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-bearing mouse i.v. with a prepared drug-containing formulation.</li>
		<li>Anesthetize the mouse at designated time-points (e.g., 15 min) by giving inhalable 2% isoflurane in a sealed chamber.</li>
		<li>Lay the anesthetized mouse on its back and put its nose through a nosepiece that constantly supplies 2% isoflurane. 
		NOTE: To ensure the mouse undergoes deep anesthesia, gently pinch fore limbs of the mouse and look for any twitching movement.</li>
		<li>Thoroughly clean the chest and abdomen regions using 70% ethanol and then perform a terminal procedure of cardiac puncture on the deep anesthetized mice using a heparinized 1 mL syringe and a 23 G needle.</li>
		<li>Collect the whole blood into a labeled sodium heparin sprayed plastic tube and gently swirl the tube to ensure collected whole blood comes into contact with the coated heparin of the tube wall. Collect a minimum of 50 &#181;L of whole blood. Always keep the samples on ice.</li>
		<li>Tape all four limbs of the mouse to secure it and open the abdominal cavity and ribcage of the mouse using a pair of scissors and forceps. Shift the intestines to the side and push the liver upward to sufficiently expose the portal vein. Cut the portal vein for the blood drainage.</li>
		<li>Perfuse the whole mouse body with 50 mL of ice-cold 0.9% saline through the heart using a 10 mL syringe with a 25 G needle. 
		NOTE: Bend the needle at 90&#176; for guiding the syringe into the portal vein.</li>
		<li>Excise organs in the following order: heart, lung, liver, spleen, kidneys. Then, separate the breast tumor from the surrounding connective tissues using a pair of incision scissors at the right mammary fat pad of the mouse. Collect all organs individually into 1.5 mL polypropylene tubes and quickly freeze them in liquid nitrogen. 
		&#8203;NOTE: Separate the gallbladder from the liver.</li>
		<li>Store the whole blood at 4 &#176;C and excised tissues in the -80 &#176;C freezer until later HPLC analysis.</li>
	</ol>
	</li>
	<li>Extract DOX, MMC and DOXol from biological matrices.
	<ol>
		<li>Weigh all frozen dissected tissues quickly and transfer them into a 13 mL rounded-bottom conical tube. To avoid possible drug metabolism or degradation, keep the samples on ice.</li>
		<li>Add 1-5 mL of ice-cold cell lysis buffer into the tube. 
		NOTE: The volume of buffer to use depends on the tissue weight based on the tissue-buffer ratio of 1 g: 5 mL (w/v); for small organs, such as heart and spleen, the ratio is 1 g: 2 mL.</li>
		<li>Use an up-down stroke motion to homogenize the tissue samples on ice at a speed of 18,000 rpm using an electric hand homogenizer. 
		NOTE: Completed homogenization requires approximately 3 to 5 iterations of a short homogenization process of less than 15 s, followed by tissue cooling over ice between each short homogenization.</li>
		<li>Wash the 10 mm saw-tooth generator probe of the homogenizer with distilled deionized (DDI) H2O, 70% ethanol, and then DDI H2O between each tissue sample to avoid cross-contamination.</li>
		<li>Transfer 50 &#181;L of tissue homogenate or whole blood into a 1.5 mL polypropylene micro-centrifuge tube and spike with 5 &#181;L of an internal standard (I.S.) 4-methylumbelliferone (4-MU) (2000 ng/mL) into the tube. 
		NOTE: 4-MU solution was prepared in methanol here.</li>
		<li>Add 250 &#181;L of an ice-cold extraction solvent into the tube containing whole blood or tissue homogenate. 
		NOTE: The extraction solvent consists of 60% acetonitrile (ACN) and 40% ammonium acetate (5 mM) with pH adjusted to pH = 3.5 using 0.05% formic acid. Use a 1:5 (v/v) sample: extraction solvent to volume ratio.</li>
		<li>Vigorously vortex the mixture for 2 min, centrifuge at 3,000 x g force at 4 oC for 10 min and pipet 200 &#181;L supernatant into another pre-chilled fresh micro-centrifuge tube.</li>
		<li>Evaporate supernatant at 60 &#176;C under a slow stream of nitrogen gas with protection from light.</li>
		<li>Reconstitute the dried residue with 100 &#181;L of ice-cold methanol, vigorously vortex for 30 s and centrifuge at 3000 x g at 4 &#176;C for another 5 min.</li>
		<li>Transfer the supernatant into a HPLC vial insert and place sample vials into an autosampler tray for injection.</li>
	</ol>
	</li>
</ol>
2. HPLC Instrumentation and Operation Parameters
<ol>
	<li>Prepare HPLC mobile-phase with consistent reproducibility
	<ol>
		<li>Measure 500 mL of HPLC-grade H2O using a graduated cylinder.</li>
		<li>Measure 500 mL of HPLC-grade acetonitrile (ACN) using a separate graduated cylinder.</li>
		<li>Carefully add 0.5 mL of trifluoroacetic acid (TFA) (CAUTION) into each of 500 mL of H2O and ACN to obtain the mobile phase of H2O and ACN containing 0.1% TFA, respectively. 
		NOTE: TFA is corrosive and toxic and should be handled under a laboratory fume hood. All solvent mixtures are prepared at room temperature.</li>
		<li>Filter mobile phases through a nylon membrane filter with a 0.45 &#181;m pore size and transfer it into clean HPLC reservoir bottles.</li>
	</ol>
	</li>
	<li>Set-up HPLC instrumentation for simultaneous detection of DOX, MMC, and DOXol and I.S. 4-MU.
	<ol>
