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Quantification of DOX bioavailability in biological samples of mice by sensitive and precise HPLC assay a
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Chan Zhang , Yifan Wu , Yuefeng Dong , Hongying Xu & Liangqi Zhao
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School of Environment and Safety, Taiyuan University of Science and Technology, Taiyuan, China, b
College of Life Science, Shanxi University, Taiyuan, China, and
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Shanxi Institute of Medicine and Life Science, Taiyuan, China Published online: 20 May 2015.
To cite this article: Chan Zhang, Yifan Wu, Yuefeng Dong, Hongying Xu & Liangqi Zhao (2015): Quantification of DOX bioavailability in biological samples of mice by sensitive and precise HPLC assay, Pharmaceutical Biology To link to this article: http://dx.doi.org/10.3109/13880209.2015.1014918
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http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–7 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2015.1014918
ORIGINAL ARTICLE
Quantification of DOX bioavailability in biological samples of mice by sensitive and precise HPLC assay Chan Zhang1, Yifan Wu2, Yuefeng Dong3, Hongying Xu1, and Liangqi Zhao2 School of Environment and Safety, Taiyuan University of Science and Technology, Taiyuan, China, 2College of Life Science, Shanxi University, Taiyuan, China, and 3Shanxi Institute of Medicine and Life Science, Taiyuan, China
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Abstract
Keywords
Context: Doxorubicin (DOX)-loaded folate-targeted poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) [P(HB-HO)] nanoparticles [DOX/FA-PEG-P(HB-HO) NPs] were prepared by the W1/O/W2 solvent extraction/evaporation method for applications in cancer treatment. However, the biodistribution, pharmacokinetics, and targeting of the nanoparticles (NPs) have not yet been studied. Objective: The biodistribution, pharmacokinetics, and targeting of DOX/FA-PEG-P(HB-HO) NPs were evaluated in female BALB/c nude mice bearing HeLa tumors. Materials and methods: Three DOX formulations were injected into the tail vein of the mice at a dosage of 5 mg/kg. At each time point, blood and various tissues were collected. All samples were then processed and analyzed by a validated high performance liquid chromatographic (HPLC) method. Results: The t1/2 values of DOX/P(HB-HO) NPs and DOX/FA-PEG-P(HB-HO) NPs were 2.7- and 3.5-times higher than that of free DOX. No significant difference (p40.05) was found in Cmax between the NPs and free DOX. The Tmax values of the two NPs were prolonged from 0.25 to 1 h. The AUC0–t values were 1.55- and 3.05-folds higher than that of free DOX, and MRT increased to 15.99 h for DOX/P(HB-HO) NPs and 25.14 h for DOX/FA-PEG-P(HB-HO) NPs. For DOX/FA-PEG-P(HB-HO) NPs, the DOX content in the tumors were 10.81- and 3.33-times higher than those for free DOX and DOX/P(HB-HO) NPs at 48 h, respectively. Discussion and conclusions: DOX/FA-PEG-P(HB-HO) NPs displayed reduced cardiac toxicity and improved bioavailability. Moreover, the NPs exhibited a significant extent of DOX accumulation in the tumors, thus suggesting that folate-targeted NPs could effectively transport into HeLa tumors with satisfying targeting.
Biodistribution, folate-targeted nanoparticles, high performance liquid chromatographic method, pharmacokinetics, poly(3-hydroxybutyrate-co-3-hydroxyoctanoate)
Introduction Doxorubicin (DOX), an anthracycline glycoside antibiotic (Mukhopadhyay et al., 2009), is an effective chemotherapeutic agent (Chen et al., 2001) that is widely used in treating several human carcinomas, including cervical cancer, breast cancer, acute lymphoblastic leukemia, lymphoma, etc. (Zhang et al., 2007). The anticancer mechanism of DOX may be attributed to its interactions with DNA by the intercalation model, which inhibits the synthesis of nucleic acids (Hu et al., 2009). However, cardiac toxicity and myelosuppression are the main side effects of chemotherapy (Oh et al., 1999; Octavia et al., 2012). Altered free DOX formulations (such as microspheres, NPs, micelles, and liposomes) can reduce side effects, prolong retention time, and improve bioavailability. MyocetTM (Ekab Pharmaceuticals, Cyprus, Greece), which is a liposomal formulation, has a similar efficacy and improved toxicity profile compared with DOX (Harris et al., 2002). Correspondence: Chan Zhang, School of Environment and Safety, Taiyuan University of Science and Technology, Taiyuan 030024, People’s Republic of China. Tel: +86 351 6962589. E-mail: [email protected]
History Received 26 March 2014 Revised 23 October 2014 Accepted 20 January 2015 Published online 16 April 2015
Tan et al. (2005) fabricated DOX-loaded double-walled microspheres with sustained and controlled release properties for the drug. However, the large sizes (50–300 mm) of the microspheres hinder intravenous injection. Folate receptortargeted poly(D,L-lactic-co-glycolic acid) (PLGA)-poly(ethylene glycol) (PEG) DOX micelles with superior cytotoxicity and targeting have been demonstrated by Yoo and Park (2004). Poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) [P(HBHO)] is one of the latest polyhydroxyalkanoates (PHAs) fermented by Sinorhizobium fredii Chen. (CCTCC no. AB 92049) belongs to the family of Rhizobiaceae (Zhao et al., 2006). And P(HB-HO) is used as a drug carrier because of its biodegradation (Guo et al., 2009) and biocompatibility (Dong et al., 2008). P(HB-HO) microspheres (Zhang et al., 2014) and folate-targeted NPs (Zhang et al., 2010) were prepared in our previous study. The study aims to introduce a new sensitive and precise HPLC method for the quantification of DOX and to evaluate and compare the pharmacokinetics, biodistribution, and targeting of DOX-loaded folate-targeted P(HB-HO) NPs [DOX/FA-PEG-P(HB-HO) NPs], free DOX, and DOX-loaded
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P(HB-HO) NPs [DOX/P(HB-HO) NPs] in the plasma and tissues of female BALB/c nude mice bearing HeLa tumors.
DOX formulations (5 mg/kg) 10 d following injection with HeLa cells and had a median tumor volume of 50 mm3.
