http://informahealthcare.com/drt ISSN: 1061-186X (print), 1029-2330 (electronic) J Drug Target, 2014; 22(4): 343–351 ! 2014 Informa UK Ltd. DOI: 10.3109/1061186X.2013.877467
ORIGINAL ARTICLE
Experiments and synthesis of bone-targeting epirubicin with the water-soluble macromolecular drug delivery systems of oxidized-dextran Li Yu, Lin Cai, Hao Hu, and Yi Zhang
Abstract
Keywords
Epirubicin (EPI) is a broad spectrum antineoplastic drug, commonly used as a chemotherapy method to treat osteosarcoma. However, its application has been limited by many side-effects. Therefore, targeted drug delivery to bone has been the aim of current anti-bone-tumor drug studies. Due to the exceptional affinity of Bisphosphonates (BP) to bone, 1-amino-ethylene-1, 1-dephosphate acid (AEDP) was chosen as the bone targeting moiety for water-soluble macromolecular drug delivery systems of oxidized-dextran (OXD) to transport EPI to bone in this article. The bone targeting drug of AEDP–OXD–EPI was designed for the treatment of malignant bone tumors. The successful conjugation of AEDP–OXD–EPI was confirmed by analysis of FTIR and 1H-NMR spectra. To study the bone-seeking potential of AEDP–OXD–EPI, an in vitro hydroxyapatite (HAp) binding assay and an in vivo experiment of bone-targeting capacity were established. The effectiveness of AEDP–OXD–EPI was demonstrated by inducing apoptosis and necrosis of MG-63 tumor cell line. The obtained experimental data indicated that AEDP–OXD–EPI is an ideal bone-targeting anti-tumor drug.
Anti-tumor drug, bisphosphonates, bone-targeting, epirubicin, oxidized-dextran
Introduction Osteosarcoma is one of the most common malignant tumors of the skeletal system and more likely to occur at metaphysis of long bones in adolescents. The tumor frequently develops distant metastasis early and has poor prognosis because of the high degree of malignancy [1]. In recent years, comprehensive treatment measures, including new adjuvant chemotherapy and tumor surgical resection, elevates the 5-year survival rate of osteosarcoma from 20–30% to 60–80% [2,3]. Epirubicin (EPI, Scheme 1), a broad-spectrum anti-tumor drug, is commonly used to treat osteosarcoma as a chemotherapy drug because of its high anti-tumor activity [4]. However, its application has been limited by its side-effects, including cardiac toxicity, bone marrow suppression and so on [5]. In order to improve the concentration of anti-tumor drugs in the bone tumor tissue and decrease the drug’s toxicity, the bone targeting drugs have been a major research focus [6]. Water-soluble macromolecular drug delivery systems have been studied extensively for their applications in cancer
Address for correspondence: Lin Cai, Department of Orthopeadics, Zhongnan Hospital of Wuhan University, No. 169 Donghu Road, Wuhan 430071, Hubei, China. E-mail: [email protected]
History Received 3 September 2013 Revised 8 December 2013 Accepted 17 December 2013 Published online 9 January 2014
chemotherapy [7]. The drug delivery systems had many advantages, such as longer half-life in circulation, passive targeting to solid tumor, improved water-solubility of hydrophobic drugs and increased accumulation of the drug at the target location [8]. Oxidized-Dextran (OXD), a water-soluble macromolecular polymer, has been used as universal vehicles for achieving controlled drug release and drug targeting [9]. In particular, various dextran-antitumor drug conjugates enhance the effectiveness and improve the cytotoxic effects of chemotherapeutic agents [10]. Bisphosphonates (BP), which has a simple structure and relative chemical stability, are widely used in the synthesis of bone targeting drugs as bone-targeting moiety [11]. These agents, by virtue of their P–C–P backbone structure and ability to chelate calcium ions, target rapidly to bone mineral. Bisphosphonate-targeted liposomes [12] and bisphosphonatetargeted taxane [13] have previously been successfully prepared. Besides enhancing bone density and inhibit osteoclast activity, recent studies show that BP can prevent bone metastases and eliminate the tumor cells directly [14,15]. Therefore, we designed and synthesized a water-soluble polymeric bone-targeting drug, whose macromolecular delivery was OXD, bone-targeting moiety was 1-aminoethylene-1, 1-dephosphate acid (AEDP, Scheme 2) and bone therapeutic agent was EPI. It was evaluated using in vitro and in vivo experiments that the bone-targeting drug had a remarkable attraction to bone and a synergistic antitumor activity.
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Department of Orthopeadics, Zhongnan Hospital of Wuhan University, Wuhan, China
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Scheme 1. Chemical structure of EPI.
Scheme 2. Chemical structure of AEDP.
Materials and methods Materials EPI was provided by Hisun Pharmaceutical Co., Ltd. (Zhejiang, China). AEDP was offered by Chemistry and Molecular Sciences College of Wuhan University (Wuhan, China). Sephadex G-50 was purchased from Shanghai Kai Yang Biotechnology Co., Ltd. (Shanghai China). Dextran (Dex-T40, MW 40 kDa), sodium periodate, hydroxyapatite (HAp) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other chemical reagents were of analytical grade and obtained from commercial sources. Annexin V/PI was purchased from Beckman Coulter, Inc. (Brea, CA). Dialysis bag of Cutoff molecular weight 3500 was purchased from Viskase Companies, Inc. (Darien, IL). Human OS MG-63 cell line was obtained from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). MG-63 cells were cultured in MEM/ EBSS (Hyclone, Logan, UT) supplemented with 10% heatinactivated fetal bovine serum (FBS, Hyclone), 50 U/mL penicillin and 50 mg/mL streptomycin in a humidified incubator with 5% CO2 at 37 C. 12 Kunming mice (SPF) were provided by the animal center of Medicine School of Wuhan University. Synthesis and characterization of bone-targeting EPI (AEDP–OXD–EPI) Dex-T40 (10 g, 62.5 mmol of glucose units) and sodium periodate (13.5 g) were dissolved in 200 and 150 mL pH 4.4 phosphate buffer, respectively. The sodium periodate solution was slowly dropped into the dextran solution. The mixture
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was vigorously stirred (1500 rpm) in the dark at room temperature until a clear orange solution was obtained (6–8 h). Then, 4.5 mL of glycerol was added in the mixture to stop the reaction followed by extensive dialysis (Cutoff molecular weight 3500) against double deionized water (DDW) for 48 h and at 4 C. Purified OXD was freeze-dried to obtain a white powder in 5060% average yield. The content of formyl group in OXD was determined by the hydroxylamine hydrochloride method [16]. About 82 mg AEDP (0.4 mmol) was dissolved in 10 mL DDW and 100 mg OXD was dissolved in 5 mL distilled water. The AEDP solution was dropped into the OXD solution slowly, and then the mixture liquid was incubated at 4 C for 24 h. About 20 mg EPI was dissolved in 10 mL DDW. The EPI solution was dropped into the above reaction solution and stirred at 4 C for 24 h in dark. Finally, the reaction solution was purified by a gel column of Sephadex G-50. The resulting purified solution was then freeze-dried to give a red powder. Scheme 3 illustrated the synthesis route of bone-targeting EPI. The formation of the intermediates and AEDP–OXD– EPI were confirmed by FTIR spectroscopy (Shimadzu IR Prestige 21) and 1H-NMR spectroscopy measurement (INOVA 600 MHz). Determination of drug-loading dose of AEDP–OXD–EPI About 15 mg/mL EPI, 0.25 mg/mL AEDP–OXD–EPI and 2 mg/mL AEDP–OXD solution (solvent deionized water, 20.024.0 C) were scanned from 200 to 800 nm wavelength range using an ultraviolet-visible light spectrophotometer (Hach Company, Loveland, CO). The results showed that EPI maximum absorption was at 233 and 480 nm, and AEDP– OXD had no absorption at 480 nm (Figure 1). Therefore, 480 nm was chosen as the measurement wavelength for avoiding interference. DDW was used as a blank control to measure EPI content in 0.25 mg/mL AEDP–OXD–EPI. Drugloading (DL) dose of AEDP–OXD–EPI was calculated according to the following formula: DL ðdrugÞ ¼ WE =WD 100%, WD: AEDP–OXD–EPI concentration; WE: the measured concentration of EPI. Induced apoptosis and necrosis of AEDP–OXD–EPI in human osteosarcoma cell line MG-63 The MG-63 cells suspension (5 105/mL) were added into 6-well plates, each well 3 mL, and placed in incubator for 24 h. And then the culture fluids was changed with the cellculture medium contained 18.83 mg/mL AEDP–OXD–EPI (1 mg/mL EPI equivalent), 1 mg/mL EPI, 100 mg/mL OXD and 40 mg/mL AEDP, respectively (n ¼ 3). The negative control group without any drug was set up. After 24 h culture with the drugs, the apoptosis and necrosis of MG-63 cells were analyzed using a flow cytometer with Annexin V-FITC (fluorescein isothiocyanate) and propidium iodide (PI) staining. Apoptosis Detection Kit (KeyGen Biotech Co. Ltd., Najing, China) was applied in the present study. Cells were collected and washed twice in ice cold PBS and resuspended in 300 ml of binding buffer at 2 105 cells/mL.