		<li>Switch on the gradient pump, de-gasser, auto-sampler, photodiode array detector, and multi &#955; fluorescence detector.</li>
		<li>Input the initial conditions of mobile-phase composition to 16.5% H2O (0.1% TFA) and 83.5% ACN (0.1% TFA) (v/v).</li>
		<li>Set the UV detector on two channels, one at 310 nm for 4-MU (I.S.) and the other at 360 nm for MMC.</li>
		<li>Set the fluorescence detector on two channels, one at &#955;ex/&#955;em =365/445 nm for 4-MU and the other at &#955;ex/ &#955;em=480 nm/560 nm for DOX and DOXol, respectively.</li>
		<li>Set an isocratic flow rate of 1.0 mL/min.</li>
		<li>Equilibrate a preinstalled reverse phase C18 column (4.6 mm x 250 mm, 5 &#181;m) at room temperature for 10 min for baseline establishment.</li>
	</ol>
	</li>
	<li>Separate drugs (DOX, MMC, DOXol and 4-MU) using gradient mobile-phase condition.
	<ol>
		<li>Inject 15 &#181;L of extracted and re-concentrated samples using the auto-sampler.</li>
		<li>Gradually change the initial mobile-phase condition (refer to protocol step 2.2.2) to 100% ACN (0.1% TFA) over 18 min using the automated gradient pump. 
		NOTE: During the separation process, four channels (two UV absorbent and two fluorescent) appear simultaneously with each channel displaying one drug compound (refer to Protocol step 2.2.3 and 2.2.4).</li>
		<li>Maintain 100% of ACN (0.1% TFA) for 1 min and then return to the initial mobile phase condition within 1 min.</li>
		<li>Re-condition the column with the initial mobile phase at flow rate of 1.5 mL/min for 4 min for the next sample injection.</li>
	</ol>
	</li>
</ol>
3. HPLC Validation
<ol>
	<li>Prepare working standards of DOX, MMC and DOXol, and 4-MU (I.S.).
	<ol>
		<li>Weigh separately 1 mg of DOX and MMC drug powder (CAUTION) and 4-MU on a fresh small weighing paper (3 x 3 inches2). 
		Note All anticancer drugs are considered a health hazard that can cause acute toxicity and germ cell mutagenicity on inhalation or ingestion. They should be handled carefully with gloves and masks.</li>
		<li>Transfer the weighed DOX, MMC and 4-MU into a new individual 1.5 mL polypropylene micro-centrifuge tube.</li>
		<li>Add 1 mL of methanol and vortex briefly to obtain 1 mg/mL concentration of DOX and MMC.</li>
		<li>Add 1 mL of methanol into a vial containing pre-weighed 1 mg of DOXol (CAUTION) and vortex briefly to obtain 1 mg/mL concentration of DOXol. 
		NOTE: DOXol is a cardio-toxic metabolite and should be handled carefully.</li>
		<li>Pipette 20 &#181;L of prepared stock solutions of DOX, MMC, DOXol and 4-MU into a new separate 1.5 mL polypropylene micro-centrifuge tube and add 980 &#181;L of methanol to obtain a working standard of 20 &#181;g/mL of each drug.</li>
		<li>Dilute 20 &#181;g/mL of DOX, MMC and DOXol using methanol to obtain the working standards of 50 ng - 20 &#181;g /mL for DOX, MMC, and DOXol and 2000 ng/mL for I.S. 4-MU.</li>
		<li>Seal the cap of the tube of working solutions with a narrow piece of paraffin film covering to prevent methanol evaporation, wrap the entire tube with aluminum foil to avoid exposure to direct light and store at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Determine the linearity, precision, and accuracy of DOX, MMC and DOXol in biological matrices (i.e., whole blood and tumor homogenate).
	<ol>
		<li>Simultaneously spike 5 &#181;L of working standards of DOX and DOXol (50 ng/mL - 20 &#181;g/mL), MMC (1,000 ng/mL - 16 &#181;g/mL), and 4-MU (2 &#181;g/mL) into 50 &#181;L of blank whole blood or tissue homogenate in polypropylene micro-centrifuge tubes to obtain the standard concentration curve ranging from 5 - 2000 ng/mL for drug compounds and 200 ng/mL for 4-MU (I.S.).</li>
		<li>Perform the drug extraction assay described in Protocol 1.2.</li>
		<li>Use low, median and high concentrations of DOX and DOXol (50, 500, and 2,000 ng/mL) and MMC (100, 1000, 2,000 ng/mL) for intra- and inter-day precision and accuracy. 
		NOTE: Prepare fresh standard concentrations on the day of analysis.</li>
	</ol>
	</li>
	<li>Analysis of samples
	<ol>
		<li>Inject 15 &#181;L of the sample using the auto-sampler.</li>
		<li>Gradually change the mobile phase over 0 to 18 min, increasing the composition of ACN over the interval.</li>
		<li>After 18 min, hold the mobile phase condition for 1 min.</li>
		<li>Return to the initial condition over the next 2 min, then re-equilibrate for 4 min before the next injection.</li>
		<li>After each sample run, note that the peaks of drug compounds with their retention time are shown as follow: MMC, DOXol, 4-MU (I.S.) and DOX.</li>
		<li>Integrate the peak area under curve (AUC) of drug compounds using HPLC software.</li>
		<li>Calculate the AUC ratio between individual drug compound and I.S. (Equation 1) and use the standard curves prepared under the same extraction procedures to determine the drug concentrations of DOX, MMC and DOXol in DMPLN formulation. 
		<img alt="Equation 1" src="/files/ftp_upload/56159/56159eq1.jpg" /></li>
		<li>Calculate the drug recovery percentage (Equation 2) by comparing the drug concentrations reconstituted using methanol from the extracts of spiked biological samples to that of the standard (&#34;neat&#34;) drug solution in methanol. 
		<img alt="Equation 2" src="/files/ftp_upload/56159/56159eq2.jpg" /></li>
	</ol>
	</li>
</ol>