Materials and methods
Pharmacokinetics, biodistribution, and tumor targeting of nanoparticles in vivo
Chemicals and reagents
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P(HB-HO) (Mw 1.85 105) containing 10% mol of 3-hydroxyoctanoate units was fermented by Sinorhizobium fredii. DOX (C27H29NO11.HCl, Mw 579.98) was purchased from Wanle Pharmaceuticals Inc. (Shenzhen, China). HPLC grade acetonitrile was acquired from DIMA Technology Inc. (Lake Forest, CA). Other chemicals were of analytical grade. Distilled water was produced by the Milli-Q Plus System (Millipore Corporation, Bedford, MA). DOX/P(HB-HO) NPs (223.2 ± 5.1 nm) and DOX/FA-PEGP(HB-HO) NPs (241.6 ± 9.3 nm) for cancer treatment applications were prepared by the W1/O/W2 solvent extraction/ evaporation method, as described previously (Zhang et al., 2010). Briefly, P(HB-HO) [or FA-PEG-P(HB-HO)] was dissolved in chloroform containing emulsifiers. DOX in aqueous solution (W1) was added to the P(HB-HO) [or FAPEG-P(HB-HO)] solution (O), and the mixture was sonicated using a sonicator. This primary emulsion (W1/O) was then added to an aqueous phase containing an emulsifier (W2), and the mixture was sonicated to form the W1/O/W2 emulsion. The resulting double emulsion was stirred and centrifuged. Finally, the sample was washed and freeze-dried to obtain the NPs powder. Chromatographic conditions The chromatographic system (Waters 1525 series, Westbrook, CT) primarily consisted of a Waters 1525 binary pump and a Waters 2487 dual l absorbance detector set at 254 nm. The analytical column was a Thermo Hypersil Gold (250 mm 4.6 mm, 5 mm, Waltham, MA). The mobile phase consisted of acetonitrile and distilled water (adjusted to pH 3.0 with phosphoric acid) with a volumetric ratio of 32/68. The flow rate was 1.0 mLmin1, and the column temperature was 30 C. Experimental animals All experiments were carried out in compliance with the ethical standards required by law and according to the guidelines for the use of experimental animals in China. The protocol approval number of the certification for using and caring the experimental animals is SCXK (jing) 2009-0004. Female BALB/c nude mice (4–6 weeks of age) were purchased from Chinese Academy of Medical Sciences (Beijing, China) and were maintained under a pathogen-free condition. These mice were subcutaneously inoculated with HeLa cells (a human cervical carcinoma cell line provided by Cell Bank of the Chinese Academy of Sciences) in the right limb armpits. HeLa cells with overexpressed folate receptors on the cell surfaces were incubated in RPMI 1640 medium supplemented with 10% FBS and 1% antibiotics (penicillin– streptomycin solution) at 37 C, 5% CO2. The cells were briefly trypsinized and washed three times with PBS, resuspended in normal saline at 1 107 cells/20 mL, and then injected into the armpits. Mice were administered with
The pharmacokinetics, biodistribution, and tumor targeting of DOX/FA-PEG-P(HB-HO) NPs were studied in HeLa tumorbearing mice. On the 10th day after inoculation of tumor cells, 54 mice were randomly divided into three treated groups. Various formulations of DOX (group I: DOX/P(HBHO) NPs; group II: DOX/FA-PEG-P(HB-HO) NPs; group III: free DOX) were injected into the tail veins of the tumorbearing mice at a dosage of 5 mg/kg. Blood was collected from three mice and centrifuged at 3000 rpm for 10 min to separate the plasma at selected times (t ¼ 0.25, 1, 4, 12, 24, and 48 h). The animals were then sacrificed, and the tissues (heart, liver, spleen, lungs, kidneys, and tumor) were collected, weighted, blotted with filter paper towel to remove the residual blood, and homogenized in 1 mL of PBS (pH 7.4). All samples were stored at 20 C until analysis. Plasma and tissues from non-dosed mice (n ¼ 9) were also collected for the preparation of standard curves. Sample preparation DOX extraction from plasma (0.5 mL) and tissues (0.5 mL homogenate) was performed by adding 3 mL of chloroform– methanol (4:1, v/v) mixture with continuous stirring for 2 min. After centrifugation (3000 rpm, 10 min), the organic phases were collected and evaporated to dryness at 30 C under a flow of nitrogen. The dry residues were dissolved in 200 mL of methanol and then centrifuged. 30 mL of the sample was injected into the HPLC column for analysis. Foundation of DOX content determination Aqueous stock solutions of DOX were prepared and stored at 4 C. The working solutions of DOX were prepared by appropriate dilutions of the stock solutions at various concentrations. A total of 50 mL of each working solution was added to each normal untreated mice plasma (0.5 mL) or tissue homogenate (0.5 mL). The final concentrations of the calibration samples used to draw the standard-curves in plasma and tissues ranged from 2.22 to 40 mg/mL. The samples were then processed as described in the ‘‘Sample preparation’’ section. The peak areas of DOX (A) and the concentrations of DOX (C) were utilized to plot the calibration curves. To assess the intra- and inter-day precision of the assay, blank plasma or tissue homogenate samples with three DOX solutions at low, medium, and high concentrations (3.03, 18.18, and 36.36 mg/mL or 2.22, 13.33, and 26.67 mg/mL) were prepared according to the steps described in the Section of ‘‘Sample preparation’’. The intra-day precision was determined by analyzing each concentration five times. The inter-day precision was determined by analyzing the samples for five consecutive days. The precision of the assay was assessed by calculating the relative standard deviation (RSD) for each concentration level. DOX solutions with different concentrations were added into the blank plasma or tissue homogenate samples.
DOI: 10.3109/13880209.2015.1014918
The samples were then processed as mentioned above and were analyzed five times for each concentration using HPLC. The recovery was calculated as follows: measured amount of DOX Recoveryð%Þ ¼ 100% added amount of DOX
Results Foundation of DOX content determination
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components in the plasma and tissues were observed at the retention time (Figure 1B–H). The linearity of the method was statistically confirmed and was validated for DOX in plasma and various tissues over the concentration ranges of 3.03–40 and 2.22–30 mg/mL, respectively. The standard curves generated over these concentration ranges were linear with correlation coefficients (R) greater than 0.9970 (Table 1). As previously defined, the limit of detection (LOD) was approximately 50 ng/mL. The intra- and the inter-day precision of the method were evaluated using three DOX solutions at low, medium, and high concentrations (3.03, 18.18, and 36.36 mg/mL for plasma or 2.22, 13.33, and 26.67 mg/mL for tissues), and the results are listed in Table 2. The intra- and inter-day RSD values
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The typical HPLC chromatograms of the DOX samples are shown in Figure 1. Under the above chromatographic conditions, DOX was eluted at 4.5 min during the 10 min run-time, and no interfering peaks of endogenous
Biodistribution and pharmacokinetic studies of nanoparticles
Figure 1. HPLC chromatograms of DOX in different samples. (A) DOX solution; (B) DOX in plasma sample; (C) DOX in heart sample; (D) DOX in liver sample; (E) DOX in spleen sample; (F) DOX in lung sample; (G) DOX in kidney sample; (H) DOX in tumor sample.
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Table 1. Calibration curve of DOX in plasma and different tissues.
