Article pubs.acs.org/bc
Hydroxycamptothecin Nanorods Prepared by Fluorescently Labeled Oligoethylene Glycols (OEG) Codendrimer: Antitumor Efficacy in Vitro and in Vivo Yifei Guo,† Yanna Zhao,† Ting Wang, Ran Li, Meihua Han, Zhengqi Dong, Chunyan Zhu,* and Xiangtao Wang* Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 151, Malianwa North Road, Haidian District, Beijing 100193, China S Supporting Information *
ABSTRACT: Nanorods based on dendrimers were explored as excellent candidates for nanoscale drug delivery system. In this study, fluorescently labeled PAMAM-b-oligoethylene glycols codendrimer (POC) was utilized as carrier to prepare 10-hydroxycamptothecin (HCPT) loaded nanorods (HCPT NRs) via antisolvent precipitation method augmented by ultrasonication with the optimized drug-loading content (∼90.6%) and positive charged surface. The nanorods presented high stability, and the release of HCPT nanorods showed a sustained release manner and was completed within 48 h. The nanorods presented higher cytotoxicity against HepG2 and 4T1 cells than HCPT injections, and the cellular uptake mechanism was proved to involve clathrin-mediated endocytosis and macropincytosis-dependent endobytosis. Importantly, the HCPT nanorods resulted in strong antitumor efficacy on the H22 liver tumor model, and no significant adverse effects was observed. Besides, in vivo studies also showed that HCPT NRs possessed better tumor accumulation over HCPT injection at the equivalent concentration. According to the high drug-loading content, enhanced antitumor efficacy, and appropriate particle size, HCPT NRs as the safe and efficient drug delivery systems could have potential application for cancer chemotherapy in clinic.
1. INTRODUCTION Cancer is one of the most common causes of death in the world, and chemotherapy is still a commonly used strategy for cancer treatment. 10-Hydroxycamptothecin (HCPT) exerts effective antitumor activity in a wide range of cancers;1−4 however, the therapeutic potential has been inhibited due to the poor solubility of the lactone form, resulting in low antitumor efficiency.5,6 To overcome these drawbacks, tremendous efforts have been made to design and employ the nanoscale drug delivery systems to maintain the stability of the active lactone, enhance their accumulation in tumor tissue, and optimize the antitumor activity,7 including micelles,8,9 liposomes,10,11 nanoparticles,12,13 nanosuspensions,14,15 and polymeric conjugates.16−18 These nanoscale drug delivery systems present relatively small particles (mean diameter 0.05) in comparison
Figure 3. Cumulative HCPT release from HCPT injection, nanorods, and bulk powder in PBS solution (pH 7.4) at 37 °C within 48 h (n = 3).
that HCPT diffused completely through the dialysis membrane within 4 h due to the carboxylate form of HCPT in injection that was water-soluble. Meanwhile, HCPT powder exhibited a slow release property; only 40% HCPT was released at the end of the experiments. For HCPT-loaded nanorods, the sustained release for 48 h was detected, approximately 50% HCPT was released from HCPT NRs within the initial 12 h, and then 40% HCPT was released during the following 36 h. The significantly different release properties between nanorods and injection might be explained by the structure of HCPT NRs; the carrier POC extended in the periphery of HCPT NRs and formed the hydrophilic shell to hinder the diffusion of HCPT from inner core to outside dialysis medium.40 Compared with HCPT powder, the enhanced release rate of HCPT from nanorods can be attributed to the increased surface area of the nanoscale particles and enhanced solubility, which may further improve the bioavailability of HCPT. 2.5. In Vitro Cytotoxicity. The hemolytic activity of HCPT NRs on rat RBC was researched to study its toxicity to red blood cells (Supporting Information, Figure S8). After incubation with the 2% (w/v) RBC suspension at 37 °C for 5 h, the hemolysis rates were below 6% in concentration
Figure 2. Particle size and SEM images of the storage stability (a, b) at 4 °C after 14 days and plasma stability (c, d) at 37 °C after 24 h. Scale bar: 1 μm.
