeutic includes evaluation of physicochemical properties,sterility and pyrogenicity assessment, biodistribution (ADME orabsorption, distribution, metabolism and excretion) and toxicitycharacterization – which includes both in vitro tests and in vivo animal studies. The NCL has tailored each of these tiers of arational characterization cascade so that they are relevant tonanoparticles. By Michael Berger. Copyright 2008 Nanowerk LLC