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The preparation and characterization of lactone form of 10-hydroxycamptothecin-layered double hydroxide nanohybrids
Applied Clay Science 104 (2015) 128–134
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Applied Clay Science journal homepage: www.elsevier.com/locate/clay
Research paper
The preparation and characterization of lactone form of 10-hydroxycamptothecin-layered double hydroxide nanohybrids Xiujiang Pang a,⁎, Junmei Cheng b, Li Chen b, Dongxiang Li a,⁎ a b
State Key Laboratory Base of Eco-chemical Engineering, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China Key Laboratory of Rubber-Plastics, Ministry of Education/Shandong Provincial Key Laboratory of Rubber-plastics, Qingdao University of Science and Technology, Qingdao 266042, PR China
a r t i c l e
i n f o
Article history: Received 1 July 2014 Received in revised form 12 November 2014 Accepted 15 November 2014 Available online 1 December 2014 Keywords: 10-Hydroxycamptothecin Layered double hydroxides Microreactor Co-precipitation Dilute acetic acid
a b s t r a c t 10-Hydroxycamptothecin (HCPT) as a hydrophobic anticancer drug brings many challenges in the clinical applications due to its poor water solubility and its facile structure transformation to inactive structure. In this work, the lactone form of HCPT–LDH nanohybrids with high drug loading was prepared by a two-step method. Firstly, the carboxylate form of HCPT-intercalated LDH was prepared by a coprecipitation method using a microchannel reactor. Secondly, dilute acetic acid was added into the as-precipitates to perform a structure recovery of HCPT in the nanohybrids from carboxylate form to bioactive lactone form because the lactone–carboxylate equilibrium of HCPT is reversible and pH-dependent. The dispersity of HCPT–LDH nanohybrids was obviously improved as compared with particles prepared by conventional method, and the average particle size increased with the drug loading. It was found that the content of HCPT in the nanohybrids with bioactive lactone form reached 95% after treated with acetic acid. For cancer cells, the lactone form of HCPT–LDH was found to be significantly more potent than raw HCPT, and it provided an important foundation for the development and application of nanohybrid delivery system. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The pentacyclic alkaloid, 10-hydroxylcamptothecin (HCPT), isolated from the Chinese tree, Camptotheca acuminata, inhibits the activity of topoisomerase I and has a broad spectrum of anticancer activity in vitro and in vivo (Ping et al., 2006). However, as shown in Scheme 1, HCPT has a poor water solubility and more importantly, it facilely transits from the bioactive lactone form (I) into the inactive carboxylate structure form (II) via hydrolysis even at physiological conditions (Kunadharaju and Savva, 2008). Therefore, the efficient delivery of HCPT is an extreme challenge up to now. Nanosized vehicles including liposomes (Chang et al., 2009; Volodkin et al., 2009), water soluble polymers (Liu et al., 2009), dendrimers (Guillaudeu et al., 2008), vesicles (Soussan et al., 2009), polymernanoparticles (Tong and Cheng, 2008), and some inorganic materials (Lu et al., 2009; Pang et al., 2013) are investigated as the carriers of the hydrophobic drugs to greatly enhance their water solubility and stability, prolong their circulation in blood compartments, and target cancerous tissues by passive accumulation via tumors' enhanced permeability and retention (EPR) effect (Shen et al., 2010).
⁎ Corresponding authors. Tel.: +86 0532 84022681; fax: +86 0532 84023927. E-mail addresses: [email protected] (X. Pang), [email protected] (D. Li).
http://dx.doi.org/10.1016/j.clay.2014.11.019 0169-1317/© 2014 Elsevier B.V. All rights reserved.
Layered double hydroxides (LDH), with structurally positively charged layers and interlayer balancing anions, were applied widely in different fields (Tong et al., 2010; Basu et al., 2014). In recent years they were frequently investigated as drug delivery nanocarriers because their interlayer gallery could be employed as drug vessels with controlled releasing properties (Ambrogi et al., 2001; Rives et al., 2014). The water soluble anionic drugs can be intercalated into the hydrophilic gallery of pristine LDH via ion exchange (Meyn et al., 1990), reconstruction (Dong et al., 2010) and co-precipitation (Liu et al., 2008), while the charge-neutral or poorly water-soluble drugs, such as HCPT can be intercalated into the hydrophobic gallery of modified LDH usually with surfactants (You et al., 2001; Bruna et al., 2006). However, the LDH, its sole role is to make the vehicles, is the major component while the bioactive drug is the minor component in nanomedicines. In nanoparticles, the drug contents are generally not greater than 10% (Tyner et al., 2004). The HCPT–LDH nanohybrids with high drug loading can be prepared by a coprecipitation method (Liu et al., 2008), but the loaded HCPT in nanohybrids showed a poor bioactivity. Herein, HCPT–LDH nanohybrid was firstly prepared with coprecipitation method as reported (Liu et al., 2008), and then the sediment was treated with diluted acetic acid to enhance the bioactivity of the loaded drug. Because the lactone–carboxylate equilibrium (Scheme 1) is reversible and pH-dependent, the treatment in the presence of acetic acid promotes a conversion from the inactive carboxylate structure (II) into bioactive lactone form (I) (Kunadharaju and Savva, 2008).
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Scheme 1. The conversion of the lactone form of HCPT into its carboxylate form.
2. Experimental
2.4. HPLC analysis of HCPT
2.1. Synthesis of HCPTC–LDH and HCPTL–LDH
The lactone and carboxylate forms of HCPT were separated and quantified with the Dionex Summit high performance liquid chromatography (HPLC) System equipped with P680A HPG-2 High-Pressure Gradient Pump and UVD 170U HPLC UV–vis Detector. Samples were dispersed in dimethyl sulfoxide (DMSO) and stirred for 12 h, and then the dispersion was filtered through a 0.22 μm syringe filter. The concentration of HCPT is about 0.15 mg/ml. The mobile eluent phase was 70% phosphate buffer (pH value = 6.5, 25 mM), 23% methanol, and 7% tetrahydrofuran with the detection wavelength of 382 nm. The lactone fraction was calculated by the equation of flactone = Alactone/Atotal, in which Alactone refers to the area of the lactone peak and Atotal is the total peak area of the lactone and carboxylate forms.
