http://informahealthcare.com/drt ISSN: 1061-186X (print), 1029-2330 (electronic) J Drug Target, 2014; 22(5): 428–438 ! 2014 Informa UK Ltd. DOI: 10.3109/1061186X.2013.879386
ORIGINAL ARTICLE
Octreotide-conjugated PAMAM for targeted delivery to somatostatin receptors over-expressed tumor cells Jianqing Peng1*, Xiaole Qi1*, Yi Chen1, Ning Ma1, Ziwei Zhang1, Jiaxu Xing1, Xuehua Zhu1, Zhengrong Li2, and Zhenghong Wu1,3 Journal of Drug Targeting Downloaded from informahealthcare.com by University of Newcastle on 08/29/14 For personal use only.
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Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Nanjing, Jiangsu, PR China, 2Department of Cell Biology, Nanjing Medical University, Nanjing, Jiangsu, PR China, and 3Yangtze River Pharmaceutical Group, State Key Laboratory for Advanced Formulation Technologies, Taizhou, Jiangsu, PR China Abstract
Keywords
Purpose: An octreotide-conjugated polyamidoamine (PAMAM) dendrimer was synthesized and employed as nanocarriers of methotrexate (MTX), for targeting to the somatostatin receptors over-expressed tumor cells. Methods: PAMAM–PEG–octreotide (PPO) and PAMAM–PEG (PPG) were synthesized and characterized. The cellular uptake of fluorescein isothiocyanate (FITC)-labeled PPO (PPO-FITC) and PPG (PPG-FITC) were investigated. The cytotoxicity of MTX and MTX nanoparticles were conducted in the MCF-7 cells. Besides, the pharmacokinetics studies on MTX nanoparticles were carried out in rats. Results: The structure of PPO was verified by NMR detection and the diameter was 11.05 ± 1.80 nm, with the amount of MTX encapsulated by PPO was 30 (molecule/molecule). MTX nanoparticles possessed significantly higher cytotoxicity against MCF-7 cells compared with free MTX, especially the PPO/MTX nanoparticles. Correspondingly, the PPO-FITC carrier had higher cellular uptake efficiency compared to PPG-FITC. In addition, pharmacokinetics studies showed that PPO/MTX nanoparticles increased mean residence time and bioavailability of MTX distinctly. Discussion and conclusion: With further cellular uptake test of FITC-labeled carriers, the enhanced cytotoxicity of PPO/MTX nanoparticles was reasonable to ascribe to the specific receptor-mediated endocytosis induced by octreotide. The present study suggests that this PAMAM–PEG–octreotide nanocarrier opens a new path for treating cancer with higher efficacy.
Methotrexate, octreotide, polyamidoamine, receptor-mediated endocytosis, tumor targeting
Introduction Cancer chemotherapy with impressive drug delivery sitespecificity and less systemic side effects was achieved via the development of targeted drug delivery systems (DDS). The design of this kind of DDS was based on the effect of enhanced permeability and retention (EPR) and specific receptor-mediated endocytosis, which was considered to be more effective to inhibit the growth of tumor cells [1,2]. Macromolecules have shown potential characteristics as targeted drug delivery carries. Polyamidoamine (PAMAM) dendrimer, a kind of hyperbranched macromolecules, with uniform size, monodispersity and high density of peripheral functional end groups, is an important classification of drug
*These authors contributed equally to this work. Address for correspondence: Zhenghong Wu, Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Nanjing, Jiangsu 210009, PR China. Tel: +86-15062208341. Fax: +86-025-83179703. E-mail: [email protected] Zhengrong Li, Department of Cell Biology, Nanjing Medical University, Nanjing, 210029, PR China. Tel: +86 25 86863153. Fax: +86 25 86862731. E-mail: [email protected]
History Received 14 October 2013 Revised 26 November 2013 Accepted 25 December 2013 Published online 17 January 2014
carriers. However, cationic PAMAM with large number of peripheral primary amino groups performed higher cytotoxicity and rapid clearance from the circulation after intravenous administration [3,4]. Hence, polyethylene glycol (PEG), a generally used hydrophilic chain, was conjugated to PAMAM in order to prolong the systemic circulation time, avoid clearance of reticuloendothelial system (RES) and reduce hemolytic toxicity [5,6]. The synthesized PAMAM–PEG conjugate leading to passive-targeted drug delivery via EPR effect have been employed as carrier for methotrexate (MTX) [7], doxorubicin (DOX) [8] and fluorouracil (5-FU) [9]. Whereas, passive-targeted DDS with non-specific binding sites pales in the face of receptor-mediate active targeting. It has been reported that somatostatin receptors (SSTRs) are over-expressed on various tumors, especially the hormonesecreting tissue tumors compared with normal tissues [10]. The SSTRs were consisted of five G-protein-coupled receptors (SSTR-1 to SSTR-5) with a high degree of sequence similarity, and the majority of tumor cells express SSTR-2, followed by the other four subtypes of SSTRs [11]. Among hundreds of somatostatin analogs that have been synthesized, octreotide is in common clinical use for tumor diagnosis and
Octreotide-conjugated PAMAM
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DOI: 10.3109/1061186X.2013.879386
therapy. With specific affinity to SSTR-2 and SSTR-5 [12], octreotide has been employed as tumor targeting ligand to develop prodrug and effective drug carriers, such as octreotide-polyethylene glycol monostearate (OPMS) and N-octyl-N-succinyl-O-carboxymethyl chitosan (OSCC)assembled supramolecular nanocarriers [13], octreotidemodified nanostructured lipid carriers (NLC) loaded with hydroxycamptothencine [14], octreotide-liposomes for cancer imaging [15] and octreotide-modified chitosan [16]. Most of the researches showed the effective targeting capability of octreotide-carriers to tumor cells that possessed a high density of SSTRs. However, compared with these carriers, PAMAM with nanometer structure and abundant periphery functional groups make it an ideal carrier that possesses higher stability, smaller diameter and simpler modification. Hence, octreotide is conjugated to PAMAM via PEG chain to prepare a novel site-specific targeting drug carrier for breast cancer cell line MCF-7 which possesses over-expressed SSTRs [17]. MTX, a generally used antitumor drug with low solubility in water, was used as model drug in our study. With two carboxyl groups, MTX molecule is liable to combine with the abundant amine groups of PAMAM via electrostatic interactions. Both of the periphery sites and interior region of PAMAM possesses 250 amine groups in total. It has been reported that PAMAM was apt to encapsulate the hydrophobic drug molecules into its core region [18]. However, the size, pKa and structure of drugs also have an effect on the binding mode between PAMAM and drug [19]. Thus, MTX nanoparticles prepared by the complexation between MTX and PAMAM possibly included MTX around the surface and in the interior. The MTX nanoparticles are used for breast cancer therapy in this study. Herein, the PPO and PPG carriers have been synthesized via covalent bonds and characterized. Then, the encapsulation of MTX by these carriers and in vitro release profiles of prepared MTX nanoparticles were investigated. Further, the cytotoxicity of blank carriers and MTX nanoparticles on tumor cells has been determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Fluorescein isothiocyanate (FITC) was conjugated to the periphery of PAMAM to evaluate the tumor targeting efficiency of the novel carriers in vitro. Besides, the pharmacokinetics studies on MTX nanoparticles were carried out in rats. To the best of our knowledge, it the first time that octreotide-conjugated PAMAM dendrimer has been developed as drug carriers for targeted cancer therapy.
