Method Article

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

DOI:

10.3791/56159

⸱

October 5th, 2017

 ,  ,  ,  ,  ,  ,  , 
1Department of Pharmaceutical Sciences, University of Toronto, 2Departments of Medical Biophysics and Radiation Oncology, University of Toronto, Ontario Cancer Institute, University Health Network

In This Article

Summary

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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.

Abstract

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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.

Introduction

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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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Protocol

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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

  1. 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)
    1. Inject a breast tumor-bearing mouse i.v. with a prepared drug-containing formulation.
    2. Anesthetize the mouse at designated time-points (e.g., 15 min) by giving inhalable 2% isoflurane in a sealed chamber.
    3. 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.
    4. 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.
    5. 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 µL of whole blood. Always keep the samples on ice.
    6. 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.
    7. 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° for guiding the syringe into the portal vein.
    8. 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.
      ​NOTE: Separate the gallbladder from the liver.
    9. Store the whole blood at 4 °C and excised tissues in the -80 °C freezer until later HPLC analysis.
  2. Extract DOX, MMC and DOXol from biological matrices.
    1. 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.
    2. 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.
    3. 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.
    4. 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.
    5. Transfer 50 µL of tissue homogenate or whole blood into a 1.5 mL polypropylene micro-centrifuge tube and spike with 5 µL 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.
    6. Add 250 µL 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.
    7. Vigorously vortex the mixture for 2 min, centrifuge at 3,000 x g force at 4 oC for 10 min and pipet 200 µL supernatant into another pre-chilled fresh micro-centrifuge tube.
    8. Evaporate supernatant at 60 °C under a slow stream of nitrogen gas with protection from light.
    9. Reconstitute the dried residue with 100 µL of ice-cold methanol, vigorously vortex for 30 s and centrifuge at 3000 x g at 4 °C for another 5 min.
    10. Transfer the supernatant into a HPLC vial insert and place sample vials into an autosampler tray for injection.

2. HPLC Instrumentation and Operation Parameters

  1. Prepare HPLC mobile-phase with consistent reproducibility
    1. Measure 500 mL of HPLC-grade H2O using a graduated cylinder.
    2. Measure 500 mL of HPLC-grade acetonitrile (ACN) using a separate graduated cylinder.
    3. 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.
    4. Filter mobile phases through a nylon membrane filter with a 0.45 µm pore size and transfer it into clean HPLC reservoir bottles.
  2. Set-up HPLC instrumentation for simultaneous detection of DOX, MMC, and DOXol and I.S. 4-MU.
    1. Switch on the gradient pump, de-gasser, auto-sampler, photodiode array detector, and multi λ fluorescence detector.
    2. Input the initial conditions of mobile-phase composition to 16.5% H2O (0.1% TFA) and 83.5% ACN (0.1% TFA) (v/v).
    3. Set the UV detector on two channels, one at 310 nm for 4-MU (I.S.) and the other at 360 nm for MMC.
    4. Set the fluorescence detector on two channels, one at λex/λem =365/445 nm for 4-MU and the other at λex/ λem=480 nm/560 nm for DOX and DOXol, respectively.
    5. Set an isocratic flow rate of 1.0 mL/min.
    6. Equilibrate a preinstalled reverse phase C18 column (4.6 mm x 250 mm, 5 µm) at room temperature for 10 min for baseline establishment.
  3. Separate drugs (DOX, MMC, DOXol and 4-MU) using gradient mobile-phase condition.
    1. Inject 15 µL of extracted and re-concentrated samples using the auto-sampler.
    2. 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).
    3. Maintain 100% of ACN (0.1% TFA) for 1 min and then return to the initial mobile phase condition within 1 min.
    4. 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.

3. HPLC Validation

  1. Prepare working standards of DOX, MMC and DOXol, and 4-MU (I.S.).
    1. 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.
    2. Transfer the weighed DOX, MMC and 4-MU into a new individual 1.5 mL polypropylene micro-centrifuge tube.
    3. Add 1 mL of methanol and vortex briefly to obtain 1 mg/mL concentration of DOX and MMC.
    4. 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.
    5. Pipette 20 µL 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 µL of methanol to obtain a working standard of 20 µg/mL of each drug.
    6. Dilute 20 µg/mL of DOX, MMC and DOXol using methanol to obtain the working standards of 50 ng - 20 µg /mL for DOX, MMC, and DOXol and 2000 ng/mL for I.S. 4-MU.
    7. 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 °C.
  2. Determine the linearity, precision, and accuracy of DOX, MMC and DOXol in biological matrices (i.e., whole blood and tumor homogenate).
    1. Simultaneously spike 5 µL of working standards of DOX and DOXol (50 ng/mL - 20 µg/mL), MMC (1,000 ng/mL - 16 µg/mL), and 4-MU (2 µg/mL) into 50 µL 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.).
    2. Perform the drug extraction assay described in Protocol 1.2.
    3. 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.
  3. Analysis of samples
    1. Inject 15 µL of the sample using the auto-sampler.
    2. Gradually change the mobile phase over 0 to 18 min, increasing the composition of ACN over the interval.
    3. After 18 min, hold the mobile phase condition for 1 min.
    4. Return to the initial condition over the next 2 min, then re-equilibrate for 4 min before the next injection.
    5. 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.
    6. Integrate the peak area under curve (AUC) of drug compounds using HPLC software.
    7. 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.
      Peak area ratio formula AUC<sub>drug</sub>/AUC<sub>IS</sub>; analytical chemistry equation.
    8. 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 ("neat") drug solution in methanol.
      Drug recovery percentage formula: Peak Area Ratio calculation; scientific analysis equation.