HCPTC (carboxylate form of HCPT)-LDH nanohybrids were synthesized by coprecipitation method. A mixed salt solution containing 5.94 g (0.020 mol) of Zn(NO3)2·6H2O and 3.75 g (0.010 mol) of Al(NO3) 3·9H2O in 60 ml of deionized H2O was prepared. A series of HCPT with different weight (0.0089–0.890 g) (Hubei Haobo Co., Ltd., China) were respectively dissolved by dipping 0.1 mol/l aqueous solution of NaOH and then water was added to reach a solution volume of 60 ml. Then the mixed salt solution and alkaline solution containing HCPT were mixed by a microchannel reactor and a suspension was obtained. The precipitate was aged for 3 h in the mother solution at 40 °C and then centrifuged and washed with deionized water for two times. The centrifuged product held in a glass bottle was peptized at a constant temperature of 60 °C in an oven for about 24 h to obtain HCPTC–LDH nanohybrid. HCPTC–LDH nanohybrid was dispersed in deionized water and then 5% (by volume) diluted acetic acid was added and the pH value of suspension was adjusted to 5.5. After stirring at 60 °C for 12 h, the precipitate was centrifuged and then dried, and the so-obtained sample was denoted as HCPTL (lactone form of HCPT)–LDH. The drug loading of the sample changed little (b11%) before and after the treatment by dilute acetic acid.
2.2. Determination of HCPT loading The amount of HCPT loaded into the hybrids, Ain, was determined by UV–vis spectroscopy using the following method. 10 mg of the hybrid sample was dissolved in 10 ml HCl solution (1 mol/l), which was followed by addition of 40 ml ethanol. The concentration of HCPT in solution was determined by monitoring the absorbance at λmax = 380 nm with a UV–vis absorption spectrophotometer, and the concentration was calculated by regression analysis according to the standard curve obtained from a series of standard solution of HCPT. The Ain value was obtained according to the weight ratio of the HCPT in the solution and the nanohybrid used.
2.3. Determination of the release rate of HCPT from the nanohybrids The HCPT release examinations were performed at 37 °C in 0.1 M phosphate buffer solution (PBS) with a pH value of 7.2 or ethanol– water mixture (3:7 by volume). 50 mg of the HCPT–LDH nanohybrid was placed into 500 ml buffer solution or ethanol–water mixture under stirring at 37 °C. Aliquots (4 ml) of the suspension were withdrawn at predetermined time intervals and filtered through a 0.45 μm syringe filter. The absorbance was measured at λmax = 380 nm by a UV–vis spectrophotometer to obtain the HCPT concentration, and then to calculate the release percentage (Xt) of HCPT. The release percentages of HCPT from the composite were plotted versus time (t) to examine the release rate of HCPT from the nanocomposite.
2.5. In vitro cytotoxicity against HCT-116 and Colo-205 cells For the cytotoxicity analysis of free HCPT, pristine LDH and HCPT–LDH against HCT-116 and Colo-205 cells, cells were seeded in a 96-well plate at a density of 104 cells per well and cultured in 5% CO2 at 37 °C for 72 h. Then, free HCPT dispersed in DMSO and PBS, respectively, pristine LDH and HCPT–LDH dispersed in PBS, and then the cells were incubated in 5% CO2 at 37 °C for 72 h. The concentrations of free HCPT and encapsulated HCPT in nanohybrids were 0.001, 0.004, 0.012, 0.037, 0.111, 0.333, 1.0 and 3.0 μg/ml, respectively. The concentrations of pristine LDH were 0.5, 1.4, 4.1, 12.3, 37, 111, 333 and 1000 μg/ml, respectively. Cell viability was determined by CCK8 assay. Each data point is represented as mean value ± standard deviation (SD) of eight independent experiments (n = 8, n indicates the number of wells in a plate for each experimental condition). IC50 values were used to value the cytotoxic activity of different samples. 2.6. Characterizations and Instrumentations Powder X-ray diffraction (XRD) patterns were obtained on a D/ max-rA model diffractometer with CuKα radiation (40 kV and 80 mA). Transmission electron microscopy (TEM) images were obtained on a JEM-2100 transmission electron microscope. Fourier transform infrared (FT-IR) spectra were recorded in KBr dispersion on a Bruker Vector 22 spectrometer in air at room temperature. 3. Results and discussion 3.1. Preparation of HCPTC–LDH and HCPTL–LDH nanohybrids In the past years, nonionic and poorly water-soluble drug was usually first encapsulated in an anionic micelle and then the negative charge on the surfactant allows the uptake of the drug-loaded micelle between the sheets of the LDH by an ion exchange process (Tyner et al., 2004; Han et al., 2005). By these methods, drugs could be intercalated into the gallery of LDH successfully, but drug loading is quite low. In this
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work, to obtain HCPT–LDH nanohybrids with high drug loading, the carboxylate form of HCPT-intercalated LDH nanohybrids was prepared by co-precipitation followed by treating in dilute acetic acid. As illustrated in Scheme 1, the reversion of the lactone–carboxylate equilibrium is pH-dependent, and the lactone form dominates in the acidic condition (Kunadharaju and Savva, 2008). So the lactone form of HCPTintercalated LDH nanohybrids can be obtained through treating carboxylate of HCPT-intercalated LDH with weak acid. To avoid the dissolution of the LDH, the weak acid in this work was dilute acetic acid with a volume concentration of 5%. 