Materials and methods Materials and reagents PAMAM Generation 4 (PAMAM G4, 14215 Da) was purchased from Chenyuan Corporation (Shandong, China). Maleimide PEG NHS (MAL-PEG-NHS, 3400 Da) was obtained from Yare Biotech Inc. (Shanghai, China) and octreotide acetate (1019.24 Da) was from GL Biochem (Shanghai) Ltd. (Shanghai, China). MTX (454.44 Da) was purchased from Nanjing Ze Long Agriculture Development Co. Ltd. (Jiangsu, China). 2-Iminothiolane hydrochloride (Traut’s reagent, 137.63 Da) was purchased from SigmaAldrich (Ontario, Canada). Most of the other chemicals were
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obtained from Nanjing Chemical Reagent Corporation (Jiangsu, China). All organic solvents were of analytical grade and purchased from Yuwang Group (Shandong, China). Water used in all experiments was of HPLC grade. Cell line and animals MCF-7 breast tumor cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). All cell culture media and reagents were purchased from Gibco Inc (New York, NY). Male Sprague-Dawley rats (200 ± 20 g) supplied by the Qinglong Mountain Animal Centre, were housed on standard laboratory diet at an ambient temperature and humidity in airconditioned chambers for 1 week before experiment. All animal experiments were conducted in full compliance with the approval of the Institutional Animal Care and Use Committee at China Pharmaceutical University. Synthesis of modified PAMAM dendrimers Conjugation of octreotide to PEG Octreotide was conjugated to MAL-PEG-NHS through the activated NHS group. MAL-PEG-NHS (2.5 mmol, 8.9 mg) was incubated with octreotide acetate (25 mmol, 31 mg) in dimethylformamide solution, adjusting pH to 10.0 with triethylamine [20]. The reaction was performed under moderate stirring for 24 h at room temperature. Then, the reaction mixture was dialyzed via dialysis bag [molecular weight cutoff (MWCO) 2 kDa] against deionized water for 48 h to remove unreacted octreotide. After dried by lyophilization, the MAL–PEG–octreotide was obtained. Synthesis of PAMAM–PEG–octreotide and PAMAM–PEG 2-Iminothiolane hydrochloride (0.7 mg, 5 mmol) was added to the solution of G4 PAMAM (0.5 mmol, 7.1 mg) in 2 mL pH 9.0 borate buffer (0.2 M, containing 5 mM EDTA) and allowed to stir slowly for 40 min at 20 C. The mixture was subjected to ultrafiltration (MWCO 3 kDa, Millipore Corporation, Billerica, MA) to remove the surplus 2-iminothiolane hydrochloride with pH 7.0 phosphate-buffered saline (PBS) buffer (0.05 M, containing 5 mM EDTA) as substitution solution. Then, the PAMAM-SH solution was obtained. The amount of thiol groups was determined by Ellmann’s Reagent [21] with the ration of 8.5:1 (thiol group to PAMAM). Lyophilized MAL–PEG–octreotide (11.15 mg, 2.5 mmol) was added to 2 mL PAMAM-SH pH 7.0 PBS solutions (0.25 mM). The mixture was stirred for 24 h at room temperature. The solution was subjected to ultrafiltration (MWCO 10 kDa, Millipore Corporation, Billerica, MA) to remove the impurities. The resulting solution was dried by lyophilization to obtain PPO carrier (Figure 1). PPG was synthesized in the same process. MALPEG-NHS (2.5 mmol, 8.9 mg) was added to 2 mL PAMAMSH pH 7.0 PBS solution (0.25 mM). The mixture was stirred and subjected to ultrafiltration. After lyophilized, the obtained PPG and PPO were confirmed by 1H-NMR analysis at 300 MHz (Bruker, Karlsruhe, Germany). The number of conjugate moieties was calculated by the following formula: I PAMAM N ¼ IPEGðoctÞ HHPEGðoctÞ , N is the number of conjugate PAMAM moieties; IPEG(oct) and IPAMAM represent the integration of
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Figure 1. Schematic representation of the reactions involved in the synthesis of PAMAM–PEG–octreotide.
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DOI: 10.3109/1061186X.2013.879386
characteristic proton signal of PEG (or octreotide) and PAMAM; HPAMAM and HPEG(oct) represent the corresponding proton number of PEG (or octreotide) and PAMAM.
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Conjugation of FITC to modified PAMAM dendrimers FITC (0.973 mg, 2.5 mmol) dissolved in dimethyl sulfoxide (DMSO) (50 mL) was slowly added to a solution of the PPO (0.25 mM) or PPG (0.25 mM) in 2 mL distilled water. After stirring overnight, the mixtures were purified by dialysis (MWCO 3.5 kDa) against distilled water in a dark room for 48 h. The solutions were lyophilized to obtain orange powders. UV-Visible spectrophotometer (Agilent 8453, Agilent Technology, Santa Clara, CA) was used to estimate the amount of FITC conjugated to each PPG and PPO molecule (Supplementary Figure 1) [22,23].
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In vitro drug release of MTX nanoparticles Drug release profile of MTX nanoparticles was analyzed by a membrane dialysis technique. In order to stimulate the microenvironment of tumor, three phosphate buffer at pH 7.4, pH 6.5 and pH 5.4 were employed as release media. Besides, pH 7.4 PBS (containing 0.15 M NaCl) was also used as medium to mimic high ionic concentration environment. The MTX nanoparticles dissolved in different media (1 mL) were placed into the dialysis bag (MWCO 3.5 kDa). Then the bag was tightened and soaked in 50 mL of corresponding phosphate buffer media. The experiments were carried at 37 C in a shaking air bath for 12 h. At determined intervals, 0.5 mL was withdrawn and the same amount of fresh medium was replenished. The concentration of MTX was determined by HPLC/UV detection (LC10AT VP, Shimadzu, Kyoto, Japan).
Characterization and morphology study The particle size, its distribution indicated by polydispersity index (PDI) and zeta potential of PAMAM, PPG, PPO, PPGFITC and PPO-FITC were measured at room temperature with Zetasizer Nano ZS (Malvern, Worcestershire, UK). The samples were diluted by pH 7.4 PBS (0.01 M) to the final concentration of 0.2 mM before measured. The morphologic study was performed under transmission electron micrograph (TEM, alpha300R, WITec, Ulm, Germany). About 20 mL of PAMAM and PPO were placed on carbon-coated Formvar copper grids and air-dried. Negative staining was performed by adding a drop of 2% phosphotungstic acid solution to the copper grid containing the samples. The samples were determined after they were air-dried. In vitro hemolytic activity The hemolytic studies were performed following the previous studies [24]. Briefly, the rabbit red blood cells (RBCs) suspension was dispersed in distilled water and normal saline (0.9% w/v). Distilled water and normal saline was considered as 100% hemolytic and non-hemolytic, respectively. PAMAM, PPG and PPO were added to a 2% w/v solution of freshly prepared RBCs in normal saline and incubated for 1 h at 37 C in a shaking air bath. The samples were centrifuged at 1500g for 10 min and the supernatants were assayed via UV-Visible spectrophotometer (Qinghua Instruments, Shanghai, China) at ¼ 540 nm. Encapsulation of MTX by modified PAMAM dendrimers Various amounts of MTX were added to 0.1 mmol PPG or PPO in deionized water, respectively. The molar ratios of MTX/PPG (or PPO) were 20:1, 30:1, 40:1 and 60:1. The mixture was stirred at room temperature for 3 h. After centrifuged at 4000g for 15 min, the supernatant solution was filtrated through a cellulose acetate filtrate membrane (Millipore Corporation, Billerica, MA; 0.45 mm). The PPO/ MTX and PPG/MTX nanoparticles were obtained after lyophilization. In order to detect the amount of MTX encapsulated, the MTX nanoparticles were dissolved in 0.01 M NaOH and measured by UV-Visible spectrophotometer at ¼ 303 nm [25].
Cytotoxicity of blank carriers and MTX nanoparticles The MCF-7 cells were maintained at 37 C in DMEM medium supplemented with glutamine (0.29 mg/mL), 1% bovine fetal serum and 10 000 U/mL of penicillin/streptomycin, in an atmosphere of 5% CO2 with 90% relative humidity. To detect the cytotoxicity of blank carriers and MTX nanoparticles, the cells were seeded at a density of 2 104 cells/well in 96-well plates. After 24 h, cells were incubated with fresh media containing a series of concentrations of PAMAM, PPG, PPO, MTX, PPG/MTX and PPO/MTX. The cells were cultured for 48 h and wash three times with PBS. About 20 mL MTT solution (5 mg/ mL in PBS) was added to each well and cells was incubated for 4 h. The medium was removed and 100 mL DMSO was added into the wells. Then, the plate was agitated for 10 min and the optical ratio was measured at 570 nm via Enzyme Immunoassay Instrument (ELx800, BioTek Instruments, Winooski, VT). Cell viability was calculated by the following formula: Cell viability ð%Þ ¼
absorbance of sample group 100% absorbance of control group
Cellular uptake of FITC-labeled PAMAM dendrimers The MCF-7 cells were seeded at a density of 1 105 cells/ well in 6-well plates. After 24 h, cells were incubated with FITC, PPG-FITC and PPO-FITC (final concentration of 15 mM for both PPG-FITC and PPO-FITC) in the presence of serum free medium for 4 h at 37 C. For the competitive binding study, cells were pre-incubated with octreotide (50 mM) for 30 min before PPO-FITC were added. After removing the supernatant and washing the well three times with pH 7.4 PBS buffer, the cells were trypsinized using a standard protocol, and resuspended in 0.4 mL DMEM. The cellular uptake of the samples were examined by flow cytometry using a fluorescence-activated cell sorting (FACS) Caliber flow cytometer (Becton Dickinson, Franklin Lakes, NJ), while the samples were visualized and photographed under a fluorescent microscope (Olympus IX 51, Osaka, Japan).
