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<script type="text/javascript" src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.boots.min.js"> </script><title>Radioactive Blood Partitioning Assay - National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols - NCBI Bookshelf</title>
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<meta name="citation_inbook_title" content="National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet]">
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<meta name="citation_title" content="Radioactive Blood Partitioning Assay">
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<meta name="citation_publisher" content="National Cancer Institute (US)">
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<meta name="citation_date" content="2017/06">
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<meta name="citation_author" content="Stephan T. Stern">
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<meta name="citation_pmid" content="39013056">
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id="jr-fip-info-p"><a id="jr-fip-prev" class="wsprkl btn" title="Jump to previuos match">◀</a><button id="jr-fip-matches">no matches yet</button><a id="jr-fip-next" class="wsprkl btn" title="Jump to next match">▶</a></nav></nav></div><div id="jr-epub-interstitial" class="hidden"></div><div id="jr-content"><article data-type="main"><div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><div class="fm-sec"><h1 id="_NBK604956_"><span class="label">NCL Method PHA-1</span><span class="title" itemprop="name">Radioactive Blood Partitioning Assay</span></h1><div class="subtitle whole_rhythm">Version 1.1</div><p class="contribs">Stern ST.</p><p class="fm-aai"><a href="#_NBK604956_pubdet_">Publication Details</a></p></div></div><div class="jig-ncbiinpagenav body-content whole_rhythm" data-jigconfig="allHeadingLevels: ['h2'],smoothScroll: false" itemprop="text"><div id="ncipr23.s1"><h2 id="_ncipr23_s1_">1. Introduction</h2><p>Characterization of drug release under physiological conditions is very important for prediction of drug release in vivo, and can be used for formulation optimization. This assay uses a blood partitioning technique to measure the release of free drug from a nanoparticle-encapsulated drug complex. This assay is based on the principle that the free drug will partition between the plasma and red blood cell (RBC) fractions, whereas the hydrophilic nanoparticle platform is generally retained in the plasma fraction.</p><p>The protocol is written for analysis of a dual radioactively labeled drug-nanoparticle platform complex (i.e. <sup>14</sup>C-labeled drug and <sup>3</sup>H-labeled nanoparticle platform). The protocol can also be adapted to use non-radioactive materials, provided that suitable analytical methods can be developed to measure both the drug and platform components independently in blood and plasma matrix (i.e. ICP-MS for the nanoparticle platform component and HPLC for the drug component). The same principles outlined in the study design below could be used for these non-radioactive analytical methods.</p></div><div id="ncipr23.s2"><h2 id="_ncipr23_s2_">2. Principles</h2><p>The purpose of this study is to determine the extent of RBC partitioning of a dual radiolabel <sup>14</sup>C-drug:<sup>3</sup>H-platform nanoparticle complex, following incubation of whole blood with the dual labeled nanoparticle complex. By measuring both <sup>14</sup>C-drug:<sup>3</sup>H-platform signal in the whole blood and plasma fractions, the extent of component partitioning into plasma and RBC fractions of whole blood can be calculated by the equation:
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<div class="pmc_disp_formula whole_rhythm clearfix" id="ncipr23.deq1"><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow ten_col"><math id="ncipr23.eq1" display="block"><mrow><msub><mi>F</mi><mi>p</mi></msub><mi>%</mi><mo>=</mo><mrow><mo>(</mo><mrow><msub><mi>C</mi><mi>p</mi></msub><mo>/</mo><msub><mi>C</mi><mi>b</mi></msub></mrow><mo>)</mo></mrow><mo>×</mo><mrow><mo>(</mo><mrow><mn>1</mn><mo>−</mo><msub><mi>H</mi><mi>c</mi></msub></mrow><mo>)</mo></mrow><mo>×</mo><mn>100</mn></mrow></math>
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</div><div class="inline_block pmc_inline_block pmc_va_middle pmc_hide_overflow last bk_equ_label "><div><span class="nowrap">Equation 1</span></div></div></div>
