logo

SCIENCE CHINA Life Sciences, Volume 61, Issue 4: 436-447(2018) https://doi.org/10.1007/s11427-017-9274-9

Synthesis and evaluation of a paclitaxel-binding polymeric micelle for efficient breast cancer therapy

More info
  • ReceivedNov 10, 2017
  • AcceptedDec 15, 2017
  • PublishedMar 19, 2018

Abstract

Paclitaxel (PTX) is one of the most effective anticancer drugs for the treatment of various solid tumors, but its clinical use is limited by its poor solubility, low bioavailability, and severe systemic toxicity. Encapsulation of PTX in polymeric nanoparticles is used to overcome these problems but these micelles still need improvements in stability, pharmacokinetics, therapeutic efficacy, and safety profiles. In this study, we demonstrate a facile fabrication of a stable PTX-binding micelle made from poly(ethylene glycol)-block-dendritic polylysine, whose primary amines were reacted with phenethyl isothiocyanate (PEITC), a hydrophobic anticancer agent under clinical study. The amphiphilic conjugate (PEG-Gx-PEITC; Gx, the generation of the polylysine dendron) formed well-defined micelles whose core was composed of phenyl groups and thiourea groups binding PTX via π-π stacking and hydrogen bonding. Compared with the PTX-loaded poly(ethylene glycol)-block-poly(D,L-lactide) (PEG-PDLLA/PTX) micelles in clinical use, PTX-loaded PEG-Gx-PEITC third-generation (PEG-G3-PEITC/PTX) micelles showed slowed blood clearance, enhanced tumor accumulation, and thus much improved in vivo therapeutic efficacy in both subcutaneous and orthotopic human breast cancer xenografts. Therefore, PEG-G3-PEITC is a promising drug delivery system for PTX in the treatment of breast cancer.


Acknowledgment

This work was supported by the National Natural Science Foundation of China (U1501243, 51603181), the National Basic Research Program (2014CB931900), the National Natural Science Foundation of China (51603181) and the Fundamental Research Funds for the Central Universities (2016QNA4024) for financial support.


Interest statement

The author(s) declare that they have no conflict of interest.


Supplement

SUPPORTING INFORMATION

Figure S1ƒ1H NMR spectra of PEG-G4-PEITC in DMSO-d6.

Figure S2ƒThe CMC values of polymers measured with a Nile red fluorescent method.

Figure S3ƒStability of PEG-G3-PEITC/PTX micelles.

Figure S4ƒ1H NMR spectra of PTX in CDCl3, PEG-G3-PEITC in DMSO-d6, and PEG-G3-PEITC/PTX micelles in D2O.

Figure S5ƒCytotoxicity of PEG-G3-PEITC blank micelles and PEG-PDLLA blank micelles.

Figure S6ƒChange of body weight.

Figure S7ƒHistopathologic analysis.

The supporting information is available online at http://life.scichina.com and https://link.springer.com. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.


References

[1] Berglund, M., Dalence-Guzman, M.F., Skogvall, S., and Sterner, O. (2008). SAR studies of capsazepinoid bronchodilators. Part 2: chlorination and catechol replacement in the A-ring. Bioorgan Med Chem 16, 2513–2528. Google Scholar

[2] Burt H.M., Zhang X., Toleikis P., Embree L., Hunter W.L.. Development of copolymers of poly(D,L-lactide) and methoxypolyethylene glycol as micellar carriers of paclitaxel. Colloids Surface B, 1999, 16: 161-171 CrossRef Google Scholar

[3] Carstens M.G., de Jong P.H.J.L.F., van Nostrum C.F., Kemmink J., Verrijk R., de Leede L.G.J., Crommelin D.J.A., Hennink W.E.. The effect of core composition in biodegradable oligomeric micelles as taxane formulations. Eur J Pharm Biopharm, 2008, 68: 596-606 CrossRef PubMed Google Scholar