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Pharmacokinetic studies of MTX nanoparticles
Statistical analysis
To determine the pharmacokinetic profiles of the MTX nanoparticles, male Sprague-Dawley rats were randomized into three groups and treated with free MTX, PPG/MTX and PPO/MTX nanoparticles, respectively. All the preparations were administrated intravenously into the tail vein of rats as a dose of MTX 5 mg/kg, and blood samples were taken with a heparinized syringe at 5, 15, 30, 60, 120, 180 and 240 min. The blood samples were centrifuged at 1500g for 10 min at 4 C. About 20 mL folic acid (75 mg/mL) as internal standard was added the blood samples (300 mL). The mixtures were vortexed for 20 s, and 0.6 mL methanol was added. Then the contents were vortexed for 90 s and centrifuged at 4000g for 12 min. About 20 mL of supernatant was analyzed by HPLC at ¼ 303 nm. The plasma concentrations versus time data were analyzed by WinNonlin 5.2.1 (Pharsight Corporation, Sunnyvale, CA).
The data were presented as mean ± standard deviations (SD) of at least three independent experiments. Significant differences were performed by one-way analysis of variance (ANOVA) and followed by Bonferroni’s post hoc test. p value 0.05 was considered to be statistically significant.
Results Synthesis of modified PAMAM dendrimers The PAMAM-based carriers-PPG and PPO were synthesized and confirmed by 1H-NMR spectrum (Figure 2). Both of the spectra contain the characteristic peaks of PAMAM, which were d ¼ 2.35–2.45 ppm (br, Hc), d ¼ 2.55–2.65 ppm (br, He), d ¼ 2.75–2.85 ppm (br, Hd), d ¼ 2.64 ppm (br, Hd) and d ¼ 3.25–3.40 ppm (m, Ha, Hb, Hf). For the synthesis of PPG, the MAL-PEG-NHS was conjugated to PAMAM with MAL group. Thus, the signals of PEG in the spectrum of PPG
Figure 2. 1H-NMR spectra for PPG (A) and PPO (B) in D2O.
Octreotide-conjugated PAMAM
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were d ¼ 2.52 ppm (t, NHS) and d ¼ 3.50–3.80 ppm (m, CH2CH2O). The average number of PEG chains attached to the PAMAM measured by the specific signals was 4.34. However, the NHS group of PEG connected to octreotide disappeared in the spectrum of PPO. As excessive octreotide was added to ensure complete reaction in the preparation of MAL–PEG–octreotide, the characteristic signals of octreotide was used to detect the number of MAL–PEG–octreotide conjugated to PAMAM. The specific signals of benzene on octreotide was d ¼ 7.05–7.40 ppm (m, ArH). Hence, the average number of MAL–PEG–octreotide on PPO was 4.64. The characteristic peaks of FITC in 1H-NMR spectrum was at d ¼ 6.44 and 7.05 ppm which overlapped with the signals of octreotide. For this reason, the number of FITC molecules conjugated to the surface of PAMAM was determined by the UV-Visible spectrometer (Figure 3). The spectrum showed that the characteristic peak of FITC (501 nm) on PPO-FITC and PPG-FITC shifted 9 nm compared with free FITC (492 nm), which consistent with
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previous study [22]. Since PPG and PPO give no absorbance after 400 nm, the absorbance value of PPG-FITC and PPOFITC were proportional to the number of conjugated FITC at ¼ 492 nm. Hence, a calibration curve was established by free FITC, and the number of FITC conjugated to each PPG and PPO molecule were 3.33 and 3.11, respectively. Characterization and safety evaluation in vitro The particle size, PDI and zeta potential of modified PAMAM dendrimers are listed in Table 1. The conjugation of PEG and MAL–PEG–octreotide to the surface of PAMAM resulted in increasing of diameters. Meanwhile, the zeta potential of modified PAMAM carriers were significantly decreased compared with PAMAM, due to the presence of PEG chains on the surface that masked the positive charge of PAMAM [26]. Although the absolute value of zeta potential of nanoparticles below 10 mV was liable to agglomerate in the dispersion [27], the conjugated PEG chains could provide additional steric hindrance for sufficient electrostatic stabilization of vectors. The TEM morphology of PAMAM and PPO were showed in the Figure 4. It could be observed that the PAMAM appeared as tiny particles with near-spherical shapes, while the PPO with long PEG chains on the surface exhibited regular spherical shapes. Both of the carriers were loosely associated. The safety of PAMAM dendrimers was evaluated by the hemolytic activity (Figure 5). PAMAM with high cationic Table 1. Characterizations of synthesized PAMAM dendrimers. Preparations PAMAM PPG PPO PPG-FITC PPO-FITC
Figure 3. UV-Visible spectra of FITC-labeled PPG and PPO carriers. The arrows at 492 and 501 nm indicate the presence of FITC.
Diameter (nm)
PDI
Zeta potential (mV)
5.77 ± 0.25 9.52 ± 1.27 11.05 ± 1.80 15.18 ± 2.01 17.02 ± 2.16
0.23 ± 0.02 0.31 ± 0.04 0.34 ± 0.04 0.43 ± 0.11 0.39 ± 0.10
15.47 ± 1.80 4.19 ± 1.00 2.59 ± 1.53 2.60 ± 0.50 2.35 ± 1.04
Data are represented as mean ± SD (n ¼ 3).
Figure 4. The TEM of PAMAM (A) and PPO (B).
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Figure 5. The hemolytic toxicity of the synthesized PAMAM dendrimers at different concentrations. Data are represented as mean ± SD (n ¼ 3).
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Figure 6. UV-visible spectra of MTX, blank carriers and MTX nanoparticles. The arrows at 258, 303 and 373 nm represent the characteristic peaks of MTX.
charge induced 430% hemolysis at 20 mM, which was too toxic to be employed as carrier. However, all the modified dendrimers with PEG on the surface showed510% hemolysis even at 20 mM, which was in line with the previous studies that PEGylation would significantly decreased the hemolytic toxicity of PAMAM [7]. Preparation of MTX nanoparticles The UV-Visible spectra of MTX, PAMAM, PPG, PPO, PPG/ MTX and PPO/MTX were detected at first (Figure 6). The peak of MTX at 258 nm was much weaker and the peak at 373 nm moves to 350 nm with decreased intensity in the spectra of PPG/MTX and PPO/MTX. According to the previous studies, MTX molecules were supposed to be encapsulated into the interior region of carrier or combined on the surface with non-covalent bonds including: hydrogen bonds, electrostatic and hydrophobic interactions [28], and previous study showed that the physical interactions between PAMAM and curcumin induced changes on the UV spectra [29]. Therefore, the changes of the peaks might be attributed to the interactions between MTX and carriers. However, the peak of MTX at 303 nm was invariant for MTX nanoparticles. Hence, the amount of MTX encapsulated by PPG and PPO was determined by UV-Visible spectrometer at 303 nm, utilizing the calibration curve of free MTX. The influence of time on the encapsulation of MTX by PPG and PPO has been studied previously, which showed that the encapsulated MTX reached the maximum amount after 3 h stirring (Supplementary Figure 2). Thus, the preparation of MTX nanoparticles took 3 h mix. As shown in Figure 7, the number of MTX encapsulated by carriers increased with the increasing MTX/PPG (or PPO) molar ratio from 20:1 to 60:1. However, the number of encapsulated MTX was almost invariant when the ratio was above 40:1. The maximum amount of MTX encapsulated by PPG and PPO was 33 and 30 (molecule/molecule), respectively, and the MTX nanoparticles prepared at molar ratio of 60:1 was used in the cytotoxicity study.
Figure 7. Encapsulation of MTX by PPG and PPO at different molar ratios. Data are represented as mean ± SD (n ¼ 3).
In vitro release of MTX The in vitro release profiles were depicted in Figure 8. Free MTX dissolved in the four media was capable to release completely within 2 h (data not shown). For both of PPG/MTX and PPO/MTX nanoparticles, the drug release profiles exhibited the same characteristics. Firstly, MTX was almost completely released to the outer phase within 8 h. Moreover, the release profiles showed conspicuous distinctions in different media. The order of drug release of MTX nanoparticles in four media was pH 7.4 (containing 0.2 M NaCl)4pH 5.44pH 6.5 & pH 7.4., and PPO/MTX nanoparticles exhibited faster release at the initial stage. Cytotoxicity on MCF-7 cells The cytotoxicity of PAMAM, PPG and PPO against MCF-7 tumor cells was measured using MTT assay. It was shown that the toxic activity was proportional to the concentration of carriers, and PEG conjugated PAMAM (PPG and PPO) showed obviously lower cytotoxicity than PAMAM
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Figure 8. The release profiles of MTX from PPG/MTX (A) and PPO/MTX (B) in PBS buffer with various pH values and different ionic concentrations. Data are represented as mean ± SD (n ¼ 3).