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Scheme 3. Conjugation of AEDP–OXD–EPI.
(n ¼ 5). The amount of drugs not bound to HAp were quantified by ultraviolet-visible light spectrophotometer (measurement wavelength: 480 nm) in the supernatant after centrifugation of incubated HAp samples at 6000 rpm for 60 min. The initial binding kinetics of AEDP–OXD–EPI was obtained by the same method. The degree of HAP binding was assessed as mean of five independent experiments according to the following formula: HAp binding ð%Þ ¼ Figure 1. UV spectra of EPI, AEDP–OXD–EPI and AEDP–OXD.
The samples were incubated with 5 ml of Annexin V-FITC and 5 ml PI in the dark for 15 min at room temperature. Finally, samples were analyzed by flow cytometry. Bone-targeting capacity of AEDP–OXD–EPI in vitro HAp-binding assay described by Shinoda et al. [17] and Fujisawa et al. [18] was set up to evaluate the bone-targeting capacity of AEDP–OXD–EPI in vitro. Briefly, 15 mg Nanocrystalline HAp was incubated with 1 mL solution of 0.5 mg/mL AEDP–OXD–EPI, 25 mg/mL EPI, 1 mg/mL OXD and 1 mg/mL AEDP–OXD overnight at 25 C, respectively
ðX YÞ 100% X
X is the initial concentration of drug and Y is the drug concentration of supernatant after centrifugation. Bone-targeting capacity of AEDP–OXD–EPI in vivo Twelve Kunming mice, (18.7 ± 2.1) g, were divided into four groups (n ¼ 3). Group A (AEDP–OXD–EPI) were injected with 0.2 mL of solution 28.8 mg/mL AEDP–OXD–EPI (1.5 mg/mL EPI equivalent) via the tail vein; B group (EPI), were injected with 0.2 mL of 1.5 mg/mL EPI; C group (AEDP–OXD) group were injected 0.2 mL of 27.3 mg/mL AEDP–OXD; D groups (control group) were injected with 0.9% saline 0.2 mL. All the animals remained active with normal food and water consumption after the injection.
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Twenty-four hours later, animals were euthanized and then distal femora were resected en bloc, fixed in acetone for 24 h. The slices of hard tissue were made, and observed by fluorescence microscope (Leica DM4000B). All the photos were shot at the same conditions (Exposure time 1/3 s and sensitivity 400) and were analyzed fluorescence intensity of the slices using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD).
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Statistical analysis Variables used in this study were expressed as the mean ± SEM. The data were analyzed by SPSS 16.0 software (SPSS Inc., Chicago, IL). Statistical significance was determined by one-way analysis of variance (ANOVA) and Tukey’s multiple-comparison. p value 50.05 was considered statistically significant.
Results and discussion Characterization of bone-targeting EPI The conjugation scheme of AEDP–OXD–EPI was confirmed by analysis of FTIR and 1H-NMR spectra. Figure 2 shows the
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FTIR spectra of AEDP (a), Dex (b), OXD (c), AEDP-OXD (d), AEDP-OXD-EPI (e) and EPI (f). The spectra of OXD showed an absorption band at 1728.16 cm1, which assigned to the aldehyde groups (–C ¼ O), while the spectra of Dex did not have the characteristic band. The content of formyl group in OXD was (10.24 ± 0.20) mmol/g (n ¼ 5) via the hydroxylamine hydrochloride method. Therefore, Dex was oxidized successfully to OXD, which had the structure of polyaldehyde. The absorption band, which appeared at 1585.75 cm1 on the spectra of AEDP–OXD, was ascribed to the –HC ¼ N of Schiff Base, because the absorption band of AEDP aminogroup appeared at 1521.47 cm1. About 1021.99 cm1 vibration absorption peak in AEDP–OXD of was ascribed to P–O, which was brought in by AEDP, and it can also be found in the FTIR spectrum of AEDP–OXD–EPI. Comparing with the FTIR of AEDP–OXD, a new carbonyl absorption band of EPI at 1728.16 cm1, but not the aldehyde band at 1723.78 cm1, was observed in the spectra of AEDP–OXD– EPI. The absorption band of AEDP–OXD–EPI from 1200 to 1000 cm1 was different from the AEDP–OXD. The result may be due to the superposition of hydroxyl groups’ absorption bands from OXD and EPI. The synthesis of AEDP–OXD–EPI was also confirmed by 1 H-NMR spectroscopy. The multiple peaks at dH ¼ 1.23, 1.52, 1.55, 1.57 ppm corresponded to the methyl protons of AEDP and EPI. The peaks at dH ¼ 2.10, 2.12, 2.17 corresponded to the –CH2, –CH protons of EPI. The double peaks at dH ¼ 7.80, 7.95 ppm correspond to the benzene ring protons of EPI. The peak at dH ¼ 7.69 ppm was the proton peak of Schiff base structure (–HC ¼ N–). The small peak at d9.62 corresponded to the aldehyde protons of oxidized dextran, which were not completely reacted with AEDP and EPI. (Figure 3) Therefore, it can be seen, from the spectroscopy of FTIR and 1H-NMR, that the synthesis of AEDP–OXD–EPI was successful. DL dose of AEDP–OXD–EPI
Figure 2. FTIR spectra of raw materials, intermediate and final products.
