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The tumor immune microenvironment in gastroenteropancreatic neuroendocrine neoplasms
Article Journal of Nanoscience and Nanotechnology
Copyright © 2019 American Scientific Publishers All rights reserved Printed in the United States of America
Vol. 19, 5463–5468, 2019 www.aspbs.com/jnn
Ultrasound-Triggered Gas-Generating Doxorubicin Poly(lactic-co-glycolic acid)-Nanoparticles for Cancer Therapy Nan Zhang, Xinyang Liu, Ruirui Hou, Jiangnan Zhang, Jia Li, Pengchao Huo, Yue Xu, and Zhenzhong Zhang∗ School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China The purpose of current research is to develop ultrasound-triggered gas-generating Doxorubicin PLGA nanoparticle for cancer therapy. Method: pH-sensitive PLGA nanoparticles (PLGANPs) was fabricated to deliver doxorubicin (DOX) and sodium bicarbonate (NaHCO3 using the water-in-oilin-water (w/o/w) double emulsion method. Result: The nanoparticle with the size (650 nm) and high drug loading (15.8 ± 2.3%) were successfully prepared and showed pH-responsive release characteristics. In vitro results indicate that DOX/NaHCO3 @PLGANPs with ultrasound had higher inhibition and cell uptake on MCF-7 cells than free DOX and other formulation. In vivo animal experiments showed that after treatment of DOX/NaHCO3 @PLGANPs with ultrasound, the relative tumor volume (0.63) ofIP: S180-tumor-bearing was than that of without ultrasound (0.81), 79.110.28.36 On:mice Sun, 25lower Aug 2019 13:49:52 DOX@PLGANPs (1.00) and Free DOX (1.12). Moreover, safety evaluation result indicated that Copyright: American Scientific Publishers free DOX. In conclusion, the DOX/NaHCO3 @PLGANPs DOX/NaHCO3 @PLGANPs was safer than Delivered by Ingenta was successfully developed and evaluated in vitro and in vivo. This drug delivery system will be a promising strategy for cancer therapy and diagnosis.
Keywords: Doxorubicin, Sodium Bicarbonate (NaHCO3 , Drug Delivery System, Anticancer.
1. INTRODUCTION Doxorubicin (DOX), one of the most active single-agent drugs, is widely used for the treatment of various cancers, such as breast cancer, lung cancer and leukemia because of its broad spectrum of antitumor activity.1 However, the clinical use of DOX has been severely limited by its critical cardiotoxicity, myelosuppression, renal toxicity and narrow therapeutic window.2 Pegylated liposome doxorubicin (PLD; Doxil/Caelyx) is regarded as the first approved anticancer nanoparticle, which shows improved patient tolerability than conventional chemotherapy.3 This delivery system has long-circulation and passive targeting, but not active targeting. To increase the local concentration of chemotherapeutics, nanoparticles with stimulus-responsive properties have great potential to further improve the therapeutic efficiency. Usually tumor site shows acidic microenvironment due to the abnormal energy metabolism of tumor cells.4 ∗
Author to whom correspondence should be addressed.
J. Nanosci. Nanotechnol. 2019, Vol. 19, No. 9
Manipulating the response to pH changes is a viable strategy because active-targeting pH-triggered carrier disruption and drug release can be promoted in the acidic tumor environment.5 A smart gas-generating nano-lipid encapsulated system has been reported.6 The key component (NaHCO3 was encapsulated in the delivery system for the generation of CO2 .7 When the delivery systems are trafficked into tumor sites, the carbonate anion (HCO3− reacts with the acid medium, which generates CO2 bubbles to lead to the formation of pores in shells, the collapse of carriers, the prompt release of drugs, and ultimately the death of cancer cells.8 However, the encapsulations of both drug and NaHCO3 in one system is still challenging for formulation scientists. Poly(lactic-co-glycolic acid) (PLGA) is a kind of biodegradable polymer. In the United States, PLGA is approved by the FDA and is officially used as a medicinal material in the United States Pharmacopoeia.9 10 It has good biological compatibility, non-toxic, and drug-encapsulating performance, which is commonly
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used in pharmaceutical and medical engineering 2.2. Fabrication of PLGANPs materials.11 PLGANPs were prepared using the water-in-oil-in-water Ultrasound is extensively used for tumor treatment (w/o/w) double method. NaHCO3 (2.5 mg) and DOX because it can produce cavitation.12 It also can be used (1 mg) were dissolved and mixed with 1 mL of aqueous as a sonoporation inducer for tumor therapy because this PVA (10 mg/mL), then stirred at 500 rpm for 15 min at cavitation power disrupts the carriers, leading to a rapid room temperature (25 C). Thereafter, the admixture was drug release and allowing the drug to penetrate into the emulsified with 2 mL of PLGA solution (5 mg/mL in tumor cells.13 14 So the combination of ultrasound and dichloromethane (DCM)) using an ultrasonicator (2772AS chemotherapeutic drugs have drawn much attention in Ultrasonic single comprehensive fields (1 W, 1 Hz, 5 min, tumor treatment researches because ultrasonic can proChattanooga Company, American)) in an ice bath at 100 W mote the release of drugs and further improve tumor cell to obtain the primary w/o emulsion. Next, 6 mL of TPGS killing effect.12 15 Image-guided drug delivery with bubble(0.3 mg/mL) was added to the primary emulsion and generating can be performed by the combination of both homogenized at 5000 rpm for 30 min in an ice bath using drugs and bubbles. The latter has the advantage of reduced a homogenizer (T25 digital ULTRA TURRAX, Germany) damage to healthy tissues. Theranostic approach to drug to form the w/o/w double-emulsion. To evaporate DCM delivery using bubbles was presented by Zhang et al. who and solidify the particles, the resulted emulsion was added prepared