Figure 9 (A) The cytotoxicity profiles of blank carriers in MCF-7 cells at different concentrations measured by MTT assays. Data are represented as mean ± SD (n ¼ 4). (B) The cell viability profiles of free MTX and MTX nanoparticles in MCF-7 cells at different concentrations of MTX. Data are represented as mean ± SD (n ¼ 4). ** and * indicate p50.001 and 0.05 versus free MTX; ## and # indicate p50.001 and 0.01 versus PPG/MTX.
(Figure 9A). The cell viability of modified PAMAM dendrimers was 460% at 20 mM. Hence, the concentration of PPG and PPO in the MTX nanoparticles used in the following experiments was 520 mM. Then, the cytotoxicity of free MTX and MTX nanoparticles was detected against the MCF-7 cells to evaluate the antitumor activity of MTX nanoparticles in vitro (Figure 9B). The cyotoxicity of free MTX showed extremely low activity against MCF-7 cells even at 500 mM. However, both of the PPG/MTX and PPO/MTX nanoparticles exhibited remarkably higher cytotoxicity than free MTX. Moreover, it was noteworthy that the cytotoxicity of PPO/MTX nanoparticles were significantly higher than PPG/MTX nanoparticles, especially at highest concentration (p50.001), indicating that the conjugation of octreotide enhanced the cytotoxicity of MTX nanoparticles.
Cellular uptake study The cellular uptake of PPO-FITC and PPG-FITC was visualized by florescence microscope (Figure 10). After incubated with MCF-7 cells for 4 h, free FITC showed basically no fluorescent intensity in cells, while fluorescent intensity of PPO-FITC was superior to that of PPG-FITC. To testify the specificity of the uptake approach, octreotide at a concentration of 50 mM was used to combine with the SSTRs on MCF-7 cells. As shown in Figure 10(C and D), the pre-incubation of octreotide significantly decreased the fluorescent intensity of PPO-FITC. Furthermore, quantification for uptake of above preparations by MCF-7 cells was conducted by flow cytometry analysis. The result was consistent with the observation under florescence microscope that PPO-FITC showed the highest uptake in MCF-7 cells.
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Figure 10. The fluorescent microscopy images of MCF-7 cells after incubation with FITC (A), PPG-FITC (B), PPO-FITC (C) and PPO-FITC with octreotide pre-incubation (D). Evaluation of the uptake of the four preparations above determined by flow cytometry analysis (E).
Pharmacokinetics studies The pharmacokinetic studies on free MTX and MTX nanoparticles were carried out to evaluate the differences on metabolism process of MTX in vivo. After intravenous administration, all the preparations were eliminated very quickly (Figure 11). The free MTX eliminated much faster than MTX nanoparticles, while no evident distinction was found between PPG/MTX and PPO/MTX plasma concentration–time curves. Moreover, the major pharmacokinetics parameters of the preparations were listed in Table 2. A significant increase on mean residence time (MRT) of MTX nanoparticles were observed, confirming the prolonged retention of MTX in circulation. And the bioavailability of MTX was notably increased after prepared as nanoparticles, which indicated by the area under the concentration–time curve (AUC). Based on one-way ANOVA, there were obvious differences in MRT and AUC between MTX and MTX nanoparticles. However, no conspicuous distinctions have been found between PPG/MTX and PPO/MTX.
Figure 11. Concentration–time curves of MTX preparations in rat plasma after i.v. administration. Data are represented as mean ± SD (n ¼ 3). ** and * indicate p50.01 and 0.05 versus free MTX.
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Table 2. The main pharmacokinetic parameters of MTX preparations after i.v. administration. Parameters
Free MTX
PPG/MTX
PPO/MTX
MRT (h) AUC0!1 (mg h/L) CL (L/h kg) Vss (L/kg)
0.60 ± 0.05 4.40 ± 0.38 1.14 ± 0.10 0.69 ± 0.10
0.92 ± 0.03** 6.42 ± 0.43* 0.78 ± 0.05 0.72 ± 0.07
0.82 ± 0.06* 6.43 ± 0.45* 0.78 ± 0.05 0.64 ± 0.09
Data are represented as mean ± SD (n ¼ 3). ** and * indicate p50.001 and 0.01 versus free MTX.
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Discussion In this study, octreotide-modified PAMAM dendrimer was successfully synthesized and employed as a carrier for antitumor drug MTX to prepare receptor-mediate targeted drug delivery nanoparticles. The PPO-FITC carrier could be effectively internalized into the tumor cells and MTX nanoparticles increased the inhibition activity of MTX impressively due to the receptor-mediated endocytosis. The ideal size of nanoparticles should range from 10 to 100 nm, in order to efficiently extravasate from the fenestrations of leaky vasculature and avoid the filtration by the kidneys and the specific capture by the liver [30,31]. The size of PAMAM carriers was within the ideal range of nanoparticles. The zeta potential of modified PAMAM dendrimers were decreased to 5 mV below, which increasingly decreased the chance of renal elimination in comparison with PAMAM. Although low zeta potential increased the risk of agglomeration of nanoparticles, the conjugated PEG chains could provide steric stabilization for the PEGylated PAMAM dendrimer. It has been reported that the encapsulation efficiency of MTX by PAMAM was enhanced after PEGylation. In accordance with previous study, the maximum amount of encapsulated MTX molecules was 430 per PEGylated PAMAM dendrimer molecule, which was higher than the maximum encapsulation efficiency of PAMAM [7]. The conjugated PEG was supposed to enlarge the interior region of PAMAM, which was capable to accommodate more drug molecules in the ‘‘extended space’’. Therefore, the MTX in the nanoparticles might be encapsulated in the interior region of PAMAM, interact with the periphery groups of PAMAM and incorporated in the ‘‘extended space’’ with electrostatic interactions, hydrophobic and hydrogen bonds [25,28,32]. Besides, the maximum amount of encapsulated MTX by PPO was slightly less than PPG possibly owing to the block of octreotide on outside of PPO vector. From the release behavior of MTX nanoparticles in different media, both of pH value and ionic concentration influence the interaction between MTX and carriers. Previous work demonstrated that PAMAM dendrimer go through a pH induced conformation change from a ‘‘dense core’’ (high pH) to a ‘‘dense shell’’ (low pH) [33], which is a critical character for development of a pH-sensitive DDS. However, the pH responsive characteristics of MTX nanoparticles are not conspicuous compared with the release profiles of PAMAM– PEG–T7/DOX nanoparticles [34], suggesting that most of MTX was combined on the peripheral ends of PAMAM but not in the interior region. Moreover, the ionic
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concentration-sensitive release behavior further confirmed that the major forces between MTX and PAMAM were electrostatic interactions. In consistent with the results of hemolytic activity detection, the cytotoxicity of PAMAM against MCF-7 cells was significantly decreased after PEG conjugation. The increased safety of carriers was attributed to the shielded cationic charges on the surface of PAMAM which interacted with the cell membrane. Within safety concentration of carriers, both of MTX nanoparticles increased the cytotoxicity of MTX, especially the PPO/MTX nanoparticles, suggesting that PPO/MTX nanoparticles had higher affinity to MCF-7 cells and the carriers promoted the endocytosis of MTX. Furthermore, the cellular uptake study proved that octreotide strongly facilitated the uptake of PPO-FITC by MCF-7 cells, and the specificity of the uptake process was demonstrated by the competition experiment. Thus, the increased antitumor activity of MTX should be attributed to receptor-mediated endocytosis of PPO/MTX nanoparticles. Similar results were also observed in other researches that octreotide modification were effective in MCF-7 cells inhibition [35–37]. Whereas, the quick release of MTX limits the total amount of drug uptake by cells, thus, the antitumor activity of MTX nanoparticles is less than our expect. Besides, pharmacokinetics studies show that the MRT and bioavailability of MTX nanoparticles are conspicuous increased compared with free MTX. It’s reasonable to infer that the hydrophilic PEG chains modification on the PAMAM prevents the phagocytosis of nanoparticles by RES, which sustains the circulation time of MTX in vivo.
Conclusions In this present work, PAMAM–PEG–octreotide was successfully synthesized and employed as nanocarrier for MTX. The model drug, MTX, could be easily encapsulated by this novel nanocarrier with electrostatic interactions, hydrophobic and hydrogen bonds. The PPO/MTX nanoparticles showed improved antitumor activity against MCF-7 cells compared with free MTX and the uptake of FITC-PPO demonstrated that octreotide-modified carrier enhanced targeting to MCF-7 cells via receptor-mediated endocytosis. Hence, it suggested that PAMAM–PEG–octreotide can be used as potential carrier for SSTR over-expressed tumors targeting DDS. Further study is still in progress.
Declaration of interest The authors have no conflicts of interest. This work was supported by the Major State Basic Research Development Program of the National Science and Technology of China for new drugs development (Program No. 2012CB724002), Natural Science Foundation of Jiangsu Province (BK20130663 and BK2011771) and Key project of Nanjing Medical University of Science and Technology Development Fund (2010 NJMUZ25).