According to the standard curve equation of EPI at 480 nm, 0.25 mg AEDP–OXD–EPI contained (13.28 ± 0.51 mg) EPI, and then the drug loading of AEDP–OXD–EPI was (5.31 ± 0.20%), calculated by the drug loading formula. Theoretically, after 100 mg OXD (containing aldehyde 1.02 mmol) reacted with 82 mg AEDP (0.4 mmol), the remained aldehyde groups of OXD can be combined with 360 mg EPI (0.62 mmol, MW 579.99). According to the analysis, the theoretical drug loading of AEDP–OXD–EPI should be 66.42%, but the actual drug loading just was 5.31%. The result showed that the majority of the aldehyde groups had not reacted with EPI. The 1H-NMR spectroscopy of AEDP–OXD–EPI also clearly illustrated that the proton peak of aldehyde group appeared at dH ¼ 9.62 ppm. The following reasons may explain the incomplete reaction: (1) the status of OXD longer carbon chains in solution can lead to inadequate exposure of aldehyde [19] and (2) after binding with aldehyde, the molecule drugs can occupy more space so that the closed aldehyde group cannot participate in the reaction [20]. Therefore, we used the gel column of Sephadex G-50 to remove the unbound AEDP and EPI.
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Figure 3. 1H-NMR spectrum of AEDP–OXD–EPI. a: The multiple peaks of methyl protons in EPI; b: the multiple peaks of methyl protons in AEDP; c: the protons peaks of –CH2, –CH in EPI; d: the proton peak of Schiff base (–HC¼N–); e: the peaks of benzene ring protons in EPI; f: the proton peak of aldehyde in OXD.
Induced apoptosis and necrosis of AEDP–OXD–EPI in MG-63 cell line The results measured by Annexin/PI double staining showed that the necrosis and apoptosis ratio of MG-63 cell in AEDP– OXD–EPI group was significantly higher than EPI group, at the same concentration of EPI (1 mg/mL) (p50.05). We observed that AEDP also can induce the apoptosis and necrosis of osteosarcoma cells, but OXD had not obvious effects on apoptosis (Figure 4). Over the past two decades, BP is widely used for the prevention and treatment of diseases in which there is an increase in the number or activity of osteoclasts, such as osteoporosis, cancer metastasis and Paget’s disease of bone [21]. BP can reduce bone resorption and increase bone mineral density by inducing osteoclast apoptosis. Recent studies also suggest that bisphosphonates have direct effects on tumor cells, including inhibiting tumor cells adhesion, invasion and proliferation [15,22] and inducing their apoptosis [23]. Additionally, BP may inhibit angiogenesis [24] and the release of promote cancer cell growth factors [25], regulate the immune effects of cdT cells [26] and enhance the antitumor activities of cytotoxic agents [27]. Segal et al. [28] synthesized a bone-targeting nano-scaled N-(2-hydroxypropyl) methacrylamide (HPMA)
copolymer-alendronate (ALN)-TNP-470 conjugate and proved the conjugate revealed superior anti-tumor activity and decreased organ-related toxicities of the conjugate compared with the combination of free ALN plus TNP-470. Our experiment also confirmed that AEDP also could significantly induce the apoptosis of osteosarcoma cell line MG-63. The bone-targeting drug, AEDP–OXD–EPI, had superior anti-tumor activity in vitro than free EPI or AEDP. We hypothesize that the result was due to that the anti-tumor effect and synergistic anti-tumor effect of AEDP enhanced the drug effect of EPI. The result was similar with Segal’s experiments [29]. Therefore, the effects of BP in bonetargeting drug were not only the oriented agent, but also the therapeutic agent which could kill tumor cells directly. Bone-targeting capacity of AEDP–OXD–EPI According to the HAp binding formula, the HAp binding rates of OXD, AEDP–OXD, AEDP–OXD–EPI and EPI were (3.81 ± 2.07)%, (69.87 ± 3.36)%, (85.47 ± 1.27)%, (12.88 ± 5.10)%, respectively (Figure 5). AEDP–OXD–EPI had a better ability to bind with HAp than EPI in vitro. The binding of the AEDP–OXD–EPI to the surface of HA was observed to occur very quickly. The result of the initial binding kinetic was shown in Figure 6. The binding of
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Figure 4. The necrosis and apoptosis ratio of MG-63 cell induced by AEDP–OXD–EPI, EPI, AEDP and OXD. (A) AEDP–OXD–EPI was (96.51 ± 3.25)%; (B) EPI was (84.55 ± 3.97)%; (C) AEDP was (33.99 ± 7.3)% and (D) OXD was (15.19 ± 3.48)%. Cell necrosis and apoptosis ratio in AEDP–OXD–EPI is significantly higher (p 5 0.05 versus B, C and D, E), EPI is higher than C and D (p 50.05), AEDP is higher than OXD (p50.05). Note: The lower left quadrant (LL), normal cells; upper left quadrant (UL), necrotic cells; lower right quadrant (LR), early apoptotic cells; upper right quadrant (UR), late apoptotic cells.
Figure 5. The binding of OXD, EPI, AEDP–OXD and AEDP–OXD–EPI to hydroxyapatite. Conjugates were dissolved in phosphate buffered saline (pH ¼ 7.4). Background correction was applied. Data are shown as the mean SD from five different measurements (F ¼ 866.16, p50.01).
AEDP–OXD–EPI reached a plateau in 5 min with 82.16% of the conjugate bound to HAp. Prolonged incubation of the conjugate with HAp did not significantly improve binding efficiency (overnight, 85.47%, Figure 5). In vivo bone binding experiment demonstrated that the fluorescence of group C and group D is extremely weak
Figure 6. The initial binding kinetics of AEDP–OXD–EPI to hydroxyapatite. AEDP–OXD–EPI was dissolved in phosphate buffered saline (pH ¼ 7.4) with a concentration of 0.5 mg/mL. Data are shown as the mean SD from triplicate measurements.
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Figure 7. The in vivo binding of AEDP–OXD–EPI to the bone, 5. The autofluorescence of EPI can be observed in AEDP–OXD–EPI (A) and EPI (B). Figure 6(A) showed that the autofluorescence of EPI was located in cortical and cancellous bone of femoral metaphyseal and there was no red fluorescence in the epiphysis because of its own barrier function. The fluorescence IOD of AEDP–OXD–EPI was stronger than EPI. There was only very tiny autofluorescence of bone tissue in AEDP–OXD (C) and saline injection (D).