3.2. XRD patterns of HCPTC–LDH and HCPTL–LDH Fig. 1 shows the powder XRD patterns of the pristine LDH, HCPTC–LDH and HCPTL–LDH. As can be seen, the XRD patterns of the pristine LDH sample (Fig. 1a) exhibited all reflections of hydrotalcite (JCPDS card No. 51-1528), indicating that the pristine LDH sample has a wellcrystallized structure (Drits and Bookin, 2001). The d003 value of 0.87 nm corresponding to NO− 3 ions between the layers was expected (Li et al., 2009). The adjacent reflection with d003 value of 0.76 nm could be attributed to CO2− ions (Kang et al., 2009) due to the dissolu3 tion of CO2 in air. For HCPTC–LDH nanohybrid (Fig. 1b), the observed basal spacing, d003, was 3.10 nm. Given that the thickness of the LDH basal layers is about 0.48 nm (Li et al., 2009), the interlayer space of the HCPTC–LDH sample is about 2.62 nm. The length, width and thickness of HCPT molecule calculated by the method of molecular mechanics (Dewar et al., 1985) were about 1.40, 0.72 and 0.34 nm, respectively; it was assumed that the carboxylate form of HCPT arranges in LDH gallery with two layers. When the above nanohybrid was treated with dilute acetic acid at 60 °C for 12 h, the pattern of the so-obtained HCPTL–LDH nanohybrid was shown in Fig. 1c. For HCPTL–LDH nanohybrid, the d003 value was 2.5 nm, indicating a change in the arrangement of drug molecules, and a probable mechanism for drug molecules in LDH gallery may be illustrated in Scheme 2. Moreover, such raw patterns of b and c indicated a low crystallization of LDH particles, and probably implied a disordered structure of HCPT–LDH. 3.3. FT-IR of HCPTC–LDH and HCPTL–LDH nanohybrids FT-IR spectra of raw HCPT and HCPTL–LDH nanohybrid treated in dilute acetic acid for different times were displayed in Fig. 2. The broad strong band at around 3500 cm−1 corresponds to the stretching vibration (νO\H) of the H2O existing in the samples. In the spectrum
Fig. 1. XRD patterns of (a) ZnAl-LDH, (b) HCPTC–LDH, and (c) HCPTL–LDH.
of pristine HCPT (Fig. 2a), the strong absorption peaks at 1723 and 1655 cm−1 can be attributed to the stretching vibrations of C_O ketone group of lactone ring and amide, respectively. For HCPTC–LDH nanohybrid without being treated with dilute acetic acid, compared with Fig. 2a, the characteristic peak at 1655 cm−1 also existed, but no peak existed at 1723 cm−1, indicating that HCPT existed as its carboxylate structure in the LDH gallery. An obvious characteristic peak appeared at 1723 cm−1 after treating HCPTC–LDH hybrids with dilute acetic acid for 6, 12 and 24 h, indicating the existence of lactone form of HCPT in the nanohybrids. Moreover, the absorption intensity at 1723 cm−1 increased with the treating time of dilute acid. 3.4. Lactone fraction of HCPT in HCPTL–LDH nanohybrid HCPT with lactone form and carboxylate form could be well separated and characterized by high-performance liquid chromatography (HPLC). The obtained HPLC data were shown in Fig. 3A. The retention times for the lactone and carboxylate forms were about 13.8 and 2.5 min, respectively. As reported, the ratio of lactone form and carboxylate form of HCPT was proportional to the peak area (Ci et al., 2013), so the lactone fraction could be calculated from the chromatography data. The changes in the lactone fraction upon time were presented in Fig. 3B. The results showed that the content of lactone fraction of HCPT increased with the treating time, and the lactone fraction in HCPT after being treated for 6 h, 12 h, and 24 h was 82%, 93.8% and 95.6%, respectively, which coincided with the results of FT-IR. 3.5. Drug loading and release from HCPTC–LDH and HCPTL–LDH nanohybrids To explore the effect of drug loading on controlled release property, a series of HCPTC–LDH nanohybrids were prepared by co-precipitation method, and both the drug loading and controlled release were examined. From Fig. 4A, drug loading initially increased linearly, then slowly increased with HCPT/(Zn2+ + Al3+) molar ratio before and after the drug loading of 36.5%. Fig. 4B showed the drug release profiles of HCPTC–LDH with different drug loadings in PBS with a pH value of 7.2. The results showed that the higher the drug loading, the poorer the controlled release property. Combined with the result of Fig. 4A, the most possible reason was that the drug absorbed on the surface of nanoparticles increased with the HCPT/(Zn2+ + Al3+) molar ratio while the washing time was maintained constant during the preparation process. It needs to be noted that the HCPT molecules' loading on the nanohybrid may exist in two possible statuses: one is the intercalating form and the other is the adsorbing form on the surface of LDH particle. In order to distinguish the relative amount of the above two morphologies of HCPT molecules, a contrast experiment was performed, in which Zn2Al–CO3 LDH prepared with a microchannel reactor was used as adsorbent under the same experiment conditions. Taken a series of 0.004 mol/l HCPT sodium hydroxide solution with different volumes and mixed with 0.1 g dried Zn2Al–CO3 LDH according to a certain molar ratio of HCPT/(Zn2 + + Al3 +) and stirred for 2 h at 30 °C, and then the mixture was centrifuged followed by washing two times with deionized water and drying at 60 °C. The drug loading of the so-obtained HCPT–Zn2Al–CO3 LDH nanocomposite was 20.35%, and its XRD pattern (Fig. 5) showed that no HCPT was intercalated in the gallery of LDH. The HCPT adsorbing amount of the so-obtained product was also shown in Fig. 4A. The results showed that its absorption amount was obviously lower than that prepared by co-precipitation and the absorption amount increased with the molar ratio of HCPT/(Zn2+ + Al3+), which was in agreement with the results of Fig. 4B. Fig. 6 showed the in vitro release profiles for HCPT from the HCPTL–LDH nanohybrid in PBS with a pH value of 7.2 and ethanol– water (3:7 by volume) mixed at a volume ratio of 3:7, respectively. The HCPTL–LDH nanohybrid with a drug loading of 22.0% was obtained by dilute acetic acid-treated HCPTC–LDH nanohybrid with a drug
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Scheme 2. Schematic structure change of HCPT in the gallery of LDH treated by dilute acetic acid.
