Name: sample extraction and simultaneous chromatographic quantitation of doxorubicin and mitomycin c following drug combination delivery in nanoparticles to tumor-bearing mice
Uploaded: 2017-10-05T13:00:00
Description: Watch this Scientific Journal Video about Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-b

The authors gratefully acknowledge the equipment grant from the Natural Science and Engineering Research (NSERC) Council of Canada for HPLC, the operating grant from the Canadian Institute of Health Research (CIHR) and Canadian Breast Cancer Research (CBCR) Alliance to X.Y. Wu, and the University of Toronto Scholarship to R.X. Zhang and T. Zhang.

All animal experiments were approved by the Animal Care Committee of University Health Network at the Ontario Cancer Institute and conducted in accordance with the Canadian Council on Animal Care Guidelines.
1. Biological Sample Preparation
<ol>
	<li>Collect the whole blood, major organs, and breast tumor at predetermined time-points after intravenous (i.v.) administration of drug-containing formulations (e.g., DMPLN, liposomal DOX)
	<ol>
		<li>Inject a breast tumor...

Compared to other chromatographic methods that enable the detection of a single drug species at a time, the present HPLC protocol is able to simultaneously quantitate three drug compounds (DOX, MMC, and DOXol) in the same biological matrix without the need to change the mobile phase. This preparation and analysis method has been successfully applied to determine the pharmacokinetics and bio-distribution of two nanoparticle-based drug delivery systems (i.e., liposomal DOX and DMPLN)22. Sin...

Chemotherapy is a primary treatment modality for many cancers yet it is often associated with severe adverse effects and limited efficacy due to drug resistance and other factors1,2,3. To improve the outcome of chemotherapy, drug combination regimens have been applied in the clinic based on considerations such as non-overlapping toxicities, different mechanisms of drug action, and non-cross drug resistance4,5,6. In clinical trials, a better tumor response rate was often observed using simultaneously administered drug combinations compared to a regimen of sequential drug delivery7,8. However, due to sub-optimal bio-distribution of free drug forms, simultaneous injection of multiple drugs can cause prominent normal tissue toxicity that outweighs the therapeutic effect9,10,11. Nanocarrier-based drug delivery has been shown to alter the pharmacokinetics and bio-distribution of encapsulated drugs, enhancing tumor-targeted accumulation12,13,14. As reviewed in our recent articles, nanoparticles co-loaded with synergistic drug combinations have demonstrated the capability to mitigate the problems encountered by free drug combinations, due to their controlled temporal and spatial co-delivery of multiple drugs to tumor tissue, enabling synergistic drug effects against cancer cells4,15,16. As a result, superior therapeutic efficacy and low toxicity have been demonstrated in both pre-clinical and clinical studies4,17,18.
Our previous in vitro studies found that the combination of two anticancer drugs, doxorubicin (DOX) and mitomycin C (MMC), produced a synergistic effect against several breast cancer cells lines and, furthermore, co-loading DOX and MMC within polymer-lipid hybrid nanoparticles (DMPLN) overcame various multi-drug resistant associated efflux pumps (e.g., P-glycoprotein and breast cancer resistant protein)19,20,21. In vivo, DMPLN enabled spatial-temporal co-delivery of DOX and MMC to tumor sites and increased bioavailability of drugs within cancer cells, as indicated by moderation of the formation of the DOX metabolite doxorubicinol (DOXol)22. As a result, the DMPLN enhanced tumor cell apoptosis, tumor growth inhibition, and prolonged host survival compared to free DOX and MMC combination or a liposomal DOX formulation22,23,24,25.
Analyzing the actual amount of drugs co-delivered by a nanocarrier is critical for designing effective nanoparticle formulations. Many methods have been developed to analyze the plasma level of single DOX or MMC doses using high performance liquid chromatography (HPLC) alone or in combination with mass spectrometry (MS)26,27,28,29,30,31,32,33,34. However, these methods are often time-consuming and impractical for combination therapy as a large number of biological samples need to be prepared separately for analysis of multiple drugs (sometimes including drug metabolites). In addition to the strong plasma protein binding of DOX and MMC, red blood cells also have a great capacity to bind and concentrate many anticancer drugs35,36. Thus, plasma analysis for DOX or MMC may obfuscate actual blood drug concentrations. The present work (Figure 1) describes a simple and robust multiple drug analysis method using reverse phase HPLC to simultaneously extract and quantitate DOX, MMC and the DOX metabolite doxorubicinol (DOXol) from whole blood and various tissues (e.g., tumors). It has been successfully applied to determine the pharmacokinetics and bio-distribution of DOX and MMC as well as the formation of DOXol after drug delivery via free solutions or nanoparticle forms (i.e., DMPLN and liposomal DOX) in an orthotopically implanted murine breast-tumor mouse model after intravenous (i.v.) injection22.