[4] Chen H., Kim S., He W., Wang H., Low P.S., Park K., Cheng J.X.. Fast release of lipophilic agents from circulating PEG-PDLLA micelles revealed by in vivo förster resonance energy transfer imaging. Langmuir, 2008, 24: 5213-5217 CrossRef PubMed Google Scholar

[5] Fang R.H., Aryal S., Hu C.M.J., Zhang L.. Quick synthesis of lipid-polymer hybrid nanoparticles with low polydispersity using a single-step sonication method. Langmuir, 2010, 26: 16958-16962 CrossRef PubMed Google Scholar

[6] Guo Z., Zou Y., He H., Rao J., Ji S., Cui X., Ke H., Deng Y., Yang H., Chen C., et al. Bifunctional platinated nanoparticles for photoinduced tumor ablation. Adv Mater, 2016, 28: 10155-10164 CrossRef PubMed Google Scholar

[7] Gupta, P., Wright, S.E., Kim, S.H., and Srivastava, S.K. (2014). Phenethyl isothiocyanate: a comprehensive review of anti-cancer mechanisms. Biochim Biophys Acta 1846, 405–424. Google Scholar

[8] Kim S.C., Kim D.W., Shim Y.H., Bang J.S., Oh H.S., Kim S.W., Seo M.H.. In vivo evaluation of polymeric micellar paclitaxel formulation: toxicity and efficacy. J Control Release, 2001, 72: 191-202 CrossRef Google Scholar

[9] Liu K., Cang S., Ma Y., Chiao J.W.. Synergistic effect of paclitaxel and epigenetic agent phenethyl isothiocyanate on growth inhibition, cell cycle arrest and apoptosis in breast cancer cells. Cancer Cell Int, 2013, 13: 10 CrossRef PubMed Google Scholar

[10] Mishra, A., Dwivedi, J., Shukla, K., and Malviya, P. (2014). X-Ray diffraction and Fourier transformation infrared spectroscopy studies of copper (II) thiourea chloro and sulphate complexes. Journal of Physics Conference Series. Google Scholar

[11] Muthoosamy K., Abubakar I.B., Bai R.G., Loh H.S., Manickam S.. Exceedingly higher co-loading of curcumin and paclitaxel onto polymer-functionalized reduced graphene oxide for highly potent synergistic anticancer treatment. Sci Rep, 2016, 6: 32808 CrossRef PubMed ADS Google Scholar

[12] Saeed A., Bolte M., Erben M.F., Pérez H.. Intermolecular interactions in crystalline 1-(adamantane-1-carbonyl)-3-substituted thioureas with Hirshfeld surface analysis. Crystengcomm, 2015, 17: 7551-7563 CrossRef Google Scholar

[13] Shao S., Zhou Q., Si J., Tang J., Liu X., Wang M., Gao J., Wang K., Xu R., Shen Y.. A non-cytotoxic dendrimer with innate and potent anticancer and anti-metastatic activities. Nat Biomed Eng, 2017, 1: 745-757 CrossRef Google Scholar

[14] Singla A.K., Garg A., Aggarwal D.. Paclitaxel and its formulations. Int J Pharm, 2002, 235: 179-192 CrossRef Google Scholar

[15] Skog S., He Q., Khoshnoud R., Fornander T., Rutqvist L.E.. Genes related to growth regulation, DNA repair and apoptosis in an oestrogen receptor-negative (MDA-231) versus an oestrogen receptor-positive (MCF-7) breast tumour cell line. Hum Hered, 2004, 25: 41-47 CrossRef PubMed Google Scholar

[16] Stirland D.L., Nichols J.W., Miura S., Bae Y.H.. Mind the gap: a survey of how cancer drug carriers are susceptible to the gap between research and practice. J Control Release, 2013, 172: 1045-1064 CrossRef PubMed Google Scholar

[17] Sun Q., Radosz M., Shen Y.. Challenges in design of translational nanocarriers. J Control Release, 2012, 164: 156-169 CrossRef PubMed Google Scholar

[18] Tang B., Fang G., Gao Y., Liu Y., Liu J., Zou M., Cheng G.. Liprosomes loading paclitaxel for brain-targeting delivery by intravenous administration: in vitro characterization and in vivo evaluation. Int J Pharm, 2014, 475: 416-427 CrossRef PubMed Google Scholar