loading of 19.9%. As expected, HCPTL–LDH hybrids showed low drug release rate and less than 80% of the loaded HCPT was released from the nanohybrids within over 1500 min in PBS with a pH value of 7.2 (Fig. 6a), which was obviously lower than that in ethanol–water solution (Fig. 6b). And the possible reason is that solubility of HCPT with lactone form in ethanol–water mixture is higher than that in PBS. 3.6. Morphology and DLS measurements Fig. 7A showed the TEM images of HCPTL–LDH nanohybrid with drug loading of 5.2%, and the sample consisted of many nanosheets with a diameter about 50 to 100 nm. The z-average particle size with different drug loadings was shown in Fig. 7B. As can be seen, particle size increased with the drug loading. But compared with samples prepared in beakers, the average particle size of samples prepared using a microchannel reactor was apparently smaller, which facilitated the potential clinical application of nanohybrid. 3.7. Cell viability To test the therapeutic effect of the synthesized HCPTL–LDH nanohybrid with a drug loading of 22.0% and the potential toxicity of
Fig. 2. FT-IR spectra of (a) raw HCPT and HCPT–LDH treated with acetic acid for different times (b) 0 h, (c) 6 h, (d) 12 h and (e) 24 h.
Fig. 3. (A) Representative HPLC chromatograms of HCPT under indicated conditions. (a) raw HCPT, HCPTC–LDH treated with acetic acid for different times, (b) 0 h, (c) 6 h, (d) 12 h, and (e) 24 h and (B) the dependence of lactone fraction upon time.
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Fig. 6. In vitro release profiles for HCPT from HCPTL–LDH nanohybrid in (a) pH 7.2 PBS, (b) ethanol–water (3:7 by volume).
Fig. 4. (A) The effect of the molar ratio of HCPT and (Zn2++Al3+) on drug loading and (B) release profiles of HCPTC–LDH with different drug loadings in pH 7.2 PBS.
the pristine LDH, we studied cytotoxicity of the synthesized samples against HCT-116 and Colo-205 cells lines using a CCK8 method. For HCT-116 and Colo-205 cells, no obvious cytotoxicity was observed when the concentration of LDH is below 12 μg/ml. However, when the LDH concentration was above 110 μg/ml, cell survival percentage decreases with the increase of LDH concentration (Fig. 8). So the existence of LDH as drug carrier had no effect on results in our tests. When the cells were treated with either the dispersion of HCPTL–LDH nanohybrids or a DMSO solution of HCPT, growth inhibition and killing of cancer cells were obviously observed. As shown in Fig. 9A and B, the synthesized HCPTL–LDH nanohybrids demonstrated a dose-dependent cytotoxic effect against HCT-116 and Colo-205 cells and the cytotoxicity was almost equivalent to the drug dissolved in DMSO. As shown in Table 1, the IC50 values of HCPT L –LDH nanohybrids in incubation with HCT-116 and Colo-205 cells were respectively 0.071 μg/ml and 0.017 μg/ml. However, those values of HCPTC–LDH nanohybrids for HCT-116 and Colo-205 cells were 0.293 μg/ml and 0.049 μg/ml. It denoted that HCPTL–LDH nanohybrids was more effective in anticancer performance than HCPTC–LDH nanohybrids. It should be noticed that no significant cytotoxicity to the cell of HCT-116 and Colo-205 was observed in the concentration range involved in the experiment when the raw HCPT was suspended into the PBS solution and incubated with the cancer cells. This suggested that the raw HCPT could not be internalized into the cancer cells, which was in agreement with the reported results (Chen et al., 2009).
4. Conclusion
Fig. 5. XRD pattern of Zn2Al–CO3 LDH absorbed HCPT.
HCPT-intercalated LDH nanohybrids were prepared by a microchannel reactor via a coprecipitation method, and then treated in dilute acetic acid to obtain HCPT-intercalated LDH with biologically active lactone form. The intercalated HCPT in LDH could keep the lactone form because the reversible lactone–carboxylate equilibrium is pH value-dependent and the lactone form was dominated at acidic condition. The HCPT with bioactive lactone form in the drug–LDH nanohybrids was found to be significantly more effective than the raw HCPT. The soobtained HCPTL–LDH nanohybrids showed high drug loadings as well as obvious controlled release effects, and the pH control method can be used in intercalating other pH sensitive drugs.
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Fig. 7. (A) TEM image of HCPTL–LDH with 5.2% drug loading and (B) the relationship between drugs loaded amount and z-average particle size of samples (a) prepared using microchannel reactor and (b) samples prepared in beaker.
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Fig. 9. Survival percentage of (A) HCT-116 and (B) Colo-205 cell after 72 h treatment with raw HCPT, HCPTC–LDH and HCPTL–LDH.
Acknowledgments This research was supported by the National Natural Science Foundation of China (No. 21173135 and 20903059), the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20110131130008) and the Doctoral Fund of Qingdao University of Science and Technology, China (No. 0022610).
Table 1 IC50 values of HCPT, HCPTC–LDH and HCPTL–LDH samples for human colorectal carcinoma HCT-116 and Colo-205 cells. Samples
IC50 (μg/ml) HCT-116 colo-205
Fig. 8. Survival percentage of HCT-116 and Colo-205 cell after treatment with pristine LDH.
