Antitumor effect of iRGD-modified liposomes containing conjugated linoleic acid–paclitaxel (CLA-PTX) on B16-F10 melanoma

Abstract

In the present study, we prepared a novel delivery system of iRGD (CRGDK/RGPD/EC)-modified sterically stabilized liposomes (SSLs) containing conjugated linoleic acid–paclitaxel (CLA-PTX). The anti-tumor effect of iRGD-SSL-CLA-PTX was investigated on B16-F10 melanoma in vitro and in vivo. The in vitro targeting effect of iRGD-modified SSLs was investigated in a real-time confocal microscopic analysis experiment. An endocytosis-inhibition assay was used to evaluate the endocytosis pathways of the iRGD-modified SSLs. In addition, the in vitro cellular uptake and in vitro cytotoxicity of iRGD-SSL-CLA-PTX were evaluated in B16-F10 melanoma cells. In vivo biodistribution and in vivo antitumor effects of iRGD-SSL-CLA-PTX were investigated in B16-F10 tumor-bearing mice. The induction of apoptosis by iRGD-SSL-CLA-PTX was evaluated in tumor-tissue sections. Real-time confocal microscopic analysis results indicated that the iRGD-modified SSLs internalized into B16-F10 cells faster than SSLs. The identified endocytosis pathway of iRGD-modified SSLs indicated that energy- and lipid raft-mediated endocytosis played a key role in the liposomes’ cellular uptake. The results of the cellular uptake experiment indicated that the increased cellular uptake of CLA-PTX in the iRGD-SSL-CLA-PTX-treated group was 1.9-, 2.4-, or 2.1-fold compared with that in the CLA-PTX group after a 2-, 4-, or 6-hour incubation, respectively. In the biodistribution test, the CLA-PTX level in tumor tissues from iRGD-SSL-CLA-PTX-treated mice at 1 hour (1.84±0.17 μg/g) and 4 hours (1.17±0.28 μg/g) was 2.3- and 2.0-fold higher than that of CLA-PTX solution at 1 hour (0.79±0.06 μg/g) and 4 hours (0.58±0.04 μg/g). The value of the area under the curve for the first 24 hours in the tumors of iRGD-SSL-CLA-PTX-treated mice was significantly higher than that in the SSL-CLA-PTX and CLA-PTX solution-treated groups ( P <0.01). The in vivo antitumor results indicated that iRGD-SSL-CLA-PTX significantly inhibited the growth of B16-F10 tumors compared with the SSL-CLA-PTX or CLA-PTX solution-treatment groups ( P <0.01). The results of tumor-cell apoptosis showed that tumors from the iRGD-SSL-CLA-PTX-treated group exhibited more advanced cell apoptosis compared with the control, CLA-PTX solution-, and SSL-CLA-PTX-treated groups. In conclusion, the antitumor effect of iRGD-SSL-CLA-PTX was confirmed on B16-F10 melanoma in vitro and in vivo.

A self-assembling nanomedicine of conjugated linoleic acid-paclitaxel conjugate (CLA-PTX) with higher drug loading and carrier-free characteristic

“…CLA-PTX also exhibits a higher cellular uptake in C6 glioma and B16-F10 melanoma cells compared with paclitaxel. In addition, the anti-tumor activity of a CLA-PTX microemulsion and CLA-PTX-loaded iRGD modified liposomes was also confirmed in in vitro and in vivo experiments1921.…”

“…HPLC analysis was carried out using an HPLC system involving an LC-20AT liquid chromatograph (SHIMADZU, Japan) and an SPD-M20A diode array detector (SHIMADZU, Japan)1821. Briefly, the determination was carried out on an ODS 3 C18 analytical column (5 μm, 250 × 4.6 mm; Phenomenex, Torrance, CA, USA) gradient eluting with mobile phases of acetonitrile (mobile phase A) and water (mobile phase B) in the mode (0–10 min, phase A, 60%; 10–12 min, phase A 60%→100%; 12–28 min, phase A 100%; 28–30 min, phase A 100%→60%; 30–40 min, phase A 60%) at the flow rate of 1.0 ml/min.…”

“…Then, cells were treated with free CLA-PTX solution or CLA-PTX@PEG NPs (10 μM) for 2, 4 or 6 h at 37 °C. The cellular uptake efficiency was determined as in our previous report2122. In detail, after incubation, cells were washed three times with cold phosphate-buffered saline (PBS, pH 7.4), and lysed with 0.1% sodium dodecyl sulfate (SDS, 0.1 ml).…”

“…The in vitro cytotoxicity of CLA-PTX@PEG NPs was evaluated in B16-F10 cells, MDA-MB-231 cells and U87-MG cells using the sulfonylrhodamine B (SRB) assay182123. The half-maximal inhibitory concentration (IC 50 ) was calculated according to the dose-effect curves using GraphPad Prism 6 software.…”

The Janus Facet of Nanomaterials

“…We are well aware that understanding NM toxicity needs more comprehensive, complex, and novel multi- and interdisciplinary approaches [ 9 – 23 ]. These are driven in many cases by furthering imaging techniques through more specific labeling and detection of the cellular fate of NMs as illustrated by (i) in vitro/in vivo fluorescence ([ 22 , 24 ]; Figure 1 ), synchrotron radiation-based (SR) Fourier transform infrared spectroscopy (FTIR) or X-ray fluorescence microscopy [ 25 ], or single photon emission computed tomography combined with X-ray computed tomography (SPECT-CT) imaging to study NM biodistribution at organ levels ( Figure 2 ); (ii) small-angle X-ray (SAXS; Figure 3 ) or neutron scattering [ 26 – 28 ], freeze-fracture combined transmission electron microscopy (FF-TEM) and sum-frequency generation (SFG) vibrational spectroscopy for determination of structure or membrane interactions of NMs [ 13 ], and in situ high-resolution TEM [ 29 ]; (iii) application of new sets of methodologies built on basic instrumentation and related expertise in combination with NM surface modifications and toxicity assaying.…”