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Results

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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 ...

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Discussion

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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...

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Disclosures

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

Acknowledgements

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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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Doxorubicin Polymed Theraeutics111023Anticancer drug
Mitomycin CPolymed Theraeutics060814Anticancer drug
Doxorubicinol (DOXol)Toronto Research ChemicalsD558020Metabolite of DOX
4-Methylumbelliferone sodium salt Sigma-AldrichM1508Internal standard
Myristic AcidSigma-Aldrich544-63-8  Materials for poly-lipid hybrid nanoparticles
Polyoxyethylene (100) StearateSpectrumM1402Materials for poly-lipid hybrid nanoparticles
Polyoxyethylene (40) StearateSigma-AldrichP3440Materials for poly-lipid hybrid nanoparticles
Pluronic F68 (PF68)BASF Corp.9003-11-6Materials for poly-lipid hybrid nanoparticles
Ultrasonication (UP100H)Hielscher, Ultrasound TechnologyNANanoparticle preparation
Water Bath (ISOTEMP 3016HS)Fisher ScientificNANanoparticle preparation
Liposomal Doxorubicin  (Caelyx)JanssenPurchased from the pharmacy Princess Margaret HospitalClinically-approved nanoparticle formulation 
HPLC-graded MethanolCaledon Chemicals6701-7-40HPLC mobile phase composition
HPLC-graded H2OCaledon Chemicals8801-7-40HPLC mobile phase composition
HPLC-graded Acetonitrile Caledon Chemicals1401-7-40HPLC mobile phase composition
Trifluoroacetic AcidSigma-Aldrich302031HPLC mobile phase composition
0.45 μm Nylon Membrane Filter PaperWhatmanWHA7404004HPLC mobile phase preparation
1cc Plastic SyringesBecton, Dickinson and Company2606-309659Treatment injection
5cc Plastic SyringesBecton, Dickinson and Company2608-309646Tissue collections
30G 1/2 NeedlesBecton, Dickinson and Company305106Treatment injection
25G 5/8 NeedlesBecton, Dickinson and Company305122Tissue collections
Sterile 0.9% SalineUniveristy of Toronto House Brand1011Tissue perfusion
13 ml Rounded-bottom conical tube SARSTEDT62.515.006Prolyprolene, tissue homogenization
Alpha Minimum Essential Medium (MEM) Gibco12571063Cell medium
1 x Phosphate Buffer SalineGibco10010023Tissue homogenization
Triton X-100Sigma-AldrichX100-100 MLTissue homogenization
Formic acidCaledon Chemicals1/5/3840Adjust pH for extraction solvent
Sodium heparin sprayed plastic tubesBecton, Dickinson and Company367878Blood collection
Analytical Weigh Balance Sartorius CPA225DNA
pH meters Fisher Scientific13-637-671accumet BASIC
Vortex MixterFisher Scientific02-215-365Vortexing samples at desired speed
1.5 ml  Microcentrifuge TubeFisherbrand2043-05408129Prolyprolene
Model 1000 homogenizerFisher Scientific08-451-672Tissue homogenization
Centrifuge 5702REppendorf5702RExtraction preparation
Heated Evaporator SystemGlas-ColNASample reconstitution
HPLC Screw Thread VialsDIKMA5320HPLC sample injection
HPLC Screw Caps with PTFE White Silicone SeptaDIKMA5325HPLC sample injection
HPLC Polypropylene Insert  Agilent Technologies5182-0549Maximum volume 250 μl, HPLC sample injection
Xbridge C18 ColumnWaters Corporation186003117Drug analysis
Gradient pump Waters CorporationW600Drug analysis
Auto-samplerWaters CorporationW2707Drug analysis
Photodiode array detector Waters CorporationW2998Drug analysis
Multi λ fluoresence detector Waters CorporationW2475Drug analysis
EMPOWER 2Waters CorporationNAData analysis software
ScientistMicromathNAPharmacokinetic analysis
Female Balb/c MiceJackson Laboratory001026In vivo
EMT6/WT Breast Cancer CellsProvided by Dr. Ian Tannock; Ontario Cancer InstituteNAIn vivo

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Doxorubicin Mitomycin CHPLC QuantitationDrug Combination AnalysisNanoparticle Drug DeliveryReverse Phase HPLCBiological Matrix ExtractionInternal Standard MethodSimultaneous Drug DetectionPharmacokinetics StudyTumor Tissue Analysis

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