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		<col style="width:188pt" width="250" />
		<col style="width:122pt" width="162" />
		<col style="width:89pt" width="119" />
		<col style="width:236pt" width="315" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Doxorubicin&#160;</td>
			<td class="xl20" style="width:122pt" width="162">Polymed Theraeutics</td>
			<td class="xl20" style="width:89pt" width="119">111023</td>
			<td class="xl20" style="width:236pt" width="315">Anticancer drug</td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Mitomycin C</td>
			<td class="xl20" style="width:122pt" width="162">Polymed Theraeutics</td>
			<td class="xl20" style="width:89pt" width="119">060814</td>
			<td class="xl20" style="width:236pt" width="315">Anticancer drug</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl20" height="42" style="width:188pt;height:31.5pt" width="250">Doxorubicinol (DOXol)</td>
			<td class="xl20" style="width:122pt" width="162">Toronto Research Chemicals</td>
			<td class="xl20" style="width:89pt" width="119">D558020</td>
			<td class="xl21">Metabolite of DOX</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">4-Methylumbelliferone sodium salt&#160;</td>
			<td class="xl20" style="width:122pt" width="162">Sigma-Aldrich</td>
			<td class="xl20" style="width:89pt" width="119">M1508</td>
			<td class="xl21">Internal standard</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Myristic Acid</td>
			<td class="xl20" style="width:122pt" width="162">Sigma-Aldrich</td>
			<td class="xl20" style="width:89pt" width="119">544-63-8&#160;&#160;</td>
			<td class="xl21">Materials for poly-lipid hybrid nanoparticles</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Polyoxyethylene (100) Stearate</td>
			<td class="xl20" style="width:122pt" width="162">Spectrum</td>
			<td class="xl20" style="width:89pt" width="119">M1402</td>
			<td class="xl21">Materials for poly-lipid hybrid nanoparticles</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Polyoxyethylene (40) Stearate</td>
			<td class="xl20" style="width:122pt" width="162">Sigma-Aldrich</td>
			<td class="xl20" style="width:89pt" width="119">P3440</td>
			<td class="xl21">Materials for poly-lipid hybrid nanoparticles</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Pluronic F68 (PF68)</td>
			<td class="xl20" style="width:122pt" width="162">BASF Corp.</td>
			<td class="xl20" style="width:89pt" width="119">9003-11-6</td>
			<td class="xl21">Materials for poly-lipid hybrid nanoparticles</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl20" height="42" style="width:188pt;height:31.5pt" width="250">Ultrasonication (UP100H)</td>
			<td class="xl20" style="width:122pt" width="162">Hielscher, Ultrasound Technology</td>
			<td class="xl20" style="width:89pt" width="119">NA</td>
			<td class="xl21">Nanoparticle preparation</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Water Bath (ISOTEMP 3016HS)</td>
			<td class="xl20" style="width:122pt" width="162">Fisher Scientific</td>
			<td class="xl20" style="width:89pt" width="119">NA</td>
			<td class="xl21">Nanoparticle preparation</td>
		</tr>
		<tr height="105" style="height:78.75pt">
			<td class="xl20" height="105" style="width:188pt;height:78.75pt" width="250">Liposomal Doxorubicin&#160; (Caelyx)</td>
			<td class="xl20" style="width:122pt" width="162">Janssen</td>
			<td class="xl20" style="width:89pt" width="119">Purchased from the pharmacy Princess Margaret Hospital</td>
			<td class="xl21">Clinically-approved nanoparticle formulation&#160;</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">HPLC-graded Methanol</td>
			<td class="xl21">Caledon Chemicals</td>
			<td class="xl20" style="width:89pt" width="119">6701-7-40</td>
			<td class="xl21">HPLC mobile phase composition</td>
		</tr>
		<tr height="25" style="height:18.75pt">
			<td class="xl20" height="25" style="width:188pt;height:18.75pt" width="250">HPLC-graded H2O</td>
			<td class="xl21">Caledon Chemicals</td>
			<td class="xl20" style="width:89pt" width="119">8801-7-40</td>
			<td class="xl21">HPLC mobile phase composition</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">HPLC-graded Acetonitrile&#160;</td>
			<td class="xl20" style="width:122pt" width="162">Caledon Chemicals</td>
			<td class="xl20" style="width:89pt" width="119">1401-7-40</td>
			<td class="xl21">HPLC mobile phase composition</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Trifluoroacetic Acid</td>
