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

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.