[19] Tang Z., He C., Tian H., Ding J., Hsiao B.S., Chu B., Chen X.. Polymeric nanostructured materials for biomedical applications. Prog Polymer Sci, 2016, 60: 86-128 CrossRef Google Scholar

[20] Tyrrell Z.L., Shen Y., Radosz M.. Fabrication of micellar nanoparticles for drug delivery through the self-assembly of block copolymers. Prog Polymer Sci, 2010, 35: 1128-1143 CrossRef Google Scholar

[21] Wang J., Sun X., Mao W., Sun W., Tang J., Sui M., Shen Y., Gu Z.. Tumor redox heterogeneity-responsive prodrug nanocapsules for cancer chemotherapy. Adv Mater, 2013, 25: 3670-3676 CrossRef PubMed Google Scholar

[22] Wang Y.J., Wang C., Gong C.Y., Wang Y.J., Guo G., Luo F., Qian Z.Y.. Polysorbate 80 coated poly (ɛ-caprolactone)-poly (ethylene glycol)-poly (ɛ-caprolactone) micelles for paclitaxel delivery. Int J Pharm, 2012, 434: 1-8 CrossRef PubMed Google Scholar

[23] Wei Y., Li L., Xi Y., Qian S., Gao Y., Zhang J.. Sustained release and enhanced bioavailability of injectable scutellarin-loaded bovine serum albumin nanoparticles. Int J Pharm, 2014, 476: 142-148 CrossRef PubMed Google Scholar

[24] Wu, X., Zhou, Q., and Xu, K. (2009). Are isothiocyanates potential anti-cancer drugs? Acta Pharmacol Sin 30, 501–512. Google Scholar

[25] Xi X., Hu S., Zhou Z., Liu X., Tang J., Shen Y.. Dendrimers with the protocatechuic acid building block for anticancer drug delivery. J Mater Chem B, 2016, 4: 5236-5245 CrossRef Google Scholar

[26] Xiao K., Luo J., Fowler W.L., Li Y., Lee J.S., Xing L., Cheng R.H., Wang L., Lam K.S.. A self-assembling nanoparticle for paclitaxel delivery in ovarian cancer. Biomaterials, 2009, 30: 6006-6016 CrossRef PubMed Google Scholar

[27] Yano S., Takehara K., Kishimoto H., Tazawa H., Urata Y., Kagawa S., Bouvet M., Fujiwara T., Hoffman R.M.. In vivo selection of intermediately- and highly- malignant variants of triple-negative breast cancer in orthotopic nude mouse models. Anticancer Res, 2016, 36: 6273-6278 CrossRef PubMed Google Scholar

[28] Zhang P., Huang Y., Liu H., Marquez R.T., Lu J., Zhao W., Zhang X., Gao X., Li J., Venkataramanan R., et al. A PEG-Fmoc conjugate as a nanocarrier for paclitaxel. Biomaterials, 2014, 35: 7146-7156 CrossRef PubMed Google Scholar

[29] Zhang P., Huang Y., Kwon Y.T., Li S.. PEGylated fmoc-amino acid conjugates as effective nanocarriers for improved drug delivery. Mol Pharm, 2015, 12: 1680-1690 CrossRef PubMed Google Scholar

[30] Zhao B., Zhou Z., Shen Y.. Effects of chirality on gene delivery efficiency of polylysine. Chin J Polym Sci, 2016, 34: 94-103 CrossRef Google Scholar

[31] Zhao L., Zhou Y., Gao Y., Ma S., Zhang C., Li J., Wang D., Li X., Li C., Liu Y., et al. Bovine serum albumin nanoparticles for delivery of tacrolimus to reduce its kidney uptake and functional nephrotoxicity. Int J Pharm, 2015, 483: 180-187 CrossRef PubMed Google Scholar

