Mitoxantrone Loaded Superparamagnetic Nanoparticles for Drug Targeting: A Versatile and Sensitive Method for Quantification of Drug Enrichment in Rabbit Tissues Using HPLC-UV

In medicine, superparamagnetic nanoparticles bound to chemotherapeutics are currently investigated for their feasibility in local tumor therapy. After intraarterial application, these particles can be accumulated in the targeted area by an external magnetic field to increase the drug concentration in the region of interest (Magnetic-Drug-Targeting). We here present an analytical method (HPLC-UV), to detect pure or ferrofluid-bound mitoxantrone in a complex matrix even in trace amounts in order to perform biodistribution studies. Mitoxantrone could be extracted in high yields from different tissues. Recovery of mitoxantrone in liver tissue (5000 ng/g) was 76 ± 2%. The limit of quantification of mitoxantrone standard was 10 ng/mL ±12%. Validation criteria such as linearity, precision, and stability were evaluated in ranges achieving the FDA requirements. As shown for pilot samples, biodistribution studies can easily be performed after application of pure or ferrofluid-bound mitoxantrone.


Introduction
Application of chemotherapeutic agents bound to magnetic nanoparticles is a promising approach for site specific drug deposition. Magnetic-Drug-Targeting (MDT) is intended to elevate the drug concentration in the region of interest, because the drug loaded particles are enriched by a focused external magnetic field to defined body compartments after application [1][2][3][4]. This method can lead to both higher potency of antitumor treatments and reduction of negative side effects. In our model, drug loading was realized with mitoxantrone (MTO) (Figure 1), an anthracendion derivative which inhibits the DNA and RNA synthesis and causes DNA strand breaks by intercalation [5,6]. This active component was adsorptively bound to the iron oxide nanoparticles. This agent is used extensively in clinical trials to treat fatal diseases including leukemia, lymphoma, cancers of the breast and prostate [7], and to treat multiple sclerosis [8,9].
A quantitative determination of the biodistribution of magnetic nanoparticles can be achieved by magnetorelax-ometry. The applicability of this technique for the quantification of ferrofluids (FF) in tissue has been demonstrated previously for an extracted tumor slice model [10], a specific artery model [11], and is even in process for the whole body distribution. MTO enrichment might be different, so we are interested in developing an easy multifunctional method to quantify the amount of MTO, especially if it is bound to coated nanoparticles.
Various methods have been described to extract MTO from blood plasma [12][13][14][15][16] and tissues [17] on its own. Moreover there are established protocols to determine MTO by HPLC measurements in context of drug delivery systems like liposome carriers [18] and nanosphere vehicles [19]. Protein precipitation with 0.5 M hydrochloric acid: acetonitrile (90 : 10, v/v), as described by Johnson et al. [18], leads to a recovery of pure MTO in porcine muscle tissue of more than 85% (Table 1). Nevertheless, measurement of MTO bound to iron oxide nanoparticles is more difficult, and extracting whole organs of white New Zealand rabbits this way was unfeasible.  [19], which has been applied in the form of bovine serum albumin (BSA) nanospheres and revealed also promising results in our context. But in order to reliably capture even trace amounts of MTO, an optimization of the extraction mode has been necessary, as well as the development of a subsequent solid phase extraction (SPE) method. Here we present a newly developed method to determine MTO in different tissues and of different binding statuses.
The ferrofluids were synthesized according to a protocol by Hodenius [20].

HPLC. The HPLC analyses were performed by a Waters
Alliance model consisting of a separation module (2695 series) and a dual wavelength absorbance detector (2487 series). The eluate was monitored at 254 nm. The separation was carried out using a 3.0 × 100 mm X-Bridge Phenyl column (Waters, Germany) with a particle diameter of 3.5 μm; the guard column consisted of the same material and was 3.0 × 20 mm of size. The column temperature was 55 • C, the mobile phase was made up of buffer (80 nM sodium formate and formic acid, pH 3.0) and methanol (80 : 20 v/v). Flow was 1 mL/min, the injection volume was 50 μL at room temperature. All measurements were performed after an HPLC-method validation including selectivity, linearity, limit of quantification, precision, accuracy, recovery rate, and stability according to a validation protocol [21,22]. Prior to the sample measurements, a calibration curve with MTOconcentrations of 10 ng/mL-20 000 ng/mL was done.

