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Posted: Jul 18, 2008

Analyzing biocompatibility of medical nanotechnology applications with blood

(Nanowerk Spotlight) Any drug intended for systemic administration and all medical devices which will contact blood (e.g. oxygenators, tubing, catheters, artificial hearts) must undergo thorough biocompatibility testing. These tests include an in vitro assay to determine the material's potential to damage red blood cells (hemolysis). Hemolysis, the abnormal breakdown of red blood cells either in the blood vessels (intravascular hemolysis) or elsewhere in the body (extravascular), can lead to anemia or other pathological conditions. In the pharmaceutical industry, hematocompatibility testing is harmonized through the use of internationally recognized standard protocols. ASTM F756 – Standard Practice for Assessment of Hemolytic Properties of Materials – is a widely used standard for blood-damage testing. Another standard, ISO 10993-4, recommends investigating red blood cell (erythrocyte) damage as a way to study a material's compatibility with blood.
Nanotechnology-based medical devices and drug carriers are emerging as alternatives to conventional small-molecule drugs, and in vitro evaluation of their biocompatibility with blood components is a necessary part of early preclinical development. Many research papers have reported nanoparticle hemolytic properties but, so far, no in vitro hemolysis protocol has been available that is specific to nanoparticles.
A new study published this month describes in vitro assays to study nanoparticle hemolytic properties, identifies nanoparticle interferences with these in vitro tests and provides the first comprehensive insight to potential sources of this interference, demonstrates the usefulness of including nanoparticle-only controls, and illustrates the importance of physicochemical characterization of nanoparticle formulations and visually monitoring test samples to avoid false-positive or false-negative results.
"Since biomedical nanoparticle engineering is a rapidly growing field, we realized that having a protocol designed specifically to work with nanomaterials would be useful to many parties" Dr. Marina A. Dobrovolskaia tells Nanowerk. "We started from an existing standard (ASTM 756-00) for evaluating medical devices. The first task to adapt this to nanoparticles was to minimize material requirements, because biomedical nanoparticles are often expensive and complicated to produce and not available in gram-quantities. The next task was to test various nanoparticles representing different classes of materials and to identify whether or not our method was generally applicable. The main finding of our study is that nanoparticles have unique physicochemical properties that can lead to 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 Cancer Institute (NCI), the U.S. Food and Drug Administration (FDA), and the National Institute of Standards and Technology (NIST).
The NCL was established to accelerate the transition of basic nanotechnology research into clinical applications to effectively treat and diagnose cancer. The NCL performs preclinical characterization of nanoparticles intended for cancer therapeutics and diagnostics. It is a free resource available to investigators from academia, industry, and government laboratories. The NCL is comprised of staff from the fields of chemistry, physics, immunology, cell biology and toxicology, and NCL staff is now familiar with a wide variety of nanoparticle types, such as dendrimers, liposomes, gold colloids, fullerenes, and polymers. One of the NCL's objectives is to establish a standardized assay cascade which would aid researchers and regulatory agencies in understanding nanoparticle properties that affect biocompatibility.
In their paper in Nano Letters ("Method for Analysis of Nanoparticle Hemolytic Properties in Vitro"), 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 red blood cells and the use of this method to study a variety of nanoparticles. Specifically, the study describes approaches to identify nanoparticle/erythrocyte interferences, when they occur and how to resolve them to get accurate results (i.e. to avoid false-positive or false-negative results).
The NCL team's assay leverages the ASTM F-756-00 standard for analysis of hemolytic properties of medical devices. "We scaled this standard practice to a 96-well plate format assay and conducted a one month validation aimed at determining its reproducibility, precision, and accuracy, as well as qualification of negative and positive nanoparticle-relevant controls" explains Dobrovolskaia. "We subsequently used our assay to analyze various types of nanomaterials including polymers, gold nanoshells, nanoliposomes, nanoemulsions, fullerene derivatives, gold colloids, and dendrimers. This second phase was conducted over a two year period and included identification and resolution of nanoparticle interference with the assay, in addition to evaluation of reproducibility, precision, accuracy, and control qualification."
"Several previous studies evaluating nanoparticle hemolytic properties in vitro have appeared in the literature" says Dobrovolskaia. "However, all these tests were conduced using different methods, and even when similar approaches were used, factors such as plasma anti-coagulant, blood incubation times, centrifugal forces, assay detection wavelength varied from study to study. This made it difficult to compare results."
She points out that, most importantly, none of these earlier studies included special controls to identify nanoparticle interference, and therefore it was really impossible to conclude what nanoparticle properties were responsible for the hemolysis – i.e. did it depend on size, charge, surface groups or was due to the particle absorbance?
The problem therefore was to develop a hemolysis assay that is applicable to a wide variety of nanoparticles so that data can be compared between the many labs testing nanoparticle biocompatibility.
Consequently, the NCL scientists specifically designed their assay to be applicable to various nanoparticles. That way, it that it could be used by different investigators working with different nanoparticles, and allow comparison of the results.
The protocol described in this paper is one of a set of assays developed specifically for use with nanoparticles and available to the public through the NCL web site (Assay Cascade Protocols).
When used in early preclinical drug development, this particular method may help differentiate strong candidates from ones with properties that need further tuning before in vivo applications. If studies from numerous laboratories on a wide variety of nanoparticles are conducted using the same method, it will increase confidence in the quality of data and conclusions drawn from that data regarding nanoparticle biocompatibility.
Hemolysis is not the only traditional biocompatibility test that nanoparticles interfere with. Nanoparticles are intricate, often delicate systems with unique properties, and their characterization is challenging.
Dobrovolskaia mentions that, for instance, many nanoparticles have catalytic properties and can enhance assays that rely on enzymatic reactions, generating false-positive results. "Assays routine to the preclinical characterization of conventional pharmaceuticals, such as the Limulus amebocyte lysate (LAL) test for endotoxin contamination detection may yield spurious results when applied to nanoparticle samples. Multifunctional nanoparticles have to be characterized quite rigorously, as there are multiple components that must work in concert to achieve functionality."
She adds that a thorough characterization of a nanoparticle-based therapeutic includes evaluation of physicochemical properties, sterility and pyrogenicity assessment, biodistribution (ADME or absorption, distribution, metabolism and excretion) and toxicity characterization – which includes both in vitro tests and in vivo animal studies. The NCL has tailored each of these tiers of a rational characterization cascade so that they are relevant to nanoparticles.
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