[32] Zhou Z., Ma X., Jin E., Tang J., Sui M., Shen Y., Van Kirk E.A., Murdoch W.J., Radosz M.. Linear-dendritic drug conjugates forming long-circulating nanorods for cancer-drug delivery. Biomaterials, 2013, 34: 5722-5735 CrossRef PubMed Google Scholar

[33] Zhou Z., Ma X., Murphy C.J., Jin E., Sun Q., Shen Y., Van Kirk E.A., Murdoch W.J.. Molecularly precise dendrimer-drug conjugates with tunable drug release for cancer therapy. Angew Chem Int Ed, 2014, 53: 10949-10955 CrossRef PubMed Google Scholar

[34] Zhou Z., Liu X., Zhu D., Wang Y., Zhang Z., Zhou X., Qiu N., Chen X., Shen Y.. Nonviral cancer gene therapy: delivery cascade and vector nanoproperty integration. Adv Drug Deliver Rev, 2017, 115: 115-154 CrossRef PubMed Google Scholar

[35] Zhu A., Miao K., Deng Y., Ke H., He H., Yang T., Guo M., Li Y., Guo Z., Wang Y., et al. Dually pH/reduction-responsive vesicles for ultrahigh-contrast fluorescence imaging and thermo-chemotherapy-synergized tumor ablation. ACS Nano, 2015, 9: 7874-7885 CrossRef Google Scholar

  • Figure 1

    Schematic representation of self-assembly of PTX-binding polymeric micelles and the intermolecular interaction between drug and carrier. PEG-G3-PEITC/PTX micelles have improved serum stability, slowed blood clearance, and enhanced tumor accumulation.

  • Figure 2

    Characterizations of PEG-Gx-PEITC. A, 1H NMR spectrum of PEG-G3-PEITC measured in DMSO-d6. B, MALDI-TOF MS spectra. C, GPC traces (DMF, 50°C, 0.8 mL min−1).

  • Figure 3

    Characterizations of micelles. A, Sizes of PEG-G3-PEITC blank micelles, PEG-G3-PEITC/PTX, PEG-PDLLA blank micelles, and PEG-PDLLA/PTX measured by DLS in phosphate-buffered saline solution (PBS). B, TEM images of micelles. Scale bar, 200 nm.

  • Figure 4

    Study of interactions between PEG-G3-PEITC and PTX. A, 13C NMR of PTX and PTX mixed with PEG-G3-PEITC in CDCl3. The signal shift index (Δ) was defined as the chemical shift change of the carbon atom before and after mixing with carriers compared with free PTX. Sum was calculated as cumulative absolute values of the changes of all the benzene-ring carbon atoms’ chemical shifts. PTX was mixed with PEG-G3-PEITC at a molar ratio of 1:2 in accordance with the ratio in micelles. B, Fourier-transform infrared analysis (FTIR) spectra of PTX, PEG-G3-PEITC, and PEG-G3-PEITC/PTX micelles. C, X-ray diffraction (XRD) patterns of PTX, PEG-G3-PEITC, and PEG-G3-PEITC/PTX micelles.

  • Figure 5

    The drug release profiles of PEG-G3-PEITC/PTX and PEG-PDLLA/PTX micelles in mediums of PBS (0.01 mol L−1, pH 7.4 or 5.4). Data are depicted as mean±SD.

  • Figure 6

    In vitro cytotoxicity. Cytotoxicity of PEG-G3-PEITC/PTX, PEG-PDLLA/PTX micelles, and PTX against HeLa (A), MCF-7 (B), and MDA-MB-231 cells (C) after 48 h incubation. Data are depicted as mean±SD (n=6).

  • Figure 7

    Plasma pharmacokinetic profiles and biodistribution. A, Blood clearance profiles of PEG-PDLLA/PTX and PEG-G3-PEITC/PTX micelles. ICR mice (n=3) received an equivalent dose of 10 mg kg−1 PTX by intravenous injection. Blood samples were collected at 3 min and 0.5, 1, 2, 6, 12, 24 and 36 h. The concentration in the plasma was normalized as the percentage of PTX concentration at 3 min. Data are depicted as mean±SD. B, In vivo real-time biodistribution study. Nude mice bearing MCF-7 tumors received Cy5-labeled micelles (eq. 10 mg kg−1 PTX). Animals under anesthesia underwent imaging at predetermined intervals after injection (5 min and 1, 2, 5, 8, 12, 24 and 36 h). Dotted circle outlines the tumor. The urinary bladder is indicated by arrows.

