Paclitaxel (Ptx) has been established as one of the most important components of first line chemotherapy regimen in the treatment of lung cancer. However, the poor solubility of Ptx makes it employ Cremophor as a solvent, which greatly limits its application due to the severe adverse effect. Encapsulation of Ptx into nanoparticles substantially increases the solubility of Ptx, therefore eradicating the necessity of Cremophor involvement. Here we report on a simple way of preparing Ptx-loaded nanoparticles formed by amphiphilic poly(N-vinylpyrrolidone)-block-poly(
Lung cancer, one of the most common cancers worldwide, accounts for the most cancer related deaths [
Amphiphilic copolymers attract more and more attention as a promising drug delivery system for chemotherapy. In recent years, there are plenty of studies demonstrating the effective antitumor efficacy of Ptx-loaded nanoparticles with amphiphilic copolymers poly(caprolactone)-
Though PEG possesses several advantages as drug carrier such as good biocompatibility, hydrophilicity, and the absence of antigenicity and immunogenicity [
Recently it has been reported in several studies that PVP is utilized as the hydrophilic part of the amphiphilic copolymers as drug carriers [
In the current study, we utilized PVP-
N-Vinylpyrrolidone (98%, Acros) was purified by fractional distillation.
PVP-
Mean diameter and size distribution were measured before lyophilization by photon correlation spectroscopy (dynamic light scattering (DLS)) using a Brookhaven BI-9000 AT instrument (Brookhaven Instruments Corporation, Holtsville, NY, USA). Zeta potential was measured by the laser Doppler anemometry (Zeta Plus, Zeta Potential Analyzer; Brookhaven Instruments Corporation, Holtsville, NY, USA).
The solution of Ptx-NPs was stored at room temperature. Particle sizes were measured by DLS every 2 days for 15 days to evaluate stability.
The concentration of Ptx was assayed on a Shimadzu LC-10AD (Shimadzu, Japan) HPLC system equipped with a Shimadzu UV detector and an Agilent C-18, 5
For in vitro release detection, 5 mg freeze-dried Ptx-NPs were dissolved in 1 mL PBS and then put into a dialysis bag (12 kd cutoff, Sigma), followed by immersion in PBS with slight agitation. At each time point 1 mL samples were taken from the medium outside and quantified for Ptx concentrations by HPLC. After sampling, equal volume of fresh PBS was immediately added into the incubation medium. The concentration of Ptx was expressed as a percentage of the total Ptx in the nanoparticles and plotted as a function of time, respectively.
Cytotoxicity of Ptx-NPs against NCI1975 and A549 cells was assessed by MTT assay as reported in our previous study [
NCI1975 cells were treated with 40
The expression of related proteins was examined by western blot analysis as reported in previous studies [
Cells were cultured under the same conditions as in the western blot analysis. Caspase-3 activity was measured by the caspase colorimetric protease assay kit (Keygen Biotech, Nanjing, China) by following the manufacturer’s instruction. The optical density was measured at 405 nm. The obtained values were expressed as folds of controls.
The significance of observed differences between groups were analyzed by one-way analysis of variance (ANOVA) followed by taking multiple comparison test using SPSS 11.5 software package (SPSS Inc., IL, USA). Where two groups were compared, independent sample
In our previous reports, gel permission chromatography was adopted to detect the molecular weight of PVP-
Mean particle size and drug load efficiency of Ptx-NPs.
Feeding ratio | Particle size (nm)a | Zeta potential (Mv) | DLC (%)b | EE (%)c |
---|---|---|---|---|
15% | 115.5 ± 13.3 | −3.2 ± 0.3 | 17.2 ± 0.5 | 91.2 ± 2.3 |
aThe SD value was for the mean particle size obtained from the three measurements of a single batch.
bDLC, drug loading content.
cEE, encapsulation efficiency.
Characterization of PVP-
As shown in Table
In the in vitro release study, Ptx was released from the nanoparticles in a sustained manner. The initial burst release in the first 5 hours was mainly attributed to the drugs adhering to the surface of nanoparticles. After that, sustained release of Ptx was observed due to the continuous release of encapsulated Ptx from the core of nanoparticles (Figure
The viability of NCI 1975 and A549 cells exposed to a series of concentrations of Ptx or Ptx-NPs was detected by MTT assay. Blank nanoparticles were almost nontoxic to two kinds of lung adenocarcinoma cells as shown in Figure
Cytotoxicity of blank PVP-
Figure
Cytotoxicity of Ptx and Ptx-NPs on NCI1975 and A549 cells. (a) Dose-dependent cytotoxicity of free Ptx and Ptx-NPs against NCI1975 cells. (b) Dose-dependent cytotoxicity of free Ptx and Ptx-NPs against A549 cells. Data are presented as mean ± SD (
To further evaluate the apoptosis-inducing and antiproliferative effect of Ptx-NPs, cells were dual-stained by Hoechst 33342 and Edu. As to Hoechst 33342 staining, live cells have normal blue nucleuses while apoptotic cells appear to have bright blue nucleuses with distinctive condensed or fragmented chromatin. On the contrary, Edu incorporation assay was performed to detect the proliferation of cells exposed to different agents [
Dual staining of NCI1975 cells by Hoechst 33342 and Edu when treated with the equivalent dose of Ptx and Ptx-NPs. (a), (e) Lower and higher magnification fluorescent field of cells in the control group. (b), (f) Lower and higher magnification fluorescent field of cells treated with blank nanoparticles. (c), (g) Lower and higher magnification fluorescent field of cells treated with free Ptx at a dose of 40
The expression of antiapoptotic protein Bcl-2 and proapoptotic protein Bax in NCI1975 cells exposed to the equivalent dose of Ptx or Ptx-NPs detected by western blot analysis.
It is reported that apoptosis is characterized by the imbalance of proapoptotic and antiapoptotic proteins, which leads to the activation of caspase castrade [
The extent of apoptosis could also be evaluated by the activity of caspase-3 [
Caspase-3 activities in cells treated with an equivalent dose of Ptx or Ptx-NPs.
Results from the current study demonstrated that delivery of Ptx by PVP-
In the current study, we reported a simple and stable way to construct Ptx-loaded PVP-
The authors declare no conflict of interests.
Donghui Zheng, Huiping Ye, and Can Luo contributed equally to this work.
This work was supported by the National Natural Science Foundation of China (nos. 81101902 and 81270817), A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and Guizhou Scientific Grant LG2012-068.