Sample
Linear range (mg/mL)
Equation
R
Plasma Heart Liver Spleen Lung Kidney Tumor
3.03–40 2.22–30 2.22–30 2.22–30 2.22–30 2.22–30 2.22–30
A ¼ 3297.2C + 3097.6 A ¼ 3472.4C + 2222.7 A ¼ 307.81C 472.1 A ¼ 352.03C 590.95 A ¼ 125.95C + 326.95 A ¼ 306.66C 460.02 A ¼ 3140.9C + 1141.6
0.9977 0.9990 0.9976 0.9984 0.9984 0.9986 0.9997
Table 3. Recovery of the method for determination of DOX contents. Sample
Added (mg/mL)
Recovery (%)
Mean ± SD
RSD (%)
Plasma
3.03 18.18 36.36 2.22 13.33 26.67 2.22 13.33 26.67 2.22 13.33 26.67 2.22 13.33 26.67 2.22 13.33 26.67 2.22 13.33 26.67
95.71 102.20 99.55 98.65 100.15 99.48 97.30 99.10 100.64 93.24 97.22 98.80 104.50 95.95 99.10 99.55 96.99 101.39 98.32 95.43 99.67
99.15 ± 3.26
3.29
99.43 ± 0.75
0.75
99.01 ± 1.67
1.69
96.42 ± 2.87
2.98
99.85 ± 4.32
4.33
99.31 ± 2.21
2.23
97.81 ± 2.17
2.22
Heart
Liver
A is the peak areas of DOX, C is the concentrations of DOX. Spleen Table 2. Accuracy and precision of the method for determination of DOX contents. Lung Intra-day (n ¼ 5)
Inter-day (n ¼ 5)
Accuracy Precision Accuracy Precision Sample C (mg/mL) (mean ± SD) (RSD %) (mean ± SD) (RSD %)
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Plasma
Heart
Liver
Spleen
Lung
Kidney
Tumor
3.03 18.18 36.36 2.22 13.33 26.67 2.22 13.33 26.67 2.22 13.33 26.67 2.22 13.33 26.67 2.22 13.33 26.67 2.22 13.33 26.67
2.88 ± 0.01 19.08 ± 0.07 35.44 ± 0.06 1.88 ± 0.04 13.65 ± 0.23 26.44 ± 0.16 2.18 ± 0.11 13.11 ± 0.16 26.9 ± 0.16 1.97 ± 0.08 12.84 ± 0.08 26.25 ± 0.07 2.61 ± 0.06 12.77 ± 0.03 26.46 ± 0.32 1.89 ± 0.13 12.8 ± 0.02 27 ± 0.32 1.96 ± 0.03 12.86 ± 0.16 26.43 ± 0.17
0.35 0.37 0.17 2.13 1.69 0.61 5.05 1.22 0.60 4.06 0.62 0.27 2.30 0.23 1.21 6.88 0.16 1.19 1.53 1.24 0.64
2.88 ± 0.01 19.07 ± 0.07 35.48 ± 0.07 2.23 ± 0.03 13.66 ± 0.20 26.66 ± 0.21 2.23 ± 0.16 13.14 ± 0.06 26.9 ± 0.23 2 ± 0.07 12.80 ± 0.06 26.35 ± 0.09 2.58 ± 0.06 12.78 ± 0.04 26.51 ± 0.22 2.01 ± 0.03 13.33 ± 0.05 26.68 ± 0.32 1.89 ± 0.04 12.87 ± 0.23 26.35 ± 0.21
0.35 0.37 0.20 1.35 1.46 0.79 7.17 0.46 0.86 3.50 0.47 0.34 2.33 0.31 0.83 1.50 0.38 1.20 2.15 1.79 0.80
ranged from 0.16 to 6.88% and 0.2 to 7.17%, respectively. These data suggested that the method had excellent accuracy and precision. The results of the method recovery are shown in Table 3. The average recoveries of DOX in the plasma, heart, liver, spleen, lungs, kidneys and tumor were 99.15 ± 3.26%, 99.43 ± 0.75%, 99.01 ± 1.67%, 96.42 ± 2.87%, 99.85 ± 4.32%, 99.31 ± 2.21%, and 97.81 ± 2.17%, respectively. There was no significant difference (p40.05) in the recovery methods for determination of DOX contents in different biological samples. The average recoveries of DOX in the plasma and various tissues were more than 96%, and the RSD ranged from 0.75 to 4.33%. Pharmacokinetics, biodistribution, and tumor targeting of nanoparticles in vivo In this paper, the plasma and tissue levels of the DOX formulations were determined following a single injection of DOX/P(HB-HO) NPs, DOX/FA-PEG-P(HB-HO) NPs or free DOX (5 mg/kg) in female BALB/c nude mice bearing HeLa tumors. The plasma drug concentration–time curves
Kidney
Tumor
(mean ± SD) of DOX are presented in Figure 2, and the corresponding pharmacokinetic parameters are listed in Table 4. The t1/2 values of DOX/P(HB-HO) NPs and DOX/ FA-PEG-P(HB-HO) NPs were 2.7- and 3.5-times higher than that of free DOX. No significant difference (p40.05) was found in the Cmax between the NPs and free DOX. The Tmax values of the two NPs were prolonged from 0.25 to 1 h, whereas the AUC0–t values of the two NPs were 1.55- and 3.05-folds higher than that of free DOX. Moreover, MRT increased to 15.99 and 25.14 h for DOX/P(HB-HO) NPs and DOX/FA-PEG-P(HB-HO) NPs, respectively. From the above results, DOX/P(HB-HO) NPs and DOX/FA-PEG-P(HB-HO) NPs displayed improved pharmacokinetic profiles compared with free DOX in vivo. The distribution of different DOX formulations in HeLa tumor-bearing mice (Figure 3) was determined at the three time points of 4, 24, and 48 h. In the experiment, the DOX levels in the plasma and tissues gradually decreased at different rates. The DOX contents in the tumor, liver, and kidneys decreased more slowly than that in the plasma and other tissues. A significantly lower DOX contents (p50.05) in the heart was detected for the NPs groups (0.85 ± 0.37 or 0.52 ± 0.11 mg/mL) than for the free DOX group (1.86 ± 0.87 mg/mL) at 24 h (Figure 3B). The level of the free DOX in the tumors peaked at 1.74 ± 0.89 mg/mL at 4 h and dramatically decreased to 0.32 ± 0.09 mg/mL at 48 h (Figure 3A and C). For DOX/FA-PEG-P(HB-HO) NPs in the tumors, the DOX contents maximally increased at 24 h (5.39 ± 0.39 mg/mL) and then slowly decreased to 3.46 ± 0.19 mg/mL at 48 h (Figure 3B and C). However, for DOX/P(HB-HO) NPs in the tumor, the DOX level reached a peak level of 3.58 ± 0.98 mg/mL at 24 h and gradually decreased to 1.04 ± 0.38 mg/mL at 48 h (Figure 3B and C). The DOX concentrations in the tumors for the DOX/FA-PEG-P(HB-HO) NPs group were 10.81- and 3.33-times higher than those of the free DOX and DOX/ P(HB-HO) NPs groups at 48 h, respectively.
Biodistribution and pharmacokinetic studies of nanoparticles
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(A) 25
(B) 25
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free DOX DOX/P(HB-HO) NPs DOX/FA-PEG-P(HB-HO) NPs
Concentration (µg/mL)
Concentration (µg/mL)
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free DOX DOX/P(HB-HO) NPs DOX/FA-PEG-P(HB-HO) NPs
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Figure 2. Mean DOX concentration–time curves in plasma after intravenous administration of free DOX, DOX/P(HB-HO) NPs and DOX/FA-PEG-P(HB-HO) NPs to tumor-bearing mice (n ¼ 3) ((A) 0–48 h; (B) 0–12 h).
Table 4. Main pharmacokinetic parameters of DOX formulations after i.v. administration.
t1/2/h Cmax/mg mL1 Tmax/h AUC0–t/mgmL1h MRT/h
free DOX
DOX/P(HB-HO) NPs
DOX/FA-PEG-P (HB-HO) NPs
7.23 ± 1.22 20.12 ± 4.29 0.25 ± 0.03 64.62 ± 15.98 5.61 ± 1.23
19.26 ± 4.68* 20.66 ± 5.46 1.00 ± 0.01** 99.25 ± 19.66 15.99 ± 4.63*
25.08 ± 2.97** 22.78 ± 5.49 1.00 ± 0.08** 132.36 ± 25.38* 25.14 ± 7.04**
t1/2, terminal half-life; Cmax, maximum concentration; Tmax, time of maximum concentration; AUC0–t, area under plasma concentration versus time curve (0–t); MRT, mean retention time. *p50.05 compared with free DOX; **p50.01 compared with free DOX.
