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Abstract
Nanoscale drug carriers have been extensively developed to improve drug therapeutic efficiency. However, delivery of chemotherapeutic agents to tumor tissues and cells has not been favorably managed. In this study, we developed a novel “intelligent” nanoparticle, consisting of a gelatinase-cleavage peptide with poly(ethylene glycol) (PEG) and poly(ɛ-caprolactone) (PCL)-based structure for tumor-targeted docetaxel delivery (DOC-TNPs). The docetaxel-loaded PEG-PCL nanoparticles (DOC-NPs) that did not display gelatinase-stimuli behaviors were used as a control. We found clear evidence that the DOC-TNPs were transformed by gelatinases, allowing drug release and enhancing the cellular uptake of DOC (P < 0.01). In vivo biodistribution study demonstrated that targeted DOC-TNPs could accumulate and remain in the tumor regions, whereas non-targeted DOC-NPs rapidly eliminated from the tumor tissues. DOC-TNPs exhibited higher tumor growth suppression than commercialized Taxotere® (docetaxel; Jiangsu Hengrui Medicine Company, Jiangsu, China) and DOC-NPs on hepatic H22 tumor model via intravenous administration (P < 0.01). Both in vitro and in vivo experiments suggest that the gelatinase-mediated nanoscale delivery system is promising for improvement of antitumor efficacy in various overexpressed gelatinase cancers.
Acknowledgments
This work has been supported by the National Natural Science Foundation of China (Nos 30872471, 81071815, 81001408) and international cooperation plan of Nanjing Science and Technology Bureau (No 201001137). We also thank Mr Tom Morse for his helpful advice in editing the manuscript.
Disclosure
The authors report no conflicts of interest in this work.
Electronic supplementary data
Materials and methods
Docetaxel and Taxotere® were kindly provided by Jiangsu Hengrui Medicine Company (Jiangsu, China). RPMI 1640 and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were purchased from Sigma Chemical Company (St Louis, MO). Murine hepatic carcinoma cell line H22 and human colon carcinoma cell line LOVO were purchased from Shanghai Institute of Cell Biology (Shanghai, China).
Particle size and morphology characterization
1H-NMR spectra were recorded on an AVANCE 300 MHz spectrometer (Bruker, Germany) using chloroform-d1 as solvent sparately in 5 mm NMR tubes. In all the spectra tetramethylsilane (TMS) was used as the internal reference.
The nanoparticles were suspended in distilled water to achieve an appropriate level of scattering. Particle size and polydispersity of PEG-Pep-PCL and PEG-PCL were measured by dynamic light scattering (DLS) using a Brookhaven BI-9000 AT system (Brookhaven Instruments Corporation, Long Island, NY). Each sample was diluted to filtered water and measured for three times.
Drug loading content and encapsulation efficiency
The lyophilized Doc-loaded nanoparticles were dissolved in methanol (HPLC grade, Merck Sharp and Dohme, Whitehouse Station, NJ) and sonicated for 15 minutes, centrifuged at 1200 g for 5 minutes. The supernatant fractions were prepared for HPLC analysis.
Chromatographic separation was achieved using a HC-C18 column (250 × 4.6 mm, 5 μm), (Agilent Technologies, Palo Alto, CA) at 35°C. The mobile phase consisted of 50/50 double-distilled water (Millipore, Milford, MA)/acetonitrile (HPLC grade, Merck Sharp and Dohme). The flow rate was set to 1.0 mL/minute, UV detection wavelength was 230 nm. The retention time of DOC was about 3.4 minutes. The drug loading content and encapsulation efficiency were calculated by Equationequations (1)(1) and Equation(2)
(2) , respectively.