(Figure 7C and D), while very strong fluorescence was observed in A group and B group (Figure 7A and B). Via the analysis of Image-Pro Plus 6.0 software, the fluorescence integrated optical density (IOD) of group A and group B were 37.95 ± 3.97 and 15.26 ± 2.52 (p50.05), respectively. The fluorescence intensity of group A was about 2.5 times than of group B (Figure 7). Epirubicin is an anthracycline cytotoxic agent and a broad spectrum antineoplastic drug [30,31]. The main antineoplastic mechanism of EPI is interfering with the transcription process and preventing the formation of mRNA by intercalation of DNA planar rings between nucleotide base pairs [32,33]. EPI is a cell cycle phase non-specific anthracycline, which has a killing effect on a variety of tumor cell in different growth cycle. But EPI also has a strong cytotoxic effect on human body. The most common toxic reactions include: Neutropenia and thrombocytopenia in 60–80% patients; 100% of patients will endure the hair loss to varying degrees [34,35]; the cardiotoxicity often presents as ECG changes (especially change in the frequency of QRS complex) and arrhythmias, or as a cardiomyopathy leading to heart failure (sometimes presenting many years after treatment) [36]; and nausea and loss of appetite. EPI was usually used as chemotherapy drugs to treat human osteosarcoma. The 5-year disease-free and overall survival rates in patients with localized primary osteosarcoma, who were treat with EPI combined with cisplatin and ifosfamide, were 440% [4]. However, many patients suffered from the
above-mentioned cytotoxic effect throughout the whole course of chemotherapy. Dextran serves as one of the most promising macromolecular carrier candidates for a wide variety of therapeutic agents due to their excellent physico-chemical properties and physiological acceptance [10]. It is non-toxic, non-immunogenic and non-antigenic. It contains huge number of carbohydrate hydroxyl groups available for drug fixation [37]. The dextran conjugates have low clearance and relatively long plasma half-life because of large molecular weight polysaccharides, resulting in accumulation in tumor tissues [38]. Therefore, the various dextran-antitumor drug conjugates were prepared to enhance the effectiveness and improve the cytotoxic effects of chemotherapeutic agents. In particular, the dextran–anthracycline conjugates, such as dextran– Adriamycin (ADM) and dextran–EPI, showed excellent pharmacological properties [39,40]. Ueda et al. [41] proved that Adriamycin linked to oxidized dextran (ADM–OXD) via Schiff’s base showed higher activity against Walker carcinosarcoma 256 and lower acute toxicity than free Adriamycin in rat model. Phase I clinical and pharmacokinetic trial of dextran conjugated doxorubicin (DOX–OXD) proved that the maximal tolerated dose of DOX–OXD can be considered to be 40 mg/m2 doxorubicin equivalent since no toxic deaths were observed at this dose level [42]. The in vivo binding of bone-targeting conjugates to bone is much more complicated than the in vitro HAp binding mechanism. Because EPI has the character of
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autofluorescence, the in vivo bone binding assay was performed according to Wang et al. [43]. AEDP–OXD–EPI was able to arrive at the periosteum, cortical bone, cancellous bone and bone marrow via blood circulation (Figure 7A). EPI has similar molecular structure with Tetracycline (TC) which is a well-known bone-seeking drug. The structure of adjacent carbonyl (¼O) and hydroxyl (–OH), is considered to be the foundation of bone-targeting [44], exists in EPI and TC. The pro bone ability of EPI was also revealed by the vitro HAp binding and vivo bone binding assay in the article. The reason why AEDP–OXD–EPI conjugate had higher binding ratio to bone compared with AEDP–OXD may result from the bone-seeking ability of EPI. The in vivo and in vitro assay proved that AEDP–OXD– EPI had a good bone-targeting capacity, which would help increase the EPI concentration in the bone and reduce the treatment dose. Furthermore, the synergistic anti-tumor effect of AEDP could further reduce the dosage of EPI. Therefore, if AEDP–OXD–EPI can be used for the chemotherapy of bone cancer, it will be benefit to improve the chemotherapy effect of EPI and reduce its toxic and side effects. Nevertheless, animal models would strengthen the in vivo aspect of this new targeted drug.
Conclusion A new polymeric bone-targeting drug, AEDP–OXD–EPI, was prepared in the experiment. The macromolecular delivery of AEDP–OXD–EPI was OXD, bone-targeting moiety was BP and bone therapeutic agent was EPI. The successful conjugation of AEDP–OXD–EPI was confirmed by analysis of FTIR and 1H-NMR spectra. It was demonstrated by in vitro and in vivo experiments that the bone-targeting drug exhibited a remarkable affinity to bone. Induced apoptosis and necrosis in MG-63 cell line showed that AEDP–OXD–EPI had senior anti-tumor activity than EPI in vitro. However, we need to do more animal experiments to prove whether or not AEDP– OXD–EPI was an ideal anti-bone-tumor drug.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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6. Wang D, Miller SC, Kopeckova´ P, Kopecek J. Bone-targeting macromolecular therapeutics. Adv Drug Deliv Rev 2005;57: 1049–76. 7. Duncan R. The dawning era of polymer therapeutics. Nat Rev Drug Discov 2003;2:347–60. 8. Low SA, Kopecˇek J. Targeting polymer therapeutics to bone. Adv Drug Deliv Rev 2012;64:1189–204. 9. Wu H, Zhang Z, Wu D, et al. Preparation and drug release characteristics of Pingyangmycin-loaded dextran cross-linked gelatin microspheres for embolization therapy. J Biomed Mater Res B Appl Biomater 2006;78:56–62. 10. Varshosaz J. Dextran conjugates in drug delivery. Expert Opin Drug Deliv 2012;9:509–23. 11. Uludag H. Bisphosphonates as a foundation of drug delivery to bone. Curr Pharm Des 2002;8:1929–44. 12. Hengst V, Oussoren C, Kissel T, Storm G.Bone targeting potential of bisphosphonate-targeted liposomes. Preparation, characterization and hydroxyapatite binding in vitro. Int J Pharm 2007; 331:224–7. 13. Miller K, Erez R, Segal E, et al. Targeting bone metastases with a bispecific anticancer and antiangiogenic polymeralendronate-taxane conjugate. Angew Chem Int Ed Engl 2009;48: 2949–54. 14. Heymann D, Ory B, Gouin F, et al. Bisphosphonates: new therapeutic agents for the treatment of bone tumors. Trends Mol Med 2004;10:337–43. 15. Cle´zardin P, Ebetino FH, Fournier PG. Bisphosphonates and cancer-induced bone disease: beyond their antiresorptive activity. Cancer Res 2005;65:4971–4. 16. Zhao H, Heindel ND. Determination of degree of substitution of formyl groups in polyaldehyde dextran by the hydmxylamine hydroehloride method. Pharma Res 1991;8:400–2. 17. Shinoda H, Adamek G, Felix R, et al. Structure activity relationships of various bisphosphonates. Calcif Tissue Int 1983; 35:87–99. 18. Fujisawa R, Wada Y, Nodasaka Y, Kuboki Y. Acidic amino acidrich sequences as binding sites of osteonectin to hydroxyapatite crystals. Biochim Biophys Acta 1996;1292:53–60. 19. Yuan XB, Li H, Zhu XX, Woo HG. Self-aggregated nanoparticles composed of periodate-oxidized dextran and cholic acid: preparation, stabilization and in-vitro drug release. J Chem Technol Biotechnol 2006;81:746–54. 20. Chen FZ, Cai L, Sun JH, Zhang KY. Investigation on the preparation and properties of adriamycin-dextran-tetracycline conjugate. J Math Med 2006;19:294–6. 21. Rogers MJ, Crockett JC, Coxon FP, Mo¨nkko¨nen J. Biochemical and molecular mechanisms of action of bisphosphonates. Bone 2011; 49:34–41. 22. Nakajima H, Magae J, Tsuruga M, et al. Induction of mitochondriadependent apoptosis through the inhibition of mevalonate pathway in human breast cancer cells by YM529, a new third generation bisphosphonate. Cancer Lett 2007;253:89–96. 23. Cheng YY, Huang L, Lee KM, et al. Bisphosphonates induce apoptosis of stromal tumor cells in giant cell tumor of bone. Calcif Tissue Int 2004;75:71–7. 24. Edwin MP, William LD, James G. The emerging role of bisphosphonates in prostate cancer. Am J Ther 2004;11:60–73. 25. Tenta R, Sourla A, Lembessis P, et al. Bone microenviromentrelated growth factors, zoledronic acid and dexamethasone differentially modulate PTHrP expression in PC-3 prostate cancer cells. Horm Metab Res 2005;37:593–601. 26. Sanders JM, Subhash G, Julian MWC, et al. Quantitative structureactivity relationships for gammadelta T cell activation by bisphosphonates. J Med Chem 2004;47:375–84. 27. Neville-Webbe HL, Evans CA, Coleman RE, Holen I. Mechanisms of the synergistic interaction between the bisphosphonate zoledronic acid and the chemotherapy agent paclitaxel in breast cancer cells in vitro. Tumour Biol 2006;27:92–103. 28. Segal E, Pan H, Benayoun L, et al. Enhanced anti-tumor activity and safety profile of targeted nano-scaled HPMA copolymeralendronate-TNP-470 conjugate in the treatment of bone malignances. Biomaterials 2011;32:4450–63. 29. Segal E, Pan H, Ofek P, et al. Targeting angiogenesis-dependent calcified neoplasms using combined polymer therapeutics. PLoS One 2009;4:e5233.