Discussion To date, many analysis methods for DOX, such as fluorescence assay (Bibby et al., 2005; Wang et al., 2006), radiolabelled assay (Ogawara et al., 2008), LC/MS assay (Arnold et al., 2004; DiFrancesco et al., 2007), and other methods (Hu et al., 2007; Kim & Wainer, 2010), have been reported. However, several problems may be encountered in these assay methods, such as comparatively difficult procedures, complex compositions of the mobile phases, or high costs in equipment. Favorable chromatographic conditions should include the following: baseline separation of the analyte from other exogenous materials, a mobile phase with a simple composition and an appropriate pH value, brief procedure, and relatively good accuracy and precision. In this paper, the HPLC analysis of different DOX formulations in biological samples was developed and exploited. In the experiment, a simple mobile phase consisting of acetonitrile and distilled water (adjusted to pH 3.0 with phosphoric acid) was used. The chromatographic conditions and procedures were easily controlled. According to the HPLC conditions adopted, DOX was eluted at 4.5 min within 10 min without any interfering exogenous materials in the plasma and tissues. The values of the linearity and specificity demonstrated that the peak areas and DOX concentrations
displayed perfect linear relationships and that the assay had adequate specificity. In this study, the accuracy and precision values met the requirements for the determination of DOX contents in biological samples. The favorable results showed that the simple method developed for plasma and tissue handling exhibited relatively high accuracy and recovery, and was suitable for extracting and measuring the DOX contents. The applicability of the assay method was demonstrated in this study for the pharmacokinetics, biodistribution, and targeting of free DOX, DOX/P(HB-HO) NPs and DOX/FAPEG-P(HB-HO) NPs in female BALB/c nude mice bearing HeLa tumors. The three DOX formulations reached the major tissues after intravenous administration. The DOX levels in the plasma, heart, spleen, and lungs rapidly decreased compared with those in the tumors, liver, and kidneys. This phenomenon might be related to the half-life, targeting, and sustained release effects. Compared with free DOX, the circulation and action time of the two NPs in vivo was prolonged, and their bioavailability also improved (Kalaria et al., 2009). This result might be attributed to the encapsulation of the drug into NPs, protecting them from rapid elimination after administration. This protection thereby allows the drug to be transported through blood vessel walls into other tissues. The DOX contents in the heart for the NPs was lower than that for free DOX, thus indicating that the cardiac toxicity of DOX might be substantially reduced by the formulation of NPs. Similar toxicities for DOX/P(HB-HO) NPs and DOX/FA-PEG-P(HB-HO) NPs were observed compared with those shown in previous studies. It was very obvious that the distribution of DOX in the heart for liposomal DOX preparations was significantly reduced at 0.5 h compared with the free DOX preparations (Wang et al., 2006). Furthermore, significantly lower DOX contents for the DOX micelles and DOX/FOL micelles were detected in the heart than for free DOX at 24 h (Yoo & Park, 2004). This consequence was encouraging because cardiac toxicity is a unique characteristic of DOX chemotherapies. The DOX contents in the tumors reached a peak level at 4 h and dramatically decreased at 48 h for free DOX. In contrast, when DOX/P(HB-HO) NPs or DOX/FA-PEG-P(HB-HO) NPs
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4
**
2
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** **
*
* **
0 Plasma
Tumor
Heart
Liver
Spleen
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Figure 3. Biodistribution of free DOX, DOX/P(HB-HO) NPs and DOX/FA-PEG-P(HB-HO) NPs in blood, heart, liver, spleen, lung, kidney, and tumor at different times ((A) 4 h, (B) 24 h, (C) 48 h) after intravenous administration to tumor-bearing mice (n ¼ 3). *p50.05 compared with free DOX; **p50.01 compared with free DOX.
were administered, the DOX contents remained at a high level up to 48 h. NPs tend to accumulate in the solid tumors through the enhanced permeability and retention effect (EPR) (Acharya & Sahoo, 2011), thus resulting reduced DOX concentrations in the blood and heart. These results are in good agreement with those of previous studies on cardiac toxicity. The DOX contents in the tumors for the DOX/FAPEG-P(HB-HO) NP group were much higher than that for the free DOX and DOX/P(HB-HO) NP groups at 48 h. The folatetargeted NPs exhibited a greater extent of DOX accumulation in the tumors than DOX/P(HB-HO) NPs, thus suggesting that DOX/FA-PEG-P(HB-HO) NPs were more effectively transported and actively targeted into HeLa tumors than DOX/P(HB-HO) NPs because of the receptor-mediated endocytosis (Cho et al., 2008).
Conclusion A sensitive, precise, and reproducible HPLC method was developed and adopted to quantify DOX in biological specimens, including plasma, heart, liver, spleen, lungs, kidneys, and tumor. This assay utilized a brief and convenient external standard method and only involved the rapid simple pre-treatment of liquid–liquid extraction for samples. Moreover, the method displayed sufficient sensitivity and precision in determining the DOX contents in biological
samples. The HPLC method developed in the present study has been successfully applied in evaluating the pharmacokinetics, biodistribution, and targeting of DOX/FA-PEG-P(HBHO) NPs in female BALB/c nude mice bearing HeLa tumors. In vivo studies confirmed that the circulation and bioavailability of the NPs were improved, and that the folate-targeted NPs were more effectively transported and actively targeted into HeLa tumors than non-targeted NPs because of the receptor-mediated endocytosis.
Declaration of interest The authors report that they have no conflicts of interest. This work was supported by the Doctorate Science Funds of Taiyuan University of Science and Technology [20122007]; and the Natural Science Foundation for Young Scientists of Shanxi Province, China [2013021011-7].
References Acharya S, Sahoo SK. (2011). PLGA nanoparticles containing various anticancer agents and tumour delivery by EPR effect. Adv Drug Deliv Rev 63:170–83. Arnold RD, Slack JE, Straubinger RM. (2004). Quantification of doxorubicin and metabolites in rat plasma and small volume tissue samples by liquid chromatography/electrospray tandem mass spectroscopy. J Chromatogr B 808:141–52.