HCPT HCPTC–LDH HCPTL–LDH
0.053 0.293 0.071
0.015 0.049 0.017
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Applied Clay Science journal homepage: www.elsevier.com/locate/clay
Research paper
The preparation and characterization of lactone form of 10-hydroxycamptothecin-layered double hydroxide nanohybrids Xiujiang Pang a,⁎, Junmei Cheng b, Li Chen b, Dongxiang Li a,⁎ a b
State Key Laboratory Base of Eco-chemical Engineering, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, PR China Key Laboratory of Rubber-Plastics, Ministry of Education/Shandong Provincial Key Laboratory of Rubber-plastics, Qingdao University of Science and Technology, Qingdao 266042, PR China
a r t i c l e
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Article history: Received 1 July 2014 Received in revised form 12 November 2014 Accepted 15 November 2014 Available online 1 December 2014 Keywords: 10-Hydroxycamptothecin Layered double hydroxides Microreactor Co-precipitation Dilute acetic acid
a b s t r a c t 10-Hydroxycamptothecin (HCPT) as a hydrophobic anticancer drug brings many challenges in the clinical applications due to its poor water solubility and its facile structure transformation to inactive structure. In this work, the lactone form of HCPT–LDH nanohybrids with high drug loading was prepared by a two-step method. Firstly, the carboxylate form of HCPT-intercalated LDH was prepared by a coprecipitation method using a microchannel reactor. Secondly, dilute acetic acid was added into the as-precipitates to perform a structure recovery of HCPT in the nanohybrids from carboxylate form to bioactive lactone form because the lactone–carboxylate equilibrium of HCPT is reversible and pH-dependent. The dispersity of HCPT–LDH nanohybrids was obviously improved as compared with particles prepared by conventional method, and the average particle size increased with the drug loading. It was found that the content of HCPT in the nanohybrids with bioactive lactone form reached 95% after treated with acetic acid. For cancer cells, the lactone form of HCPT–LDH was found to be significantly more potent than raw HCPT, and it provided an important foundation for the development and application of nanohybrid delivery system. © 2014 Elsevier B.V. All rights reserved.
1. Introduction The pentacyclic alkaloid, 10-hydroxylcamptothecin (HCPT), isolated from the Chinese tree, Camptotheca acuminata, inhibits the activity of topoisomerase I and has a broad spectrum of anticancer activity in vitro and in vivo (Ping et al., 2006). However, as shown in Scheme 1, HCPT has a poor water solubility and more importantly, it facilely transits from the bioactive lactone form (I) into the inactive carboxylate structure form (II) via hydrolysis even at physiological conditions (Kunadharaju and Savva, 2008). Therefore, the efficient delivery of HCPT is an extreme challenge up to now. Nanosized vehicles including liposomes (Chang et al., 2009; Volodkin et al., 2009), water soluble polymers (Liu et al., 2009), dendrimers (Guillaudeu et al., 2008), vesicles (Soussan et al., 2009), polymernanoparticles (Tong and Cheng, 2008), and some inorganic materials (Lu et al., 2009; Pang et al., 2013) are investigated as the carriers of the hydrophobic drugs to greatly enhance their water solubility and stability, prolong their circulation in blood compartments, and target cancerous tissues by passive accumulation via tumors' enhanced permeability and retention (EPR) effect (Shen et al., 2010).
⁎ Corresponding authors. Tel.: +86 0532 84022681; fax: +86 0532 84023927. E-mail addresses: [email protected] (X. Pang), [email protected] (D. Li).
http://dx.doi.org/10.1016/j.clay.2014.11.019 0169-1317/© 2014 Elsevier B.V. All rights reserved.
Layered double hydroxides (LDH), with structurally positively charged layers and interlayer balancing anions, were applied widely in different fields (Tong et al., 2010; Basu et al., 2014). In recent years they were frequently investigated as drug delivery nanocarriers because their interlayer gallery could be employed as drug vessels with controlled releasing properties (Ambrogi et al., 2001; Rives et al., 2014). The water soluble anionic drugs can be intercalated into the hydrophilic gallery of pristine LDH via ion exchange (Meyn et al., 1990), reconstruction (Dong et al., 2010) and co-precipitation (Liu et al., 2008), while the charge-neutral or poorly water-soluble drugs, such as HCPT can be intercalated into the hydrophobic gallery of modified LDH usually with surfactants (You et al., 2001; Bruna et al., 2006). However, the LDH, its sole role is to make the vehicles, is the major component while the bioactive drug is the minor component in nanomedicines. In nanoparticles, the drug contents are generally not greater than 10% (Tyner et al., 2004). The HCPT–LDH nanohybrids with high drug loading can be prepared by a coprecipitation method (Liu et al., 2008), but the loaded HCPT in nanohybrids showed a poor bioactivity. Herein, HCPT–LDH nanohybrid was firstly prepared with coprecipitation method as reported (Liu et al., 2008), and then the sediment was treated with diluted acetic acid to enhance the bioactivity of the loaded drug. Because the lactone–carboxylate equilibrium (Scheme 1) is reversible and pH-dependent, the treatment in the presence of acetic acid promotes a conversion from the inactive carboxylate structure (II) into bioactive lactone form (I) (Kunadharaju and Savva, 2008).
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Scheme 1. The conversion of the lactone form of HCPT into its carboxylate form.
2. Experimental
2.4. HPLC analysis of HCPT
2.1. Synthesis of HCPTC–LDH and HCPTL–LDH
The lactone and carboxylate forms of HCPT were separated and quantified with the Dionex Summit high performance liquid chromatography (HPLC) System equipped with P680A HPG-2 High-Pressure Gradient Pump and UVD 170U HPLC UV–vis Detector. Samples were dispersed in dimethyl sulfoxide (DMSO) and stirred for 12 h, and then the dispersion was filtered through a 0.22 μm syringe filter. The concentration of HCPT is about 0.15 mg/ml. The mobile eluent phase was 70% phosphate buffer (pH value = 6.5, 25 mM), 23% methanol, and 7% tetrahydrofuran with the detection wavelength of 382 nm. The lactone fraction was calculated by the equation of flactone = Alactone/Atotal, in which Alactone refers to the area of the lactone peak and Atotal is the total peak area of the lactone and carboxylate forms.