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where <i>C</i><sub>p</sub> is the concentration in plasma, <i>C</i><sub>b</sub> is the concentration in whole blood, and <i>H</i><sub>c</sub> is the Hematocrit. <i>F</i><sub>p</sub>%, the fraction in plasma, is an estimate of the percentage in the plasma fraction. Whereas, (100–<i>F</i><sub>p</sub>%) would be an estimate of the percentage in the RBC fraction.</p><p>Another important parameter is the ratio of the <sup>14</sup>C/<sup>3</sup>H signals as a measure of platform integrity. If the nanoparticle complex is stable, then the <sup>14</sup>C/<sup>3</sup>H signal ratio should be constant in all fractions measured, and F<sub>p</sub> % should be the same for drug and platform. If the drug component partitions independent of platform, then this ratio in the blood fractions will change and F<sub>p</sub> % will be disparate for drug and platform.</p><p>Hydrophilic platforms (e.g. PEGylated materials) are generally retained in the plasma fraction. As this assay does not involve “sink” conditions, partition of the radiolabeled drug:platform complex should be compared to partitioning of free radiolabeled drug in order to determine at what <i>F</i><sub>p</sub>% equilibrium between the red blood cells and plasma occurs (this represents the maximum drug release attainable in the assay). Knowledge of the percentage of free drug in the initial nanoformulation is important in evaluating the actual drug release.</p></div><div id="ncipr23.s3"><h2 id="_ncipr23_s3_">3. Reagents, Materials, Cell Lines, and Equipment</h2><blockquote><p><i>Note: The NCL does not endorse any of the suppliers listed below; their inclusion is for informational purposes only. Equivalent supplies from alternate vendors can be substituted</i>.</p></blockquote><dl id="ncipr23.l1" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.1.</dt><dd id="ncipr23.lt1"><p class="no_top_margin">Reagents
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<dl id="ncipr23.l2" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.1.1.</dt><dd id="ncipr23.lt2"><p class="no_top_margin">15 mL female heparinized Sprague Dawley (SD) rat (or human) whole blood (rat whole blood, Innovative Research, SKU: IR1-150N Li-Heparin; human whole blood, Innovative Research, SKU: IPLA-WB1 Li-Heparin)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.2.</dt><dd id="ncipr23.lt3"><p class="no_top_margin"><sup>14</sup>C-drug/<sup>3</sup>H-platform dual labeled nanoparticle complex (Theoretical per mL activity of the reconstituted nanoparticle complex should be ~ <b>7.5</b> µ<b>Ci <sup>14</sup>C-drug/ 15</b> µ<b>Ci <sup>3</sup>H-platform/mL</b>)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.3.</dt><dd id="ncipr23.lt4"><p class="no_top_margin"><sup>14</sup>C-free drug (Theoretical per mL activity of the reconstituted nanoparticle complex should be ~ <b>7.5</b> µ<b>Ci <sup>14</sup>C-drug/mL</b>)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.4.</dt><dd id="ncipr23.lt5"><p class="no_top_margin">BTS-450 tissue solubilizer (500 mL, Beckman Coulter, P/N 580691)</p></dd></dl><dl class="bkr_refwrap"><dt>3.1.5.</dt><dd id="ncipr23.lt6"><p class="no_top_margin">Ready Organic Scintillation Cocktail (3.75L, Beckman Coulter, P/N 586600)</p></dd></dl></dl></p></dd></dl><dl class="bkr_refwrap"><dt>3.2.</dt><dd id="ncipr23.lt7"><p class="no_top_margin">Materials
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<dl id="ncipr23.l3" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.2.1.</dt><dd id="ncipr23.lt8"><p class="no_top_margin">1.5 mL Li-heparin coated-Eppendorf tubes (Fischer Scientific, 05-407-13C)</p></dd></dl></dl></p></dd></dl><dl class="bkr_refwrap"><dt>3.3.</dt><dd id="ncipr23.lt9"><p class="no_top_margin">Equipment
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<dl id="ncipr23.l4" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>3.3.1.</dt><dd id="ncipr23.lt10"><p class="no_top_margin">Barnstead Thermolyte Labquake Shaker</p></dd></dl><dl class="bkr_refwrap"><dt>3.3.2.</dt><dd id="ncipr23.lt11"><p class="no_top_margin">37<sup>°</sup>C oven</p></dd></dl><dl class="bkr_refwrap"><dt>3.3.3.</dt><dd id="ncipr23.lt12"><p class="no_top_margin">VWR Galaxy mini centrifuge</p></dd></dl><dl class="bkr_refwrap"><dt>3.3.4.</dt><dd id="ncipr23.lt13"><p class="no_top_margin">Beckman Coulter LS6500 scintillation counter</p></dd></dl></dl></p></dd></dl></dl></div><div id="ncipr23.s4"><h2 id="_ncipr23_s4_">4. Experimental Procedure</h2><blockquote><p>
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<b>
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<i>CAUTION: This procedure uses radioactivity. Radioactivity should only be handled by properly trained personnel, using all appropriate safety measures. Disposal of radioactive waste should be done in accordance with your local regulations.</i>