			<td class="xl20" style="width:122pt" width="162">Sigma-Aldrich</td>
			<td class="xl20" style="width:89pt" width="119">302031</td>
			<td class="xl21">HPLC mobile phase composition</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl20" height="42" style="width:188pt;height:31.5pt" width="250">0.45 &#956;m Nylon Membrane Filter Paper</td>
			<td class="xl20" style="width:122pt" width="162">Whatman</td>
			<td class="xl20" style="width:89pt" width="119">WHA7404004</td>
			<td class="xl21">HPLC mobile phase preparation</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl29" height="42" style="height:31.5pt">1cc Plastic Syringes</td>
			<td class="xl20" style="width:122pt" width="162">Becton, Dickinson and Company</td>
			<td class="xl20" style="width:89pt" width="119">2606-309659</td>
			<td class="xl21">Treatment injection</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl29" height="42" style="height:31.5pt">5cc Plastic Syringes</td>
			<td class="xl20" style="width:122pt" width="162">Becton, Dickinson and Company</td>
			<td class="xl20" style="width:89pt" width="119">2608-309646</td>
			<td class="xl21">Tissue collections</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl20" height="42" style="width:188pt;height:31.5pt" width="250">30G 1/2 Needles</td>
			<td class="xl20" style="width:122pt" width="162">Becton, Dickinson and Company</td>
			<td class="xl20" style="width:89pt" width="119">305106</td>
			<td class="xl21">Treatment injection</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl20" height="42" style="width:188pt;height:31.5pt" width="250">25G 5/8 Needles</td>
			<td class="xl20" style="width:122pt" width="162">Becton, Dickinson and Company</td>
			<td class="xl20" style="width:89pt" width="119">305122</td>
			<td class="xl21">Tissue collections</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl20" height="42" style="width:188pt;height:31.5pt" width="250">Sterile 0.9% Saline</td>
			<td class="xl20" style="width:122pt" width="162">Univeristy of Toronto House Brand</td>
			<td class="xl20" style="width:89pt" width="119">1011</td>
			<td class="xl21">Tissue perfusion</td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl20" height="22" style="width:188pt;height:16.5pt" width="250">13 ml Rounded-bottom conical tube&#160;</td>
			<td class="xl20" style="width:122pt" width="162">SARSTEDT</td>
			<td class="xl20" style="width:89pt" width="119">62.515.006</td>
			<td class="xl21">Prolyprolene, tissue homogenization</td>
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		<tr height="23" style="height:17.25pt">
			<td class="xl30" height="23" style="height:17.25pt">Alpha Minimum Essential Medium (MEM)&#160;</td>
			<td class="xl27">Gibco</td>
			<td class="xl28" style="width:89pt" width="119">12571063</td>
			<td class="xl21">Cell medium</td>
		</tr>
		<tr height="22" style="height:16.5pt">
			<td class="xl20" height="22" style="width:188pt;height:16.5pt" width="250">1 x Phosphate Buffer Saline</td>
			<td class="xl20" style="width:122pt" width="162">Gibco</td>
			<td class="xl20" style="width:89pt" width="119">10010023</td>
			<td class="xl21">Tissue homogenization</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Triton X-100</td>
			<td class="xl20" style="width:122pt" width="162">Sigma-Aldrich</td>
			<td class="xl20" style="width:89pt" width="119">X100-100 ML</td>
			<td class="xl21">Tissue homogenization</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Formic acid</td>
			<td class="xl20" style="width:122pt" width="162">Caledon Chemicals</td>
			<td class="xl20" style="width:89pt" width="119">1/5/3840</td>
			<td class="xl21">Adjust pH for extraction solvent</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl20" height="42" style="width:188pt;height:31.5pt" width="250">Sodium heparin sprayed plastic tubes</td>
			<td class="xl20" style="width:122pt" width="162">Becton, Dickinson and Company</td>
			<td class="xl20" style="width:89pt" width="119">367878</td>
			<td class="xl21">Blood collection</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Analytical Weigh Balance&#160;</td>
			<td class="xl20" style="width:122pt" width="162">Sartorius&#160;</td>
			<td class="xl21">CPA225D</td>
			<td class="xl21">NA</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">pH meters&#160;</td>
			<td class="xl20" style="width:122pt" width="162">Fisher Scientific</td>
			<td class="xl26">13-637-671</td>
			<td class="xl21">accumet BASIC</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Vortex Mixter</td>