The authors have no competing financial interests and conflicts of interest.

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.

<table><tbody><tr><td>Doxorubicin&amp;nbsp;</td><td>Polymed Theraeutics</td><td>111023</td><td>Anticancer drug</td></tr><tr><td>Mitomycin C</td><td>Polymed Theraeutics</td><td>060814</td><td>Anticancer drug</td></tr><tr><td>Doxorubicinol (DOXol)</td><td>Toronto Research Chemicals</td><td>D558020</td><td>Metabolite of DOX</td></tr><tr><td>4-Methylumbelliferone sodium salt&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M1508</td><td>Internal standard</td></tr><tr><td>Myristic Acid</td><td>Sigma-Aldrich</td><td>544-63-8&amp;nbsp;&amp;nbsp;</td><td>Materials for poly-lipid hybrid nanoparticles</td></tr><tr><td>Polyoxyethylene (100) Stearate</td><td>Spectrum</td><td>M1402</td><td>Materials for poly-lipid hybrid nanoparticles</td></tr><tr><td>Polyoxyethylene (40) Stearate</td><td>Sigma-Aldrich</td><td>P3440</td><td>Materials for poly-lipid hybrid nanoparticles</td></tr><tr><td>Pluronic F68 (PF68)</td><td>BASF Corp.</td><td>9003-11-6</td><td>Materials for poly-lipid hybrid nanoparticles</td></tr><tr><td>Ultrasonication (UP100H)</td><td>Hielscher, Ultrasound Technology</td><td>NA</td><td>Nanoparticle preparation</td></tr><tr><td>Water Bath (ISOTEMP 3016HS)</td><td>Fisher Scientific</td><td>NA</td><td>Nanoparticle preparation</td></tr><tr><td>Liposomal Doxorubicin&amp;nbsp; (Caelyx)</td><td>Janssen</td><td>Purchased from the pharmacy Princess Margaret Hospital</td><td>Clinically-approved nanoparticle formulation&amp;nbsp;</td></tr><tr><td>HPLC-graded Methanol</td><td>Caledon Chemicals</td><td>6701-7-40</td><td>HPLC mobile phase composition</td></tr><tr><td>HPLC-graded H&lt;span class=&quot;font10&quot;&gt;&lt;sub&gt;2&lt;/sub&gt;&lt;/span&gt;&lt;span class=&quot;font8&quot;&gt;O&lt;/span&gt;</td><td>Caledon Chemicals</td><td>8801-7-40</td><td>HPLC mobile phase composition</td></tr><tr><td>HPLC-graded Acetonitrile&amp;nbsp;</td><td>Caledon Chemicals</td><td>1401-7-40</td><td>HPLC mobile phase composition</td></tr><tr><td>Trifluoroacetic Acid</td><td>Sigma-Aldrich</td><td>302031</td><td>HPLC mobile phase composition</td></tr><tr><td>0.45 &amp;mu;m Nylon Membrane Filter Paper</td><td>Whatman</td><td>WHA7404004</td><td>HPLC mobile phase preparation</td></tr><tr><td>1cc Plastic Syringes</td><td>Becton, Dickinson and Company</td><td>2606-309659</td><td>Treatment injection</td></tr><tr><td>5cc Plastic Syringes</td><td>Becton, Dickinson and Company</td><td>2608-309646</td><td>Tissue collections</td></tr><tr><td>30G 1/2 Needles</td><td>Becton, Dickinson and Company</td><td>305106</td><td>Treatment injection</td></tr><tr><td>25G 5/8 Needles</td><td>Becton, Dickinson and Company</td><td>305122</td><td>Tissue collections</td></tr><tr><td>Sterile 0.9% Saline</td><td>Univeristy of Toronto House Brand</td><td>1011</td><td>Tissue perfusion</td></tr><tr><td>13 ml Rounded-bottom