bubble-generating nano-lipid carriers for ultrato 16 mL of deionized water and stirred overnight at room sound/CT imaging-guided tumor therapy. This drug delivtemperature. All the formulations were recollected by cenery system integrated targeting therapeutic and diagnostic trifugation (2000 rpm, 10 min), washed with deionized functions to target tumors and control drug release stawater for 3 times, and finally resuspended with 10 mL of bility in the blood circulation, and enhancement of ultraphosphate buffered saline (10% PBS with pH 6.8). sonic imaging with bubbles. Apart from the use of drug with bubble-generating loaded nano-lipid for target-tumors 2.3. Characterization of Test PLGANPs delivery, mineralized nanoparticles for drug delivery and The particle size of the test PLGAHPs was measured drug therapy has been also investigated. pH controlled gasby dynamic light scattering (Zetasizer Nano ZS-90, generating mineralized nanoparticles were developed for Malvern, UK). TEM (Tecnai G2 20, FEI) was used the drug accumulation in tumors the tumor image and minfor morphological examination. The test PLGANPs were 79.110.28.36 25 Aug 2019 13:49:52 eralized nanoparticle destruction IP: by ultrasound for On: effec-Sun, destroyed by DMSO and the encapsulated DOX concenCopyright: American Scientific Publishers tive tumor growth inhibition. tration was measured by high-performance liquid chroDelivered by Ingenta Current research aims to fabricate an ultrasoundmatography (HPLC) at 485 nm. The calculation formula triggered pH-sensitive PLGA-based DOX delivery system, of encapsulation efficiency (EE, %) and loading efficiency which can reduce cardiotoxity and myelosuppression of (LE, %) are as follows:16 17 DOX and to increase the therapeutic effect of DOX by the integration of ultrasound and chemotherapy treatment. EE (%) = We/Wt × 100% LE (%) = We/Wm × 100%
2. EXPERIMENTAL DETAILS 2.1. Materials In this study, D,L-lactide/glycolic acid (PLGA, ratio 75:25, Mw 15000-23000 Da) was purchased from Jinan Daigang Biotech Co., Ltd., China. DOX was purchased from Dalian Meilun Biological Technology Co., Ltd., China. Poly vinyl alcohol (PVA, Mw 31000 Da) was purchased from Aladdin Industrial Corporation. Vatamine E-D--tocopheryl polyethylene glycol 1000 succinate (VE-TPGS) was purchased from Shanghai Macklin Biochemical Co., Ltd., China. 3-(4.5-Dimethylthiazol-2-yl)2,5-diphenyltetrazaliumbromide (MTT) and RPMI-1640 cell culture medium were purchased from Beijing Solarbio Science & Technology Co., Ltd., China. Ethanol, acetonitrile, and dimethyl sulfoxide (DMSO) were used as analytic reagents. The water used in this work was pretreated by the Milli-Q plus system (Millipore, Bedford, MA, USA). The MCF-7 human breast cancer cell line and S180 sarcoma tumor cell line were obtained from the Chinese Academy of Science Cell Bank. 5464
(We: The weight of drug loaded; Wt: The total drug in the preparation; Wm: The total weight of the PLGANPs). 2.4. In Vitro Drug Release from Test PLGANPs The release profile of DOX from PLGANPs was obtained by a dialysis method. Briefly, the test sample (1 mL) was placed in a dialysis bag (MWCO 8000–14,000). The bag was immersed in 100 mL release medium in a thermostatic rotary shaker at 100 rpm and 37 C. One release medium was pH 7.4 PBS and the other was pH 6.0 PBS. Samples from the release medium were collected at predetermined time intervals and their fluorescence intensity was measured at 480 nm. 2.5. Cell Cultures The MCF-7 human breast cancer cell line was obtained from the Chinese Academy of Sciences Cell Bank (Catalog No. HYC3204). MCF-7 cells were cultured in normal RPMI-1640 culture medium in 5% CO2 and 95% air at J. Nanosci. Nanotechnol. 19, 5463–5468, 2019
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Ultrasound-Triggered Gas-Generating Doxorubicin PLGA-Nanoparticles for Cancer Therapy
37 C in a humidified incubator.18 The cell was then ready for the experiments outlined in Sections 2.6 and 2.7. 2.6. In Vitro Cytotoxicity The cytotoxic activity of DOX forms were evaluated using an MTT assay. For this study, cancer cells were seeded (5 × 103 cells/well) in 96-well plates containing RPMI1640 medium. Test formulations with different DOX concentrations were added to the cells and incubated for 4 h. Probe sonication (1 W, 1 Hz, 1 min) were used as the methods to release DOX from the delivery system after incubation for 4 h. The cells were cultured for 48 h and incubated in the growth medium containing 20 L of 5 mg/mL MTT reagents for an additional 4 h. After that, 150 L of DMSO was added to each well to ensure solubilization of the formazan crystals. Finally, optical density readings were obtained using a multi-well scanning spectrophotometer at a wavelength of 490 nm.
vein. After injection, ultrasound images were obtained by using a Vevo 770 US imaging system with a RMV 706 probes. The imaging intensity was determined at both experimental and control groups. Secondly, these mice were randomly divided into 5 groups as follows: (1) saline (control group), (2) free DOX, (3) DOX@PLGANPs, (4) DOX/NaHCO3 @PLGANPs, (5) DOX/NaHCO3 @PLGANPs + US treatment (1 W/cm2 , 2 min). All groups were treated by intravenous injection at every two days and 6 injections totally. To evaluate antitumor efficacy, relative tumor volume and relative body weight were recorded and calculated. At the end of treatment, the biochemical parameters were examined to observe their cardiac and renal toxicity, including routine analysis of blood and cardiac function tests (performed by Zhengzhou University Hospital). 2.10. Statistical Analysis All statistical analyses results were expressed as mean ± standard deviation. The data were compared using the GraphPad Prism 5 and P values <0.05 were deemed to be statistically significant.