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</b>
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</p></blockquote><dl id="ncipr23.l5" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>4.1.</dt><dd id="ncipr23.lt14"><p class="no_top_margin">Reconstitute the dual radiolabeled nanoparticle complex and free radiolabeled drug to 7.5 µCi <sup>14</sup>C-drug/ 15 µCi <sup>3</sup>H-platform/mL in an appropriate aqueous diluent. Immediately take a small aliquot for DLS size measurement (or other stability indicating assay), and measurement of <sup>14</sup>C/<sup>3</sup>H signal to estimate specific activity and compare to theoretical.</p></dd></dl><dl class="bkr_refwrap"><dt>4.2.</dt><dd id="ncipr23.lt15"><p class="no_top_margin">To determine actual specific activity, add 1 mL of BTS-450 to 10 µL reconstituted nanoparticle complex or free radiolabeled drug, followed by addition of 200 µL of a 30% (w/w) H<sub>2</sub>O<sub>2</sub> solution and 15 mL of Ready Organic Scintillation Cocktail. Radioactivity is measured by scintillation counting using dual <sup>14</sup>C:<sup>3</sup>H label mode (Beckman Coulter LS6500).</p></dd></dl><dl class="bkr_refwrap"><dt>4.3.</dt><dd id="ncipr23.lt16"><p class="no_top_margin">Prepare time zero samples, by spiking 100 µL reconstituted nanoparticle complex or free radiolabeled drug into 400 µL of 37<sup>°</sup>C whole blood in triplicate, vortex, and immediately collect 10 µL of whole blood (<i>This is the C<sub>b</sub> value at time zero</i>). Immediately centrifuge the remaining whole blood sample at 4<sup>°</sup>C to prepare plasma (VWR Galaxy mini, 2000x<i>g</i>, 5 min), and collect 10 µL of resulting plasma for radioactivity measurement (<i>This is the C<sub>p</sub> value at time zero</i>).</p></dd></dl><dl class="bkr_refwrap"><dt>4.4.</dt><dd id="ncipr23.lt17"><p class="no_top_margin">Prepare the 10 and 60 min time points (or any additional time points desired) by spiking 100 µL of the reconstituted nanoparticle complex or free radiolabeled drug into 400 µL of whole blood in triplicate, and vortex. Incubate the samples at 37<sup>°</sup>C on a rotary shaker (set at 8 rpm) for the desired time.</p></dd></dl><dl class="bkr_refwrap"><dt>4.5.</dt><dd id="ncipr23.lt18"><p class="no_top_margin">Following incubation, a 10 µL sample of the radiolabel:blood mixture is removed for radioactivity measurement (<i>These are the C<sub>b</sub> values at each timepoint</i>). Plasma is separated from the remaining radiolabel:blood mixture by centrifugation (VWR Galaxy mini, 2000x<i>g</i>, 5 min), and 10 µL of the resulting plasma is used for radioactivity measurement (<i>These are the C<sub>p</sub> values at each timepoint</i>).</p></dd></dl><dl class="bkr_refwrap"><dt>4.6.</dt><dd id="ncipr23.lt19"><p class="no_top_margin">Plasma and blood samples are prepared by adding 1 mL of BTS-450 to the 10 µL sample, followed by addition of 200 µL of a 30% (w/w) H<sub>2</sub>O<sub>2</sub> solution and 15 mL of Ready Organic Scintillation Cocktail. Radioactivity is measured by scintillation counting using dual <sup>14</sup>C:<sup>3</sup>H label mode (Beckman Coulter LS6500).</p></dd></dl></dl></div><div id="ncipr23.s5"><h2 id="_ncipr23_s5_">5. Calculations</h2><dl id="ncipr23.l6" class="temp-labeled-list"><dl class="bkr_refwrap"><dt>5.1.</dt><dd id="ncipr23.lt20"><p class="no_top_margin">The fraction of drug and platform in plasma (Fp) is calculated according to <a href="#ncipr23.deq1">Equation 1</a> [<a class="bibr" href="#ncipr23.ref1" rid="ncipr23.ref1">1</a>]. The estimated hematocrit value for female SD rat is (H<sub>c</sub>) is 0.44, according to the literature [<a class="bibr" href="#ncipr23.ref2" rid="ncipr23.ref2">2</a>]. Ideally, the actual hematocrit of the blood sample should be determined (especially in the case that the drug complex causes hemolysis!). The mean values and standard deviations of the triplicate measurements should be reported.</p></dd></dl></dl></div><div id="ncipr23.rl.r1"><h2 id="_ncipr23_rl_r1_">6. References</h2><dl class="temp-labeled-list"><dl class="bkr_refwrap"><dt>1.</dt><dd><div class="bk_ref" id="ncipr23.ref1">Petterino
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C, Argentino-Storino
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A. (2006) <a href="/pubmed/16343876" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">Clinical chemistry and haematology historical data in control Sprague-Dawley rats from pre-clinical toxicity studies</a>. <em>Exp Toxicol Pathol</em>. <strong>57</strong>(<strong>3</strong>):213–9.