Gelatin zymography
Gelatin zymography was used to quantify the active MMP-2 and MMP-9 extracellular levels. Briefly, 100 μL culture medium was subjected to SDS-PAGE in a gel containing 10 mg/mL of gelatin. The gels were then incubated in 2.5% Triton X-100 for 2 hours and rinsed in nanopure distilled H2O. Gels were further incubated at 37°C for 24 hours in 200 mM NaCl, 5 mM CaCl2, 0.02% Brij-35, 50 mM Tris-HCl buffer (pH 8.8), 1 μM ZnCl2, then stained with 0.2% Coomassie brilliant blue R-250, and destained in 10% acetic acid and 30% methanol in H2O. Gelatinolytic activity was detected as unstained bands on a blue background. Three independent experiments were performed.
Immunohistochemistry
Tumor samples fixed in 10% neutral buffered formalin were embedded in paraffin using automatic embedding equipment, after which 5-Am sections were prepared. Immunohistochemical analysis for MMP-9 and MMP-2 was done on paraffin-embedded H22 tumor sections of mice.
Side effects study
On Day 21 after treatment, the tissues including tumor, liver, spleen, lung, kidney and heart from the tested groups treated were dissected and fixed in 10% neutral buffered formalin. The tissues were processed routinely into paraffin, sectioned at a thickness of 5 μm, stained with H&E, and examined by optical microscope. The blood biochemistry and weights of all tested mice were also scrutinized.
Results
Figure S1 Synthesis scheme of PEG-Pep-PCL copolymers.
Abbreviations: PCL, poly(ɛ-caprolactone); PEG, poly(ethylene glycol); Pep, peptide.
Figure S2 1H nuclear magnetic resonance spectra (300 MHz, 25°C) of polymers in CDCl3: (A) PEG-Pep-PCL copolymers; (B) PEG-PCL copolymers.
Note: The insert in (A) shows the proton signal from methyl groups in peptide (0.816–1.032 ppm), thus indicating portions of peptide were successfully conjugated into the copolymers.
Abbreviations: PCL, poly(ɛ-caprolactone); PEG, poly(ethylene glycol); Pep, peptide.
Table S1 Characterization of the DOC-TNPs and DOC-NPs
Figure S3 In vitro DOC release of DOC-TNPs and DOC-NPs in phosphate buffered saline.
Abbreviations: DOC, docetaxel; DOC-NPs, docetaxel-loaded nanoparticles; DOC-TNPs, tumor-targeted docetaxel-loaded nanoparticles; PCL, poly(ɛ-caprolactone); PEG, poly(ethylene glycol); Pep, peptide.
Figure S4 Detection of gelatinases by gelatin zymography, which can quantitatively measure the activity of gelatinases (MMP2/9). Following the Coomassie blue staining, gelatinases activity is detected as a white zone on black background and quantified by densitometry. (A) Scanning images of the gelatin zymography for LLC and LOVO cancer cells. (B) The expressions of LLC and LOVO cells for MMP-2 and MMP-9 by a semi-quantitative technique.
Abbreviations: LLC, Lewis lung carcinoma; MMP, matrix metalloproteases.
Figure S5 (A) Gelatin zymography of the gelatinases (MMP2/9) expression by in H22 cells. (B) Immunohistochemical analysis of the gelatinases (MMP2/9) expression in H22 tumours (×100).
Abbreviation: MMP, matrix metalloproteases.
Figure S6 (A) Body weight curves of each group during the whole experiment. (B) Blood chemistry ALT and AST test was performed on Day 21 after initiation of the treatment. (C) Blood chemistry BUN and Cr test was performed on Day 21 after initiation of the treatment. (D) Abnormal damage was not observed in the H&E stained sections of main organs (×40). The result was consistent with the normal hepatic enzyme levels measured in the blood test (a) saline; (b) empty PEG-PCL nanoparticles; (c) empty PEG-Pep-PCL nanoparticles; (d) Taxotere®; (e) DOC-NPs; (f) Doc-TNPs.
Abbreviations: ALT, alamine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; Cr, creatinine; DOC-NP, docetaxel-loaded nanoparticle; DOC-TNP, tumor-targeted docetaxel-loaded nanoparticle; H&E, hematoxylin and eosin; PCL, poly(ɛ-caprolactone); PEG, poly(ethylene glycol); Pep, peptide.