2.7. Cellular Uptake To investigate the targeted ability of PLGANPs, coumarin-6 (with green fluorescence) was used as the marker of PLGANPs. MCF-7 cells were seeded at a density of 2 × 105 per well in 6-well plates to observe the 3. RESULTS AND DISCUSSION profile of cellular uptake with or without NaHCO3 . When 3.1. Synthesis and Characterization of the cells reached 80% confluence, the culture medium DOX/NaHCO3@PLGANPs was changed to fresh medium containing coumarin-6, IP: 79.110.28.36 On: Sun, The 25 Aug 2019 13:49:52 DOX/NaHCO @PLGANPs and DOX/NaHCO3 @ coumarin-6-PLGANPs, NaHCO3 @coumarin-6-PLGANPs Copyright: American Scientific Publishers3 PLGANPs were prepared using a double-emulsion w/o/w 1 Hz, by Ingenta and NaHCO3 @coumarin-6-PLGANPs + US (1 W, Delivered solvent-diffusion-evaporation method. Schematic diagram 1 min), respectively. The cells were incubated for 4 h. of DOX/NaHCO @PLGANPs was shown in Figure 1(a). Then the medium was removed, and the cells were washed 3 The TEM images indicated that the particles size of three times with PBS and fixed in 4% paraformaldehyde 650 nm (Fig. 1(b)). Their size ranged from 600 nm to for 15 min. Finally, the cells were imaged using a flu700 nm (Fig. 1(c)) with a zeta potential of approximately orescence microscope (Zeiss LSM 510, Thornwood, NY, −21.8 mV (Fig. 1(d)). Table I listed important characUSA). teristic parameters of the test PLGANPs. The results of DOX/NaHCO3 @PLGANPs indicated that entrapment effi2.8. Animal Model ciencies (EE) was 89.4 ± 3.5% and drug loading (DL) was Kunming (KM) mice (female, 20 ± 2 g, 4–6 weeks old) 15.8 ± 2.3%. were purchased from the Henan Laboratory Animal Center. All animal experiments were in keeping with the 3.2. In Vitro Drug Release of Test guidelines for humane and responsible use of animals DOX/NaHCO3@PLGANPs in research set by ZhengZhou University. Approximately In order to determine the in vitro release profile 2 × 106 S180 ascites cells were injected into the armpits with subcutaneous of the mice. When the tumors grow to of DOX, three formulations (DOX@PLGANPs, DOX/ the volume of 60–100 mm3 , these mice were ready for the NaHCO3 @PLGANPs and DOX/NaHCO3 @PLGANPs + experiments outlined in Section 2.9. US) were incubated in two different medium (pH 7.4 PBS and pH 6.0 PBS). From Figure 2, DOX in DOX/ NaHCO3 @PLGANPs with US treatment released more 2.9. In Vivo Antitumor Efficacy and the than without US in pH 7.4 and pH 6.0, indicating Safety Evaluation that ultrasound can promote drug released. Moreover, To investigate the target effect in vitro, degassed water DOX in DOX/NaHCO3 @PLGANPs released more than was as the negative control for ultrasonic imaging of DOX@PLGANPs at the same pH. Both two formuDOX/NaHCO3 @PLGANPs. Similar tumor-bearing mice lations release more DOX at pH 6.0 than pH 7.4. model was used for ultrasound imaging. When tumor DOX/NaHCO3 @PLGANPs release the drug completely at volume was about 100 mm3 , DOX/NaHCO3 @PLGANPs 12 h, but other formulations need more time to release the (5 mg/kg) was injected into the mice via the tail J. Nanosci. Nanotechnol. 19, 5463–5468, 2019
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Figure 1. (a) Schematic illustrations showing the structure of DOX/NaHCO3 @PLGANPs; (b) the TEM micrograph of the PLGANPs; (c) particle size of DOX/NaHCO3 @PLGANPs; (d) particle zeta potential of DOX/NaHCO3 @PLGANPs.
drugs, which indicated that DOX/NaHCO3 @PLGANPs is pH-sensitive and easy to release DOX in the microacid environment of the tumor.4
Figure 4. With the concentration increase of the above groups, the cell inhibition increases, this showed that our formulations were concentration-dependent. Moreover, the inhibition of those groups: free DOX < DOX@PLGANPs < DOX/NaHCO3 @PLGANPs < 3.3. Enhanced Cell Uptake of DOX In Vitro To investigate the uptake capacity of tumor cells, 3 @PLGANPs + US.” Cell inhibition of IP: 79.110.28.36 On: Sun, “DOX/NaHCO 25 Aug 2019 13:49:52 DOX/NaHCO @PLGANPs was better than that of formucoumarin-6 was used to mark the Copyright: green fluorescence 3 American Scientific Publishers without NaHCO3 due to pH sensitivity of NaHCO3 (control, bylation of the PLGA19 in the following groups Delivered Ingenta may enhance more DOX release into cells. Moreover, the coumarin-6, coumarin-6-PLGANPs, NaHCO3 @coumarincell inhibition of “DOX/NaHCO3 @PLGANPs + US” was 6-PLGANPs and NaHCO3 @coumarin-6-PLGANPs + US (1 W, 1 Hz, 1 min)). After 4 hours, the samples were greater than DOX/NaHCO3 @PLGANPs, which indicated taken under the inverted fluorescence microscope as that the combination of ultrasound with the preparations Figure 3. The results showed that the fluorescence intenwas able to kill tumor cells and improve drugs antitumor sity of NaHCO3 @coumarin-6-PLGANPs was stronger activity.13 14 Thus, the cavitation effect of ultrasound was than coumarin-6-PLGANPs, but control groups of and coumarin-6 group has no signal, which indicated that NaHCO3 in this delivery system plays an important role in tumor targeting. Moreover, the strongest fluorescence of “NaHCO3 @coumarin-6-PLGANPs+US” demonstrated the US can enhance more DOX release to improve the inhibition efficiency.20 3.4. In Vitro Cytotoxicity The inhibitory effects on MCF-7 cells after incubation with free DOX, DOX@PLGANPs, DOX/NaHCO3 @PLGANPs and DOX/NaHCO3 @PLGANPs + US were shown in Table I. The encapsulation efficiency (EE, %) and loading efficiency (LE, %) result. Formulation DOX@PLGANPs DOX/NaHCO3 @PLGANPs Note: PS. Mean ± standard deviation; n = 3.