HCPTC (carboxylate form of HCPT)-LDH nanohybrids were synthesized by coprecipitation method. A mixed salt solution containing 5.94 g (0.020 mol) of Zn(NO3)2·6H2O and 3.75 g (0.010 mol) of Al(NO3) 3·9H2O in 60 ml of deionized H2O was prepared. A series of HCPT with different weight (0.0089–0.890 g) (Hubei Haobo Co., Ltd., China) were respectively dissolved by dipping 0.1 mol/l aqueous solution of NaOH and then water was added to reach a solution volume of 60 ml. Then the mixed salt solution and alkaline solution containing HCPT were mixed by a microchannel reactor and a suspension was obtained. The precipitate was aged for 3 h in the mother solution at 40 °C and then centrifuged and washed with deionized water for two times. The centrifuged product held in a glass bottle was peptized at a constant temperature of 60 °C in an oven for about 24 h to obtain HCPTC–LDH nanohybrid. HCPTC–LDH nanohybrid was dispersed in deionized water and then 5% (by volume) diluted acetic acid was added and the pH value of suspension was adjusted to 5.5. After stirring at 60 °C for 12 h, the precipitate was centrifuged and then dried, and the so-obtained sample was denoted as HCPTL (lactone form of HCPT)–LDH. The drug loading of the sample changed little (b11%) before and after the treatment by dilute acetic acid.
2.2. Determination of HCPT loading The amount of HCPT loaded into the hybrids, Ain, was determined by UV–vis spectroscopy using the following method. 10 mg of the hybrid sample was dissolved in 10 ml HCl solution (1 mol/l), which was followed by addition of 40 ml ethanol. The concentration of HCPT in solution was determined by monitoring the absorbance at λmax = 380 nm with a UV–vis absorption spectrophotometer, and the concentration was calculated by regression analysis according to the standard curve obtained from a series of standard solution of HCPT. The Ain value was obtained according to the weight ratio of the HCPT in the solution and the nanohybrid used.
2.3. Determination of the release rate of HCPT from the nanohybrids The HCPT release examinations were performed at 37 °C in 0.1 M phosphate buffer solution (PBS) with a pH value of 7.2 or ethanol– water mixture (3:7 by volume). 50 mg of the HCPT–LDH nanohybrid was placed into 500 ml buffer solution or ethanol–water mixture under stirring at 37 °C. Aliquots (4 ml) of the suspension were withdrawn at predetermined time intervals and filtered through a 0.45 μm syringe filter. The absorbance was measured at λmax = 380 nm by a UV–vis spectrophotometer to obtain the HCPT concentration, and then to calculate the release percentage (Xt) of HCPT. The release percentages of HCPT from the composite were plotted versus time (t) to examine the release rate of HCPT from the nanocomposite.
2.5. In vitro cytotoxicity against HCT-116 and Colo-205 cells For the cytotoxicity analysis of free HCPT, pristine LDH and HCPT–LDH against HCT-116 and Colo-205 cells, cells were seeded in a 96-well plate at a density of 104 cells per well and cultured in 5% CO2 at 37 °C for 72 h. Then, free HCPT dispersed in DMSO and PBS, respectively, pristine LDH and HCPT–LDH dispersed in PBS, and then the cells were incubated in 5% CO2 at 37 °C for 72 h. The concentrations of free HCPT and encapsulated HCPT in nanohybrids were 0.001, 0.004, 0.012, 0.037, 0.111, 0.333, 1.0 and 3.0 μg/ml, respectively. The concentrations of pristine LDH were 0.5, 1.4, 4.1, 12.3, 37, 111, 333 and 1000 μg/ml, respectively. Cell viability was determined by CCK8 assay. Each data point is represented as mean value ± standard deviation (SD) of eight independent experiments (n = 8, n indicates the number of wells in a plate for each experimental condition). IC50 values were used to value the cytotoxic activity of different samples. 2.6. Characterizations and Instrumentations Powder X-ray diffraction (XRD) patterns were obtained on a D/ max-rA model diffractometer with CuKα radiation (40 kV and 80 mA). Transmission electron microscopy (TEM) images were obtained on a JEM-2100 transmission electron microscope. Fourier transform infrared (FT-IR) spectra were recorded in KBr dispersion on a Bruker Vector 22 spectrometer in air at room temperature. 3. Results and discussion 3.1. Preparation of HCPTC–LDH and HCPTL–LDH nanohybrids In the past years, nonionic and poorly water-soluble drug was usually first encapsulated in an anionic micelle and then the negative charge on the surfactant allows the uptake of the drug-loaded micelle between the sheets of the LDH by an ion exchange process (Tyner et al., 2004; Han et al., 2005). By these methods, drugs could be intercalated into the gallery of LDH successfully, but drug loading is quite low. In this
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work, to obtain HCPT–LDH nanohybrids with high drug loading, the carboxylate form of HCPT-intercalated LDH nanohybrids was prepared by co-precipitation followed by treating in dilute acetic acid. As illustrated in Scheme 1, the reversion of the lactone–carboxylate equilibrium is pH-dependent, and the lactone form dominates in the acidic condition (Kunadharaju and Savva, 2008). So the lactone form of HCPTintercalated LDH nanohybrids can be obtained through treating carboxylate of HCPT-intercalated LDH with weak acid. To avoid the dissolution of the LDH, the weak acid in this work was dilute acetic acid with a volume concentration of 5%. 