conical tube&amp;nbsp;</td><td>SARSTEDT</td><td>62.515.006</td><td>Prolyprolene, tissue homogenization</td></tr><tr><td>Alpha Minimum Essential Medium (MEM)&amp;nbsp;</td><td>Gibco</td><td>12571063</td><td>Cell medium</td></tr><tr><td>1 x Phosphate Buffer Saline</td><td>Gibco</td><td>10010023</td><td>Tissue homogenization</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100-100 ML</td><td>Tissue homogenization</td></tr><tr><td>Formic acid</td><td>Caledon Chemicals</td><td>1/5/3840</td><td>Adjust pH for extraction solvent</td></tr><tr><td>Sodium heparin sprayed plastic tubes</td><td>Becton, Dickinson and Company</td><td>367878</td><td>Blood collection</td></tr><tr><td>Analytical Weigh Balance&amp;nbsp;</td><td>Sartorius&amp;nbsp;</td><td>CPA225D</td><td>NA</td></tr><tr><td>pH meters&amp;nbsp;</td><td>Fisher Scientific</td><td>13-637-671</td><td>accumet BASIC</td></tr><tr><td>Vortex Mixter</td><td>Fisher Scientific</td><td>02-215-365</td><td>Vortexing samples at desired speed</td></tr><tr><td>1.5 ml&amp;nbsp; Microcentrifuge Tube</td><td>Fisherbrand</td><td>2043-05408129</td><td>Prolyprolene</td></tr><tr><td>Model 1000 homogenizer</td><td>Fisher Scientific</td><td>08-451-672</td><td>Tissue homogenization</td></tr><tr><td>Centrifuge 5702R</td><td>Eppendorf</td><td>5702R</td><td>Extraction preparation</td></tr><tr><td>Heated Evaporator System</td><td>Glas-Col</td><td>NA</td><td>Sample reconstitution</td></tr><tr><td>HPLC Screw Thread Vials</td><td>DIKMA</td><td>5320</td><td>HPLC sample injection</td></tr><tr><td>HPLC Screw Caps with PTFE White Silicone Septa</td><td>DIKMA</td><td>5325</td><td>HPLC sample injection</td></tr><tr><td>HPLC Polypropylene Insert&amp;nbsp;&amp;nbsp;</td><td>Agilent Technologies</td><td>5182-0549</td><td>Maximum volume 250 &amp;mu;l, HPLC sample injection</td></tr><tr><td>Xbridge C&lt;span class=&quot;font10&quot;&gt;&lt;sub&gt;18&lt;/sub&gt;&lt;/span&gt;&lt;span class=&quot;font8&quot;&gt; Column&lt;/span&gt;</td><td>Waters Corporation</td><td>186003117</td><td>Drug analysis</td></tr><tr><td>Gradient pump&amp;nbsp;</td><td>Waters Corporation</td><td>W600</td><td>Drug analysis</td></tr><tr><td>Auto-sampler</td><td>Waters Corporation</td><td>W2707</td><td>Drug analysis</td></tr><tr><td>Photodiode array detector&amp;nbsp;</td><td>Waters Corporation</td><td>W2998</td><td>Drug analysis</td></tr><tr><td>Multi &amp;lambda; fluoresence detector&amp;nbsp;</td><td>Waters Corporation</td><td>W2475</td><td>Drug analysis</td></tr><tr><td>EMPOWER 2</td><td>Waters Corporation</td><td>NA</td><td>Data analysis software</td></tr><tr><td>Scientist</td><td>Micromath</td><td>NA</td><td>Pharmacokinetic analysis</td></tr><tr><td>Female Balb/c Mice</td><td>Jackson Laboratory</td><td>001026</td><td>In vivo</td></tr><tr><td>EMT6/WT Breast Cancer Cells</td><td>Provided by Dr. Ian Tannock; Ontario Cancer Institute</td><td>NA</td><td>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 ...

