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 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.
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.
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
2. HPLC Instrumentation and Operation Parameters
3. HPLC Validation
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 tissues showed concentration linearity with correlation coefficients (R2) ranging from 0.98 to 1.00 (Figure 3 and Table 1). The lower limit of quantitation (LLOQ) of DOX, DOXol and MMC was 10 ng/mL, 10 ng/mL and 100 ng/mL in whole blood and 25 ng/mL, 25 ng/mL and 200 ng/mL in various tissues, respectively (Table 1). The HPLC method developed displayed less than 15% variation in terms of intra- and inter-day precision and accuracy for DOX, MMC and DOXol in whole blood and various biological matrices (e.g., heart, lungs, liver, spleen, and kidneys), indicating excellent reproducibility (Tables 2 and 3). More than 85% of DOX and MMC was recovered from whole blood after extraction (Table 4).
The multidrug analysis procedures using one-step de-proteinization by an acidified extraction solvent followed by employing a multichannel HPLC method was successfully applied to determine the pharmacokinetics and bio-distribution of long-circulating or PEGylated nanoparticle-based drug delivery of both DOX alone or in combination with MMC in an orthotopic breast tumor murine model (Figures 4 and 5). Figure 4 shows at least 6-fold higher drug concentrations in the blood over-time delivered by nanoparticles (i.e., liposomal DOX and DMPLN) than the equivalent free drug solutions (i.e., free DOX or free DOX-MMC) (Figure 4). Because of prolonged systemic circulation, nanoparticles were able to exploit the enhanced permeability and retention effects of the tumor, resulting in increased DOX and MMC accumulation in the breast tumor (Figure 5A)37. Meanwhile, the quantitatively determined formation of DOX metabolite DOXol in breast tumors over 24 h indicates a difference in drug bio-availability for various drug formulations (Figure 5B).
Figure 1: Illustration of the analysis processes for the simultaneous determination of DOX and MMC delivered by nanoparticles in vivo. (A) Preparation of DMPLN using a one-step ultra-sonication method followed by a self-assembly process; (B) Biological sample collections from an orthotopic breast tumor murine model; (C) Drug extraction from biological matrices and drug reconstitution; (D) Gradient HPLC for separation of DOX, MMC and DOXol. Please click here to view a larger version of this figure.
Figure 2: Comparison of chromatograms of blank whole blood and drug mixtures in blood. Comparison of chromatograms of blank whole blood and drug mixtures in blood using HPLC coupled to UV and fluorescence detectors at (A) UV 360 nm for MMC; (B) UV 310 nm for I.S. 4-MU; (C) Fluorescence at λex/em = 480/560 nm for DOX and DOXol; (D) Fluorescence at λex/em = 365/445 nm for I.S. 4-MU. AU is absorbance unit and EU is fluorescence unit. The injected concentrations of MMC, DOX, and DOXol and their I.S. 4-MU were 100 ng/mL, 50 ng/mL, 50 ng/mL and 200 ng/mL, respectively. Please click here to view a larger version of this figure.
Figure 3: Representation of standard curves for MMC, DOX and DOXol in whole blood. The concentration ranges were from 100 ng/mL to 2000 ng/mL for MMC (A), from 5 ng/mL to 2000 ng/mL for low DOX concentration (B), and from 5 ng/mL to 50 ng/mL for DOXol (C). Please click here to view a larger version of this figure.
Figure 4: Application of the multiple drug analysis system, using a multichannel and gradient HPLC method, to study the pharmacokinetics of long-circulating nanoparticle-based drug delivery. (A) Time-blood concentration profiles of DOX in mono-therapy using free drug solutions (free DOX) or a liposomal formulation (see Table of Materials); (B) Time-blood concentration profiles of DOX and MMC as a free drug combination (free DOX-MMC) or DMPLN. Whole blood was collected at various time points up to 24 h after a single i.v. injection to mice bearing an orthotopic murine breast tumor. All mice were treated with 9.2 mg/kg DOX alone or in combination with 2.9 mg/kg MMC. Because the LLOD of MMC was 100 ng/mL using the HPLC coupled UV detector, MMC concentration after 6 h post-injection was determined by mass spectrometry. The figure has been modified from Zhang et al. Nanomedicine with permission22. All the data points are presented as mean ± standard deviation (SD) with n = 3. Please click here to view a larger version of this figure.