with their original size, however, the particle size increased at 14th day probably, which could be explained by Ostwald ripening phenomenon.38 Meanwhile, the morphology of HCPT NRs did not present significant change (Figure 2b). The storage stability of HCPT NRs was also detected at 25 °C, and the similar results are observed (Supporting Information, Figure S4). The particle size change of HCPT NRs in plasma at 37 °C was investigated furthermore to evaluate their stability in the physiological environment. The mean hydrodynamic diameter (Figure 2c) and morphology (Figure 2d) of nanorods showed no significant change over 24 h when compared to initial size (p > 0.05), suggesting the high stability in vivo (Supporting Information Figure S5). After lyophilization directly without any protector, the rodlike morphology and the particle size of HCPT NRs were C
DOI: 10.1021/acs.bioconjchem.6b00536 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Bioconjugate Chemistry ranging from 0.01 to 3 mg mL−1 (HCPT equivalent concentration), suggesting no RBC membrane related toxicity. To investigate in vitro antitumor efficacy of HCPT NRs, the viability of two different cancer cell lines including 4T1 cells (mouse breast cancer cells) and HepG2 cells (human liver hepatocellular carcinoma cells) was measured with MTT assay (Figure 4) and the half maximal inhibitory concentrations
Figure 4. Cytotoxicities of HCPT NRs toward 4T1 cells (a) and HepG2 cells (b) after incubation for 48 h (n = 5).
(IC50) are determined from the concentration-dependent cell inhibition curves. Codendrimer POC showed no significant cytotoxicity toward both cell lines. Compared with HCPT injections, HCPT NRs presented a significantly enhanced cytotoxicity against both cancer cell lines in a concentrationdependent manner after incubation for 48 h. IC50 values were 0.69 and 2.80 μg mL−1 against 4T1 cells for nanorods and HCPT injections, respectively (Figure 4a); nanorods showed approximately 3-fold enhanced cytotoxicity (p < 0.01). The similar results were obtained against HepG2 cells; nanorods exhibited about 1-fold higher cytotoxicity than HCPT injections (IC50 8.74 μg mL−1 vs 16.72 μg mL−1, p < 0.05, Figure 4b). The enhanced cytotoxicity of nanorods was generally due to the nonspecifically internalized into cells via endocytosis, phagocytosis, or pinocytosis after accumulating on the surface of the cells, while free drugs were transported by passive diffusion. Besides, it had been reported that positively charged particles were selectively accumulated in angiogenic endothelial cells of tumor.12,41,42 2.6. Cellular Uptake. Cellular uptake of HCPT NRs and HCPT injection (mixed with cy5.5) were characterized against HepG2 cell lines using fluorescent microcopy imaging system at the equivalent cy5.5 and HCPT concentration. After repeated washing, the HCPT injection treated cells showed weak HCPT and cy5.5 fluorescence signals in HepG2 cells (Figure 5, injection). On the contrary, HCPT NRs treated cells exhibited greater fluorescence signals (Figure 5, nanorods), indicating that the HCPT NRs were taken up well by cancer cells. Furthermore, it could be seen that more intense fluorescence could be detected from the HepG2 cells with prolonged the incubation time from 0.5 to 6 h (Supporting Information, Figure S9). These results demonstrated that nanorods could be preferentially internalized via endocytosis pathway, while free HCPT was transported into cells by passive diffusion. To verify the endocytosis pathway taking part in the internalization process, HepG2 cells were incubated with various endocytosis inhibitors of caveolae-mediated endocytosis, clathrin-mediated endocytosis, and macropincytosisdependent endocytosis (Figure 5).43 After incubating for 3 h, methyl-β-cyclodextrin (MβCD, inhibitor of caveolae-mediated endocytosis) resulted in no significant decrease in the
Figure 5. Representative fluorescent microscopy images of HCPT injection and HCPT NRs incubated with HepG2 cells for 3 h. Inhibitors: methyl-β-cyclodextrin (MβCD), sucrose, cytochalasin D (CCD). All images were taken under identical instrument conditions. Blue: HCPT. Red: cy5.5.