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EE (%)
LE (%)
92.3 ± 4.8 89.4 ± 3.5
16.3 ± 1.7 15.8 ± 2.3
Figure 2. In vitro pH-triggered release profiles of DOX from DOX@PLGAHPs and DOX/NaHCO3 @PLGAHPs incubated at pH = 6.0 and 7.4. Data were presented as mean ± standard deviation (n = 3).
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Ultrasound-Triggered Gas-Generating Doxorubicin PLGA-Nanoparticles for Cancer Therapy
Figure 3. Qualitative results of cell uptake. (a: Control; b: Coumarin-6; c: Coumarin-6-PLGANPs; d: NaHCO3 @coumarin-6-PLGANPs; e: NaHCO3 @coumarin-6-PLGANPs + US (4 h)).
very useful for the DOX/NaHCO3 @PLGANPs delivery system to kill the tumor cells. 3.5. In Vivo Antitumor Efficacy After injection 30 min, control group with distilled water could not be observed clear signals, but DOX/NaHCO3 @PLGANPs clearly showed excellent enhancement (Fig. 5(a)) because the CO2 bubbles could be observed from DOX/NaHCO3 @PLGANPs in the microacid environment of tumor by ultrasound. Therefore, our result revealed that DOX/NaHCO3 @PLGANPs had good tumor targeting.
The change in relative tumor volumes in S180 tumorbearing mice is considered indices of tumor inhibition efficiency. Figure 5(b) showed that the relative tumor volume of the control group (Saline) increased greatly, while free DOX and other formulations could effectively inhibit the tumor growth. When the tumorbearing mice were subjected to ultrasound energy with DOX/NaHCO3 @PLGANPs, the tumor suppression effect was significantly improved. There are two possible reasons. On the one hand, the pH-sensitive tumor targeting effect promoted higher efficiency than other non-targeted formulations. Large amounts of CO2 bubbles from NaHCO3 in microacidic environment of tumor bursts PLGA shell to fast release DOX into the tumor cells and effectively inhibit tumor growth. On the other hand, cavitation forces by ultrasound also damage vascular endothelia to strengthen tumor killing ability,21 22 which is considered the appropriate way to achieve the maximum therapeutic
IP: 79.110.28.36 On: Sun, 25 Aug 2019 13:49:52 Copyright: American Scientific Publishers Delivered by Ingenta Figure 4. In vitro cytotoxicity of a various formulations (grouped as shown above) against MCF-7 cells at different concentrations of DOX for 12 h with or without the US (1 W, 1 Hz, 2 min).
Figure 5. (a) In vivo ultrasound image of tumors before and after intra tumor injection DOX/NaHCO3 @PLGANPs into a tumor-bearing mouse model for 30 min; (b) tumor growth of mice in different treatment groups within 12 days. Data were presented as mean ± standard deviation (n = 6).
J. Nanosci. Nanotechnol. 19, 5463–5468, 2019
Figure 6. (a) Changes of relative body weight of mice in different groups during treatment; Data were presented as mean ± standard deviation (n = 6); (b) tumor-bearing mice blood routine examination; (c) liver function test in tumor-bearing mice.
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Acknowledgments: I would like to express heartfelt thanks to the Professor Xia Xu Group for their helpful discussions. We also appreciate School of Pharmaceutical Sciences of Zhengzhou University for providing financial support.
References and Notes 1. D. L. Li, Z. V. Wang, G. Ding, W. Tan, X. Luo, A. Criollo, M. Xie, N. Jiang, H. May, V. Kyrychenko, J. W. Schneider, T. G. Gillette, and J. A. Hill, Circulation 133, 1668 (2016). 2. W. Ma, Q. Guo, Y. Li, X. Wang, J. Wang, and P. Tu, Eur. J. Pharm. Biopharm. 112, 209 (2017). 3. J. M. Gibson, S. Alzghari, C. Ahn, H. Trantham, and N. M. La-Beck, Oncologist 18, 1022 (2013). 4. J. Zhu, L. Liao, X. Bian, J. Kong, P. Yang, and B. Liu, Small 8, 2715 (2012). 5. X. L. He, F. Liang, F. Wang, L. S. Zou, J. Wang, C. K. Tang, K. Y. Figure 7. Schematic illustration is showing the biofunctions of Zhao, and D. S. Wei, J. Mater. Sci. 53, 2326 (2018). DOX/NaHCO3 @PLGANPs. 6. N. Zhang, J. Li, R. Hou, J. Zhang, P. Wang, X. Liu, and Z. Zhang, Int. J. Pharm. 534, 251 (2017). 7. Y. Niu, F. J. Stadler, J. Song, and S. Chen, Colloid Surface B effect. The combination of ultrasonic and chemotherapy is 153, 160 (2017). an effective treatment strategy in cancer therapy. 8. C. J. Ke, Y. J. Lin, Y. C. Hu, W. L. Chiang, K. J. Chen, W. C. Yang, H. L. Liu, C. C. Fu, and H. W. Sung, Biomaterials 33, 5156 (2012). 9. B. Tyler, D. Gullotti, A. Mangraviti, T. Utsuki, and H. Brem, Adv. 3.6. In Vivo Safety Evaluation Drug Deliver. Rev. 107, 163 (2016). The change in relative body weight is considered indices 10. M. Kanamala, W. R. Wilson, M. Yang, B. D. Palmer, and Z. Wu, of safety evaluation.23 24 Although free DOX demonBiomaterials 85, 152 (2016). 11. S. Acharya and S. K. Sahoo, Adv. Drug. Deliver. Rev. 63, 170 (2011). strated tumor inhibition effects, weight loss of positive 12. F. Kiessling, S. Fokong, J. Bzyl, W. Lederle, M. Palmowski, and control group was more than others groups by 12 days T. Lammers, Adv. Drug. Deliver. Rev. 72, 15 (2014). (Fig. 6(a)), indicating serious sideIP:effects. Other testOn: for-Sun, 13. 