Method Development/Optimization.
For the extraction experiments, we used porcine muscle tissue and liver tissue which were doped with defined amounts of pure MTO or MTO bound to FF. This occurred by adding dissolved MTO at defined values (see Tables 1 and 2) to tissue homogenates and incubating overnight at room temperature. Due to the complexity of liver tissue and the successful determination here and moreover in muscle tissue, this method could be suitable to perform a complete biodistribution of MTO in rabbits. The extraction was a result of an optimization process. Liver or muscle tissue, doped with MTO, was homogenized for 1 minute using an Ultra Turrax apparatus (Ika, Staufen, Germany) to lyse the cell membranes prior to sonication. Several extraction mixtures containing hydrochloric acid or phosphoric acid in combination with MeOH were tested for extraction efficiency and their compatibility with the SPE procedures (see Tables 1 and 3). For the extraction, simple shaking for 4 or 24 hours was compared to vortex mixing and 1 to 4-fold ultrasonic treatment with a duration of 1 hour each using an ultrasonic bath (US) (Bandelin Sonorex TK 52, Berlin, Germany, without temperature control) ( Table 2).
To sum up, each 0.5 g MTO-containing tissue was treated 4 times for 1 hour with ultrasound in the extraction mixture (500 μL water, 50 μL ascorbic acid (20%) in citrate buffer (pH 3.0) 200 μL methanole (MeOH), 200 μL formic acid, 100 μL, 20% trichloroacetic acid, 400 μL chloroform) followed by centrifugation for 10 minutes at 8000 × G (Jouan MR 23 I, Germany). The combined supernatant was concentrated via a Bond Elut Plexa (200 mg, 6 mL, Varian, Darmstadt, Germany) cartridge after conditioning with acetonitrile (MeCN) and 2% formic acid in water. The analyte was eluted with 5 mL 2% formic acid in MeCN and dried under airstream. The water resolved residue was measured by HPLC as described above to display the efficiency of the extraction.

Optimization of the Extraction.
In terms of drug delivery with nanoparticles, only traces of MTO are embedded in different tissues so that the whole organ has to be processed. The extraction of MTO using 1 N hydrochloric acid (HCl),  (Table 1). When it was bound to FF, the yield decreased significantly. Since a onephase-extraction led to turbid, protein-rich supernatants, protein precipitation by trichloroacetic acid (TCA) as well as Carrez-clearing [24] were used. Unfortunately, both methods did not show promising results. Especially, Carrez-clearing even leads to a blue coprecipitate of MTO.
A new point of view offered the extraction mode of Lu et al. [19] with a two-phase-extraction system to separate interfering lipophilic substances in organs, especially liver tissue. This method gave also the possibility to precipitate the protein fraction and save MTO from decomposition. Nevertheless, we modified this method because more tissue and less MTO required an optimization. We used homogenization for 1 minute via Ultra Turrax (Ika) to dehisce the cell membranes prior to sonication. Simple vortexing and just one single extraction cycle obtained only 26% recovery ( Table 2). Ultrasonic extraction was superior to simple vortexing, and multistep-extraction even increased the efficiency. A 4-fold extraction each for 1 hour was nearly quantitative (88 ± 4%) ( Figure 2).
Of course, the accelerated solvent extraction (ASE) ( Table 2) method led to high extraction rates in short time but this is very expensive. Moreover, ASE tends to require also a useful SPE processing afterwards.

Combination of Extraction and Cartridge Procedure.
To quantify the MTO amount of a whole organ, a useful SPE method was necessary. Unfortunately all the extraction modes described in Table 1 lead to turbid, protein-rich supernatants clogging the SPE cartridge. Using the RP-18 Cartridge (Sep-Pak-Plus C18, 360 mg, Waters) and a standard elution procedure with water and MeOH, recovery of MTO was not sufficient although pretesting with pure MTO gave promising results (>90%) ( Table 3). A recovery rate of 76% by extraction of MTO in porcine liver tissue with phosphoric acid was adequate (Table 3). It is also possible to extract, fixate, and elute MTO bound to FF when using phosphoric acid or phosphate; but in the presence of liver tissue, the recovery of MTO connected to FF was scarce after the elution from the cartridge. In that case, extraction mixtures lead at the most to 17% recovery and almost no reproducibility. This was also true when hydrochloric acid was used for the extraction. The FF dissolved under these conditions and interestingly only traces of MTO could be detected, no matter what kind of conditioning mode was used for the SPE.
The choice of the right cartridge was essential. The widespread used RP-18 cartridge seemed to be not useful in our context. Most of the tested cartridges perform the fixation and elution of released MTO bound to FF, but in presence of liver tissue only Varian Bond Elut Plexa gave appropriate yields with more than 80% recovery (Table 4).
To sum up, the whole method entails a 1-hour ultrasonic extraction repeated 3× of tissue with the 2-phase extractionsolution followed by concentration and purification via Varian Bond Elut Plexa cartridge and the consecutive HPLC measurement (Figure 3).