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ORIGINAL ARTICLE
Octreotide-conjugated PAMAM for targeted delivery to somatostatin receptors over-expressed tumor cells Jianqing Peng1*, Xiaole Qi1*, Yi Chen1, Ning Ma1, Ziwei Zhang1, Jiaxu Xing1, Xuehua Zhu1, Zhengrong Li2, and Zhenghong Wu1,3 Journal of Drug Targeting Downloaded from informahealthcare.com by University of Newcastle on 08/29/14 For personal use only.
1
Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Nanjing, Jiangsu, PR China, 2Department of Cell Biology, Nanjing Medical University, Nanjing, Jiangsu, PR China, and 3Yangtze River Pharmaceutical Group, State Key Laboratory for Advanced Formulation Technologies, Taizhou, Jiangsu, PR China Abstract
Keywords
Purpose: An octreotide-conjugated polyamidoamine (PAMAM) dendrimer was synthesized and employed as nanocarriers of methotrexate (MTX), for targeting to the somatostatin receptors over-expressed tumor cells. Methods: PAMAM–PEG–octreotide (PPO) and PAMAM–PEG (PPG) were synthesized and characterized. The cellular uptake of fluorescein isothiocyanate (FITC)-labeled PPO (PPO-FITC) and PPG (PPG-FITC) were investigated. The cytotoxicity of MTX and MTX nanoparticles were conducted in the MCF-7 cells. Besides, the pharmacokinetics studies on MTX nanoparticles were carried out in rats. Results: The structure of PPO was verified by NMR detection and the diameter was 11.05 ± 1.80 nm, with the amount of MTX encapsulated by PPO was 30 (molecule/molecule). MTX nanoparticles possessed significantly higher cytotoxicity against MCF-7 cells compared with free MTX, especially the PPO/MTX nanoparticles. Correspondingly, the PPO-FITC carrier had higher cellular uptake efficiency compared to PPG-FITC. In addition, pharmacokinetics studies showed that PPO/MTX nanoparticles increased mean residence time and bioavailability of MTX distinctly. Discussion and conclusion: With further cellular uptake test of FITC-labeled carriers, the enhanced cytotoxicity of PPO/MTX nanoparticles was reasonable to ascribe to the specific receptor-mediated endocytosis induced by octreotide. The present study suggests that this PAMAM–PEG–octreotide nanocarrier opens a new path for treating cancer with higher efficacy.
Methotrexate, octreotide, polyamidoamine, receptor-mediated endocytosis, tumor targeting
Introduction Cancer chemotherapy with impressive drug delivery sitespecificity and less systemic side effects was achieved via the development of targeted drug delivery systems (DDS). The design of this kind of DDS was based on the effect of enhanced permeability and retention (EPR) and specific receptor-mediated endocytosis, which was considered to be more effective to inhibit the growth of tumor cells [1,2]. Macromolecules have shown potential characteristics as targeted drug delivery carries. Polyamidoamine (PAMAM) dendrimer, a kind of hyperbranched macromolecules, with uniform size, monodispersity and high density of peripheral functional end groups, is an important classification of drug
*These authors contributed equally to this work. Address for correspondence: Zhenghong Wu, Key Laboratory of Modern Chinese Medicines, China Pharmaceutical University, Nanjing, Jiangsu 210009, PR China. Tel: +86-15062208341. Fax: +86-025-83179703. E-mail: [email protected] Zhengrong Li, Department of Cell Biology, Nanjing Medical University, Nanjing, 210029, PR China. Tel: +86 25 86863153. Fax: +86 25 86862731. E-mail: [email protected]
History Received 14 October 2013 Revised 26 November 2013 Accepted 25 December 2013 Published online 17 January 2014
carriers. However, cationic PAMAM with large number of peripheral primary amino groups performed higher cytotoxicity and rapid clearance from the circulation after intravenous administration [3,4]. Hence, polyethylene glycol (PEG), a generally used hydrophilic chain, was conjugated to PAMAM in order to prolong the systemic circulation time, avoid clearance of reticuloendothelial system (RES) and reduce hemolytic toxicity [5,6]. The synthesized PAMAM–PEG conjugate leading to passive-targeted drug delivery via EPR effect have been employed as carrier for methotrexate (MTX) [7], doxorubicin (DOX) [8] and fluorouracil (5-FU) [9]. Whereas, passive-targeted DDS with non-specific binding sites pales in the face of receptor-mediate active targeting. It has been reported that somatostatin receptors (SSTRs) are over-expressed on various tumors, especially the hormonesecreting tissue tumors compared with normal tissues [10]. The SSTRs were consisted of five G-protein-coupled receptors (SSTR-1 to SSTR-5) with a high degree of sequence similarity, and the majority of tumor cells express SSTR-2, followed by the other four subtypes of SSTRs [11]. Among hundreds of somatostatin analogs that have been synthesized, octreotide is in common clinical use for tumor diagnosis and
Octreotide-conjugated PAMAM
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DOI: 10.3109/1061186X.2013.879386
therapy. With specific affinity to SSTR-2 and SSTR-5 [12], octreotide has been employed as tumor targeting ligand to develop prodrug and effective drug carriers, such as octreotide-polyethylene glycol monostearate (OPMS) and N-octyl-N-succinyl-O-carboxymethyl chitosan (OSCC)assembled supramolecular nanocarriers [13], octreotidemodified nanostructured lipid carriers (NLC) loaded with hydroxycamptothencine [14], octreotide-liposomes for cancer imaging [15] and octreotide-modified chitosan [16]. Most of the researches showed the effective targeting capability of octreotide-carriers to tumor cells that possessed a high density of SSTRs. However, compared with these carriers, PAMAM with nanometer structure and abundant periphery functional groups make it an ideal carrier that possesses higher stability, smaller diameter and simpler modification. Hence, octreotide is conjugated to PAMAM via PEG chain to prepare a novel site-specific targeting drug carrier for breast cancer cell line MCF-7 which possesses over-expressed SSTRs [17]. MTX, a generally used antitumor drug with low solubility in water, was used as model drug in our study. With two carboxyl groups, MTX molecule is liable to combine with the abundant amine groups of PAMAM via electrostatic interactions. Both of the periphery sites and interior region of PAMAM possesses 250 amine groups in total. It has been reported that PAMAM was apt to encapsulate the hydrophobic drug molecules into its core region [18]. However, the size, pKa and structure of drugs also have an effect on the binding mode between PAMAM and drug [19]. Thus, MTX nanoparticles prepared by the complexation between MTX and PAMAM possibly included MTX around the surface and in the interior. The MTX nanoparticles are used for breast cancer therapy in this study. Herein, the PPO and PPG carriers have been synthesized via covalent bonds and characterized. Then, the encapsulation of MTX by these carriers and in vitro release profiles of prepared MTX nanoparticles were investigated. Further, the cytotoxicity of blank carriers and MTX nanoparticles on tumor cells has been determined by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Fluorescein isothiocyanate (FITC) was conjugated to the periphery of PAMAM to evaluate the tumor targeting efficiency of the novel carriers in vitro. Besides, the pharmacokinetics studies on MTX nanoparticles were carried out in rats. To the best of our knowledge, it the first time that octreotide-conjugated PAMAM dendrimer has been developed as drug carriers for targeted cancer therapy.