			<td class="xl20" style="width:122pt" width="162">Fisher Scientific</td>
			<td class="xl20" style="width:89pt" width="119">02-215-365</td>
			<td class="xl21">Vortexing samples at desired speed</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">1.5 ml&#160; Microcentrifuge Tube</td>
			<td class="xl20" style="width:122pt" width="162">Fisherbrand</td>
			<td class="xl20" style="width:89pt" width="119">2043-05408129</td>
			<td class="xl21">Prolyprolene</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Model 1000 homogenizer</td>
			<td class="xl22">Fisher Scientific</td>
			<td class="xl20" style="width:89pt" width="119">08-451-672</td>
			<td class="xl21">Tissue homogenization</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Centrifuge 5702R</td>
			<td class="xl20" style="width:122pt" width="162">Eppendorf</td>
			<td class="xl20" style="width:89pt" width="119">5702R</td>
			<td class="xl21">Extraction preparation</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Heated Evaporator System</td>
			<td class="xl20" style="width:122pt" width="162">Glas-Col</td>
			<td class="xl20" style="width:89pt" width="119">NA</td>
			<td class="xl21">Sample reconstitution</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">HPLC Screw Thread Vials</td>
			<td class="xl20" style="width:122pt" width="162">DIKMA</td>
			<td class="xl20" style="width:89pt" width="119">5320</td>
			<td class="xl21">HPLC sample injection</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl20" height="42" style="width:188pt;height:31.5pt" width="250">HPLC Screw Caps with PTFE White Silicone Septa</td>
			<td class="xl20" style="width:122pt" width="162">DIKMA</td>
			<td class="xl20" style="width:89pt" width="119">5325</td>
			<td class="xl21">HPLC sample injection</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">HPLC Polypropylene Insert&#160;&#160;</td>
			<td class="xl20" style="width:122pt" width="162">Agilent Technologies</td>
			<td class="xl20" style="width:89pt" width="119">5182-0549</td>
			<td class="xl21">Maximum volume 250 &#956;l, HPLC sample injection</td>
		</tr>
		<tr height="25" style="height:18.75pt">
			<td class="xl20" height="25" style="width:188pt;height:18.75pt" width="250">Xbridge C18 Column</td>
			<td class="xl23">Waters Corporation</td>
			<td class="xl20" style="width:89pt" width="119">186003117</td>
			<td class="xl21">Drug analysis</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Gradient pump&#160;</td>
			<td class="xl23">Waters Corporation</td>
			<td class="xl20" style="width:89pt" width="119">W600</td>
			<td class="xl21">Drug analysis</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Auto-sampler</td>
			<td class="xl23">Waters Corporation</td>
			<td class="xl20" style="width:89pt" width="119">W2707</td>
			<td class="xl21">Drug analysis</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Photodiode array detector&#160;</td>
			<td class="xl23">Waters Corporation</td>
			<td class="xl20" style="width:89pt" width="119">W2998</td>
			<td class="xl21">Drug analysis</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Multi &#955; fluoresence detector&#160;</td>
			<td class="xl23">Waters Corporation</td>
			<td class="xl20" style="width:89pt" width="119">W2475</td>
			<td class="xl21">Drug analysis</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">EMPOWER 2</td>
			<td class="xl23">Waters Corporation</td>
			<td class="xl20" style="width:89pt" width="119">NA</td>
			<td class="xl21">Data analysis software</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Scientist</td>
			<td class="xl20" style="width:122pt" width="162">Micromath</td>
			<td class="xl20" style="width:89pt" width="119">NA</td>
			<td class="xl21">Pharmacokinetic analysis</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl20" height="21" style="width:188pt;height:15.75pt" width="250">Female Balb/c Mice</td>
			<td class="xl20" style="width:122pt" width="162">Jackson Laboratory</td>
			<td class="xl20" style="width:89pt" width="119">001026</td>
			<td class="xl21">In vivo</td>
		</tr>
		<tr height="63" style="height:47.25pt">
			<td class="xl20" height="63" style="width:188pt;height:47.25pt" width="250">EMT6/WT Breast Cancer Cells</td>
			<td class="xl20" style="width:122pt" width="162">Provided by Dr. Ian Tannock; Ontario Cancer Institute</td>
			<td class="xl20" style="width:89pt" width="119">NA</td>
			<td class="xl21">In vivo</td>
		</tr>
	</tbody>
</table>