Selectivity.
Chromatograms obtained from processed blank liver tissue did not show interfering peaks at the retention time of pure MTO measured by HPLC as seen in Figure 4. Selectivity was proved from 6 blank animals using their liver, lung, kidneys and muscle tissue. Each extraction was measured 5 times via HPLC. The results have to be observed in detail. Kidney, lung, and muscle tissue show a baseline chromatographic resolution of at least 1.5 from all other sample components and can be declared as fully separated. This criterion cannot be achieved for liver. The deficient baseline resolution could influence the analyte response. Experiments were performed to evaluate to what extend the impurity peak will affect the final assay result. 6 extraction solutions doted with MTO at the same amount, as the impurity peak is responding to (100 ng/mL), were concomitant analysed with identical amount of 100 ng/mL MTO in mobile phase solution. T-test of these 2 different population means gave the result that the impurity peak influences the respond significantly. To meet the specific criteria that nonseparable impurity peaks influence the result at most for 0.5% MTO [25], amounts more than 250 ng/mL can seriously be quantified.     unlike the LLOQ. The deviation of each reading point meets the FDA specification for linearity of ±15% (LLOQ ±20%). For quantification of unknown samples, a linear equation without any weighting was used. Goodness of fit was not evaluated, because the correlation coefficient with more than 0.999 guarantees a sufficient linear correlation between MTO concentration and analytical response over the whole range ( Figure 5). The LLOQ was determined according to the guidelines of FDA for bioanalytical method validation and is defined as the 5-time response compared to blank samples. This applies to the level of 10 ng/mL [21].

Precision.
Intraassay precision is determined for 3 concentrations of pure MTO (5000; 10000 and 15000 ng/mL). Each level was measured 5 times. The Level for 5000 ng/mL gave 1.0% relative standard deviation, the according data for the 10000 ng/mL and 15000 ng/mL levels are 0.9% and 1.0%, respectively. Interassay precision was performed on 5 different days for the same values mentioned for intraassay. The relative standard deviations were: 1.4% for 5000 ng/mL, 8.6% for 10000 ng/mL and 2.4% for 15000 ng/mL.

Accuracy.
Intraassay accuracy is determined for 3 concentrations of unbound MTO (5000, 10000, and 15000 ng/mL). Each level was measured 5 times. The Level for 5000 ng/mL gave a deviation of the mean from the true value of 1.4%; for 10000 ng/mL measurements gave 1.7% and 1.8% for 15000 ng/mL, respectively and so meet the criteria which are 80-120% for the target concentration.
The same experiments have been performed as interassay accuracy. Measurements mentioned above were arranged on 5 different days. The accuracy values (deviation of the mean from the true value) were 2.1% for 5000 ng/mL, 5.6% for 10000 ng/mL, and 0.3% for 15000 ng/mL. The total recovery rate (extraction and SPE) for MTO in liver tissue for series A (5000 ng/g) was 76 ± 2% for series B (500 ng/g) 67 ± 5% and for series C (50 ng/g) 68 ± 4%. The experiments were performed for 3 independent samples keeping in mind that the recovery can be different when using other tissues than liver. For biodistribution, recovery depends on the total weight of organs. The bigger the organs are the more MTO can be extracted in comparison to smaller organs having the same drug concentration.

Pilot Experiments in order to Perform Biodistribution
Studies. We used the method, described above with 4 × 1 hour ultrasonic extraction (Figure 3) After 24 hours, the animals were sacrificed and organs harvested. Complete organs of liver, lung, kidneys, and a muscle tissue sample were processed and treated in a 100 mL glass flask (Duran, Germany) with the respective amount of extraction solution increased to the amount for the weight of the entire organ as described above followed by SPE.

Suitability of the Method to Quantify Ferrofluid-Bound
MTO. The application of this newly developed method has been proven suitable in a starting in vivo pilot study with New Zealand White rabbits. First experiments with ferrofluid-bound MTO showed that the highest enrichment could be found in the kidneys. The results exhibited the wide range of MTO amounts in different tissues and proved the suitability of the method for different kinds of biological materials (Table 5).

Discussion
To the best of our knowledge, none of the existing protocols, concerning the analysis of mitoxantrone in tissues, have been feasible for our approach of ferrofluid-bound MTO. Moreover, there is especially a lack of techniques to detect small amounts in large scaled tissues. With our method connecting efficient extraction procedures with enrichment strategies towards detectable extraction solutions, we are able to perform biodistribution studies. Monitoring MTO enrichment in different tissues in case of FF-bound drug delivery and overlapping the results with magnetorelaxometric measurements, complex biodistribution patterns after application of MTO-bound iron oxide nanoparticles become low hanging fruits. Current developments in chemotherapy increasingly base on nanoscaled delivery systems. Nanoparticles, liposomes, encapsulations, micelles, and other nanostructures carrying chemotherapeutics require newly developed or modulated analytical methods. With this protocol in hand further detailed analytical instructions can be developed for adsorptive drug delivery systems using well-established and prevalent applied HPLC.

Conclusion
Here we present an easy-to-perform and versatile applicable new method to determine even small amounts of mitoxantrone, regardless if it is in pure or ferrofluid-bound condition in tissues or other biological matrices. Established procedures have not been useful in our context to detect mitoxantrone, especially in presence of FF and contained in tissue samples weighing more than 100 g which especially is necessary for medical application studies in cancer research. We applied this method to evaluate the biodistribution of MTO in rabbit tissue. Within first experiments, we could easily perform the measurements of whole organs and showed the applicability of this new method and its widespread possibilities and analytical applications.