Materials and methods Materials and reagents PAMAM Generation 4 (PAMAM G4, 14215 Da) was purchased from Chenyuan Corporation (Shandong, China). Maleimide PEG NHS (MAL-PEG-NHS, 3400 Da) was obtained from Yare Biotech Inc. (Shanghai, China) and octreotide acetate (1019.24 Da) was from GL Biochem (Shanghai) Ltd. (Shanghai, China). MTX (454.44 Da) was purchased from Nanjing Ze Long Agriculture Development Co. Ltd. (Jiangsu, China). 2-Iminothiolane hydrochloride (Traut’s reagent, 137.63 Da) was purchased from SigmaAldrich (Ontario, Canada). Most of the other chemicals were
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obtained from Nanjing Chemical Reagent Corporation (Jiangsu, China). All organic solvents were of analytical grade and purchased from Yuwang Group (Shandong, China). Water used in all experiments was of HPLC grade. Cell line and animals MCF-7 breast tumor cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). All cell culture media and reagents were purchased from Gibco Inc (New York, NY). Male Sprague-Dawley rats (200 ± 20 g) supplied by the Qinglong Mountain Animal Centre, were housed on standard laboratory diet at an ambient temperature and humidity in airconditioned chambers for 1 week before experiment. All animal experiments were conducted in full compliance with the approval of the Institutional Animal Care and Use Committee at China Pharmaceutical University. Synthesis of modified PAMAM dendrimers Conjugation of octreotide to PEG Octreotide was conjugated to MAL-PEG-NHS through the activated NHS group. MAL-PEG-NHS (2.5 mmol, 8.9 mg) was incubated with octreotide acetate (25 mmol, 31 mg) in dimethylformamide solution, adjusting pH to 10.0 with triethylamine [20]. The reaction was performed under moderate stirring for 24 h at room temperature. Then, the reaction mixture was dialyzed via dialysis bag [molecular weight cutoff (MWCO) 2 kDa] against deionized water for 48 h to remove unreacted octreotide. After dried by lyophilization, the MAL–PEG–octreotide was obtained. Synthesis of PAMAM–PEG–octreotide and PAMAM–PEG 2-Iminothiolane hydrochloride (0.7 mg, 5 mmol) was added to the solution of G4 PAMAM (0.5 mmol, 7.1 mg) in 2 mL pH 9.0 borate buffer (0.2 M, containing 5 mM EDTA) and allowed to stir slowly for 40 min at 20 C. The mixture was subjected to ultrafiltration (MWCO 3 kDa, Millipore Corporation, Billerica, MA) to remove the surplus 2-iminothiolane hydrochloride with pH 7.0 phosphate-buffered saline (PBS) buffer (0.05 M, containing 5 mM EDTA) as substitution solution. Then, the PAMAM-SH solution was obtained. The amount of thiol groups was determined by Ellmann’s Reagent [21] with the ration of 8.5:1 (thiol group to PAMAM). Lyophilized MAL–PEG–octreotide (11.15 mg, 2.5 mmol) was added to 2 mL PAMAM-SH pH 7.0 PBS solutions (0.25 mM). The mixture was stirred for 24 h at room temperature. The solution was subjected to ultrafiltration (MWCO 10 kDa, Millipore Corporation, Billerica, MA) to remove the impurities. The resulting solution was dried by lyophilization to obtain PPO carrier (Figure 1). PPG was synthesized in the same process. MALPEG-NHS (2.5 mmol, 8.9 mg) was added to 2 mL PAMAMSH pH 7.0 PBS solution (0.25 mM). The mixture was stirred and subjected to ultrafiltration. After lyophilized, the obtained PPG and PPO were confirmed by 1H-NMR analysis at 300 MHz (Bruker, Karlsruhe, Germany). The number of conjugate moieties was calculated by the following formula: I PAMAM N ¼ IPEGðoctÞ HHPEGðoctÞ , N is the number of conjugate PAMAM moieties; IPEG(oct) and IPAMAM represent the integration of
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Figure 1. Schematic representation of the reactions involved in the synthesis of PAMAM–PEG–octreotide.
Octreotide-conjugated PAMAM
DOI: 10.3109/1061186X.2013.879386
characteristic proton signal of PEG (or octreotide) and PAMAM; HPAMAM and HPEG(oct) represent the corresponding proton number of PEG (or octreotide) and PAMAM.
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Conjugation of FITC to modified PAMAM dendrimers FITC (0.973 mg, 2.5 mmol) dissolved in dimethyl sulfoxide (DMSO) (50 mL) was slowly added to a solution of the PPO (0.25 mM) or PPG (0.25 mM) in 2 mL distilled water. After stirring overnight, the mixtures were purified by dialysis (MWCO 3.5 kDa) against distilled water in a dark room for 48 h. The solutions were lyophilized to obtain orange powders. UV-Visible spectrophotometer (Agilent 8453, Agilent Technology, Santa Clara, CA) was used to estimate the amount of FITC conjugated to each PPG and PPO molecule (Supplementary Figure 1) [22,23].
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In vitro drug release of MTX nanoparticles Drug release profile of MTX nanoparticles was analyzed by a membrane dialysis technique. In order to stimulate the microenvironment of tumor, three phosphate buffer at pH 7.4, pH 6.5 and pH 5.4 were employed as release media. Besides, pH 7.4 PBS (containing 0.15 M NaCl) was also used as medium to mimic high ionic concentration environment. The MTX nanoparticles dissolved in different media (1 mL) were placed into the dialysis bag (MWCO 3.5 kDa). Then the bag was tightened and soaked in 50 mL of corresponding phosphate buffer media. The experiments were carried at 37 C in a shaking air bath for 12 h. At determined intervals, 0.5 mL was withdrawn and the same amount of fresh medium was replenished. The concentration of MTX was determined by HPLC/UV detection (LC10AT VP, Shimadzu, Kyoto, Japan).
Characterization and morphology study The particle size, its distribution indicated by polydispersity index (PDI) and zeta potential of PAMAM, PPG, PPO, PPGFITC and PPO-FITC were measured at room temperature with Zetasizer Nano ZS (Malvern, Worcestershire, UK). The samples were diluted by pH 7.4 PBS (0.01 M) to the final concentration of 0.2 mM before measured. The morphologic study was performed under transmission electron micrograph (TEM, alpha300R, WITec, Ulm, Germany). About 20 mL of PAMAM and PPO were placed on carbon-coated Formvar copper grids and air-dried. Negative staining was performed by adding a drop of 2% phosphotungstic acid solution to the copper grid containing the samples. The samples were determined after they were air-dried. In vitro hemolytic activity The hemolytic studies were performed following the previous studies [24]. Briefly, the rabbit red blood cells (RBCs) suspension was dispersed in distilled water and normal saline (0.9% w/v). Distilled water and normal saline was considered as 100% hemolytic and non-hemolytic, respectively. PAMAM, PPG and PPO were added to a 2% w/v solution of freshly prepared RBCs in normal saline and incubated for 1 h at 37 C in a shaking air bath. The samples were centrifuged at 1500g for 10 min and the supernatants were assayed via UV-Visible spectrophotometer (Qinghua Instruments, Shanghai, China) at ¼ 540 nm. Encapsulation of MTX by modified PAMAM dendrimers Various amounts of MTX were added to 0.1 mmol PPG or PPO in deionized water, respectively. The molar ratios of MTX/PPG (or PPO) were 20:1, 30:1, 40:1 and 60:1. The mixture was stirred at room temperature for 3 h. After centrifuged at 4000g for 15 min, the supernatant solution was filtrated through a cellulose acetate filtrate membrane (Millipore Corporation, Billerica, MA; 0.45 mm). The PPO/ MTX and PPG/MTX nanoparticles were obtained after lyophilization. In order to detect the amount of MTX encapsulated, the MTX nanoparticles were dissolved in 0.01 M NaOH and measured by UV-Visible spectrophotometer at ¼ 303 nm [25].
Cytotoxicity of blank carriers and MTX nanoparticles The MCF-7 cells were maintained at 37 C in DMEM medium supplemented with glutamine (0.29 mg/mL), 1% bovine fetal serum and 10 000 U/mL of penicillin/streptomycin, in an atmosphere of 5% CO2 with 90% relative humidity. To detect the cytotoxicity of blank carriers and MTX nanoparticles, the cells were seeded at a density of 2 104 cells/well in 96-well plates. After 24 h, cells were incubated with fresh media containing a series of concentrations of PAMAM, PPG, PPO, MTX, PPG/MTX and PPO/MTX. The cells were cultured for 48 h and wash three times with PBS. About 20 mL MTT solution (5 mg/ mL in PBS) was added to each well and cells was incubated for 4 h. The medium was removed and 100 mL DMSO was added into the wells. Then, the plate was agitated for 10 min and the optical ratio was measured at 570 nm via Enzyme Immunoassay Instrument (ELx800, BioTek Instruments, Winooski, VT). Cell viability was calculated by the following formula: Cell viability ð%Þ ¼
absorbance of sample group 100% absorbance of control group
Cellular uptake of FITC-labeled PAMAM dendrimers The MCF-7 cells were seeded at a density of 1 105 cells/ well in 6-well plates. After 24 h, cells were incubated with FITC, PPG-FITC and PPO-FITC (final concentration of 15 mM for both PPG-FITC and PPO-FITC) in the presence of serum free medium for 4 h at 37 C. For the competitive binding study, cells were pre-incubated with octreotide (50 mM) for 30 min before PPO-FITC were added. After removing the supernatant and washing the well three times with pH 7.4 PBS buffer, the cells were trypsinized using a standard protocol, and resuspended in 0.4 mL DMEM. The cellular uptake of the samples were examined by flow cytometry using a fluorescence-activated cell sorting (FACS) Caliber flow cytometer (Becton Dickinson, Franklin Lakes, NJ), while the samples were visualized and photographed under a fluorescent microscope (Olympus IX 51, Osaka, Japan).