3.2. XRD patterns of HCPTC–LDH and HCPTL–LDH Fig. 1 shows the powder XRD patterns of the pristine LDH, HCPTC–LDH and HCPTL–LDH. As can be seen, the XRD patterns of the pristine LDH sample (Fig. 1a) exhibited all reflections of hydrotalcite (JCPDS card No. 51-1528), indicating that the pristine LDH sample has a wellcrystallized structure (Drits and Bookin, 2001). The d003 value of 0.87 nm corresponding to NO− 3 ions between the layers was expected (Li et al., 2009). The adjacent reflection with d003 value of 0.76 nm could be attributed to CO2− ions (Kang et al., 2009) due to the dissolu3 tion of CO2 in air. For HCPTC–LDH nanohybrid (Fig. 1b), the observed basal spacing, d003, was 3.10 nm. Given that the thickness of the LDH basal layers is about 0.48 nm (Li et al., 2009), the interlayer space of the HCPTC–LDH sample is about 2.62 nm. The length, width and thickness of HCPT molecule calculated by the method of molecular mechanics (Dewar et al., 1985) were about 1.40, 0.72 and 0.34 nm, respectively; it was assumed that the carboxylate form of HCPT arranges in LDH gallery with two layers. When the above nanohybrid was treated with dilute acetic acid at 60 °C for 12 h, the pattern of the so-obtained HCPTL–LDH nanohybrid was shown in Fig. 1c. For HCPTL–LDH nanohybrid, the d003 value was 2.5 nm, indicating a change in the arrangement of drug molecules, and a probable mechanism for drug molecules in LDH gallery may be illustrated in Scheme 2. Moreover, such raw patterns of b and c indicated a low crystallization of LDH particles, and probably implied a disordered structure of HCPT–LDH. 3.3. FT-IR of HCPTC–LDH and HCPTL–LDH nanohybrids FT-IR spectra of raw HCPT and HCPTL–LDH nanohybrid treated in dilute acetic acid for different times were displayed in Fig. 2. The broad strong band at around 3500 cm−1 corresponds to the stretching vibration (νO\H) of the H2O existing in the samples. In the spectrum
Fig. 1. XRD patterns of (a) ZnAl-LDH, (b) HCPTC–LDH, and (c) HCPTL–LDH.
of pristine HCPT (Fig. 2a), the strong absorption peaks at 1723 and 1655 cm−1 can be attributed to the stretching vibrations of C_O ketone group of lactone ring and amide, respectively. For HCPTC–LDH nanohybrid without being treated with dilute acetic acid, compared with Fig. 2a, the characteristic peak at 1655 cm−1 also existed, but no peak existed at 1723 cm−1, indicating that HCPT existed as its carboxylate structure in the LDH gallery. An obvious characteristic peak appeared at 1723 cm−1 after treating HCPTC–LDH hybrids with dilute acetic acid for 6, 12 and 24 h, indicating the existence of lactone form of HCPT in the nanohybrids. Moreover, the absorption intensity at 1723 cm−1 increased with the treating time of dilute acid. 3.4. Lactone fraction of HCPT in HCPTL–LDH nanohybrid HCPT with lactone form and carboxylate form could be well separated and characterized by high-performance liquid chromatography (HPLC). The obtained HPLC data were shown in Fig. 3A. The retention times for the lactone and carboxylate forms were about 13.8 and 2.5 min, respectively. As reported, the ratio of lactone form and carboxylate form of HCPT was proportional to the peak area (Ci et al., 2013), so the lactone fraction could be calculated from the chromatography data. The changes in the lactone fraction upon time were presented in Fig. 3B. The results showed that the content of lactone fraction of HCPT increased with the treating time, and the lactone fraction in HCPT after being treated for 6 h, 12 h, and 24 h was 82%, 93.8% and 95.6%, respectively, which coincided with the results of FT-IR. 3.5. Drug loading and release from HCPTC–LDH and HCPTL–LDH nanohybrids To explore the effect of drug loading on controlled release property, a series of HCPTC–LDH nanohybrids were prepared by co-precipitation method, and both the drug loading and controlled release were examined. From Fig. 4A, drug loading initially increased linearly, then slowly increased with HCPT/(Zn2+ + Al3+) molar ratio before and after the drug loading of 36.5%. Fig. 4B showed the drug release profiles of HCPTC–LDH with different drug loadings in PBS with a pH value of 7.2. The results showed that the higher the drug loading, the poorer the controlled release property. Combined with the result of Fig. 4A, the most possible reason was that the drug absorbed on the surface of nanoparticles increased with the HCPT/(Zn2+ + Al3+) molar ratio while the washing time was maintained constant during the preparation process. It needs to be noted that the HCPT molecules' loading on the nanohybrid may exist in two possible statuses: one is the intercalating form and the other is the adsorbing form on the surface of LDH particle. In order to distinguish the relative amount of the above two morphologies of HCPT molecules, a contrast experiment was performed, in which Zn2Al–CO3 LDH prepared with a microchannel reactor was used as adsorbent under the same experiment conditions. Taken a series of 0.004 mol/l HCPT sodium hydroxide solution with different volumes and mixed with 0.1 g dried Zn2Al–CO3 LDH according to a certain molar ratio of HCPT/(Zn2 + + Al3 +) and stirred for 2 h at 30 °C, and then the mixture was centrifuged followed by washing two times with deionized water and drying at 60 °C. The drug loading of the so-obtained HCPT–Zn2Al–CO3 LDH nanocomposite was 20.35%, and its XRD pattern (Fig. 5) showed that no HCPT was intercalated in the gallery of LDH. The HCPT adsorbing amount of the so-obtained product was also shown in Fig. 4A. The results showed that its absorption amount was obviously lower than that prepared by co-precipitation and the absorption amount increased with the molar ratio of HCPT/(Zn2+ + Al3+), which was in agreement with the results of Fig. 4B. Fig. 6 showed the in vitro release profiles for HCPT from the HCPTL–LDH nanohybrid in PBS with a pH value of 7.2 and ethanol– water (3:7 by volume) mixed at a volume ratio of 3:7, respectively. The HCPTL–LDH nanohybrid with a drug loading of 22.0% was obtained by dilute acetic acid-treated HCPTC–LDH nanohybrid with a drug
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Scheme 2. Schematic structure change of HCPT in the gallery of LDH treated by dilute acetic acid.