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Mukhopadhyay P, Rajesh M, Ba´tkai S, et al. (2009). Role of superoxide, nitric oxide, and neroxynitrite in doxorubicin-induced cell death in vivo and in vitro. Am J Physiol-Heart C 296: H1466–83. Octavia Y, Tocchetti CG, Gabrielson KL, et al. (2012). Doxorubicininduced cardiomyopathy: From molecular mechanisms to therapeutic strategies. J Mol Cell Cardiol 52:1213–25. Ogawara K, Un K, Minato K, et al. (2008). Determinants for in vivo anti-tumor effects of PEG liposomal doxorubicin: Importance of vascular permeability within tumors. Int J Pharm 359:234–40. Oh I, Lee K, Kwon HY, et al. (1999). Release of adriamycin from poly(g-benzyl-L-glutamate)/poly(ethylene oxide) nanoparticles. Int J Pharm 181:107–15. Tan EC, Lin R, Wang CH. (2005). Fabrication of double-walled microspheres for the sustained release of doxorubicin. J Colloid Interface Sci 291:135–43. Wang JC, Liu XY, Lu WL, et al. (2006). Pharmacokinetics of intravenously administered stealth liposomal doxorubicin modulated with verapamil in rats. Eur J Pharm Biopharm 62:44–51. Yoo HS, Park TG. (2004). Folate receptor targeted biodegradable polymeric doxorubicin micelles. J Control Release 96: 273–83. Zhang C, Dong YF, Zhao LQ. (2014). Preparation and characterization of novel microparticles based on poly (3-hydroxybutyarte-co-3hydroxyoctanoate). J Microencapsul 31:9–15. Zhang C, Zhao LQ, Dong YF, et al. (2010). Folate-mediated poly(3hydroxybutyrate-co-3-hydroxyoctanoate) nanoparticles for targeting drug delivery. Eur J Pharm Biopharm 76:10–16. Zhang ZP, Lee SH, Feng SS. (2007). Folate-decorated poly(lactideco-glycolide)-vitamin E TPGS nanoparticles for targeted drug delivery. Biomaterials 28:1889–99. Zhao LQ, Xiao JF, Feng T, et al. (2006). Synthesis of poly (3-hydroxybutyrate-co-3-hydroxyoctanoate) by a Sinorhizobium fredii strain. Lett Appl Microbiol 42:344–9.
Pharmaceutical Biology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/iphb20
Quantification of DOX bioavailability in biological samples of mice by sensitive and precise HPLC assay a
b
c
a
Chan Zhang , Yifan Wu , Yuefeng Dong , Hongying Xu & Liangqi Zhao
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a
School of Environment and Safety, Taiyuan University of Science and Technology, Taiyuan, China, b
College of Life Science, Shanxi University, Taiyuan, China, and
c
Shanxi Institute of Medicine and Life Science, Taiyuan, China Published online: 20 May 2015.
To cite this article: Chan Zhang, Yifan Wu, Yuefeng Dong, Hongying Xu & Liangqi Zhao (2015): Quantification of DOX bioavailability in biological samples of mice by sensitive and precise HPLC assay, Pharmaceutical Biology To link to this article: http://dx.doi.org/10.3109/13880209.2015.1014918
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http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–7 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2015.1014918
ORIGINAL ARTICLE
Quantification of DOX bioavailability in biological samples of mice by sensitive and precise HPLC assay Chan Zhang1, Yifan Wu2, Yuefeng Dong3, Hongying Xu1, and Liangqi Zhao2 School of Environment and Safety, Taiyuan University of Science and Technology, Taiyuan, China, 2College of Life Science, Shanxi University, Taiyuan, China, and 3Shanxi Institute of Medicine and Life Science, Taiyuan, China
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Abstract
Keywords
Context: Doxorubicin (DOX)-loaded folate-targeted poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) [P(HB-HO)] nanoparticles [DOX/FA-PEG-P(HB-HO) NPs] were prepared by the W1/O/W2 solvent extraction/evaporation method for applications in cancer treatment. However, the biodistribution, pharmacokinetics, and targeting of the nanoparticles (NPs) have not yet been studied. Objective: The biodistribution, pharmacokinetics, and targeting of DOX/FA-PEG-P(HB-HO) NPs were evaluated in female BALB/c nude mice bearing HeLa tumors. Materials and methods: Three DOX formulations were injected into the tail vein of the mice at a dosage of 5 mg/kg. At each time point, blood and various tissues were collected. All samples were then processed and analyzed by a validated high performance liquid chromatographic (HPLC) method. Results: The t1/2 values of DOX/P(HB-HO) NPs and DOX/FA-PEG-P(HB-HO) NPs were 2.7- and 3.5-times higher than that of free DOX. No significant difference (p40.05) was found in Cmax between the NPs and free DOX. The Tmax values of the two NPs were prolonged from 0.25 to 1 h. The AUC0–t values were 1.55- and 3.05-folds higher than that of free DOX, and MRT increased to 15.99 h for DOX/P(HB-HO) NPs and 25.14 h for DOX/FA-PEG-P(HB-HO) NPs. For DOX/FA-PEG-P(HB-HO) NPs, the DOX content in the tumors were 10.81- and 3.33-times higher than those for free DOX and DOX/P(HB-HO) NPs at 48 h, respectively. Discussion and conclusions: DOX/FA-PEG-P(HB-HO) NPs displayed reduced cardiac toxicity and improved bioavailability. Moreover, the NPs exhibited a significant extent of DOX accumulation in the tumors, thus suggesting that folate-targeted NPs could effectively transport into HeLa tumors with satisfying targeting.
Biodistribution, folate-targeted nanoparticles, high performance liquid chromatographic method, pharmacokinetics, poly(3-hydroxybutyrate-co-3-hydroxyoctanoate)
Introduction Doxorubicin (DOX), an anthracycline glycoside antibiotic (Mukhopadhyay et al., 2009), is an effective chemotherapeutic agent (Chen et al., 2001) that is widely used in treating several human carcinomas, including cervical cancer, breast cancer, acute lymphoblastic leukemia, lymphoma, etc. (Zhang et al., 2007). The anticancer mechanism of DOX may be attributed to its interactions with DNA by the intercalation model, which inhibits the synthesis of nucleic acids (Hu et al., 2009). However, cardiac toxicity and myelosuppression are the main side effects of chemotherapy (Oh et al., 1999; Octavia et al., 2012). Altered free DOX formulations (such as microspheres, NPs, micelles, and liposomes) can reduce side effects, prolong retention time, and improve bioavailability. MyocetTM (Ekab Pharmaceuticals, Cyprus, Greece), which is a liposomal formulation, has a similar efficacy and improved toxicity profile compared with DOX (Harris et al., 2002). Correspondence: Chan Zhang, School of Environment and Safety, Taiyuan University of Science and Technology, Taiyuan 030024, People’s Republic of China. Tel: +86 351 6962589. E-mail: [email protected]
History Received 26 March 2014 Revised 23 October 2014 Accepted 20 January 2015 Published online 16 April 2015
Tan et al. (2005) fabricated DOX-loaded double-walled microspheres with sustained and controlled release properties for the drug. However, the large sizes (50–300 mm) of the microspheres hinder intravenous injection. Folate receptortargeted poly(D,L-lactic-co-glycolic acid) (PLGA)-poly(ethylene glycol) (PEG) DOX micelles with superior cytotoxicity and targeting have been demonstrated by Yoo and Park (2004). Poly(3-hydroxybutyrate-co-3-hydroxyoctanoate) [P(HBHO)] is one of the latest polyhydroxyalkanoates (PHAs) fermented by Sinorhizobium fredii Chen. (CCTCC no. AB 92049) belongs to the family of Rhizobiaceae (Zhao et al., 2006). And P(HB-HO) is used as a drug carrier because of its biodegradation (Guo et al., 2009) and biocompatibility (Dong et al., 2008). P(HB-HO) microspheres (Zhang et al., 2014) and folate-targeted NPs (Zhang et al., 2010) were prepared in our previous study. The study aims to introduce a new sensitive and precise HPLC method for the quantification of DOX and to evaluate and compare the pharmacokinetics, biodistribution, and targeting of DOX-loaded folate-targeted P(HB-HO) NPs [DOX/FA-PEG-P(HB-HO) NPs], free DOX, and DOX-loaded
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P(HB-HO) NPs [DOX/P(HB-HO) NPs] in the plasma and tissues of female BALB/c nude mice bearing HeLa tumors.