79.110.28.36 25 Aug 2019 P. Huang, X. 13:49:52 Qian, Y. Chen, L. Yu, H. Lin, L. Wang, Y. Zhu, and Copyright: mulation groups showed relatively small changesAmerican in body Scientific J. Shi, Publishers J. Am. Chem. Soc. 139, 1275 (2017). Ingenta S. Gao, G. Wang, Z. Qin, X. Wang, G. Zhao, Q. Ma, and L. Zhu, weight. Furthermore, blood tests and cardiac Delivered function by14. Biomaterials 112, 324 (2017). tests (Figs. 6(b and c)) showed that the targeted formula15. S. R. Sirsi and M. A. Borden, Adv. Drug. Deliver. Rev. 72, 3 (2014). tions could effectively reduce the side effect of DOX on 16. L. Jiang, L. Li, X. He, Q. Yi, B. He, J. Cao, W. Pan, and Z. Gu, the heart and bone marrow. This is probably caused by Biomaterials 52, 126 (2015). reduced accumulation of drugs at non-tumor sites. In sum17. P. Xu, H. Zuo, B. Chen, R. Wang, A. Ahmed, Y. Hu, and J. Ouyang, Sci. Rep. 7, 42632 (2017). mary, the DOX/NaHCO3 @PLGANPs delivery system can 18. I. P. Pogribny, J. N. Filkowski, V. P. Tryndyak, A. Golubov, S. I. reduce the adverse reactions of DOX. Shpyleva, and O. Kovalchuk, Int. J. Cancer 127, 1785 (2010). 19. S. S. Feng, L. Mei, P. Anitha, C. W. Gan, and W. Zhou, Biomaterials 30, 3297 (2009). 4. CONCLUSION 20. D. Zhu, W. Tao, H. Zhang, G. Liu, T. Wang, L. Zhang, X. Zeng, In summary, we have success prepared the pH-respond and L. Mei, Acta Biomater. 30, 144 (2016). DOX delivery system-DOX/NaHCO3 @PLGANPs. In vitro 21. S. Hernot and A. L. Klibanov, Adv. Drug. Deliver. Rev. 60, 1153 (2008). and vivo studies indicated that our drug delivery system 22. V. Frenkel, Adv. Drug. Deliver. Rev. 60, 1193 (2008). not only improves antitumor ability, but also reduces side 23. C. T. Chen, Z. H. Wang, C. C. Hsu, H. H. Lin, and J. H. Chen, effect as show in Figure 7. Our smart drug delivery system J. Food Drug Anal. 25, 872 (2017). is a potential candidate for the next generation of effica24. C. Y. Lien, T. Y. Chuang, C. H. Hsu, C. L. Lin, S. E. Wang, S. J. Sheu, C. T. Chien, and C. H. Wu, Oncotargets Ther. 8, 1193 (2015). cious tumor therapies.
Received: 1 March 2018. Accepted: 14 May 2018.
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Copyright © 2019 American Scientific Publishers All rights reserved Printed in the United States of America
Vol. 19, 5463–5468, 2019 www.aspbs.com/jnn
Ultrasound-Triggered Gas-Generating Doxorubicin Poly(lactic-co-glycolic acid)-Nanoparticles for Cancer Therapy Nan Zhang, Xinyang Liu, Ruirui Hou, Jiangnan Zhang, Jia Li, Pengchao Huo, Yue Xu, and Zhenzhong Zhang∗ School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou 450001, China The purpose of current research is to develop ultrasound-triggered gas-generating Doxorubicin PLGA nanoparticle for cancer therapy. Method: pH-sensitive PLGA nanoparticles (PLGANPs) was fabricated to deliver doxorubicin (DOX) and sodium bicarbonate (NaHCO3 using the water-in-oilin-water (w/o/w) double emulsion method. Result: The nanoparticle with the size (650 nm) and high drug loading (15.8 ± 2.3%) were successfully prepared and showed pH-responsive release characteristics. In vitro results indicate that DOX/NaHCO3 @PLGANPs with ultrasound had higher inhibition and cell uptake on MCF-7 cells than free DOX and other formulation. In vivo animal experiments showed that after treatment of DOX/NaHCO3 @PLGANPs with ultrasound, the relative tumor volume (0.63) ofIP: S180-tumor-bearing was than that of without ultrasound (0.81), 79.110.28.36 On:mice Sun, 25lower Aug 2019 13:49:52 DOX@PLGANPs (1.00) and Free DOX (1.12). Moreover, safety evaluation result indicated that Copyright: American Scientific Publishers free DOX. In conclusion, the DOX/NaHCO3 @PLGANPs DOX/NaHCO3 @PLGANPs was safer than Delivered by Ingenta was successfully developed and evaluated in vitro and in vivo. This drug delivery system will be a promising strategy for cancer therapy and diagnosis.
Keywords: Doxorubicin, Sodium Bicarbonate (NaHCO3 , Drug Delivery System, Anticancer.
1. INTRODUCTION Doxorubicin (DOX), one of the most active single-agent drugs, is widely used for the treatment of various cancers, such as breast cancer, lung cancer and leukemia because of its broad spectrum of antitumor activity.1 However, the clinical use of DOX has been severely limited by its critical cardiotoxicity, myelosuppression, renal toxicity and narrow therapeutic window.2 Pegylated liposome doxorubicin (PLD; Doxil/Caelyx) is regarded as the first approved anticancer nanoparticle, which shows improved patient tolerability than conventional chemotherapy.3 This delivery system has long-circulation and passive targeting, but not active targeting. To increase the local concentration of chemotherapeutics, nanoparticles with stimulus-responsive properties have great potential to further improve the therapeutic efficiency. Usually tumor site shows acidic microenvironment due to the abnormal energy metabolism of tumor cells.4 ∗
Author to whom correspondence should be addressed.