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30. Lown JW. Anthracycline and anthraquinone anticancer agents: current status and recent developments. Pharmacol Ther 1993;60: 185–214. 31. Nielsen D, Maare C, Skovsgaard T. Cellular resistance to anthracyclines. Gen Pharmacol 1996;27:251–5. 32. Gewirtz DA. A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin. Biochem Pharmacol 1999;57: 727–41. 33. Rivera E. Implications of anthracycline-resistant and taxaneresistant metastatic breast cancer and new therapeutic options. Breast J 2010;16:252–63. 34. Roche´ H, Fumoleau P, Spielmann M, et al. Sequential adjuvant epirubicin-based and docetaxel chemotherapy for node-positive breast cancer patients: the FNCLCC PACS 01 Trial. J Clin Oncol 2006;24:5664–71. 35. Cersosimo RJ, Hong WK. Epirubicin: a review of the pharmacology, clinical activity, and adverse effects of anadriamycin analogue. J Clin Oncol 1986;4:425–39. 36. Kremer LC, van Dalen EC, Offringa M, et al. Anthracyclineinduced clinical heart failure in a cohort of 607 children: long-term follow-up study. J Clin Oncol 2001;19:191–6. 37. Vert M. Polyvalent polymeric drug carriers. Crit Rev Ther Drug Carrier Syst 1986;2:291–327.
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38. Mehvar R. Recent trends in the use of polysaccharides for improved delivery of therapeutic agents: pharmacokinetic and pharmacodynamic perspectives. Curr Pharm Biotechnol 2003;4: 283–302. 39. Lam W, Leung CH, Chan HL, Fong WF. Toxicity and DNA binding of dextran-doxorubicin conjugates in multidrug-resistant KB-V1 cells: optimization of dextran size. Anticancer Drugs 2000;11: 377–84. 40. Bisht S, Maitra A. Dextran-doxorubicin/chitosan nanoparticles for solid tumor therapy. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2009;1:415–25. 41. Ueda Y, Munechika K, Kikukawa A, et al. Comparison of efficacy, toxicity and pharmacokinetics of free adriamycin and adriamycin linked to oxidized dextran in rats. Chem Pharm Bull (Tokyo) 1989; 37:1639–41. 42. Danhauser-Riedl S, Hausmann E, Schick HD, et al. Phase I clinical and pharmacokinetic trial of dextran conjugated doxorubicin (AD-70, DOX-OXD). Invest New Drugs 1993;11: 187–95. 43. Wang D, Miller S, Sima M, et al. Synthesis and evaluation of watersoluble polymeric bone-targeted drug delivery systems. Bioconjug Chem 2003;14:853–9. 44. Perrin RA. Binding of tetracycline to bone. Nature 1965;208: 787–8.
ORIGINAL ARTICLE
Experiments and synthesis of bone-targeting epirubicin with the water-soluble macromolecular drug delivery systems of oxidized-dextran Li Yu, Lin Cai, Hao Hu, and Yi Zhang
Abstract
Keywords
Epirubicin (EPI) is a broad spectrum antineoplastic drug, commonly used as a chemotherapy method to treat osteosarcoma. However, its application has been limited by many side-effects. Therefore, targeted drug delivery to bone has been the aim of current anti-bone-tumor drug studies. Due to the exceptional affinity of Bisphosphonates (BP) to bone, 1-amino-ethylene-1, 1-dephosphate acid (AEDP) was chosen as the bone targeting moiety for water-soluble macromolecular drug delivery systems of oxidized-dextran (OXD) to transport EPI to bone in this article. The bone targeting drug of AEDP–OXD–EPI was designed for the treatment of malignant bone tumors. The successful conjugation of AEDP–OXD–EPI was confirmed by analysis of FTIR and 1H-NMR spectra. To study the bone-seeking potential of AEDP–OXD–EPI, an in vitro hydroxyapatite (HAp) binding assay and an in vivo experiment of bone-targeting capacity were established. The effectiveness of AEDP–OXD–EPI was demonstrated by inducing apoptosis and necrosis of MG-63 tumor cell line. The obtained experimental data indicated that AEDP–OXD–EPI is an ideal bone-targeting anti-tumor drug.
Anti-tumor drug, bisphosphonates, bone-targeting, epirubicin, oxidized-dextran
Introduction Osteosarcoma is one of the most common malignant tumors of the skeletal system and more likely to occur at metaphysis of long bones in adolescents. The tumor frequently develops distant metastasis early and has poor prognosis because of the high degree of malignancy [1]. In recent years, comprehensive treatment measures, including new adjuvant chemotherapy and tumor surgical resection, elevates the 5-year survival rate of osteosarcoma from 20–30% to 60–80% [2,3]. Epirubicin (EPI, Scheme 1), a broad-spectrum anti-tumor drug, is commonly used to treat osteosarcoma as a chemotherapy drug because of its high anti-tumor activity [4]. However, its application has been limited by its side-effects, including cardiac toxicity, bone marrow suppression and so on [5]. In order to improve the concentration of anti-tumor drugs in the bone tumor tissue and decrease the drug’s toxicity, the bone targeting drugs have been a major research focus [6]. Water-soluble macromolecular drug delivery systems have been studied extensively for their applications in cancer
Address for correspondence: Lin Cai, Department of Orthopeadics, Zhongnan Hospital of Wuhan University, No. 169 Donghu Road, Wuhan 430071, Hubei, China. E-mail: [email protected]
History Received 3 September 2013 Revised 8 December 2013 Accepted 17 December 2013 Published online 9 January 2014
chemotherapy [7]. The drug delivery systems had many advantages, such as longer half-life in circulation, passive targeting to solid tumor, improved water-solubility of hydrophobic drugs and increased accumulation of the drug at the target location [8]. Oxidized-Dextran (OXD), a water-soluble macromolecular polymer, has been used as universal vehicles for achieving controlled drug release and drug targeting [9]. In particular, various dextran-antitumor drug conjugates enhance the effectiveness and improve the cytotoxic effects of chemotherapeutic agents [10]. Bisphosphonates (BP), which has a simple structure and relative chemical stability, are widely used in the synthesis of bone targeting drugs as bone-targeting moiety [11]. These agents, by virtue of their P–C–P backbone structure and ability to chelate calcium ions, target rapidly to bone mineral. Bisphosphonate-targeted liposomes [12] and bisphosphonatetargeted taxane [13] have previously been successfully prepared. Besides enhancing bone density and inhibit osteoclast activity, recent studies show that BP can prevent bone metastases and eliminate the tumor cells directly [14,15]. Therefore, we designed and synthesized a water-soluble polymeric bone-targeting drug, whose macromolecular delivery was OXD, bone-targeting moiety was 1-aminoethylene-1, 1-dephosphate acid (AEDP, Scheme 2) and bone therapeutic agent was EPI. It was evaluated using in vitro and in vivo experiments that the bone-targeting drug had a remarkable attraction to bone and a synergistic antitumor activity.