DOX formulations (5 mg/kg) 10 d following injection with HeLa cells and had a median tumor volume of 50 mm3.
Materials and methods
Pharmacokinetics, biodistribution, and tumor targeting of nanoparticles in vivo
Chemicals and reagents
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P(HB-HO) (Mw 1.85 105) containing 10% mol of 3-hydroxyoctanoate units was fermented by Sinorhizobium fredii. DOX (C27H29NO11.HCl, Mw 579.98) was purchased from Wanle Pharmaceuticals Inc. (Shenzhen, China). HPLC grade acetonitrile was acquired from DIMA Technology Inc. (Lake Forest, CA). Other chemicals were of analytical grade. Distilled water was produced by the Milli-Q Plus System (Millipore Corporation, Bedford, MA). DOX/P(HB-HO) NPs (223.2 ± 5.1 nm) and DOX/FA-PEGP(HB-HO) NPs (241.6 ± 9.3 nm) for cancer treatment applications were prepared by the W1/O/W2 solvent extraction/ evaporation method, as described previously (Zhang et al., 2010). Briefly, P(HB-HO) [or FA-PEG-P(HB-HO)] was dissolved in chloroform containing emulsifiers. DOX in aqueous solution (W1) was added to the P(HB-HO) [or FAPEG-P(HB-HO)] solution (O), and the mixture was sonicated using a sonicator. This primary emulsion (W1/O) was then added to an aqueous phase containing an emulsifier (W2), and the mixture was sonicated to form the W1/O/W2 emulsion. The resulting double emulsion was stirred and centrifuged. Finally, the sample was washed and freeze-dried to obtain the NPs powder. Chromatographic conditions The chromatographic system (Waters 1525 series, Westbrook, CT) primarily consisted of a Waters 1525 binary pump and a Waters 2487 dual l absorbance detector set at 254 nm. The analytical column was a Thermo Hypersil Gold (250 mm 4.6 mm, 5 mm, Waltham, MA). The mobile phase consisted of acetonitrile and distilled water (adjusted to pH 3.0 with phosphoric acid) with a volumetric ratio of 32/68. The flow rate was 1.0 mLmin1, and the column temperature was 30 C. Experimental animals All experiments were carried out in compliance with the ethical standards required by law and according to the guidelines for the use of experimental animals in China. The protocol approval number of the certification for using and caring the experimental animals is SCXK (jing) 2009-0004. Female BALB/c nude mice (4–6 weeks of age) were purchased from Chinese Academy of Medical Sciences (Beijing, China) and were maintained under a pathogen-free condition. These mice were subcutaneously inoculated with HeLa cells (a human cervical carcinoma cell line provided by Cell Bank of the Chinese Academy of Sciences) in the right limb armpits. HeLa cells with overexpressed folate receptors on the cell surfaces were incubated in RPMI 1640 medium supplemented with 10% FBS and 1% antibiotics (penicillin– streptomycin solution) at 37 C, 5% CO2. The cells were briefly trypsinized and washed three times with PBS, resuspended in normal saline at 1 107 cells/20 mL, and then injected into the armpits. Mice were administered with
The pharmacokinetics, biodistribution, and tumor targeting of DOX/FA-PEG-P(HB-HO) NPs were studied in HeLa tumorbearing mice. On the 10th day after inoculation of tumor cells, 54 mice were randomly divided into three treated groups. Various formulations of DOX (group I: DOX/P(HBHO) NPs; group II: DOX/FA-PEG-P(HB-HO) NPs; group III: free DOX) were injected into the tail veins of the tumorbearing mice at a dosage of 5 mg/kg. Blood was collected from three mice and centrifuged at 3000 rpm for 10 min to separate the plasma at selected times (t ¼ 0.25, 1, 4, 12, 24, and 48 h). The animals were then sacrificed, and the tissues (heart, liver, spleen, lungs, kidneys, and tumor) were collected, weighted, blotted with filter paper towel to remove the residual blood, and homogenized in 1 mL of PBS (pH 7.4). All samples were stored at 20 C until analysis. Plasma and tissues from non-dosed mice (n ¼ 9) were also collected for the preparation of standard curves. Sample preparation DOX extraction from plasma (0.5 mL) and tissues (0.5 mL homogenate) was performed by adding 3 mL of chloroform– methanol (4:1, v/v) mixture with continuous stirring for 2 min. After centrifugation (3000 rpm, 10 min), the organic phases were collected and evaporated to dryness at 30 C under a flow of nitrogen. The dry residues were dissolved in 200 mL of methanol and then centrifuged. 30 mL of the sample was injected into the HPLC column for analysis. Foundation of DOX content determination Aqueous stock solutions of DOX were prepared and stored at 4 C. The working solutions of DOX were prepared by appropriate dilutions of the stock solutions at various concentrations. A total of 50 mL of each working solution was added to each normal untreated mice plasma (0.5 mL) or tissue homogenate (0.5 mL). The final concentrations of the calibration samples used to draw the standard-curves in plasma and tissues ranged from 2.22 to 40 mg/mL. The samples were then processed as described in the ‘‘Sample preparation’’ section. The peak areas of DOX (A) and the concentrations of DOX (C) were utilized to plot the calibration curves. To assess the intra- and inter-day precision of the assay, blank plasma or tissue homogenate samples with three DOX solutions at low, medium, and high concentrations (3.03, 18.18, and 36.36 mg/mL or 2.22, 13.33, and 26.67 mg/mL) were prepared according to the steps described in the Section of ‘‘Sample preparation’’. The intra-day precision was determined by analyzing each concentration five times. The inter-day precision was determined by analyzing the samples for five consecutive days. The precision of the assay was assessed by calculating the relative standard deviation (RSD) for each concentration level. DOX solutions with different concentrations were added into the blank plasma or tissue homogenate samples.
DOI: 10.3109/13880209.2015.1014918
The samples were then processed as mentioned above and were analyzed five times for each concentration using HPLC. The recovery was calculated as follows: measured amount of DOX Recoveryð%Þ ¼ 100% added amount of DOX
Results Foundation of DOX content determination
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components in the plasma and tissues were observed at the retention time (Figure 1B–H). The linearity of the method was statistically confirmed and was validated for DOX in plasma and various tissues over the concentration ranges of 3.03–40 and 2.22–30 mg/mL, respectively. The standard curves generated over these concentration ranges were linear with correlation coefficients (R) greater than 0.9970 (Table 1). As previously defined, the limit of detection (LOD) was approximately 50 ng/mL. The intra- and the inter-day precision of the method were evaluated using three DOX solutions at low, medium, and high concentrations (3.03, 18.18, and 36.36 mg/mL for plasma or 2.22, 13.33, and 26.67 mg/mL for tissues), and the results are listed in Table 2. The intra- and inter-day RSD values
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The typical HPLC chromatograms of the DOX samples are shown in Figure 1. Under the above chromatographic conditions, DOX was eluted at 4.5 min during the 10 min run-time, and no interfering peaks of endogenous
Biodistribution and pharmacokinetic studies of nanoparticles
Figure 1. HPLC chromatograms of DOX in different samples. (A) DOX solution; (B) DOX in plasma sample; (C) DOX in heart sample; (D) DOX in liver sample; (E) DOX in spleen sample; (F) DOX in lung sample; (G) DOX in kidney sample; (H) DOX in tumor sample.