Figure 5: Application of multiple drug analysis using the gradient HPLC method to study the tumor bio-distribution of a nanoparticle-based drug delivery system. (A) Total DOX and MMC concentrations of free DOX-MMC or DMPLN in breast tumors; (B) Total DOXol metabolite formation in breast tumors treated with mono- or combination DOX chemotherapy. All mice were treated with 9.2 mg/kg DOX alone or in combination with 2.9 mg/kg MMC. Because free DOX-MMC had a low tumor accumulation that was out of LLOD of MMC using the HPLC coupled UV detector, MMC concentration in free DOX-MMC was determined by mass spectrometry. The figure has been modified from Zhang et al. Nanomedicine with permission22. All the data points are presented as mean ± SD with n = 3. Please click here to view a larger version of this figure.
Table 1: Linearity and LLOQ of DOX, MMC and DOXol in various biological matrices. The data represent mean ± SD for n = 3.
Table 2: Intra- and inter-day precision and accuracy of DOX, MMC and DOXol in mouse whole blood (n = 3).
Table 3: Intra- and inter-day precision and accuracy of DOX, MMC and DOXol in breast tumors (n = 3).
Table 4: DOX and MMC recovery percentage in the whole blood samples after extraction (n = 3). The data represents mean ± SD for n = 3.
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. Since the PEGylated nanoparticles prolong systemic circulation of loaded drug resulting in high blood drug concentrations over a long period of time (>24 h), the described chromatographic method is cost-effective for large scale sample analysis of nanoparticle co-loaded drug combinations in pre-clinical studies22. The pharmacokinetics of free DOX solution and liposomal DOX shown in Figure 4A is consistent with reported literature data in rodents38,39, further supporting the validity of the current method. Although the UV photodiode-array detector allows multiple-channels to simultaneously detect and display drugs using variable wavelengths (i.e., 310 nm for 4-MU and 360 nm for MMC), the intrinsic detection limit of UV detector is less sensitive than the fluorescence detector. Thus, for fast eliminating, non-fluorescent drugs like MMC delivered in free solutions, the drug concentrations at later time points may fall below the LLOQ of UV detector.
In general, a short chromatography column (e.g., 5 cm) would be used for HPLC analysis to minimize the elution time and operating time. Yet, in the case of analyzing multiple drug compounds, especially those with very similar molecular structures (e.g., DOX and its metabolite DOXol), it is difficult to achieve full separation using the short column due to intrinsic column efficiency. Thus, both a longer column (e.g., 25 cm, used in the current protocol) and an optimization of HPLC parameters during the development of the method are needed to achieve a good peak resolution. Although DOX, DOXol and 4-MU were eluted at close retention time, the interference between DOX/DOXol and 4-MU was not observed in the prepared sample concentration (e.g., 50 ng/mL) in the chromatographs (Figure 2). However, a high DOX concentration (e.g., ~10,000 ng/mL) delivered by nanoparticles as a result of long blood circulation time could interfere with the peak detection of itself and other compounds (e.g., DOXol) and may result in self-quenching of DOX fluorescence40,41. In this case, a proper sample dilution using blank whole blood may be required before sample analysis to determine drug concentrations.
Extraction methods can further complicate the analysis of chemotherapeutic drug combinations. Although DOX or MMC can be extracted using various extraction methods, including solid phase extraction (SPE) or liquid-solid extraction, these procedures are time-consuming and expensive. Some of the extraction methods result in poor recovery and possible drug degradation when hydrochloric acid is added to the extraction solvent42,43,44. For large scale biological sample preparations, the present extraction method is simple, quick and only requires the addition of small amounts of non-hazardous organic solvent for efficient de-proteinization followed by systematic sample reconstitution using methanol. High recovery rates (>85%) were achieved for all drug samples of varying concentrations by systematically applying the same extraction protocol. Although variability still exists, the differences are statistically insignificant. To further reduce the variation, the optimization of individual sample extraction at low and high drug concentrations may be required. Note that the extraction method used does not distinguish free released drug from drug remaining in the nanoparticle as nanoparticles were completely dissolved after addition of organic solvents. Thus, the true pharmacokinetics of nanoparticles alone vs. free drug alone requires the development of a numerical deconvolution method using mathematical modelling to predict their behavior in vivo45.
In summary, a simple and selective HPLC method was developed for simultaneous determination of DOX, MMC and Doxol in vivo. The present method shows robustness, selectivity, precision and accuracy over a broad range of drug concentrations for the mouse whole blood and tissues. This method has been successfully applied to obtain the blood concentration-time profile of DOX and MMC and the bio-distribution of DOX, DOXol and MMC in breast tumors and other major organs (e.g., heart). This protocol provides a useful tool for elucidating macro- and microscopic in vivo mechanisms of nanoparticle-delivered DOX containing drug combination chemotherapy.
The authors have nothing to disclose.
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.