J. Nanosci. Nanotechnol. 2019, Vol. 19, No. 9
Manipulating the response to pH changes is a viable strategy because active-targeting pH-triggered carrier disruption and drug release can be promoted in the acidic tumor environment.5 A smart gas-generating nano-lipid encapsulated system has been reported.6 The key component (NaHCO3 was encapsulated in the delivery system for the generation of CO2 .7 When the delivery systems are trafficked into tumor sites, the carbonate anion (HCO3− reacts with the acid medium, which generates CO2 bubbles to lead to the formation of pores in shells, the collapse of carriers, the prompt release of drugs, and ultimately the death of cancer cells.8 However, the encapsulations of both drug and NaHCO3 in one system is still challenging for formulation scientists. Poly(lactic-co-glycolic acid) (PLGA) is a kind of biodegradable polymer. In the United States, PLGA is approved by the FDA and is officially used as a medicinal material in the United States Pharmacopoeia.9 10 It has good biological compatibility, non-toxic, and drug-encapsulating performance, which is commonly
1533-4880/2019/19/5463/006
doi:10.1166/jnn.2019.16521
5463
Ultrasound-Triggered Gas-Generating Doxorubicin PLGA-Nanoparticles for Cancer Therapy
Zhang et al.
used in pharmaceutical and medical engineering 2.2. Fabrication of PLGANPs materials.11 PLGANPs were prepared using the water-in-oil-in-water Ultrasound is extensively used for tumor treatment (w/o/w) double method. NaHCO3 (2.5 mg) and DOX because it can produce cavitation.12 It also can be used (1 mg) were dissolved and mixed with 1 mL of aqueous as a sonoporation inducer for tumor therapy because this PVA (10 mg/mL), then stirred at 500 rpm for 15 min at cavitation power disrupts the carriers, leading to a rapid room temperature (25 C). Thereafter, the admixture was drug release and allowing the drug to penetrate into the emulsified with 2 mL of PLGA solution (5 mg/mL in tumor cells.13 14 So the combination of ultrasound and dichloromethane (DCM)) using an ultrasonicator (2772AS chemotherapeutic drugs have drawn much attention in Ultrasonic single comprehensive fields (1 W, 1 Hz, 5 min, tumor treatment researches because ultrasonic can proChattanooga Company, American)) in an ice bath at 100 W mote the release of drugs and further improve tumor cell to obtain the primary w/o emulsion. Next, 6 mL of TPGS killing effect.12 15 Image-guided drug delivery with bubble(0.3 mg/mL) was added to the primary emulsion and generating can be performed by the combination of both homogenized at 5000 rpm for 30 min in an ice bath using drugs and bubbles. The latter has the advantage of reduced a homogenizer (T25 digital ULTRA TURRAX, Germany) damage to healthy tissues. Theranostic approach to drug to form the w/o/w double-emulsion. To evaporate DCM delivery using bubbles was presented by Zhang et al. who and solidify the particles, the resulted emulsion was added prepared bubble-generating nano-lipid carriers for ultrato 16 mL of deionized water and stirred overnight at room sound/CT imaging-guided tumor therapy. This drug delivtemperature. All the formulations were recollected by cenery system integrated targeting therapeutic and diagnostic trifugation (2000 rpm, 10 min), washed with deionized functions to target tumors and control drug release stawater for 3 times, and finally resuspended with 10 mL of bility in the blood circulation, and enhancement of ultraphosphate buffered saline (10% PBS with pH 6.8). sonic imaging with bubbles. Apart from the use of drug with bubble-generating loaded nano-lipid for target-tumors 2.3. Characterization of Test PLGANPs delivery, mineralized nanoparticles for drug delivery and The particle size of the test PLGAHPs was measured drug therapy has been also investigated. pH controlled gasby dynamic light scattering (Zetasizer Nano ZS-90, generating mineralized nanoparticles were developed for Malvern, UK). TEM (Tecnai G2 20, FEI) was used the drug accumulation in tumors the tumor image and minfor morphological examination. The test PLGANPs were 79.110.28.36 25 Aug 2019 13:49:52 eralized nanoparticle destruction IP: by ultrasound for On: effec-Sun, destroyed by DMSO and the encapsulated DOX concenCopyright: American Scientific Publishers tive tumor growth inhibition. tration was measured by high-performance liquid chroDelivered by Ingenta Current research aims to fabricate an ultrasoundmatography (HPLC) at 485 nm. The calculation formula triggered pH-sensitive PLGA-based DOX delivery system, of encapsulation efficiency (EE, %) and loading efficiency which can reduce cardiotoxity and myelosuppression of (LE, %) are as follows:16 17 DOX and to increase the therapeutic effect of DOX by the integration of ultrasound and chemotherapy treatment. EE (%) = We/Wt × 100% LE (%) = We/Wm × 100%
2. EXPERIMENTAL DETAILS 2.1. Materials In this study, D,L-lactide/glycolic acid (PLGA, ratio 75:25, Mw 15000-23000 Da) was purchased from Jinan Daigang Biotech Co., Ltd., China. DOX was purchased from Dalian Meilun Biological Technology Co., Ltd., China. Poly vinyl alcohol (PVA, Mw 31000 Da) was purchased from Aladdin Industrial Corporation. Vatamine E-D--tocopheryl polyethylene glycol 1000 succinate (VE-TPGS) was purchased from Shanghai Macklin Biochemical Co., Ltd., China. 3-(4.5-Dimethylthiazol-2-yl)2,5-diphenyltetrazaliumbromide (MTT) and RPMI-1640 cell culture medium were purchased from Beijing Solarbio Science & Technology Co., Ltd., China. Ethanol, acetonitrile, and dimethyl sulfoxide (DMSO) were used as analytic reagents. The water used in this work was pretreated by the Milli-Q plus system (Millipore, Bedford, MA, USA). The MCF-7 human breast cancer cell line and S180 sarcoma tumor cell line were obtained from the Chinese Academy of Science Cell Bank. 5464
(We: The weight of drug loaded; Wt: The total drug in the preparation; Wm: The total weight of the PLGANPs). 2.4. In Vitro Drug Release from Test PLGANPs The release profile of DOX from PLGANPs was obtained by a dialysis method. Briefly, the test sample (1 mL) was placed in a dialysis bag (MWCO 8000–14,000). The bag was immersed in 100 mL release medium in a thermostatic rotary shaker at 100 rpm and 37 C. One release medium was pH 7.4 PBS and the other was pH 6.0 PBS. Samples from the release medium were collected at predetermined time intervals and their fluorescence intensity was measured at 480 nm. 2.5. Cell Cultures The MCF-7 human breast cancer cell line was obtained from the Chinese Academy of Sciences Cell Bank (Catalog No. HYC3204). MCF-7 cells were cultured in normal RPMI-1640 culture medium in 5% CO2 and 95% air at J. Nanosci. Nanotechnol. 19, 5463–5468, 2019
Zhang et al.