sample extraction and simultaneous chromatographic quantitation of doxorubicin and mitomycin c following drug combination delivery in nanoparticles to tumor-bearing mice

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its therapeutic benefit. Nanoparticle-based drug combination has been shown to mitigate the problems encountered by free drug combination therapy. Our previous studies have shown that the combination of two anticancer drugs, doxorubicin (DOX) and mitomycin C (MMC), produced a synergistic effect against both murine and human breast cancer cells in vitro. DOX and MMC co-loaded polymer-lipid hybrid nanoparticles (DMPLN) bypassed various efflux transporter pumps that confer multidrug resistance and demonstrated enhanced efficacy in breast tumor models. Compared to conventional solution forms, such superior efficacy of DMPLN was attributed to the synchronized pharmacokinetics of DOX and MMC and increased intracellular drug bioavailability within tumor cells enabled by the nanocarrier PLN. 
To evaluate the pharmacokinetics and bio-distribution of co-administered DOX and MMC in both free solution and nanoparticle forms, a simple and efficient multi-drug analysis method using reverse-phase high performance liquid chromatography (HPLC) was developed. In contrast to previously reported methods that analyzed DOX or MMC individually in the plasma, this new HPLC method is able to simultaneously quantitate DOX, MMC and a major cardio-toxic DOX metabolite, doxorubicinol (DOXol), in various biological matrices (e.g., whole blood, breast tumor, and heart). A dual fluorescent and ultraviolet absorbent probe 4-methylumbelliferone (4-MU) was used as an internal standard (I.S.) for one-step detection of multiple drug analysis with different detection wavelengths. This method was successfully applied to determine the concentrations of DOX and MMC delivered by both nanoparticle and solution approaches in whole blood and various tissues in an orthotopic breast tumor murine model. The analytical method presented is a useful tool for pre-clinical analysis of nanoparticle-based delivery of drug combinations.