intracellular fluorescence intensity of HCPT NRs. As the inhibitor of clathrin-mediated endocytosis, the sucrose caused dramatic decrease in the intracellular fluorescence intensity (approximately 49% decrease compared to the control). Meanwhile, the cytochalasin D (CCD)-blocking macropincytosis-dependent endocytosis pathway showed a significant effect on the uptake of HCPT NRs, exhibiting 43% decrease. These results suggested that clathrin-mediated endocytosis and macropincytosis-dependent endocytosis were the main uptake mechanism that contributed to the effective uptake of nanorods. 2.7. In Vivo Antitumor Effect of HCPT NRs. Motivated by the high in vitro anticancer effect and cellular uptake efficacy, the in vivo antitumor activitites of HCPT NRs were further investigated by using ICR mice bearing H22 murine liver tumor model via intravenous administration with the normal saline and HCPT injection as control. The concentrations of HCPT NRs were normalized to be 1.2, 2.5, and 5.0 mg kg−1 (equivalent HCPT concentration); the HCPT injection was 5.0 mg kg1−. The mice were administrated every 2 days for 4 times, and the tumor volumes and body weights were monitored daily for 7 days. As shown in Figure 6a, the time-related tumor volume increase was observed. Compared to saline control group, HCPT injection group only presented moderate antitumor efficacy, in contrast, all nanorods groups presented better antitumor efficacy than either HCPT injection group or saline group (p < 0.001). The tumor inhibition rates calculated based on the average weight of the tumors further indicated the good tumor inhibition activity of nanorods. The HCPT injection group resulted in tumor weight of 1.43 ± 0.04 g (Figure 6c), with the inhibition rate of 45.8% (Figure 6d). Meanwhile, the three concentrations of nanoparticle groups resulted in tumor weight of 1.03 ± 0.01, 0.47 ± D
DOI: 10.1021/acs.bioconjchem.6b00536 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Figure 7. Biodistribution of HCPT NRs and injection in ICR mice bearing H22 murine liver tumor model (n = 10) at 4 h (a) and 24 h (b) determined by HPLC: (#) p < 0.001 vs HCPT injection.
concentration of HCPT NRs in the tumor was enhanced by 7.3-fold at 4 h (p < 0.001) and 15.1-fold at 24 h (p < 0.001). The real-time biodistributions of HCPT injection and HCPT NRs in vivo are shown in Figure S10 (Supporting Information). The mean drug level profiles in H22 solid tumors over 24 h for injections and nanorods are shown in Figure S11 (Supporting Information). HCPT NRs displayed enhanced tumor drug concentration throughout the experiment with 3.8-fold Cmax and 4.7-fold AUC. These results indicated HCPT NRs could be accumulated in tumor tissue for a long time, generating the high antitumor efficacy. The fluorescence imaging study was carried out to further confirm the potential drug accumulation in tumor. Dir was utilized as fluorescence probe and encapsulated in HCPT NRs. The fluorescence images of mice bearing 4T1 tumor at different time after administration are shown in Figure S12 (Supporting Information), and the results are compiled in Figure 8. As shown in Figure 8a, the in vivo fluorescence signals in tumor tissue were monitored during the whole procedure, and the fluorescence intensity of nanorods group was strong and enhanced significantly; however, extremely weak signal was found in tumors from the mice treated with HCPT injection and the intensity was enhanced slightly. These results showed that the biodistribution of nanorods in tumor tissue was improved obviously. All mice were sacrificed at 24 h after intravenous injection. The tumors and main organs were removed and fluorescently imaged (Figure 8b). Compared to those in injection group, the mice treated with nanorods groups showed strong fluorescence signals in tumors (p < 0.001). Besides, the signals in hearts, lung, and kidney were strengthened. The average signals further confirmed in the ex vivo images as shown in Figure 8c. The signal intensity of nanorods in tumor tissue was approximately 8-fold that of HCPT injection. Meanwhile, for the mice in nanorods groups, strong signals were detected in livers and spleens. These results suggested that nanorods were mainly distributed in the tumor tissue as well as reticuloendothelial system (RES) organs such as liver, spleen, and kidney, which was consistent with the studies of biodistribution in tumor-bearing mice. All these results suggested that HCPT NRs could prefer to accumulate in tumor tissue, which could be attributed to the EPR effect in cancerous tumors with tortuous and leaky vasculatures. Besides, it had been reported that positively charged nanorods were selectively accumulated in angiogenic endothelial cells of tumor; the ζ potential of HCPT NRs was 24.2 ± 0.7 mV, which may explain the high distribution of nanorods in tumor further.44,45
Figure 6. Comparison of the in vivo tumor growth inhibition of HCPT NRs versus free drug HCPT (injection) and saline in the liver tumor model: tumor volume change of H22 bearing ICR mice (a), body weight change (b), tumor weight (c), tumor inhibition rate (d). For each animal, four consecutive doses were given (marked by arrows). Data represent the mean ± SD (n = 10): (∗) p < 0.001 vs saline control group, (#) p < 0.001 vs HCPT injection.