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Table 1. Calibration curve of DOX in plasma and different tissues.
Sample
Linear range (mg/mL)
Equation
R
Plasma Heart Liver Spleen Lung Kidney Tumor
3.03–40 2.22–30 2.22–30 2.22–30 2.22–30 2.22–30 2.22–30
A ¼ 3297.2C + 3097.6 A ¼ 3472.4C + 2222.7 A ¼ 307.81C 472.1 A ¼ 352.03C 590.95 A ¼ 125.95C + 326.95 A ¼ 306.66C 460.02 A ¼ 3140.9C + 1141.6
0.9977 0.9990 0.9976 0.9984 0.9984 0.9986 0.9997
Table 3. Recovery of the method for determination of DOX contents. Sample
Added (mg/mL)
Recovery (%)
Mean ± SD
RSD (%)
Plasma
3.03 18.18 36.36 2.22 13.33 26.67 2.22 13.33 26.67 2.22 13.33 26.67 2.22 13.33 26.67 2.22 13.33 26.67 2.22 13.33 26.67
95.71 102.20 99.55 98.65 100.15 99.48 97.30 99.10 100.64 93.24 97.22 98.80 104.50 95.95 99.10 99.55 96.99 101.39 98.32 95.43 99.67
99.15 ± 3.26
3.29
99.43 ± 0.75
0.75
99.01 ± 1.67
1.69
96.42 ± 2.87
2.98
99.85 ± 4.32
4.33
99.31 ± 2.21
2.23
97.81 ± 2.17
2.22
Heart
Liver
A is the peak areas of DOX, C is the concentrations of DOX. Spleen Table 2. Accuracy and precision of the method for determination of DOX contents. Lung Intra-day (n ¼ 5)
Inter-day (n ¼ 5)
Accuracy Precision Accuracy Precision Sample C (mg/mL) (mean ± SD) (RSD %) (mean ± SD) (RSD %)
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Plasma
Heart
Liver
Spleen
Lung
Kidney
Tumor
3.03 18.18 36.36 2.22 13.33 26.67 2.22 13.33 26.67 2.22 13.33 26.67 2.22 13.33 26.67 2.22 13.33 26.67 2.22 13.33 26.67
2.88 ± 0.01 19.08 ± 0.07 35.44 ± 0.06 1.88 ± 0.04 13.65 ± 0.23 26.44 ± 0.16 2.18 ± 0.11 13.11 ± 0.16 26.9 ± 0.16 1.97 ± 0.08 12.84 ± 0.08 26.25 ± 0.07 2.61 ± 0.06 12.77 ± 0.03 26.46 ± 0.32 1.89 ± 0.13 12.8 ± 0.02 27 ± 0.32 1.96 ± 0.03 12.86 ± 0.16 26.43 ± 0.17
0.35 0.37 0.17 2.13 1.69 0.61 5.05 1.22 0.60 4.06 0.62 0.27 2.30 0.23 1.21 6.88 0.16 1.19 1.53 1.24 0.64
2.88 ± 0.01 19.07 ± 0.07 35.48 ± 0.07 2.23 ± 0.03 13.66 ± 0.20 26.66 ± 0.21 2.23 ± 0.16 13.14 ± 0.06 26.9 ± 0.23 2 ± 0.07 12.80 ± 0.06 26.35 ± 0.09 2.58 ± 0.06 12.78 ± 0.04 26.51 ± 0.22 2.01 ± 0.03 13.33 ± 0.05 26.68 ± 0.32 1.89 ± 0.04 12.87 ± 0.23 26.35 ± 0.21
0.35 0.37 0.20 1.35 1.46 0.79 7.17 0.46 0.86 3.50 0.47 0.34 2.33 0.31 0.83 1.50 0.38 1.20 2.15 1.79 0.80
ranged from 0.16 to 6.88% and 0.2 to 7.17%, respectively. These data suggested that the method had excellent accuracy and precision. The results of the method recovery are shown in Table 3. The average recoveries of DOX in the plasma, heart, liver, spleen, lungs, kidneys and tumor were 99.15 ± 3.26%, 99.43 ± 0.75%, 99.01 ± 1.67%, 96.42 ± 2.87%, 99.85 ± 4.32%, 99.31 ± 2.21%, and 97.81 ± 2.17%, respectively. There was no significant difference (p40.05) in the recovery methods for determination of DOX contents in different biological samples. The average recoveries of DOX in the plasma and various tissues were more than 96%, and the RSD ranged from 0.75 to 4.33%. Pharmacokinetics, biodistribution, and tumor targeting of nanoparticles in vivo In this paper, the plasma and tissue levels of the DOX formulations were determined following a single injection of DOX/P(HB-HO) NPs, DOX/FA-PEG-P(HB-HO) NPs or free DOX (5 mg/kg) in female BALB/c nude mice bearing HeLa tumors. The plasma drug concentration–time curves
Kidney
Tumor
(mean ± SD) of DOX are presented in Figure 2, and the corresponding pharmacokinetic parameters are listed in Table 4. The t1/2 values of DOX/P(HB-HO) NPs and DOX/ FA-PEG-P(HB-HO) NPs were 2.7- and 3.5-times higher than that of free DOX. No significant difference (p40.05) was found in the Cmax between the NPs and free DOX. The Tmax values of the two NPs were prolonged from 0.25 to 1 h, whereas the AUC0–t values of the two NPs were 1.55- and 3.05-folds higher than that of free DOX. Moreover, MRT increased to 15.99 and 25.14 h for DOX/P(HB-HO) NPs and DOX/FA-PEG-P(HB-HO) NPs, respectively. From the above results, DOX/P(HB-HO) NPs and DOX/FA-PEG-P(HB-HO) NPs displayed improved pharmacokinetic profiles compared with free DOX in vivo. The distribution of different DOX formulations in HeLa tumor-bearing mice (Figure 3) was determined at the three time points of 4, 24, and 48 h. In the experiment, the DOX levels in the plasma and tissues gradually decreased at different rates. The DOX contents in the tumor, liver, and kidneys decreased more slowly than that in the plasma and other tissues. A significantly lower DOX contents (p50.05) in the heart was detected for the NPs groups (0.85 ± 0.37 or 0.52 ± 0.11 mg/mL) than for the free DOX group (1.86 ± 0.87 mg/mL) at 24 h (Figure 3B). The level of the free DOX in the tumors peaked at 1.74 ± 0.89 mg/mL at 4 h and dramatically decreased to 0.32 ± 0.09 mg/mL at 48 h (Figure 3A and C). For DOX/FA-PEG-P(HB-HO) NPs in the tumors, the DOX contents maximally increased at 24 h (5.39 ± 0.39 mg/mL) and then slowly decreased to 3.46 ± 0.19 mg/mL at 48 h (Figure 3B and C). However, for DOX/P(HB-HO) NPs in the tumor, the DOX level reached a peak level of 3.58 ± 0.98 mg/mL at 24 h and gradually decreased to 1.04 ± 0.38 mg/mL at 48 h (Figure 3B and C). The DOX concentrations in the tumors for the DOX/FA-PEG-P(HB-HO) NPs group were 10.81- and 3.33-times higher than those of the free DOX and DOX/ P(HB-HO) NPs groups at 48 h, respectively.