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Department of Orthopeadics, Zhongnan Hospital of Wuhan University, Wuhan, China
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Scheme 1. Chemical structure of EPI.
Scheme 2. Chemical structure of AEDP.
Materials and methods Materials EPI was provided by Hisun Pharmaceutical Co., Ltd. (Zhejiang, China). AEDP was offered by Chemistry and Molecular Sciences College of Wuhan University (Wuhan, China). Sephadex G-50 was purchased from Shanghai Kai Yang Biotechnology Co., Ltd. (Shanghai China). Dextran (Dex-T40, MW 40 kDa), sodium periodate, hydroxyapatite (HAp) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other chemical reagents were of analytical grade and obtained from commercial sources. Annexin V/PI was purchased from Beckman Coulter, Inc. (Brea, CA). Dialysis bag of Cutoff molecular weight 3500 was purchased from Viskase Companies, Inc. (Darien, IL). Human OS MG-63 cell line was obtained from the Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). MG-63 cells were cultured in MEM/ EBSS (Hyclone, Logan, UT) supplemented with 10% heatinactivated fetal bovine serum (FBS, Hyclone), 50 U/mL penicillin and 50 mg/mL streptomycin in a humidified incubator with 5% CO2 at 37 C. 12 Kunming mice (SPF) were provided by the animal center of Medicine School of Wuhan University. Synthesis and characterization of bone-targeting EPI (AEDP–OXD–EPI) Dex-T40 (10 g, 62.5 mmol of glucose units) and sodium periodate (13.5 g) were dissolved in 200 and 150 mL pH 4.4 phosphate buffer, respectively. The sodium periodate solution was slowly dropped into the dextran solution. The mixture
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was vigorously stirred (1500 rpm) in the dark at room temperature until a clear orange solution was obtained (6–8 h). Then, 4.5 mL of glycerol was added in the mixture to stop the reaction followed by extensive dialysis (Cutoff molecular weight 3500) against double deionized water (DDW) for 48 h and at 4 C. Purified OXD was freeze-dried to obtain a white powder in 5060% average yield. The content of formyl group in OXD was determined by the hydroxylamine hydrochloride method [16]. About 82 mg AEDP (0.4 mmol) was dissolved in 10 mL DDW and 100 mg OXD was dissolved in 5 mL distilled water. The AEDP solution was dropped into the OXD solution slowly, and then the mixture liquid was incubated at 4 C for 24 h. About 20 mg EPI was dissolved in 10 mL DDW. The EPI solution was dropped into the above reaction solution and stirred at 4 C for 24 h in dark. Finally, the reaction solution was purified by a gel column of Sephadex G-50. The resulting purified solution was then freeze-dried to give a red powder. Scheme 3 illustrated the synthesis route of bone-targeting EPI. The formation of the intermediates and AEDP–OXD– EPI were confirmed by FTIR spectroscopy (Shimadzu IR Prestige 21) and 1H-NMR spectroscopy measurement (INOVA 600 MHz). Determination of drug-loading dose of AEDP–OXD–EPI About 15 mg/mL EPI, 0.25 mg/mL AEDP–OXD–EPI and 2 mg/mL AEDP–OXD solution (solvent deionized water, 20.024.0 C) were scanned from 200 to 800 nm wavelength range using an ultraviolet-visible light spectrophotometer (Hach Company, Loveland, CO). The results showed that EPI maximum absorption was at 233 and 480 nm, and AEDP– OXD had no absorption at 480 nm (Figure 1). Therefore, 480 nm was chosen as the measurement wavelength for avoiding interference. DDW was used as a blank control to measure EPI content in 0.25 mg/mL AEDP–OXD–EPI. Drugloading (DL) dose of AEDP–OXD–EPI was calculated according to the following formula: DL ðdrugÞ ¼ WE =WD 100%, WD: AEDP–OXD–EPI concentration; WE: the measured concentration of EPI. Induced apoptosis and necrosis of AEDP–OXD–EPI in human osteosarcoma cell line MG-63 The MG-63 cells suspension (5 105/mL) were added into 6-well plates, each well 3 mL, and placed in incubator for 24 h. And then the culture fluids was changed with the cellculture medium contained 18.83 mg/mL AEDP–OXD–EPI (1 mg/mL EPI equivalent), 1 mg/mL EPI, 100 mg/mL OXD and 40 mg/mL AEDP, respectively (n ¼ 3). The negative control group without any drug was set up. After 24 h culture with the drugs, the apoptosis and necrosis of MG-63 cells were analyzed using a flow cytometer with Annexin V-FITC (fluorescein isothiocyanate) and propidium iodide (PI) staining. Apoptosis Detection Kit (KeyGen Biotech Co. Ltd., Najing, China) was applied in the present study. Cells were collected and washed twice in ice cold PBS and resuspended in 300 ml of binding buffer at 2 105 cells/mL.
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Scheme 3. Conjugation of AEDP–OXD–EPI.
(n ¼ 5). The amount of drugs not bound to HAp were quantified by ultraviolet-visible light spectrophotometer (measurement wavelength: 480 nm) in the supernatant after centrifugation of incubated HAp samples at 6000 rpm for 60 min. The initial binding kinetics of AEDP–OXD–EPI was obtained by the same method. The degree of HAP binding was assessed as mean of five independent experiments according to the following formula: HAp binding ð%Þ ¼ Figure 1. UV spectra of EPI, AEDP–OXD–EPI and AEDP–OXD.
The samples were incubated with 5 ml of Annexin V-FITC and 5 ml PI in the dark for 15 min at room temperature. Finally, samples were analyzed by flow cytometry. Bone-targeting capacity of AEDP–OXD–EPI in vitro HAp-binding assay described by Shinoda et al. [17] and Fujisawa et al. [18] was set up to evaluate the bone-targeting capacity of AEDP–OXD–EPI in vitro. Briefly, 15 mg Nanocrystalline HAp was incubated with 1 mL solution of 0.5 mg/mL AEDP–OXD–EPI, 25 mg/mL EPI, 1 mg/mL OXD and 1 mg/mL AEDP–OXD overnight at 25 C, respectively
ðX YÞ 100% X
X is the initial concentration of drug and Y is the drug concentration of supernatant after centrifugation. Bone-targeting capacity of AEDP–OXD–EPI in vivo Twelve Kunming mice, (18.7 ± 2.1) g, were divided into four groups (n ¼ 3). Group A (AEDP–OXD–EPI) were injected with 0.2 mL of solution 28.8 mg/mL AEDP–OXD–EPI (1.5 mg/mL EPI equivalent) via the tail vein; B group (EPI), were injected with 0.2 mL of 1.5 mg/mL EPI; C group (AEDP–OXD) group were injected 0.2 mL of 27.3 mg/mL AEDP–OXD; D groups (control group) were injected with 0.9% saline 0.2 mL. All the animals remained active with normal food and water consumption after the injection.