0.05, and 0.26 ± 0.03 g, respectively (Figure 6c), with the corresponding inhibition rate of 59.2%, 79.6%, and 88.7% (Figure 6d). Compared with HCPT injection groups, all three nanorods groups showed advanced antitumor efficacy (p < 0.001 for all). At the same concentration of 5 mg kg−1, the tumor inhibition rate of nanorods group was approximately 2-fold as that for HCPT injections group. Even at the concentration of 1.2 mg kg−1, nanorods led to better inhibition rate than HCPT injection (59.2% vs 45.8%). From either tumor growth curves or the final weight of the tumor, the HCPT NRs exhibited great antitumor efficacy, which was in good agreement with these results from in vitro antitumor efficacy and cellular uptake efficacy. These in vivo antitumor results suggested that the HCPT NRs based on codendrimer POC could improve antitumor efficacy significantly. Simultaneously, the changes in the body weight of mice in the whole experiment were carried out to investigate the adverse effects of the nanorods. As seen in Figure 6b, comparatively the average body weight of HCPT injections was lower; the three HCPT nanorods groups showed the similar body weight increase profile to that for normal saline group (p > 0.05), suggesting a very low level potential of systemic toxicity. In vivo studies showed that HCPT NRs possessed obviously superior antitumor efficacy than HCPT injection at the equivalent concentration, and no significant weight loss was observed. 2.8. Biodistribution in Tumor-Bearing Mice. The concentration and accumulation of HCPT in the tumor, plasma, and major organs (heart, liver, spleen, lung, kidney, and brain) were examined after administration of the HCPT injection and HCPT NRs to H22-bearing mice via tail vein at a comparable concentration of 5 mg kg−1. At 4 and 24 h after administration, HCPT nanorods led to higher drug concentration than HCPT injections in all tissues (Figure 7). HCPT NRs were mainly distributed in the tumor tissue as well as reticuloendothelial system (RES) organs such as liver, spleen, and kidney. Compared to that of HCPT injections, the drug E
DOI: 10.1021/acs.bioconjchem.6b00536 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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Figure 8. In vivo and ex vivo fluorescence images. The mice bearing 4T1 tumors were injected one time with HCPT injection and nanorods: in vivo fluorescent intensity of tumor at 0.5, 1, 3, 6, 9, 12, and 24 h after administration (a). Ex vivo fluorescence images of main tissues (up: injection group, down: nanorods group); all the organs and tumors were placed in the order of (heart, liver, spleen, lung, kidney, brain, and tumor) at 24 h after administration (b). Intensity of ex vivo fluorescent signal in organs and tumor at 24 h after administration (c): (#) p < 0.001 vs HCPT injection.
3. CONCLUSIONS In this study, fluorescently labeled codendrimer POC from PAMAM G4.0 decorated with oligoethylene glycol (OEG) dendrons was utilized to prepare hydroxycamptothecine nanorods (HCPT NRs) via antisolvent precipitation method augmented by ultrasonication, with the mean diameter of approximately 168.6 nm and a very narrow size distribution, and the drug-loading content was 90.6%. The FALT and surface element analysis results suggested the codendrimer POC was dispersed on the surface of HCPT NRs. Besides, HCPT NRs showed high stability and sustained release for 48 h in vitro. The HCPT nanorods presented obviously enhanced internalization rates compared to HCPT injection, and the cellular uptake mechanism was clathrin-mediated endocytosis and macropincytosis-dependent endocytosis. In vivo studies also showed that HCPT NRs possessed superior antitumor efficacy and better tumor accumulation over HCPT injection at the equivalent concentration, and no statistically significant weight loss was observed. These results suggested the potential advantages of HCPT nanorods as safe and efficient drug delivery system for anticancer therapy in clinic.