Doxorubicin | Polymed Theraeutics | 111023 | Anticancer drug |
Mitomycin C | Polymed Theraeutics | 060814 | Anticancer drug |
Doxorubicinol (DOXol) | Toronto Research Chemicals | D558020 | Metabolite of DOX |
4-Methylumbelliferone sodium salt | Sigma-Aldrich | M1508 | Internal standard |
Myristic Acid | Sigma-Aldrich | 544-63-8 | Materials for poly-lipid hybrid nanoparticles |
Polyoxyethylene (100) Stearate | Spectrum | M1402 | Materials for poly-lipid hybrid nanoparticles |
Polyoxyethylene (40) Stearate | Sigma-Aldrich | P3440 | Materials for poly-lipid hybrid nanoparticles |
Pluronic F68 (PF68) | BASF Corp. | 9003-11-6 | Materials for poly-lipid hybrid nanoparticles |
Ultrasonication (UP100H) | Hielscher, Ultrasound Technology | NA | Nanoparticle preparation |
Water Bath (ISOTEMP 3016HS) | Fisher Scientific | NA | Nanoparticle preparation |
Liposomal Doxorubicin (Caelyx) | Janssen | Purchased from the pharmacy Princess Margaret Hospital | Clinically-approved nanoparticle formulation |
HPLC-graded Methanol | Caledon Chemicals | 6701-7-40 | HPLC mobile phase composition |
HPLC-graded H2O | Caledon Chemicals | 8801-7-40 | HPLC mobile phase composition |
HPLC-graded Acetonitrile | Caledon Chemicals | 1401-7-40 | HPLC mobile phase composition |
Trifluoroacetic Acid | Sigma-Aldrich | 302031 | HPLC mobile phase composition |
0.45 μm Nylon Membrane Filter Paper | Whatman | WHA7404004 | HPLC mobile phase preparation |
1cc Plastic Syringes | Becton, Dickinson and Company | 2606-309659 | Treatment injection |
5cc Plastic Syringes | Becton, Dickinson and Company | 2608-309646 | Tissue collections |
30G 1/2 Needles | Becton, Dickinson and Company | 305106 | Treatment injection |
25G 5/8 Needles | Becton, Dickinson and Company | 305122 | Tissue collections |
Sterile 0.9% Saline | Univeristy of Toronto House Brand | 1011 | Tissue perfusion |
13 ml Rounded-bottom conical tube | SARSTEDT | 62.515.006 | Prolyprolene, tissue homogenization |
Alpha Minimum Essential Medium (MEM) | Gibco | 12571063 | Cell medium |
1 x Phosphate Buffer Saline | Gibco | 10010023 | Tissue homogenization |
Triton X-100 | Sigma-Aldrich | X100-100 ML | Tissue homogenization |
Formic acid | Caledon Chemicals | 1/5/3840 | Adjust pH for extraction solvent |
Sodium heparin sprayed plastic tubes | Becton, Dickinson and Company | 367878 | Blood collection |
Analytical Weigh Balance | Sartorius | CPA225D | NA |
pH meters | Fisher Scientific | 13-637-671 | accumet BASIC |
Vortex Mixter | Fisher Scientific | 02-215-365 | Vortexing samples at desired speed |
1.5 ml Microcentrifuge Tube | Fisherbrand | 2043-05408129 | Prolyprolene |
Model 1000 homogenizer | Fisher Scientific | 08-451-672 | Tissue homogenization |
Centrifuge 5702R | Eppendorf | 5702R | Extraction preparation |
Heated Evaporator System | Glas-Col | NA | Sample reconstitution |
HPLC Screw Thread Vials | DIKMA | 5320 | HPLC sample injection |
HPLC Screw Caps with PTFE White Silicone Septa | DIKMA | 5325 | HPLC sample injection |
HPLC Polypropylene Insert | Agilent Technologies | 5182-0549 | Maximum volume 250 μl, HPLC sample injection |
Xbridge C18 Column | Waters Corporation | 186003117 | Drug analysis |
Gradient pump | Waters Corporation | W600 | Drug analysis |
Auto-sampler | Waters Corporation | W2707 | Drug analysis |
Photodiode array detector | Waters Corporation | W2998 | Drug analysis |
Multi λ fluoresence detector | Waters Corporation | W2475 | Drug analysis |
EMPOWER 2 | Waters Corporation | NA | Data analysis software |
Scientist | Micromath | NA | Pharmacokinetic analysis |
Female Balb/c Mice | Jackson Laboratory | 001026 | In vivo |
EMT6/WT Breast Cancer Cells | Provided by Dr. Ian Tannock; Ontario Cancer Institute | NA | In vivo |