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Twenty-four hours later, animals were euthanized and then distal femora were resected en bloc, fixed in acetone for 24 h. The slices of hard tissue were made, and observed by fluorescence microscope (Leica DM4000B). All the photos were shot at the same conditions (Exposure time 1/3 s and sensitivity 400) and were analyzed fluorescence intensity of the slices using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD).
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Statistical analysis Variables used in this study were expressed as the mean ± SEM. The data were analyzed by SPSS 16.0 software (SPSS Inc., Chicago, IL). Statistical significance was determined by one-way analysis of variance (ANOVA) and Tukey’s multiple-comparison. p value 50.05 was considered statistically significant.
Results and discussion Characterization of bone-targeting EPI The conjugation scheme of AEDP–OXD–EPI was confirmed by analysis of FTIR and 1H-NMR spectra. Figure 2 shows the
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FTIR spectra of AEDP (a), Dex (b), OXD (c), AEDP-OXD (d), AEDP-OXD-EPI (e) and EPI (f). The spectra of OXD showed an absorption band at 1728.16 cm1, which assigned to the aldehyde groups (–C ¼ O), while the spectra of Dex did not have the characteristic band. The content of formyl group in OXD was (10.24 ± 0.20) mmol/g (n ¼ 5) via the hydroxylamine hydrochloride method. Therefore, Dex was oxidized successfully to OXD, which had the structure of polyaldehyde. The absorption band, which appeared at 1585.75 cm1 on the spectra of AEDP–OXD, was ascribed to the –HC ¼ N of Schiff Base, because the absorption band of AEDP aminogroup appeared at 1521.47 cm1. About 1021.99 cm1 vibration absorption peak in AEDP–OXD of was ascribed to P–O, which was brought in by AEDP, and it can also be found in the FTIR spectrum of AEDP–OXD–EPI. Comparing with the FTIR of AEDP–OXD, a new carbonyl absorption band of EPI at 1728.16 cm1, but not the aldehyde band at 1723.78 cm1, was observed in the spectra of AEDP–OXD– EPI. The absorption band of AEDP–OXD–EPI from 1200 to 1000 cm1 was different from the AEDP–OXD. The result may be due to the superposition of hydroxyl groups’ absorption bands from OXD and EPI. The synthesis of AEDP–OXD–EPI was also confirmed by 1 H-NMR spectroscopy. The multiple peaks at dH ¼ 1.23, 1.52, 1.55, 1.57 ppm corresponded to the methyl protons of AEDP and EPI. The peaks at dH ¼ 2.10, 2.12, 2.17 corresponded to the –CH2, –CH protons of EPI. The double peaks at dH ¼ 7.80, 7.95 ppm correspond to the benzene ring protons of EPI. The peak at dH ¼ 7.69 ppm was the proton peak of Schiff base structure (–HC ¼ N–). The small peak at d9.62 corresponded to the aldehyde protons of oxidized dextran, which were not completely reacted with AEDP and EPI. (Figure 3) Therefore, it can be seen, from the spectroscopy of FTIR and 1H-NMR, that the synthesis of AEDP–OXD–EPI was successful. DL dose of AEDP–OXD–EPI
Figure 2. FTIR spectra of raw materials, intermediate and final products.
According to the standard curve equation of EPI at 480 nm, 0.25 mg AEDP–OXD–EPI contained (13.28 ± 0.51 mg) EPI, and then the drug loading of AEDP–OXD–EPI was (5.31 ± 0.20%), calculated by the drug loading formula. Theoretically, after 100 mg OXD (containing aldehyde 1.02 mmol) reacted with 82 mg AEDP (0.4 mmol), the remained aldehyde groups of OXD can be combined with 360 mg EPI (0.62 mmol, MW 579.99). According to the analysis, the theoretical drug loading of AEDP–OXD–EPI should be 66.42%, but the actual drug loading just was 5.31%. The result showed that the majority of the aldehyde groups had not reacted with EPI. The 1H-NMR spectroscopy of AEDP–OXD–EPI also clearly illustrated that the proton peak of aldehyde group appeared at dH ¼ 9.62 ppm. The following reasons may explain the incomplete reaction: (1) the status of OXD longer carbon chains in solution can lead to inadequate exposure of aldehyde [19] and (2) after binding with aldehyde, the molecule drugs can occupy more space so that the closed aldehyde group cannot participate in the reaction [20]. Therefore, we used the gel column of Sephadex G-50 to remove the unbound AEDP and EPI.
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Figure 3. 1H-NMR spectrum of AEDP–OXD–EPI. a: The multiple peaks of methyl protons in EPI; b: the multiple peaks of methyl protons in AEDP; c: the protons peaks of –CH2, –CH in EPI; d: the proton peak of Schiff base (–HC¼N–); e: the peaks of benzene ring protons in EPI; f: the proton peak of aldehyde in OXD.
Induced apoptosis and necrosis of AEDP–OXD–EPI in MG-63 cell line The results measured by Annexin/PI double staining showed that the necrosis and apoptosis ratio of MG-63 cell in AEDP– OXD–EPI group was significantly higher than EPI group, at the same concentration of EPI (1 mg/mL) (p50.05). We observed that AEDP also can induce the apoptosis and necrosis of osteosarcoma cells, but OXD had not obvious effects on apoptosis (Figure 4). Over the past two decades, BP is widely used for the prevention and treatment of diseases in which there is an increase in the number or activity of osteoclasts, such as osteoporosis, cancer metastasis and Paget’s disease of bone [21]. BP can reduce bone resorption and increase bone mineral density by inducing osteoclast apoptosis. Recent studies also suggest that bisphosphonates have direct effects on tumor cells, including inhibiting tumor cells adhesion, invasion and proliferation [15,22] and inducing their apoptosis [23]. Additionally, BP may inhibit angiogenesis [24] and the release of promote cancer cell growth factors [25], regulate the immune effects of cdT cells [26] and enhance the antitumor activities of cytotoxic agents [27]. Segal et al. [28] synthesized a bone-targeting nano-scaled N-(2-hydroxypropyl) methacrylamide (HPMA)
copolymer-alendronate (ALN)-TNP-470 conjugate and proved the conjugate revealed superior anti-tumor activity and decreased organ-related toxicities of the conjugate compared with the combination of free ALN plus TNP-470. Our experiment also confirmed that AEDP also could significantly induce the apoptosis of osteosarcoma cell line MG-63. The bone-targeting drug, AEDP–OXD–EPI, had superior anti-tumor activity in vitro than free EPI or AEDP. We hypothesize that the result was due to that the anti-tumor effect and synergistic anti-tumor effect of AEDP enhanced the drug effect of EPI. The result was similar with Segal’s experiments [29]. Therefore, the effects of BP in bonetargeting drug were not only the oriented agent, but also the therapeutic agent which could kill tumor cells directly. Bone-targeting capacity of AEDP–OXD–EPI According to the HAp binding formula, the HAp binding rates of OXD, AEDP–OXD, AEDP–OXD–EPI and EPI were (3.81 ± 2.07)%, (69.87 ± 3.36)%, (85.47 ± 1.27)%, (12.88 ± 5.10)%, respectively (Figure 5). AEDP–OXD–EPI had a better ability to bind with HAp than EPI in vitro. The binding of the AEDP–OXD–EPI to the surface of HA was observed to occur very quickly. The result of the initial binding kinetic was shown in Figure 6. The binding of
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Figure 4. The necrosis and apoptosis ratio of MG-63 cell induced by AEDP–OXD–EPI, EPI, AEDP and OXD. (A) AEDP–OXD–EPI was (96.51 ± 3.25)%; (B) EPI was (84.55 ± 3.97)%; (C) AEDP was (33.99 ± 7.3)% and (D) OXD was (15.19 ± 3.48)%. Cell necrosis and apoptosis ratio in AEDP–OXD–EPI is significantly higher (p 5 0.05 versus B, C and D, E), EPI is higher than C and D (p 50.05), AEDP is higher than OXD (p50.05). Note: The lower left quadrant (LL), normal cells; upper left quadrant (UL), necrotic cells; lower right quadrant (LR), early apoptotic cells; upper right quadrant (UR), late apoptotic cells.