reagents and solvents were purchased at reagent grade and used without further purification. 4.2. Animals and Cell Line. Male ICR mice (20 ± 2 g), male nu/nu nude mice (14−16 g), and male Sprague-Dawley rats (200 ± 20 g) were supplied by Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All the animals were acclimatized at a temperature of 25 ± 2 °C and a relative humidity of 70 ± 5% under standard 12/12 light−dark circadian cycle condition with free access to food and water. All experimental procedures comply with the Guidelines and Policies for Ethical and Regulatory for Animal Experiments as approved by the Animal Ethics Committee of Peking Union Medical College (Beijing, China). The murine hepatocarcinoma 22 (H22) cell line, the human hepatoma (HepG2) cell line, and the mouse breast cancer (4T1) cell line were purchased from the Institute of Basic Medical Science, Chinese Academy of Medical Science (Beijing, China). 4.3. Synthesis of Codendrimer POC. Codendrimer POT. The solution of tetrahydropyranyl protected oligoethylene glycol dendron pentafluorophenol active ester (THP-OEG-Pfp, 1.00 g, 0.32 mmol) in dimethylformamide (DMF, 20 mL) was added into a solution of polyamidoamine G4.0 (PAMAM-NH2, 0.23 mg, 16.00 μmol), triethylamine (TEA, 10 mg, 0.96 mmol), and N,N-dimethylaminopyridine (DMAP, 40 mg) in mixed solvent DMF/H2O (30 mL, 1/1, v/v) at −5 °C with stirring, and the reaction temperature was allowed to rise to room temperature. After stirring for 36 h, the solvents were evaporated in vacuo, and the residue was purified by column chromatography with dichloromethane (DCM) as eluent, affording THP protected codendrimer POT as a colorless oil (0.93 g, 90%). 1H NMR (600 MHz, DMSO-d6): δ = 1.31−1.48 (br, CH2), 1.52−1.73 (br, CH2), 2.14−2.26 (br, CH2), 2.35− 2.46 (br, CH2), 2.61−2.89 (br, CH2), 3.11−3.28 (br, CH2),
4. MATERIALS AND METHODS 4.1. Materials. Tetrahydropyranyl protected oligoethylene glycol dendron active ester (THP-OEG-Pfp) was synthesized according to the previous paper.46,47 Hydroxycamptothecin (HCPT, purity >98%) was purchased from Beijing Ouhe BioTech Co., Ltd. (Beijing, China). HCPT injection (used as the sodium carboxylate in China) was obtained from Shenghe Pharmaceutical Ltd. (Sichuan, China). Acetonitrile (HPLC grade) was purchased from Fisher Scientific (Pittsburgh, PA, USA). Dimethylsulfoxide (DMSO, ACS grade) was purchased from Sigma-Aldrich Chemicals, Germany. Dialysis membrane (MWCO 14 000) was purchased from Spectrum. Other F
DOI: 10.1021/acs.bioconjchem.6b00536 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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4.6. Particle Size and ζ Potential Measurements. Dynamic light scattering (DLS) analysis was utilized to measure the particle size, size distribution, and ζ potential of the HCPT NRs (Zetasizer Nano-ZS analyzer, Malvern Instruments, U.K.), which used the integrated 4 mV He−Ne laser (λ = 633 nm) and the backscattering detection (scattering angle θ = 173°) at room temperature. Experiments were performed in triplicate, and the data were shown as the mean values plus standard deviation (±SD). 4.7. Transmission Electron Microscope. Transmission electron microscope (TEM) measurements were performed on a JEM-1400 (JEOL, Japan) by the negative dyeing method, operating at an accelerating voltage of 80 kV. A drop of HCPT NRs (0.2 mg mL−1) was placed on a carbon-coated copper grid. After 2 min, the grid was drained by filter paper to remove the aqueous solution, followed by air-drying at room temperature and then dyeing with uranyl acetate solution (2%, w/v) for 2 min. 4.8. Scanning Electron Microscope and Surface Element Analysis. The morphology and surface chemical composition assay of HCPT bulk powders and HCPT NRs were investigated by scanning electron microscopy (SEM) with energy dispersive spectrometer (SEM-EDS; S-4800, Hitachi Limited., Tokyo, Japan). A drop of HCPT NRs solution (0.1 mg mL−1) was placed on matrix and air-dried, and then these samples were sputter-coated with a conductive layer of gold− palladium (Au/Pd) for 1 min. An accelerating potential of 30 mV was used for the observation and analysis. 4.9. X-ray Diffraction Analysis. X-ray diffraction analysis was performed using graphite filtered Cu Kα radiation (λ = 1.54 Å) at 40 kV and 100 mA with a scanning rate of 8° per minute (2θ from 3° to 80°) with an X-ray diffractometer (Rigaku D/Max-2500; Rigaku Denki Co., Tokyo, Japan) at room temperature. HCPT mixed with POC in the same ratio of HCPT NRs was used for all the experiments. 