Biodistribution and pharmacokinetic studies of nanoparticles
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(A) 25
(B) 25
20
20
free DOX DOX/P(HB-HO) NPs DOX/FA-PEG-P(HB-HO) NPs
Concentration (µg/mL)
Concentration (µg/mL)
5
15
10
5
free DOX DOX/P(HB-HO) NPs DOX/FA-PEG-P(HB-HO) NPs
15
10
5
0
0 0
10
20
30
40
50
0
2
4
6
8
10
12
14
t/h
t/h
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Figure 2. Mean DOX concentration–time curves in plasma after intravenous administration of free DOX, DOX/P(HB-HO) NPs and DOX/FA-PEG-P(HB-HO) NPs to tumor-bearing mice (n ¼ 3) ((A) 0–48 h; (B) 0–12 h).
Table 4. Main pharmacokinetic parameters of DOX formulations after i.v. administration.
t1/2/h Cmax/mg mL1 Tmax/h AUC0–t/mgmL1h MRT/h
free DOX
DOX/P(HB-HO) NPs
DOX/FA-PEG-P (HB-HO) NPs
7.23 ± 1.22 20.12 ± 4.29 0.25 ± 0.03 64.62 ± 15.98 5.61 ± 1.23
19.26 ± 4.68* 20.66 ± 5.46 1.00 ± 0.01** 99.25 ± 19.66 15.99 ± 4.63*
25.08 ± 2.97** 22.78 ± 5.49 1.00 ± 0.08** 132.36 ± 25.38* 25.14 ± 7.04**
t1/2, terminal half-life; Cmax, maximum concentration; Tmax, time of maximum concentration; AUC0–t, area under plasma concentration versus time curve (0–t); MRT, mean retention time. *p50.05 compared with free DOX; **p50.01 compared with free DOX.
Discussion To date, many analysis methods for DOX, such as fluorescence assay (Bibby et al., 2005; Wang et al., 2006), radiolabelled assay (Ogawara et al., 2008), LC/MS assay (Arnold et al., 2004; DiFrancesco et al., 2007), and other methods (Hu et al., 2007; Kim & Wainer, 2010), have been reported. However, several problems may be encountered in these assay methods, such as comparatively difficult procedures, complex compositions of the mobile phases, or high costs in equipment. Favorable chromatographic conditions should include the following: baseline separation of the analyte from other exogenous materials, a mobile phase with a simple composition and an appropriate pH value, brief procedure, and relatively good accuracy and precision. In this paper, the HPLC analysis of different DOX formulations in biological samples was developed and exploited. In the experiment, a simple mobile phase consisting of acetonitrile and distilled water (adjusted to pH 3.0 with phosphoric acid) was used. The chromatographic conditions and procedures were easily controlled. According to the HPLC conditions adopted, DOX was eluted at 4.5 min within 10 min without any interfering exogenous materials in the plasma and tissues. The values of the linearity and specificity demonstrated that the peak areas and DOX concentrations
displayed perfect linear relationships and that the assay had adequate specificity. In this study, the accuracy and precision values met the requirements for the determination of DOX contents in biological samples. The favorable results showed that the simple method developed for plasma and tissue handling exhibited relatively high accuracy and recovery, and was suitable for extracting and measuring the DOX contents. The applicability of the assay method was demonstrated in this study for the pharmacokinetics, biodistribution, and targeting of free DOX, DOX/P(HB-HO) NPs and DOX/FAPEG-P(HB-HO) NPs in female BALB/c nude mice bearing HeLa tumors. The three DOX formulations reached the major tissues after intravenous administration. The DOX levels in the plasma, heart, spleen, and lungs rapidly decreased compared with those in the tumors, liver, and kidneys. This phenomenon might be related to the half-life, targeting, and sustained release effects. Compared with free DOX, the circulation and action time of the two NPs in vivo was prolonged, and their bioavailability also improved (Kalaria et al., 2009). This result might be attributed to the encapsulation of the drug into NPs, protecting them from rapid elimination after administration. This protection thereby allows the drug to be transported through blood vessel walls into other tissues. The DOX contents in the heart for the NPs was lower than that for free DOX, thus indicating that the cardiac toxicity of DOX might be substantially reduced by the formulation of NPs. Similar toxicities for DOX/P(HB-HO) NPs and DOX/FA-PEG-P(HB-HO) NPs were observed compared with those shown in previous studies. It was very obvious that the distribution of DOX in the heart for liposomal DOX preparations was significantly reduced at 0.5 h compared with the free DOX preparations (Wang et al., 2006). Furthermore, significantly lower DOX contents for the DOX micelles and DOX/FOL micelles were detected in the heart than for free DOX at 24 h (Yoo & Park, 2004). This consequence was encouraging because cardiac toxicity is a unique characteristic of DOX chemotherapies. The DOX contents in the tumors reached a peak level at 4 h and dramatically decreased at 48 h for free DOX. In contrast, when DOX/P(HB-HO) NPs or DOX/FA-PEG-P(HB-HO) NPs
C. Zhang et al. (A)
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Figure 3. Biodistribution of free DOX, DOX/P(HB-HO) NPs and DOX/FA-PEG-P(HB-HO) NPs in blood, heart, liver, spleen, lung, kidney, and tumor at different times ((A) 4 h, (B) 24 h, (C) 48 h) after intravenous administration to tumor-bearing mice (n ¼ 3). *p50.05 compared with free DOX; **p50.01 compared with free DOX.
were administered, the DOX contents remained at a high level up to 48 h. NPs tend to accumulate in the solid tumors through the enhanced permeability and retention effect (EPR) (Acharya & Sahoo, 2011), thus resulting reduced DOX concentrations in the blood and heart. These results are in good agreement with those of previous studies on cardiac toxicity. The DOX contents in the tumors for the DOX/FAPEG-P(HB-HO) NP group were much higher than that for the free DOX and DOX/P(HB-HO) NP groups at 48 h. The folatetargeted NPs exhibited a greater extent of DOX accumulation in the tumors than DOX/P(HB-HO) NPs, thus suggesting that DOX/FA-PEG-P(HB-HO) NPs were more effectively transported and actively targeted into HeLa tumors than DOX/P(HB-HO) NPs because of the receptor-mediated endocytosis (Cho et al., 2008).
Conclusion A sensitive, precise, and reproducible HPLC method was developed and adopted to quantify DOX in biological specimens, including plasma, heart, liver, spleen, lungs, kidneys, and tumor. This assay utilized a brief and convenient external standard method and only involved the rapid simple pre-treatment of liquid–liquid extraction for samples. Moreover, the method displayed sufficient sensitivity and precision in determining the DOX contents in biological
samples. The HPLC method developed in the present study has been successfully applied in evaluating the pharmacokinetics, biodistribution, and targeting of DOX/FA-PEG-P(HBHO) NPs in female BALB/c nude mice bearing HeLa tumors. In vivo studies confirmed that the circulation and bioavailability of the NPs were improved, and that the folate-targeted NPs were more effectively transported and actively targeted into HeLa tumors than non-targeted NPs because of the receptor-mediated endocytosis.
Declaration of interest The authors report that they have no conflicts of interest. This work was supported by the Doctorate Science Funds of Taiyuan University of Science and Technology [20122007]; and the Natural Science Foundation for Young Scientists of Shanxi Province, China [2013021011-7].
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DOI: 10.3109/13880209.2015.1014918
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