Two anticancer drugs, DOX and MMC, as well as the DOX metabolite, DOXol, were simultaneously detected without any biological interference under the same applied gradient HPLC condition using 4-MU as the I.S. for both the fluorescence and UV detectors. DOX, MMC, DOXol and 4-MU were well-separated from each other with retention times of 5.7 min for MMC, 10.4 min for DOXol, 10.9 min for 4-MU, and 11.1 min for DOX (Figure 2). Each drug in whole blood and various ...

Watch this Scientific Journal Video about Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-b

Sample Extraction and Simultaneous Chromatographic Quantitation of Doxorubicin and Mitomycin C Following Drug Combination Delivery in Nanoparticles to Tumor-bearing Mice

This protocol describes an efficient and convenient analytical process of sample extraction and simultaneous determination of multiple drugs, doxorubicin (DOX), mitomycin C (MMC) and a cardio-toxic DOX metabolite, doxorubicinol (DOXol), in the biological samples from a preclinical breast tumor model treated with nanoparticle formulations of synergistic drug combination.

Combination chemotherapy is frequently used in the clinic for cancer treatment; however, associated adverse effects to normal tissue may limit its ...

The overall goal of this analytical protocol is to simultaneously extract and quantitate the two anti-cancer drugs Doxorubicin and Mitomycin C, co-delivered by polymer-lipid hybrid nanoparticles, and the Doxorubicin's metabolite, Doxorubicinol, in biological matrices using a simple and robust reverse phase high performance liquid chromatography method. This method can help answer key questions in the field in nanomedicine, such as how to design an effective nanocarry system based on the nanopharmacokinetics of drug combinations. The main advantage of this technique is that it allows for the simultaneous quantitation of three drug compounds in the same biological matrix, without the need to change the mobile phase of HPLC.So, this method can provide insight into the biological behaviors of Doxorubicin, Mitomycin C, and Doxorubicinol in primary breast tumors. It can also be applied to other cancer types treated with similar drugs. Visual demonstration of this method is critical, as efficient sample preparation steps are difficult to learn.To begin this procedure, collect and freeze the whole blood, major organs, and breast tumor as outlined in the text protocol. Quickly weigh the frozen tissues and record the weight on the lab note book. Then, transfer them into a 13 milliliter round bottom conical tube.Next, add between one and five milliliters of ice cold Cell Lysis buffer. Using an electric hand homogenizer, homogenize the tissue with an up and down stroke motion on ice at 18, 000 rpm. After this, wash the homogenizer's 10 millimeter saw tooth generator probe with distilled deionized water.Wash the probe with 70%ethanol, and then again with distilled deionized water. Transfer 50 microliters of either tissue homogenate or whole blood to a 1.5 milliliter polypropylene microcentrifuge tube. Then, spike with five microliters of a 2, 000 nanogram per milliliter internal standard 4-MethylUmbelliferone.Add an ice cold extraction solvent containing 150 microliters of acetonitrile and 100 microliters of five millimolar ammonium acetate. Vigorously vortex the mixture for two minutes. Centrifuge at 3, 000 g and four degrees celsius for 10 minutes.After this, transfer 200 microliters of the supernatant to a fresh, pre-chilled microcentrifuge tube. Use a slow stream of nitrogen gas to evaporate the supernatant at 60 degrees celsius with protection from light. Next, reconstitute the dried residue with 100 microliters of ice cold methanol.Vigorously vortex for 30 seconds. Centrifuge at 3, 000 g and four degrees celsius for five minutes. Then, transfer the supernatant into an HPLC vial insert.Place the sample vials into an autosampler tray for injection. To begin, prepare the HPLC mobile phase as outlined in the text protocol. Set the initial conditions of the mobile phase composition to 16.5%H2O, and 83.5%acetonitrile.Set the Isocratic Flow Rate to 1.0 milliliters per minute. Then, separate the drugs using gradient mobile phase condition as outlined in the text protocol. Next, set the UV detector on two channels:one at 310 nanometers for internal standard 4-MethylUmbelliferone and the other at 360 nanometers for Mitomycin C.After this, set the first channel of the florescence detector to an excitation wavelength of 365 nanometers, and an emission wavelength of 445 nanometers for 4-MethylUmbelliferone.Set the second channel to an excitation wavelength of 480 nanometers, and an emission wavelength of 560 nanometers for Doxorubicin and Doxorubicinol. To begin, use the autosampler to inject 15 microliters of the sample. The programmed gradient mobile phase composition changes automatically by increasing the composition of acetonitrile over zero to 18 minutes.After 18 minutes, the mobile phase condition is reestablished to the initial condition and re-equilibrated for the next injection. After each sample is run, note the peaks of the drug compounds and their retention times. Then, use the HPLC software to integrate the peak area under the curve for the drug compounds.Calculate the drug recovery percentage and the area under the curve ratio between each drug compound and the internal standard, as outlined in the text protocol. In this procedure, two anticancer drugs, Doxorubicin and Mitomycin C, along with the Doxorubicin metabolite, Doxorubicinol, are detected without biological interference. HPLC analysis shows that each drug is well separated from the others.The HPLC method developed displays less than 15%variation in intra-and inter-day precision and accuracy for each of the drugs studied, in both whole blood and various biological matrices, indicating excellent reproducibility. The pharmacokinetic and bio distribution of long circulating or pegylated nanoparticle based co-delivery of Doxorubicin and Mitomycin C are then determined. In the blood, drug concentrations delivered by polymer-lipid hybrid nanoparticles over time are seen to be at least six fold higher than in the equivalent free drug solutions.In an orthotopic breast tumor, polymer-lipid hybrid nanoparticles can co-deliver Doxorubicin and Mitomycin C at their synergistic drug ratio. Compared to free drug solution, nanoparticles are seen to have increased tumor accumulation. This is due to the prolonged systematic circulation, which allows the nanoparticles to exploit the enhanced permeability and retention effects of the tumor.Further quantitatively determined formation of Doxorubicinol in the breast tumor over 24 hours, along with Enhanced Tumor Apoptosis, indicates that drug bioavailability is critical in designing an effective nanoparticle formulation. Using this HPLC method, it was successfully demonstrated that polymer-lipid hybrid nanoparticles can precisely deliver synergistic drug combination into cancer cells in vivo, leading to improved chemotherapy. This technique can be done in about two hours if it is performed properly.To minimize potential drug depredation, it is important to remember to avoid direct sunlight and to keep sample on ice while attempting this procedure. After its development, this technique paved the way for the researcher in the field of nanomedicine to explore nanopharmacokinetics of the drug combination, and to better design the effective nanoparticle formulation for cancer therapy. After watching this video, you should understand how to simultaneous extract and to detect Doxorubicin, Mitomycin C, and Doxorubicin's metabolite, Doxorubicinol, over a wide ranges of the biological samples containing nanoparticle loaded the drug combination.Don't forget that working with anticancer drugs and acid can be extremely hazardous, and safety precautions such as wearing gloves, goggles, and lab coats should always be applied while performing this protocol.