4.10. Measurement of the Fixed Aqueous Layer Thickness (FALT). The HCPT NRs were centrifuged at 25 °C for 20 min at 13 000 rpm to obtain the nanorods, and the pellet was washed with a phosphate buffer solution. Then the pellet was resuspended in NaCl solutions with different ion concentrations. The ζ potential was measured, and the calculation of FALT (L) was based on the linear correlation between ln ζ (zeta potential) and κ (Debye−Huckel parameter): ln ζ = ln A − κL, where A is regarded as constant and κ is Debye−Huckel parameter, expressed as κ = √C/0.3 for univalent salts where C is the molarity of electrolytes. The slope L gives the position of the slipping plane or thickness of the fixed aqueous layer in nm units. The experiments were conducted in triplicate. The data were shown as the mean values plus standard deviation (±SD). 4.11. Stability Study. For the storage stability study, HCPT NRs were stored at 4, 25 °C separately, and the particle size of the samples were measured at 0, 1, 3, 7, and 14 days. The experiments were conducted in triplicate, and the data were shown as the mean values plus standard deviation (±SD). For the solution stability study, HCPT NRs were mixed with glucose solution (5%, wt %), RPMI-1640 medium with 10% fetal calf serum, and plasma at 37 °C, and the particle size and morphology were measured after incubated 24 h. The experiments were conducted in triplicate, and the data were shown as the mean values plus standard deviation (±SD). For the lyophilized stability study, after freeze-drying, HCPT NRs were resuspended in deionized water to their original
3.30−3.64 (br, CH2), 3.66−3.80 (br, CH2), 3.83−4.09 (br, CH2), 4.25−4.32 (br, CH2), 4.46−4.53 (br, CH2), 6.56−6.59 (br, CH), 7.08−7.24 (br, CH) ppm. Codendrimer POH. The solution of p-toluenesulfonic acid (PTSA, 0.25 mg, 1.44 mmol) in methanol (MeOH, 30 mL) was added into a solution of THP protected codendrimer POT (0.43 g, 9.17 μmol) in MeOH (20 mL), and the mixture was stirred for 10 h at room tempareture. The mixture solution was transferred into a dialysis membrane (MWCO 14000), dialyzed against NaHCO3 solution (pH 8.0, 1 L × 8) and deionized water (1 L × 8) successively. After evaporation of water, codendrimer POH was afforded as colorless oil (0.28 g, 82%). 1 H NMR (600 MHz, DMSO-d6): δ = 2.24 (br, CH2), 2.43 (br, CH2), 2.63 (br, CH2), 3.12 (br, CH2), 3.18 (br, CH2), 3.30 (br, CH2), 3.42−3.81 (br, CH2) 3.96−4.18 (br, CH2), 4.75 (br, CH2), 6.65 (s, CH), 7.20 (br, CH), 7.75−8.25 (br, CH), 8.49 (br, CH) ppm. Codendrimer POC. Codendrimer POH (0.20 g, 4.26 μmol), cy5.5-NHS ester (10 mg, 8.86 μmol), TEA (10 mg, 98.8 μmol), and DMAP (10 mg, 89.1 μmol) were dissolved in DMF (20 mL) at room temperature. After stirring for 24 h, the solvents were evaporated in vacuo at room temperature, and the residue was dissolved in MeOH and transferred into a dialysis membrane (MWCO 14000), dialyzed against deionized water (1 L × 8). After evaporation of water, codendrimer POC was afforded as blue oil (0.26 g, 95%). To quantify the content of near-infrared probe cy5.5, the codendrimer POC was analyzed by HPLC (Ultimate3000, DIONEX) using a fluorescence detector (excitation/emission wavelengths 678/698 nm) and compared to a calibration curve generated from acetonitrile (y = 2.2216x + 0.9013, R2 = 0.9997). The flow rate was 1.0 mL min−1, and the sample injection volume was 20 μL. 4.4. Characterization of Codendrimers: NMR and GPC. 1 H NMR spectra were recorded on a Bruker AV 600 spectrometer with DMSO-d6 as solvent, and chemical shifts were reported as δ values (ppm) relative to internal Me4Si. Gel permeation chromatography (GPC) measurements were carried out on a Viscotek GPC VE2001 instrument with single column set equipped with refractive index detector and DMF (containing 1 g L−1 LiBr) as the eluent at 35 °C. Calibration was performed with polymethyl methacrylate standards. 4.5. Preparation of HCPT-Loaded Nanorods. HCPTloaded POC nanorods (HCPT NRs) were prepared by the antisolvent precipitation augmented by ultrasonication. Briefly, HCPT (24 mg) and POC (3 mg) were dissolved in DMF (1 mL) and then quickly injected into deionized water (5 mL) under continuous ultrasonication for 10 min. The mixed solution was dialyzed (MWCO 14 000) against deionized water (4 × 1 L) for 4 h to remove DMF and the free drug. The quantitative analysis of HCPT in the nanorods was carried out on a Thermo C18 (4.60 mm × 250 mm, 5 μm) with HPLC (UltiMate3000, DIONEX) using a UV detector operated at 384 nm and compared to a calibration curve generated from acetonitrile/water with acetic acid (0.1%) (25:75, v/v) (y = 1.7874x + 0.1561, R2 = 0.9999). The flow rate was 1.0 mL min−1, and the sample injection volume was 20 μL. The drugloading content (DLC) was calculated as follows. The experiments were conducted in triplicate, and the data were shown as the mean values plus standard deviation (±SD). ⎛ ⎞ weight of loaded drug DLC = ⎜ ⎟ × 100 ⎝ weight of drug − loaded NRs ⎠ G