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Pharmacokinetics and Pharmacodynamics

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10.8K Views.  University of Toronto. 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.

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

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past decade. However, only a small number of nanomedicines have been approved for clinical use, whereas the majority of nanomedicines under clinical development have produced disappointing results. One major obstacle to the successful clinical translation of new cancer nanomedicines is the lack of an accurate understanding of their in vivo performance. This article features a rigorous procedure to characterize the in vivo behavior of nanomedicines in tumor-bearing mice at systemic, tissue, single-cell, and subcellular levels via the integration of positron emission tomography-computed tomography (PET-CT), radioactivity quantification methods, flow cytometry, and fluorescence microscopy. Using this approach, researchers can accurately evaluate novel nanoscale formulations in relevant mouse models of cancer. These protocols may have the ability to identify the most promising cancer nanomedicines with high translational potential or to aid in the optimization of cancer nanomedicines for future translation.

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

The poor understanding of the in vivo performance of nanomedicines stymies their clinical translation. Procedures to evaluate the in vivo behavior of cancer nanomedicines at systemic, tissue, single-cell, and subcellular levels in tumor-bearing immunocompetent mice are described here. This approach may help researchers to identify promising cancer nanomedicines for clinical translation.

Inspired by the success of previous cancer nanomedicines in the clinic, researchers have generated a large number of novel formulations in the past ...