  • Figure 8

    Antitumor effect. Nude mice with MCF-7 or MDA-MB-231 tumors received PBS, PEG-PDLLA/PTX, or PEG-G3-PEITC/PTX micelles (n=5, q2×5, eq. 10 mg kg−1 PTX). Dosing schedules are indicated by black arrows. A, Tumor volumes as a function of time for mice with MCF-7 tumors. B, Images of MCF-7 tumors at the end of the experiment. Dotted circle represents mice which were tumor-free. C, Tumor volumes as a function of time for mice with MDA-MB-231 tumors. D, Images of MDA-MB-231 tumors harvested at the end of the experiment. E, Histologic features of MDA-MB-231 tumors. Tissue paraffin sections were stained with hematoxylin-eosin (H&E) and examined by light microscopy. Tumor apoptosis is indicated by arrows. Data are depicted as mean±SD. Statistical significance: *, P<0.05; **, P<0.005, and ***, P<0.0005. Scale bar, 70 µm.

  • Table 1   Characterizations of polymers and PTX-loaded micelles

    Polymers

    PEG-G3-PEITC

    PEG-G4-PEITC

    PEG-PDLLA

    Block length*

    5,000:2,143

    5,000:4,484

    2,000:2,000

    m/z**

    7,146.4

    9,251.6

    Mn, GPC

    16,000

    18,000

    Mw, GPC

    17,000

    20,000

    PDI***

    1.06

    1.09

    CMC (mg mL−1)****

    0.031

    0.025

    0.036

    Size of blank micelles (nm)*****

    28

    62

    24

    Size of PTX-loaded micelles (nm)*****

    31

    72

    39

    PDI*****

    0.13

    0.31

    0.24

    Zeta potential (mV)

    −6.69

    −4.03

    −4.85

    PTX loading content (wt.%)******

    23.7

    5.6

    22.8

    Encapsulation efficiency (wt.%)******

    98.3

    52.1

    97.4

    *, Determined by 1H NMR spectra. **, Determined by MALDI-TOF MS. ***, Polydispersity index (PDI) obtained by GPC. ****, Critical micelle concentration (CMC) was measured with a Nile red fluorescent method (Figure S2 in Supporting Information). *****, Measured by dynamic light scattering (DLS). ******, PTX content was determined by high-performance liquid chromatography (HPLC).

  • Table 2   of PEG-G3-PEITC/PTX, PEG-PDLLA/PTX micelles, and PTX on different cancer cell lines.

    Formulation

    IC50(μg PTX mL−1)

    HeLa

    MCF-7

    MDA-MB-231

    PEG-G3-PEITC/PTX

    0.244

    0.012

    >1

    PEG-PDLLA/PTX

    0.277

    0.021

    >1

    PTX

    0.211

    0.014

    >1

  • Table 3   Pharmacokinetic parameters of PEG-PDLLA/PTX and PEG-G3-PEITC/PTX micelles (ICR mice, =3, intravenously, eq. PTX)

    Parameter

    Unit

    PEG-PDLLA/PTX

    PEG-G3-PEITC/PTX

    T1/2 alpha

    h

    0.12±0.04

    0.27±0.17

    T1/2 beta

    h

    10.42±1.58

    16.85±4.29

    AUC0~t

    μg mL−1 h−1

    220.92±34.86

    445.83±178.08*

    T1/2 alpha: half-life of distribution phase. T1/2 beta: half-life of elimination phases. Data are depicted as mean±SD. Statistical significance: *, P<0.05.

Copyright 2019 Science China Press Co., Ltd. 《中国科学》杂志社有限责任公司 版权所有

京ICP备18024590号-1