DOI: 10.1021/acs.bioconjchem.6b00536 Bioconjugate Chem. XXXX, XXX, XXX−XXX
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all the images and average fluorescence intensity were recorded under the same condition. 4.15. In Vivo Antitumor Efficacy in H22-Tumor Bearing Mice. The in vivo antitumor efficacy of HCPT NRs was assessed with the H22-tumor bearing ICR mice model. Briefly, male ICR mice (18−22 g) were induced H22 tumor by subcutaneous injection of 0.2 mL of cell suspension (2 × 106 cells per mouse) in the right armpit. When tumor exceeded 400 mm3 (7 days after implantation), mice were randomly divided into 5 groups (10 mice per group). Mice were administrated with saline (control group), HCPT injection at a concentration of 5 mg kg −1 (positive group), and HCPT NRs at concentrations of 1.25, 2.5, and 5 mg kg1− (test groups) in the final volume of 0.2 mL via the tail vein every 2 days for 4 times. During the administration process, the relative body weight of the mice was monitored as an index of systemic toxicity. Tumor volume was measured daily with a caliper and calculated as tumor volume (mm3) = (the largest diameter) × (the smallest diameter)2/2. All mice were sacrificed on the seventh day, and the tumors were removed and weighed. The inhibitory rate of the tumor (IR) was calculated as follows, and the data were shown as the mean values plus standard deviation (n = 10):
concentration. The particle size and morphology were measured. The experiments were conducted in triplicate, and the data were shown as the mean values plus standard deviation (±SD). 4.12. In Vitro Studies on Release Kinetics. In vitro release characteristics of HCPT NRs were studied by the dialysis method. Briefly, HCPT injection, nanorods solution (2 mL), and bulk powder were placed in a dialysis bag (MWCO 14 000), then immersed in 50 mL of PBS solution (pH 7.4). The release studies were performed at 37 °C with continuous magnetic stirring at 100 rpm under sink conditions. At predetermined time intervals, 5 mL of external solution was withdrawn for analysis and an equal volume of fresh media was replenished. The drug release study was performed for 48 h and performed in triplicate. The amount of HCPT released was quantified by a UV−HPLC, and the data were shown as the mean values plus standard deviation (±SD). 4.13. In Vitro Cytotoxicity Assay. The rat red blood cell (RBC, 2% w/v) solution was prepared and centrifuged at 5000 rpm for 5 min. The plasma supernatant was removed, and the erythrocytes were resuspended in 5% glucose solution. The HCPT NRs were incubated with the 2% (w/v) RBC suspension at 37 °C for 5 h with different concentrations. Then the RBCs were removed by centrifugation, and 150 μL of the supernatant was measured at 540 nm using a microplate reader (Versamax tunable microplate reader). The results were expressed with the assumption that deionized water causes 100% hemolysis and normal saline solution 0% hemolysis. The experiments were conducted in triplicate, and the data were shown as the mean values plus standard deviation (±SD). The in vitro cytotoxicity against HepG2 and 4T1 cells using MTT assay was evaluated. Briefly, cells were seeded in RPMI1640 medium supplemented with 10% fetal calf serum, 100 units mL−1 penicicillin G, and streptomycin at 37 °C with 5% CO2 in 96-well plates at a density of 1 × 104 cells per well. After incubation for 48 h, the growth medium was replaced with fresh RPMI-1640. Then, HCPT NRs and HCPT injection were added into the wells. After incubation for 48 h, an amount of 20 μL of MTT solution (5 mg mL−1) was added to each well and incubation was continued for another 4 h. The medium was removed, and 200 μL of DMSO was added into each well to dissolve the formazan by pipetting up and down several times. The absorbance of solution in each well was measured using ELISA plate reader at 570 nm to determine the OD value. The cell inhibition rate was calculated as follows: cell inhibition = (1 − ODtreated/ODcontrol) × 100, where ODtreated was obtained for the cells treated by the nanorods, ODcontrol was obtained for the cells treated by the culture medium, and the other culture conditions were the same. Each experiment was done in quintuplicate. The data were shown as the mean values plus standard deviation (±SD). The half maximal inhibitory concentration (IC50 value) was calculated according to the MTT results. 