Figure 5. The binding of OXD, EPI, AEDP–OXD and AEDP–OXD–EPI to hydroxyapatite. Conjugates were dissolved in phosphate buffered saline (pH ¼ 7.4). Background correction was applied. Data are shown as the mean SD from five different measurements (F ¼ 866.16, p50.01).
AEDP–OXD–EPI reached a plateau in 5 min with 82.16% of the conjugate bound to HAp. Prolonged incubation of the conjugate with HAp did not significantly improve binding efficiency (overnight, 85.47%, Figure 5). In vivo bone binding experiment demonstrated that the fluorescence of group C and group D is extremely weak
Figure 6. The initial binding kinetics of AEDP–OXD–EPI to hydroxyapatite. AEDP–OXD–EPI was dissolved in phosphate buffered saline (pH ¼ 7.4) with a concentration of 0.5 mg/mL. Data are shown as the mean SD from triplicate measurements.
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Figure 7. The in vivo binding of AEDP–OXD–EPI to the bone, 5. The autofluorescence of EPI can be observed in AEDP–OXD–EPI (A) and EPI (B). Figure 6(A) showed that the autofluorescence of EPI was located in cortical and cancellous bone of femoral metaphyseal and there was no red fluorescence in the epiphysis because of its own barrier function. The fluorescence IOD of AEDP–OXD–EPI was stronger than EPI. There was only very tiny autofluorescence of bone tissue in AEDP–OXD (C) and saline injection (D).
(Figure 7C and D), while very strong fluorescence was observed in A group and B group (Figure 7A and B). Via the analysis of Image-Pro Plus 6.0 software, the fluorescence integrated optical density (IOD) of group A and group B were 37.95 ± 3.97 and 15.26 ± 2.52 (p50.05), respectively. The fluorescence intensity of group A was about 2.5 times than of group B (Figure 7). Epirubicin is an anthracycline cytotoxic agent and a broad spectrum antineoplastic drug [30,31]. The main antineoplastic mechanism of EPI is interfering with the transcription process and preventing the formation of mRNA by intercalation of DNA planar rings between nucleotide base pairs [32,33]. EPI is a cell cycle phase non-specific anthracycline, which has a killing effect on a variety of tumor cell in different growth cycle. But EPI also has a strong cytotoxic effect on human body. The most common toxic reactions include: Neutropenia and thrombocytopenia in 60–80% patients; 100% of patients will endure the hair loss to varying degrees [34,35]; the cardiotoxicity often presents as ECG changes (especially change in the frequency of QRS complex) and arrhythmias, or as a cardiomyopathy leading to heart failure (sometimes presenting many years after treatment) [36]; and nausea and loss of appetite. EPI was usually used as chemotherapy drugs to treat human osteosarcoma. The 5-year disease-free and overall survival rates in patients with localized primary osteosarcoma, who were treat with EPI combined with cisplatin and ifosfamide, were 440% [4]. However, many patients suffered from the
above-mentioned cytotoxic effect throughout the whole course of chemotherapy. Dextran serves as one of the most promising macromolecular carrier candidates for a wide variety of therapeutic agents due to their excellent physico-chemical properties and physiological acceptance [10]. It is non-toxic, non-immunogenic and non-antigenic. It contains huge number of carbohydrate hydroxyl groups available for drug fixation [37]. The dextran conjugates have low clearance and relatively long plasma half-life because of large molecular weight polysaccharides, resulting in accumulation in tumor tissues [38]. Therefore, the various dextran-antitumor drug conjugates were prepared to enhance the effectiveness and improve the cytotoxic effects of chemotherapeutic agents. In particular, the dextran–anthracycline conjugates, such as dextran– Adriamycin (ADM) and dextran–EPI, showed excellent pharmacological properties [39,40]. Ueda et al. [41] proved that Adriamycin linked to oxidized dextran (ADM–OXD) via Schiff’s base showed higher activity against Walker carcinosarcoma 256 and lower acute toxicity than free Adriamycin in rat model. Phase I clinical and pharmacokinetic trial of dextran conjugated doxorubicin (DOX–OXD) proved that the maximal tolerated dose of DOX–OXD can be considered to be 40 mg/m2 doxorubicin equivalent since no toxic deaths were observed at this dose level [42]. The in vivo binding of bone-targeting conjugates to bone is much more complicated than the in vitro HAp binding mechanism. Because EPI has the character of
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autofluorescence, the in vivo bone binding assay was performed according to Wang et al. [43]. AEDP–OXD–EPI was able to arrive at the periosteum, cortical bone, cancellous bone and bone marrow via blood circulation (Figure 7A). EPI has similar molecular structure with Tetracycline (TC) which is a well-known bone-seeking drug. The structure of adjacent carbonyl (¼O) and hydroxyl (–OH), is considered to be the foundation of bone-targeting [44], exists in EPI and TC. The pro bone ability of EPI was also revealed by the vitro HAp binding and vivo bone binding assay in the article. The reason why AEDP–OXD–EPI conjugate had higher binding ratio to bone compared with AEDP–OXD may result from the bone-seeking ability of EPI. The in vivo and in vitro assay proved that AEDP–OXD– EPI had a good bone-targeting capacity, which would help increase the EPI concentration in the bone and reduce the treatment dose. Furthermore, the synergistic anti-tumor effect of AEDP could further reduce the dosage of EPI. Therefore, if AEDP–OXD–EPI can be used for the chemotherapy of bone cancer, it will be benefit to improve the chemotherapy effect of EPI and reduce its toxic and side effects. Nevertheless, animal models would strengthen the in vivo aspect of this new targeted drug.
Conclusion A new polymeric bone-targeting drug, AEDP–OXD–EPI, was prepared in the experiment. The macromolecular delivery of AEDP–OXD–EPI was OXD, bone-targeting moiety was BP and bone therapeutic agent was EPI. The successful conjugation of AEDP–OXD–EPI was confirmed by analysis of FTIR and 1H-NMR spectra. It was demonstrated by in vitro and in vivo experiments that the bone-targeting drug exhibited a remarkable affinity to bone. Induced apoptosis and necrosis in MG-63 cell line showed that AEDP–OXD–EPI had senior anti-tumor activity than EPI in vitro. However, we need to do more animal experiments to prove whether or not AEDP– OXD–EPI was an ideal anti-bone-tumor drug.
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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