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[<a href="https://pubmed.ncbi.nlm.nih.gov/16343876" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16343876</span></a>]</div></dd></dl><dl class="bkr_refwrap"><dt>2.</dt><dd><div class="bk_ref" id="ncipr23.ref2">Weiss
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HM, Fresneau
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M, Camenisch
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GP, Kretz
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O and Gross
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G (2006) In vitro blood distribution and plasma protein binding of the iron chelator deferasirox (ICL670) and its iron complex Fe-[ICL670]2 for rat, marmoset, rabbit, mouse, dog, and human. <em>Drug Metab Dispos</em>
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<strong>34</strong>:971–975.
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[<a href="https://pubmed.ncbi.nlm.nih.gov/16531476" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 16531476</span></a>]</div></dd></dl></dl></div><div id="ncipr23.s6"><h2 id="_ncipr23_s6_">7. Abbreviations</h2><dl><dt id="ncipr23.abb_DL1_DI1">
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<i>C</i>
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<sub>b</sub>
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</dt><dd><p>concentration in whole blood</p></dd><dt id="ncipr23.abb_DL1_DI2">
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<i>C</i>
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<sub>p</sub>
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</dt><dd><p>concentration in plasma</p></dd><dt id="ncipr23.abb_DL1_DI3">DLS</dt><dd><p>dynamic light scattering</p></dd><dt id="ncipr23.abb_DL1_DI4"><i>F</i><sub>p</sub>%</dt><dd><p>fraction in plasma</p></dd><dt id="ncipr23.abb_DL1_DI5">GTA</dt><dd><p>general toxicity assay</p></dd><dt id="ncipr23.abb_DL1_DI6">
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<i>H</i>
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<sub>c</sub>
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</dt><dd><p>Hematocrit</p></dd><dt id="ncipr23.abb_DL1_DI7">RBC</dt><dd><p>red blood cell</p></dd><dt id="ncipr23.abb_DL1_DI8">SD</dt><dd><p>Sprague Dawley</p></dd></dl></div><div><dl class="temp-labeled-list small"><dl class="bkr_refwrap"><dt></dt><dd><div><p class="no_top_margin"><p>This protocol assumes an intermediate level of scientific competency with regard to techniques, instrumentation, and safety procedures. Rudimentary assay details have been omitted for the sake of brevity.</p></p></div></dd></dl><dl class="bkr_refwrap"><dt></dt><dd><div><p class="no_top_margin"><div>
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<span class="mixed-citation" id="ncipr23.suggestedcitation">Stern ST, NCL Method PHA-1: Radioactive Blood Partitioning Assay. <a href="https://ncl.cancer.gov/resources/assay-cascade-protocols" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">https://ncl.cancer.gov/resources/assay-cascade-protocols</a> DOI: <a href="http://dx.crossref.org/10.17917/2HFK-9E25" ref="pagearea=body&targetsite=external&targetcat=link&targettype=uri">10.17917/2HFK-9E25</a></span>
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</div></p></div></dd></dl></dl></div><div id="bk_toc_contnr"></div></div></div><div class="fm-sec"><h2 id="_NBK604956_pubdet_">Publication Details</h2><h3>Author Information and Affiliations</h3><p class="contrib-group"><h4>Authors</h4><span itemprop="author">Stephan T. Stern</span>, Ph.D.</p><h3>Publication History</h3><p class="small">Published: <span itemprop="datePublished">June 2017</span>.</p><h3>Copyright</h3><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a></div></div><h3>Publisher</h3><p><a href="https://www.cancer.gov/nano/research/ncl" ref="pagearea=page-banner&targetsite=external&targetcat=link&targettype=publisher">National Cancer Institute (US)</a>, Bethesda (MD)</p><h3>NLM Citation</h3><p>Stern ST. Radioactive Blood Partitioning Assay: Version 1.1. 2017 Jun. In: National Cancer Institute’s Nanotechnology Characterization Laboratory Assay Cascade Protocols [Internet]. Bethesda (MD): National Cancer Institute (US); 2005 May 1-. NCL Method PHA-1.<span class="bk_cite_avail"></span> doi: 10.17917/2HFK-9E25</p></div><div class="small-screen-prev"><a href="/books/n/nciprotocols/ncipr32/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M75,30 c-80,60 -80,0 0,60 c-30,-60 -30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Prev</text></svg></a></div><div class="small-screen-next"><a href="/books/n/nciprotocols/ncipr22/?report=reader"><svg xmlns="http://www.w3.org/2000/svg" viewBox="0 0 100 100" preserveAspectRatio="none"><path d="M25,30c80,60 80,0 0,60 c30,-60 30,0 0,-60"></path><text x="20" y="28" textLength="60" style="font-size:25px">Next</text></svg></a></div></article></div><div id="jr-scripts"><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/libs.min.js"> </script><script src="/corehtml/pmc/jatsreader/ptpmc_3.22/js/jr.min.js"> </script><script type="text/javascript" src="/core/mathjax/2.7.9/MathJax.js?config=TeX-AMS-MML_SVG"> </script></div></div>
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