4.14. In Vitro Cellular Uptake Efficiency. The HepG2 cells (1 × 105 per well) were seeded in a 6-well plate and cultured at 37 °C for 24 h in a humidified atmosphere with 5% CO2, and then the cy5.5 labeled agents (1 μg mL−1) were added into the 6-well plate. After 4 h of additional incubation at 37 °C, the medium was removed and the cells were washed with PBS three times and fixed with 4% of paraformaldehyde PBS solution for 15 min. The cellular uptake images were recorded with Delta Vision Microscopy Imaging Systems, and
⎛ 1 − tumor weight of tested group ⎞ IR (%) = ⎜ ⎟ × 100 ⎝ tumor weight of saline control group ⎠
4.16. Biodistribution in Tumor Bearing Mice. Male ICR mice (18−22 g) were induced with H22 tumor by subcutaneous injection of 0.2 mL of cell suspension (2 × 106 cells per mouse) in the right armpit. When tumor exceeded 400 mm3 (8 days after implantation), mice were randomly divided into 2 groups (36 mice per group). Mice were administrated with HCPT injection (control group) and HCPT NRs (test group) at a concentation of 5 mg kg−1 in the final volume of 0.2 mL via the tail vein. Six mice in each group were sacrificed after injection at 0.5, 2, 4, 6, 8, 12, and 24 h. At the predetermined time, blood samples were collected from the ocular artery after eyeball removal and placed into heparinized test tubes. The heart, liver, spleen, lung, kidney, brain, tumor were collected, washed, accurately weighed, and homogenized with normal saline solution. The HCPT in the blood and tissues was analyzed by HPLC (UltiMate3000, DIONEX) using a fluorescence detector (excitation/emission wavelengths 375/ 435 nm). The quantitative analysis was carried out on a Thermo C18 (4.60 mm × 250 mm, 5 μm) and compared to the calibration curves generated from acetonitrile/water with acetic acid (0.1%) (30/70, v/v). The flow rate was 1.0 mL min−1, and the sample injection volume was 20 μL. Calibration curves were established respectively for the tumor, organs, and plasma; all of the correlation coefficients were more than 0.99. To determine the in vivo biodistribution further, male nu/nu nude mice bearing 4T1 tumor model was established. Briefly, 2 × 106 4T1 cells were inoculated subcutaneously into the right armpit of the nude mice (4−5 weeks, 14−16 g). When the tumor exceeded 200 mm3 (10 days after implantation), the mice were randomly divided into 2 groups (6 mice per group) and treated with Dir/HCPT solution and Dir/HCPT NRs intravenously at the concentration of 10 μg kg−1, respectively. The real-time distribution and tumor accumulation of free Dir/ HCPT solution and Dir/HCPT NRs were recorded at 0.5, 1, 3, 6, 9, 12, and 24 h after injection using an in vivo imaging system (IVIS Spectrum 200, PerkinElmer Co., MA, USA). Mice H
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were sacrificed at 24 h after postinjection, and the tissues were excised and observed by the imaging system. The signal intensity of different tissues was quantified as the sum of all the detected photon counts within the region of interest (ROI) in the unit of [p s−1 cm−2 sr−1]/[μW cm−2]. 4.17. Statistical Analysis. Statistical analysis was performed with SPSS 19.0 software. Both ANOVA and Student’s t test were used to evaluate the differences between groups, and P < 0.05 was considered significant.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.6b00536. Characterization data (NMR, XRD, EDS), stability study (DLS and SEM), FALT, hemolytic activity, real-time biodistribution, and fluorescence images of tumor bearing mice (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Yifei Guo: 0000-0003-4548-5040 Author Contributions †
Y.G. and Y.Z. contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work is financially supported by PUMC Youth Fund (Grant 33320140184), National Natural Science Foundation of China (Grant U1401223), National Natural Science Foundation of China (Grant 81573622), Beijing Natural Science Foundation (Grant 7152099), and CAMS Innovation Fund for Medical Sciences (CIFMS, Grant 2016-12M-1-012).
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REFERENCES
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