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Pharmacokinetic studies of MTX nanoparticles
Statistical analysis
To determine the pharmacokinetic profiles of the MTX nanoparticles, male Sprague-Dawley rats were randomized into three groups and treated with free MTX, PPG/MTX and PPO/MTX nanoparticles, respectively. All the preparations were administrated intravenously into the tail vein of rats as a dose of MTX 5 mg/kg, and blood samples were taken with a heparinized syringe at 5, 15, 30, 60, 120, 180 and 240 min. The blood samples were centrifuged at 1500g for 10 min at 4 C. About 20 mL folic acid (75 mg/mL) as internal standard was added the blood samples (300 mL). The mixtures were vortexed for 20 s, and 0.6 mL methanol was added. Then the contents were vortexed for 90 s and centrifuged at 4000g for 12 min. About 20 mL of supernatant was analyzed by HPLC at ¼ 303 nm. The plasma concentrations versus time data were analyzed by WinNonlin 5.2.1 (Pharsight Corporation, Sunnyvale, CA).
The data were presented as mean ± standard deviations (SD) of at least three independent experiments. Significant differences were performed by one-way analysis of variance (ANOVA) and followed by Bonferroni’s post hoc test. p value 0.05 was considered to be statistically significant.
Results Synthesis of modified PAMAM dendrimers The PAMAM-based carriers-PPG and PPO were synthesized and confirmed by 1H-NMR spectrum (Figure 2). Both of the spectra contain the characteristic peaks of PAMAM, which were d ¼ 2.35–2.45 ppm (br, Hc), d ¼ 2.55–2.65 ppm (br, He), d ¼ 2.75–2.85 ppm (br, Hd), d ¼ 2.64 ppm (br, Hd) and d ¼ 3.25–3.40 ppm (m, Ha, Hb, Hf). For the synthesis of PPG, the MAL-PEG-NHS was conjugated to PAMAM with MAL group. Thus, the signals of PEG in the spectrum of PPG
Figure 2. 1H-NMR spectra for PPG (A) and PPO (B) in D2O.
Octreotide-conjugated PAMAM
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DOI: 10.3109/1061186X.2013.879386
were d ¼ 2.52 ppm (t, NHS) and d ¼ 3.50–3.80 ppm (m, CH2CH2O). The average number of PEG chains attached to the PAMAM measured by the specific signals was 4.34. However, the NHS group of PEG connected to octreotide disappeared in the spectrum of PPO. As excessive octreotide was added to ensure complete reaction in the preparation of MAL–PEG–octreotide, the characteristic signals of octreotide was used to detect the number of MAL–PEG–octreotide conjugated to PAMAM. The specific signals of benzene on octreotide was d ¼ 7.05–7.40 ppm (m, ArH). Hence, the average number of MAL–PEG–octreotide on PPO was 4.64. The characteristic peaks of FITC in 1H-NMR spectrum was at d ¼ 6.44 and 7.05 ppm which overlapped with the signals of octreotide. For this reason, the number of FITC molecules conjugated to the surface of PAMAM was determined by the UV-Visible spectrometer (Figure 3). The spectrum showed that the characteristic peak of FITC (501 nm) on PPO-FITC and PPG-FITC shifted 9 nm compared with free FITC (492 nm), which consistent with
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previous study [22]. Since PPG and PPO give no absorbance after 400 nm, the absorbance value of PPG-FITC and PPOFITC were proportional to the number of conjugated FITC at ¼ 492 nm. Hence, a calibration curve was established by free FITC, and the number of FITC conjugated to each PPG and PPO molecule were 3.33 and 3.11, respectively. Characterization and safety evaluation in vitro The particle size, PDI and zeta potential of modified PAMAM dendrimers are listed in Table 1. The conjugation of PEG and MAL–PEG–octreotide to the surface of PAMAM resulted in increasing of diameters. Meanwhile, the zeta potential of modified PAMAM carriers were significantly decreased compared with PAMAM, due to the presence of PEG chains on the surface that masked the positive charge of PAMAM [26]. Although the absolute value of zeta potential of nanoparticles below 10 mV was liable to agglomerate in the dispersion [27], the conjugated PEG chains could provide additional steric hindrance for sufficient electrostatic stabilization of vectors. The TEM morphology of PAMAM and PPO were showed in the Figure 4. It could be observed that the PAMAM appeared as tiny particles with near-spherical shapes, while the PPO with long PEG chains on the surface exhibited regular spherical shapes. Both of the carriers were loosely associated. The safety of PAMAM dendrimers was evaluated by the hemolytic activity (Figure 5). PAMAM with high cationic Table 1. Characterizations of synthesized PAMAM dendrimers. Preparations PAMAM PPG PPO PPG-FITC PPO-FITC
Figure 3. UV-Visible spectra of FITC-labeled PPG and PPO carriers. The arrows at 492 and 501 nm indicate the presence of FITC.
Diameter (nm)
PDI
Zeta potential (mV)
5.77 ± 0.25 9.52 ± 1.27 11.05 ± 1.80 15.18 ± 2.01 17.02 ± 2.16
0.23 ± 0.02 0.31 ± 0.04 0.34 ± 0.04 0.43 ± 0.11 0.39 ± 0.10
15.47 ± 1.80 4.19 ± 1.00 2.59 ± 1.53 2.60 ± 0.50 2.35 ± 1.04
Data are represented as mean ± SD (n ¼ 3).
Figure 4. The TEM of PAMAM (A) and PPO (B).
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Figure 5. The hemolytic toxicity of the synthesized PAMAM dendrimers at different concentrations. Data are represented as mean ± SD (n ¼ 3).
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Figure 6. UV-visible spectra of MTX, blank carriers and MTX nanoparticles. The arrows at 258, 303 and 373 nm represent the characteristic peaks of MTX.
charge induced 430% hemolysis at 20 mM, which was too toxic to be employed as carrier. However, all the modified dendrimers with PEG on the surface showed510% hemolysis even at 20 mM, which was in line with the previous studies that PEGylation would significantly decreased the hemolytic toxicity of PAMAM [7]. Preparation of MTX nanoparticles The UV-Visible spectra of MTX, PAMAM, PPG, PPO, PPG/ MTX and PPO/MTX were detected at first (Figure 6). The peak of MTX at 258 nm was much weaker and the peak at 373 nm moves to 350 nm with decreased intensity in the spectra of PPG/MTX and PPO/MTX. According to the previous studies, MTX molecules were supposed to be encapsulated into the interior region of carrier or combined on the surface with non-covalent bonds including: hydrogen bonds, electrostatic and hydrophobic interactions [28], and previous study showed that the physical interactions between PAMAM and curcumin induced changes on the UV spectra [29]. Therefore, the changes of the peaks might be attributed to the interactions between MTX and carriers. However, the peak of MTX at 303 nm was invariant for MTX nanoparticles. Hence, the amount of MTX encapsulated by PPG and PPO was determined by UV-Visible spectrometer at 303 nm, utilizing the calibration curve of free MTX. The influence of time on the encapsulation of MTX by PPG and PPO has been studied previously, which showed that the encapsulated MTX reached the maximum amount after 3 h stirring (Supplementary Figure 2). Thus, the preparation of MTX nanoparticles took 3 h mix. As shown in Figure 7, the number of MTX encapsulated by carriers increased with the increasing MTX/PPG (or PPO) molar ratio from 20:1 to 60:1. However, the number of encapsulated MTX was almost invariant when the ratio was above 40:1. The maximum amount of MTX encapsulated by PPG and PPO was 33 and 30 (molecule/molecule), respectively, and the MTX nanoparticles prepared at molar ratio of 60:1 was used in the cytotoxicity study.
Figure 7. Encapsulation of MTX by PPG and PPO at different molar ratios. Data are represented as mean ± SD (n ¼ 3).
In vitro release of MTX The in vitro release profiles were depicted in Figure 8. Free MTX dissolved in the four media was capable to release completely within 2 h (data not shown). For both of PPG/MTX and PPO/MTX nanoparticles, the drug release profiles exhibited the same characteristics. Firstly, MTX was almost completely released to the outer phase within 8 h. Moreover, the release profiles showed conspicuous distinctions in different media. The order of drug release of MTX nanoparticles in four media was pH 7.4 (containing 0.2 M NaCl)4pH 5.44pH 6.5 & pH 7.4., and PPO/MTX nanoparticles exhibited faster release at the initial stage. Cytotoxicity on MCF-7 cells The cytotoxicity of PAMAM, PPG and PPO against MCF-7 tumor cells was measured using MTT assay. It was shown that the toxic activity was proportional to the concentration of carriers, and PEG conjugated PAMAM (PPG and PPO) showed obviously lower cytotoxicity than PAMAM
Octreotide-conjugated PAMAM
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Figure 8. The release profiles of MTX from PPG/MTX (A) and PPO/MTX (B) in PBS buffer with various pH values and different ionic concentrations. Data are represented as mean ± SD (n ¼ 3).