loading of 19.9%. As expected, HCPTL–LDH hybrids showed low drug release rate and less than 80% of the loaded HCPT was released from the nanohybrids within over 1500 min in PBS with a pH value of 7.2 (Fig. 6a), which was obviously lower than that in ethanol–water solution (Fig. 6b). And the possible reason is that solubility of HCPT with lactone form in ethanol–water mixture is higher than that in PBS. 3.6. Morphology and DLS measurements Fig. 7A showed the TEM images of HCPTL–LDH nanohybrid with drug loading of 5.2%, and the sample consisted of many nanosheets with a diameter about 50 to 100 nm. The z-average particle size with different drug loadings was shown in Fig. 7B. As can be seen, particle size increased with the drug loading. But compared with samples prepared in beakers, the average particle size of samples prepared using a microchannel reactor was apparently smaller, which facilitated the potential clinical application of nanohybrid. 3.7. Cell viability To test the therapeutic effect of the synthesized HCPTL–LDH nanohybrid with a drug loading of 22.0% and the potential toxicity of
Fig. 2. FT-IR spectra of (a) raw HCPT and HCPT–LDH treated with acetic acid for different times (b) 0 h, (c) 6 h, (d) 12 h and (e) 24 h.
Fig. 3. (A) Representative HPLC chromatograms of HCPT under indicated conditions. (a) raw HCPT, HCPTC–LDH treated with acetic acid for different times, (b) 0 h, (c) 6 h, (d) 12 h, and (e) 24 h and (B) the dependence of lactone fraction upon time.
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Fig. 6. In vitro release profiles for HCPT from HCPTL–LDH nanohybrid in (a) pH 7.2 PBS, (b) ethanol–water (3:7 by volume).
Fig. 4. (A) The effect of the molar ratio of HCPT and (Zn2++Al3+) on drug loading and (B) release profiles of HCPTC–LDH with different drug loadings in pH 7.2 PBS.
the pristine LDH, we studied cytotoxicity of the synthesized samples against HCT-116 and Colo-205 cells lines using a CCK8 method. For HCT-116 and Colo-205 cells, no obvious cytotoxicity was observed when the concentration of LDH is below 12 μg/ml. However, when the LDH concentration was above 110 μg/ml, cell survival percentage decreases with the increase of LDH concentration (Fig. 8). So the existence of LDH as drug carrier had no effect on results in our tests. When the cells were treated with either the dispersion of HCPTL–LDH nanohybrids or a DMSO solution of HCPT, growth inhibition and killing of cancer cells were obviously observed. As shown in Fig. 9A and B, the synthesized HCPTL–LDH nanohybrids demonstrated a dose-dependent cytotoxic effect against HCT-116 and Colo-205 cells and the cytotoxicity was almost equivalent to the drug dissolved in DMSO. As shown in Table 1, the IC50 values of HCPT L –LDH nanohybrids in incubation with HCT-116 and Colo-205 cells were respectively 0.071 μg/ml and 0.017 μg/ml. However, those values of HCPTC–LDH nanohybrids for HCT-116 and Colo-205 cells were 0.293 μg/ml and 0.049 μg/ml. It denoted that HCPTL–LDH nanohybrids was more effective in anticancer performance than HCPTC–LDH nanohybrids. It should be noticed that no significant cytotoxicity to the cell of HCT-116 and Colo-205 was observed in the concentration range involved in the experiment when the raw HCPT was suspended into the PBS solution and incubated with the cancer cells. This suggested that the raw HCPT could not be internalized into the cancer cells, which was in agreement with the reported results (Chen et al., 2009).
4. Conclusion
Fig. 5. XRD pattern of Zn2Al–CO3 LDH absorbed HCPT.
HCPT-intercalated LDH nanohybrids were prepared by a microchannel reactor via a coprecipitation method, and then treated in dilute acetic acid to obtain HCPT-intercalated LDH with biologically active lactone form. The intercalated HCPT in LDH could keep the lactone form because the reversible lactone–carboxylate equilibrium is pH value-dependent and the lactone form was dominated at acidic condition. The HCPT with bioactive lactone form in the drug–LDH nanohybrids was found to be significantly more effective than the raw HCPT. The soobtained HCPTL–LDH nanohybrids showed high drug loadings as well as obvious controlled release effects, and the pH control method can be used in intercalating other pH sensitive drugs.
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Fig. 7. (A) TEM image of HCPTL–LDH with 5.2% drug loading and (B) the relationship between drugs loaded amount and z-average particle size of samples (a) prepared using microchannel reactor and (b) samples prepared in beaker.
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Fig. 9. Survival percentage of (A) HCT-116 and (B) Colo-205 cell after 72 h treatment with raw HCPT, HCPTC–LDH and HCPTL–LDH.
Acknowledgments This research was supported by the National Natural Science Foundation of China (No. 21173135 and 20903059), the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20110131130008) and the Doctoral Fund of Qingdao University of Science and Technology, China (No. 0022610).
Table 1 IC50 values of HCPT, HCPTC–LDH and HCPTL–LDH samples for human colorectal carcinoma HCT-116 and Colo-205 cells. Samples
IC50 (μg/ml) HCT-116 colo-205
Fig. 8. Survival percentage of HCT-116 and Colo-205 cell after treatment with pristine LDH.
HCPT HCPTC–LDH HCPTL–LDH
0.053 0.293 0.071
0.015 0.049 0.017
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