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Research

JoVE Journal

Cancer Research

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

Comprehensive Protocol to Sample and Process Bone Marrow for Measuring Measurable Residual Disease and Leukemic Stem Cells in Acute Myeloid Leukemia

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients have achieved complete remission (CR). Approximately half of the adult patients who entered CR will relapse within 12 months due to the outgrowth of residual AML cells in the bone marrow. The quantitation of these remaining leukemia cells, known as minimal or measurable residual disease (MRD), can be a robust biomarker for the prediction of these relapses. Moreover, retrospective analysis of several studies has shown that the presence of MRD in the bone marrow of AML patients correlates with poor survival. Not only is the total leukemic population, reflected by cells harboring a leukemia associated immune-phenotype (LAIP), associated with clinical outcome, but so is the immature low frequency subpopulation of leukemia stem cells (LSC), both of which can be monitored through flow cytometry MRD or MRD-like approaches. The availability of sensitive assays that enable detection of residual leukemia (stem) cells on the basis of disease-specific or disease-associated features (abnormal molecular markers or aberrant immunophenotypes) have drastically improved MRD assessment in AML. However, given the inherent heterogeneity and complexity of AML as a disease, methods for sampling bone marrow and performing MRD and LSC analysis should be harmonized when possible. In this manuscript we describe a detailed methodology for adequate bone marrow aspirate sampling, transport, sample processing for optimal multi-color flow cytometry assessment, and gating strategies to assess MRD and LSC to aid in therapeutic decision making for AML patients.

Detection of minimal or measurable residual disease (MRD) is an important prognostic biomarker for refining risk assessment and predicting relapse in acute myeloid leukemia (AML). These comprehensive guidelines and recommendations with best practices for consistent and accurate identification and detection of MRD, may aid in making effective AML treatment decisions.

Response criteria in acute myeloid leukemia (AML) has recently been re-established, with morphologic examination utilized to determine whether patients ...

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy against cancer. Here we provide a protocol to identify a rational combination at the preclinical step. First, we describe a cell-based assay to assess the synergism between anticancer mAb and cytotoxic drugs, that uses the combination index equation of Chou and Talalay1. This includes the measurement of tumor cell drug- and antibody-sensitivity using an MTT assay, followed by an automated computer analysis to calculate the combination index (CI) values. CI values of &lt;1 indicate synergism between tested mAbs and cytotoxic agents1. To corroborate the in vitro findings in vivo, we further describe a method to assess the combination regimen efficacy in a xenograft tumor model. In this model, the combined regimen significantly delays tumor growth, which results in a significant extended survival in comparison to single-agent controls. Importantly, the in vivo experimentation reveals that the combination regimen is well tolerated. This protocol allows the effective evaluation of anticancer drug combinations in preclinical models and the identification of rational combination to evaluate in clinical trials.