Ultrasound-Triggered Gas-Generating Doxorubicin PLGA-Nanoparticles for Cancer Therapy
37 C in a humidified incubator.18 The cell was then ready for the experiments outlined in Sections 2.6 and 2.7. 2.6. In Vitro Cytotoxicity The cytotoxic activity of DOX forms were evaluated using an MTT assay. For this study, cancer cells were seeded (5 × 103 cells/well) in 96-well plates containing RPMI1640 medium. Test formulations with different DOX concentrations were added to the cells and incubated for 4 h. Probe sonication (1 W, 1 Hz, 1 min) were used as the methods to release DOX from the delivery system after incubation for 4 h. The cells were cultured for 48 h and incubated in the growth medium containing 20 L of 5 mg/mL MTT reagents for an additional 4 h. After that, 150 L of DMSO was added to each well to ensure solubilization of the formazan crystals. Finally, optical density readings were obtained using a multi-well scanning spectrophotometer at a wavelength of 490 nm.
vein. After injection, ultrasound images were obtained by using a Vevo 770 US imaging system with a RMV 706 probes. The imaging intensity was determined at both experimental and control groups. Secondly, these mice were randomly divided into 5 groups as follows: (1) saline (control group), (2) free DOX, (3) DOX@PLGANPs, (4) DOX/NaHCO3 @PLGANPs, (5) DOX/NaHCO3 @PLGANPs + US treatment (1 W/cm2 , 2 min). All groups were treated by intravenous injection at every two days and 6 injections totally. To evaluate antitumor efficacy, relative tumor volume and relative body weight were recorded and calculated. At the end of treatment, the biochemical parameters were examined to observe their cardiac and renal toxicity, including routine analysis of blood and cardiac function tests (performed by Zhengzhou University Hospital). 2.10. Statistical Analysis All statistical analyses results were expressed as mean ± standard deviation. The data were compared using the GraphPad Prism 5 and P values <0.05 were deemed to be statistically significant.
2.7. Cellular Uptake To investigate the targeted ability of PLGANPs, coumarin-6 (with green fluorescence) was used as the marker of PLGANPs. MCF-7 cells were seeded at a density of 2 × 105 per well in 6-well plates to observe the 3. RESULTS AND DISCUSSION profile of cellular uptake with or without NaHCO3 . When 3.1. Synthesis and Characterization of the cells reached 80% confluence, the culture medium DOX/NaHCO3@PLGANPs was changed to fresh medium containing coumarin-6, IP: 79.110.28.36 On: Sun, The 25 Aug 2019 13:49:52 DOX/NaHCO @PLGANPs and DOX/NaHCO3 @ coumarin-6-PLGANPs, NaHCO3 @coumarin-6-PLGANPs Copyright: American Scientific Publishers3 PLGANPs were prepared using a double-emulsion w/o/w 1 Hz, by Ingenta and NaHCO3 @coumarin-6-PLGANPs + US (1 W, Delivered solvent-diffusion-evaporation method. Schematic diagram 1 min), respectively. The cells were incubated for 4 h. of DOX/NaHCO @PLGANPs was shown in Figure 1(a). Then the medium was removed, and the cells were washed 3 The TEM images indicated that the particles size of three times with PBS and fixed in 4% paraformaldehyde 650 nm (Fig. 1(b)). Their size ranged from 600 nm to for 15 min. Finally, the cells were imaged using a flu700 nm (Fig. 1(c)) with a zeta potential of approximately orescence microscope (Zeiss LSM 510, Thornwood, NY, −21.8 mV (Fig. 1(d)). Table I listed important characUSA). teristic parameters of the test PLGANPs. The results of DOX/NaHCO3 @PLGANPs indicated that entrapment effi2.8. Animal Model ciencies (EE) was 89.4 ± 3.5% and drug loading (DL) was Kunming (KM) mice (female, 20 ± 2 g, 4–6 weeks old) 15.8 ± 2.3%. were purchased from the Henan Laboratory Animal Center. All animal experiments were in keeping with the 3.2. In Vitro Drug Release of Test guidelines for humane and responsible use of animals DOX/NaHCO3@PLGANPs in research set by ZhengZhou University. Approximately In order to determine the in vitro release profile 2 × 106 S180 ascites cells were injected into the armpits with subcutaneous of the mice. When the tumors grow to of DOX, three formulations (DOX@PLGANPs, DOX/ the volume of 60–100 mm3 , these mice were ready for the NaHCO3 @PLGANPs and DOX/NaHCO3 @PLGANPs + experiments outlined in Section 2.9. US) were incubated in two different medium (pH 7.4 PBS and pH 6.0 PBS). From Figure 2, DOX in DOX/ NaHCO3 @PLGANPs with US treatment released more 2.9. In Vivo Antitumor Efficacy and the than without US in pH 7.4 and pH 6.0, indicating Safety Evaluation that ultrasound can promote drug released. Moreover, To investigate the target effect in vitro, degassed water DOX in DOX/NaHCO3 @PLGANPs released more than was as the negative control for ultrasonic imaging of DOX@PLGANPs at the same pH. Both two formuDOX/NaHCO3 @PLGANPs. Similar tumor-bearing mice lations release more DOX at pH 6.0 than pH 7.4. model was used for ultrasound imaging. When tumor DOX/NaHCO3 @PLGANPs release the drug completely at volume was about 100 mm3 , DOX/NaHCO3 @PLGANPs 12 h, but other formulations need more time to release the (5 mg/kg) was injected into the mice via the tail J. Nanosci. Nanotechnol. 19, 5463–5468, 2019
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Figure 1. (a) Schematic illustrations showing the structure of DOX/NaHCO3 @PLGANPs; (b) the TEM micrograph of the PLGANPs; (c) particle size of DOX/NaHCO3 @PLGANPs; (d) particle zeta potential of DOX/NaHCO3 @PLGANPs.