Figure 9 (A) The cytotoxicity profiles of blank carriers in MCF-7 cells at different concentrations measured by MTT assays. Data are represented as mean ± SD (n ¼ 4). (B) The cell viability profiles of free MTX and MTX nanoparticles in MCF-7 cells at different concentrations of MTX. Data are represented as mean ± SD (n ¼ 4). ** and * indicate p50.001 and 0.05 versus free MTX; ## and # indicate p50.001 and 0.01 versus PPG/MTX.
(Figure 9A). The cell viability of modified PAMAM dendrimers was 460% at 20 mM. Hence, the concentration of PPG and PPO in the MTX nanoparticles used in the following experiments was 520 mM. Then, the cytotoxicity of free MTX and MTX nanoparticles was detected against the MCF-7 cells to evaluate the antitumor activity of MTX nanoparticles in vitro (Figure 9B). The cyotoxicity of free MTX showed extremely low activity against MCF-7 cells even at 500 mM. However, both of the PPG/MTX and PPO/MTX nanoparticles exhibited remarkably higher cytotoxicity than free MTX. Moreover, it was noteworthy that the cytotoxicity of PPO/MTX nanoparticles were significantly higher than PPG/MTX nanoparticles, especially at highest concentration (p50.001), indicating that the conjugation of octreotide enhanced the cytotoxicity of MTX nanoparticles.
Cellular uptake study The cellular uptake of PPO-FITC and PPG-FITC was visualized by florescence microscope (Figure 10). After incubated with MCF-7 cells for 4 h, free FITC showed basically no fluorescent intensity in cells, while fluorescent intensity of PPO-FITC was superior to that of PPG-FITC. To testify the specificity of the uptake approach, octreotide at a concentration of 50 mM was used to combine with the SSTRs on MCF-7 cells. As shown in Figure 10(C and D), the pre-incubation of octreotide significantly decreased the fluorescent intensity of PPO-FITC. Furthermore, quantification for uptake of above preparations by MCF-7 cells was conducted by flow cytometry analysis. The result was consistent with the observation under florescence microscope that PPO-FITC showed the highest uptake in MCF-7 cells.
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Figure 10. The fluorescent microscopy images of MCF-7 cells after incubation with FITC (A), PPG-FITC (B), PPO-FITC (C) and PPO-FITC with octreotide pre-incubation (D). Evaluation of the uptake of the four preparations above determined by flow cytometry analysis (E).
Pharmacokinetics studies The pharmacokinetic studies on free MTX and MTX nanoparticles were carried out to evaluate the differences on metabolism process of MTX in vivo. After intravenous administration, all the preparations were eliminated very quickly (Figure 11). The free MTX eliminated much faster than MTX nanoparticles, while no evident distinction was found between PPG/MTX and PPO/MTX plasma concentration–time curves. Moreover, the major pharmacokinetics parameters of the preparations were listed in Table 2. A significant increase on mean residence time (MRT) of MTX nanoparticles were observed, confirming the prolonged retention of MTX in circulation. And the bioavailability of MTX was notably increased after prepared as nanoparticles, which indicated by the area under the concentration–time curve (AUC). Based on one-way ANOVA, there were obvious differences in MRT and AUC between MTX and MTX nanoparticles. However, no conspicuous distinctions have been found between PPG/MTX and PPO/MTX.
Figure 11. Concentration–time curves of MTX preparations in rat plasma after i.v. administration. Data are represented as mean ± SD (n ¼ 3). ** and * indicate p50.01 and 0.05 versus free MTX.
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Table 2. The main pharmacokinetic parameters of MTX preparations after i.v. administration. Parameters
Free MTX
PPG/MTX
PPO/MTX
MRT (h) AUC0!1 (mg h/L) CL (L/h kg) Vss (L/kg)
0.60 ± 0.05 4.40 ± 0.38 1.14 ± 0.10 0.69 ± 0.10
0.92 ± 0.03** 6.42 ± 0.43* 0.78 ± 0.05 0.72 ± 0.07
0.82 ± 0.06* 6.43 ± 0.45* 0.78 ± 0.05 0.64 ± 0.09
Data are represented as mean ± SD (n ¼ 3). ** and * indicate p50.001 and 0.01 versus free MTX.
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Discussion In this study, octreotide-modified PAMAM dendrimer was successfully synthesized and employed as a carrier for antitumor drug MTX to prepare receptor-mediate targeted drug delivery nanoparticles. The PPO-FITC carrier could be effectively internalized into the tumor cells and MTX nanoparticles increased the inhibition activity of MTX impressively due to the receptor-mediated endocytosis. The ideal size of nanoparticles should range from 10 to 100 nm, in order to efficiently extravasate from the fenestrations of leaky vasculature and avoid the filtration by the kidneys and the specific capture by the liver [30,31]. The size of PAMAM carriers was within the ideal range of nanoparticles. The zeta potential of modified PAMAM dendrimers were decreased to 5 mV below, which increasingly decreased the chance of renal elimination in comparison with PAMAM. Although low zeta potential increased the risk of agglomeration of nanoparticles, the conjugated PEG chains could provide steric stabilization for the PEGylated PAMAM dendrimer. It has been reported that the encapsulation efficiency of MTX by PAMAM was enhanced after PEGylation. In accordance with previous study, the maximum amount of encapsulated MTX molecules was 430 per PEGylated PAMAM dendrimer molecule, which was higher than the maximum encapsulation efficiency of PAMAM [7]. The conjugated PEG was supposed to enlarge the interior region of PAMAM, which was capable to accommodate more drug molecules in the ‘‘extended space’’. Therefore, the MTX in the nanoparticles might be encapsulated in the interior region of PAMAM, interact with the periphery groups of PAMAM and incorporated in the ‘‘extended space’’ with electrostatic interactions, hydrophobic and hydrogen bonds [25,28,32]. Besides, the maximum amount of encapsulated MTX by PPO was slightly less than PPG possibly owing to the block of octreotide on outside of PPO vector. From the release behavior of MTX nanoparticles in different media, both of pH value and ionic concentration influence the interaction between MTX and carriers. Previous work demonstrated that PAMAM dendrimer go through a pH induced conformation change from a ‘‘dense core’’ (high pH) to a ‘‘dense shell’’ (low pH) [33], which is a critical character for development of a pH-sensitive DDS. However, the pH responsive characteristics of MTX nanoparticles are not conspicuous compared with the release profiles of PAMAM– PEG–T7/DOX nanoparticles [34], suggesting that most of MTX was combined on the peripheral ends of PAMAM but not in the interior region. Moreover, the ionic
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concentration-sensitive release behavior further confirmed that the major forces between MTX and PAMAM were electrostatic interactions. In consistent with the results of hemolytic activity detection, the cytotoxicity of PAMAM against MCF-7 cells was significantly decreased after PEG conjugation. The increased safety of carriers was attributed to the shielded cationic charges on the surface of PAMAM which interacted with the cell membrane. Within safety concentration of carriers, both of MTX nanoparticles increased the cytotoxicity of MTX, especially the PPO/MTX nanoparticles, suggesting that PPO/MTX nanoparticles had higher affinity to MCF-7 cells and the carriers promoted the endocytosis of MTX. Furthermore, the cellular uptake study proved that octreotide strongly facilitated the uptake of PPO-FITC by MCF-7 cells, and the specificity of the uptake process was demonstrated by the competition experiment. Thus, the increased antitumor activity of MTX should be attributed to receptor-mediated endocytosis of PPO/MTX nanoparticles. Similar results were also observed in other researches that octreotide modification were effective in MCF-7 cells inhibition [35–37]. Whereas, the quick release of MTX limits the total amount of drug uptake by cells, thus, the antitumor activity of MTX nanoparticles is less than our expect. Besides, pharmacokinetics studies show that the MRT and bioavailability of MTX nanoparticles are conspicuous increased compared with free MTX. It’s reasonable to infer that the hydrophilic PEG chains modification on the PAMAM prevents the phagocytosis of nanoparticles by RES, which sustains the circulation time of MTX in vivo.
Conclusions In this present work, PAMAM–PEG–octreotide was successfully synthesized and employed as nanocarrier for MTX. The model drug, MTX, could be easily encapsulated by this novel nanocarrier with electrostatic interactions, hydrophobic and hydrogen bonds. The PPO/MTX nanoparticles showed improved antitumor activity against MCF-7 cells compared with free MTX and the uptake of FITC-PPO demonstrated that octreotide-modified carrier enhanced targeting to MCF-7 cells via receptor-mediated endocytosis. Hence, it suggested that PAMAM–PEG–octreotide can be used as potential carrier for SSTR over-expressed tumors targeting DDS. Further study is still in progress.
Declaration of interest The authors have no conflicts of interest. This work was supported by the Major State Basic Research Development Program of the National Science and Technology of China for new drugs development (Program No. 2012CB724002), Natural Science Foundation of Jiangsu Province (BK20130663 and BK2011771) and Key project of Nanjing Medical University of Science and Technology Development Fund (2010 NJMUZ25).
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