This protocol describes how to assess synergism between an anticancer antibody and antineoplastic drugs in preclinical models by using the combination index equation of Chou and Talalay.

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the tumor milieu can possess pro- or anti-tumor capacity. One of the important cytokines regulating neutrophil functions in a tumor context are type I interferons. In the presence of interferons, neutrophils gain anti-tumor properties, including cytotoxicity or stimulation of the immune system. Conversely, the absence of an interferon signaling results in prominent pro-tumor activity, characterized with strong stimulation of tumor angiogenesis. Recently, we could demonstrate that pro-angiogenic properties of neutrophils depend on the activation of nicotinamide phosphoribosyltransferase (NAMPT) signaling pathway in these cells. Inhibition of this pathway in tumor-associated neutrophils leads to their potent anti-angiogenic phenotype. Here, we demonstrate our newly established model allowing in vivo evaluation of tumorigenic potential of manipulated tumor-associated neutrophils (TANs). Shortly, pro-angiogenic tumor-associated neutrophils can be isolated from tumor-bearing interferon-deficient mice and repolarized into anti-angiogenic phenotype by blocking of NAMPT signaling. The angiogenic activity of these cells can be subsequently evaluated using an aortic ring assay. Anti-angiogenic TANs can be transferred into tumor-bearing wild type recipients and tumor growth should be monitored for 14 days. At day 14 mice are sacrificed, tumors removed and cut with their vascularization assessed. Overall, our protocol provides a novel tool to in vivo evaluate angiogenic capacity of primary cells, such as tumor-associated neutrophils, without a need to use artificial neutrophil cell line models. vc

Here, we show therapeutic potential of anti-angiogenic tumor-associated neutrophils after their transfer into tumor-bearing mice. This protocol can be used to manipulate neutrophil activity ex vivo and to subsequently evaluate their functionality in vivo&#160;in developing tumors. It is an appropriate model for studying potential neutrophil-based immunotherapies.

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...

Exploring Mitochondrial Energy Metabolism of Single 3D Microtissue Spheroids Using Extracellular Flux Analysis

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing cells as two-dimensional, single-cell monolayers (2D culture), spheroid cell culture promotes, regulates, and supports physiological cellular architecture and characteristics that exist in vivo, including the expression of extracellular matrix proteins, cell signaling, gene expression, protein production, differentiation, and proliferation. The importance of 3D culture has been recognized in many research fields, including oncology, diabetes, stem cell biology, and tissue engineering. Over the last decade, improved methods have been developed to produce spheroids and assess their metabolic function and fate.
Extracellular flux (XF) analyzers have been used to explore mitochondrial function in 3D microtissues such as spheroids using either an XF24 islet capture plate or an XFe96 spheroid microplate. However, distinct protocols and the optimization of probing mitochondrial energy metabolism in spheroids using XF technology have not been described in detail. This paper provides detailed protocols for probing mitochondrial energy metabolism in single 3D spheroids using spheroid microplates with the XFe96 XF analyzer. Using different cancer cell lines, XF technology is demonstrated to be capable of distinguishing between cellular respiration in 3D spheroids of not only different sizes but also different volumes, cell numbers,&#160;DNA content and type.
The optimal mitochondrial effector compound concentrations of oligomycin, BAM15, rotenone, and antimycin A are used to probe specific parameters of mitochondrial energy metabolism in 3D spheroids. This paper also discusses methods to normalize data obtained from spheroids and addresses many considerations that should be considered when exploring spheroid metabolism using XF technology. This protocol will help drive research in advanced in vitro spheroid models.

These protocols will help users probe mitochondrial energy metabolism in 3D cancer cell-line-derived spheroids using Seahorse extracellular flux analysis.

Three-dimensional (3D) cellular aggregates, termed spheroids, have become the forefront of in vitro cell culture in recent years. In contrast to culturing ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...