drugs, which indicated that DOX/NaHCO3 @PLGANPs is pH-sensitive and easy to release DOX in the microacid environment of the tumor.4
Figure 4. With the concentration increase of the above groups, the cell inhibition increases, this showed that our formulations were concentration-dependent. Moreover, the inhibition of those groups: free DOX < DOX@PLGANPs < DOX/NaHCO3 @PLGANPs < 3.3. Enhanced Cell Uptake of DOX In Vitro To investigate the uptake capacity of tumor cells, 3 @PLGANPs + US.” Cell inhibition of IP: 79.110.28.36 On: Sun, “DOX/NaHCO 25 Aug 2019 13:49:52 DOX/NaHCO @PLGANPs was better than that of formucoumarin-6 was used to mark the Copyright: green fluorescence 3 American Scientific Publishers without NaHCO3 due to pH sensitivity of NaHCO3 (control, bylation of the PLGA19 in the following groups Delivered Ingenta may enhance more DOX release into cells. Moreover, the coumarin-6, coumarin-6-PLGANPs, NaHCO3 @coumarincell inhibition of “DOX/NaHCO3 @PLGANPs + US” was 6-PLGANPs and NaHCO3 @coumarin-6-PLGANPs + US (1 W, 1 Hz, 1 min)). After 4 hours, the samples were greater than DOX/NaHCO3 @PLGANPs, which indicated taken under the inverted fluorescence microscope as that the combination of ultrasound with the preparations Figure 3. The results showed that the fluorescence intenwas able to kill tumor cells and improve drugs antitumor sity of NaHCO3 @coumarin-6-PLGANPs was stronger activity.13 14 Thus, the cavitation effect of ultrasound was than coumarin-6-PLGANPs, but control groups of and coumarin-6 group has no signal, which indicated that NaHCO3 in this delivery system plays an important role in tumor targeting. Moreover, the strongest fluorescence of “NaHCO3 @coumarin-6-PLGANPs+US” demonstrated the US can enhance more DOX release to improve the inhibition efficiency.20 3.4. In Vitro Cytotoxicity The inhibitory effects on MCF-7 cells after incubation with free DOX, DOX@PLGANPs, DOX/NaHCO3 @PLGANPs and DOX/NaHCO3 @PLGANPs + US were shown in Table I. The encapsulation efficiency (EE, %) and loading efficiency (LE, %) result. Formulation DOX@PLGANPs DOX/NaHCO3 @PLGANPs Note: PS. Mean ± standard deviation; n = 3.
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EE (%)
LE (%)
92.3 ± 4.8 89.4 ± 3.5
16.3 ± 1.7 15.8 ± 2.3
Figure 2. In vitro pH-triggered release profiles of DOX from DOX@PLGAHPs and DOX/NaHCO3 @PLGAHPs incubated at pH = 6.0 and 7.4. Data were presented as mean ± standard deviation (n = 3).
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Ultrasound-Triggered Gas-Generating Doxorubicin PLGA-Nanoparticles for Cancer Therapy
Figure 3. Qualitative results of cell uptake. (a: Control; b: Coumarin-6; c: Coumarin-6-PLGANPs; d: NaHCO3 @coumarin-6-PLGANPs; e: NaHCO3 @coumarin-6-PLGANPs + US (4 h)).
very useful for the DOX/NaHCO3 @PLGANPs delivery system to kill the tumor cells. 3.5. In Vivo Antitumor Efficacy After injection 30 min, control group with distilled water could not be observed clear signals, but DOX/NaHCO3 @PLGANPs clearly showed excellent enhancement (Fig. 5(a)) because the CO2 bubbles could be observed from DOX/NaHCO3 @PLGANPs in the microacid environment of tumor by ultrasound. Therefore, our result revealed that DOX/NaHCO3 @PLGANPs had good tumor targeting.
The change in relative tumor volumes in S180 tumorbearing mice is considered indices of tumor inhibition efficiency. Figure 5(b) showed that the relative tumor volume of the control group (Saline) increased greatly, while free DOX and other formulations could effectively inhibit the tumor growth. When the tumorbearing mice were subjected to ultrasound energy with DOX/NaHCO3 @PLGANPs, the tumor suppression effect was significantly improved. There are two possible reasons. On the one hand, the pH-sensitive tumor targeting effect promoted higher efficiency than other non-targeted formulations. Large amounts of CO2 bubbles from NaHCO3 in microacidic environment of tumor bursts PLGA shell to fast release DOX into the tumor cells and effectively inhibit tumor growth. On the other hand, cavitation forces by ultrasound also damage vascular endothelia to strengthen tumor killing ability,21 22 which is considered the appropriate way to achieve the maximum therapeutic
IP: 79.110.28.36 On: Sun, 25 Aug 2019 13:49:52 Copyright: American Scientific Publishers Delivered by Ingenta Figure 4. In vitro cytotoxicity of a various formulations (grouped as shown above) against MCF-7 cells at different concentrations of DOX for 12 h with or without the US (1 W, 1 Hz, 2 min).
Figure 5. (a) In vivo ultrasound image of tumors before and after intra tumor injection DOX/NaHCO3 @PLGANPs into a tumor-bearing mouse model for 30 min; (b) tumor growth of mice in different treatment groups within 12 days. Data were presented as mean ± standard deviation (n = 6).
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Figure 6. (a) Changes of relative body weight of mice in different groups during treatment; Data were presented as mean ± standard deviation (n = 6); (b) tumor-bearing mice blood routine examination; (c) liver function test in tumor-bearing mice.
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Acknowledgments: I would like to express heartfelt thanks to the Professor Xia Xu Group for their helpful discussions. We also appreciate School of Pharmaceutical Sciences of Zhengzhou University for providing financial support.
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Received: 1 March 2018. Accepted: 14 May 2018.
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