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Medicine

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Bioengineering

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. HerDox is formed by non-covalent self-assembly of a tumor targeted cell penetration protein with the chemotherapy agent, doxorubicin, via a small nucleic acid linker. A combination of electrophilic, intercalation, and oligomerization interactions facilitate self-assembly into round 10-20 nm particles. HerDox exhibits stability in blood as well as in extended storage at different temperatures. Systemic delivery of HerDox in tumor-bearing mice results in tumor-cell death with no detectable adverse effects to non-tumor tissue, including the heart and liver (which undergo marked damage by untargeted doxorubicin). HER2 elevation facilitates targeting to cells expressing the human epidermal growth factor receptor, hence tumors displaying elevated HER2 levels exhibit greater accumulation of HerDox compared to cells expressing lower levels, both in vitro and in vivo. Fluorescence intensity imaging combined with in situ confocal and spectral analysis has allowed us to verify in vivo tumor targeting and tumor cell penetration of HerDox after systemic delivery. Here we detail our methods for assessing tumor targeting via multimode imaging after systemic delivery.

This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here.

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous environment through self-assembly. The hydrophobic core of micelles functions as a drug reservoir and encapsulates hydrophobic drugs. The hydrophilic shell prevents the aggregation of micelles and also prolongs their systemic circulation in vivo. In this protocol, we describe a method to synthesize a doxorubicin lipophilic pro-drug, doxorubicin-palmitic acid (DOX-PA), which will enhance drug loading into micelles. A pH-sensitive hydrazone linker was used to conjugate doxorubicin with the lipid, which facilitates the release of free doxorubicin inside cancer cells. Synthesized DOX-PA was purified with a silica gel column using dichloromethane/methanol as the eluent. Purified DOX-PA was analyzed with thin layer chromatography (TLC) and 1H-Nuclear Magnetic Resonance Spectroscopy (1H-NMR). A film dispersion method was used to prepare DOX-PA loaded DSPE-PEG micelles. In addition, several methods for characterizing micelle formulations are described, including determination of DOX-PA concentration and encapsulation efficiency, measurement of particle size and distribution, and assessment of in vitro anticancer activities. This protocol provides useful information regarding the preparation and characterization of drug-loaded micelles and thus will facilitate the research and development of novel micelle-based cancer nanomedicines.

A protocol for the preparation and characterization of lipophilic doxorubicin pro-drug loaded 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] (DSPE-PEG) micelles is described.

Micelles have been successfully used for the delivery of anticancer drugs. Amphiphilic polymers form core-shell structured micelles in an aqueous ...

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...

Chemistry

Nanodrug Targeting Oncogenic miRNA-10b for Treating Metastatic Breast Cancer

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or &#34;nanodrug&#34;) administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have ...

Iron Oxide Nanoparticles for Image-Guided Therapy Delivery in Murine Glioblastoma

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

Author Spotlight: Innovative Cancer Therapies with Iron Oxide Nanoparticles for Glioblastoma Treatment

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice

Tumor and tumor vessel development, as well as tumor response to therapeutics, are highly dynamic biological processes. Histology provides static information and is often not sufficient for a correct interpretation. Intravital evaluation, in which a process is followed in time, provides extra and often unexpected information. With the creation of transgenic animals expressing cell-specific markers and live cell tracers, improvements to imaging equipment, and the development of several imaging chambers, intravital microscopy has become an important tool to better understand biological processes. This paper describes an experimental design for the investigation of tumor vessel development and of therapeutic effects in a spatial and temporal manner. Using this setup, the stage of vessel development, tip cell and lumen formation, blood flow, extravasation, an established vascular bed, and vascular destruction can be visualized and followed. Furthermore, therapeutic effects, intratumoral fate, and the localization of chemotherapeutic compounds can also be followed.

This protocol describes the intravital imaging of transgenic mice expressing cell-specific fluorescent markers. Intravital imaging provides a non-invasive method for high-resolution observations in living animals using implantable windows through which the microscopic visualization of processes is possible. This method is especially useful to study longitudinal processes.

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

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Chapters in this video

A Comprehensive Procedure to Evaluate the In Vivo Performance of Cancer Nanomedicines

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Viral Nanoparticles for In vivo Tumor Imaging

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

Preparation and Characterization of Lipophilic Doxorubicin Pro-drug Micelles

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Intravital Microscopy of Tumor-associated Vasculature Using Advanced Dorsal Skinfold Window Chambers on Transgenic Fluorescent Mice