- Nano Express
- Open Access
Nanoparticle Delivery of Artesunate Enhances the Anti-tumor Efficiency by Activating Mitochondria-Mediated Cell Apoptosis
© The Author(s). 2017
- Received: 19 February 2017
- Accepted: 29 May 2017
- Published: 12 June 2017
Artemisinin and its derivatives were considered to exert a broad spectrum of anti-cancer activities, and they induced significant anti-cancer effects in tumor cells. Artemisinin and its derivatives could be absorbed quickly, and they were widely distributed, selectively killing tumor cells. Since low concentrations of artesunate primarily depended on oncosis to induce cell death in tumor cells, its anti-tumor effects were undesirable and limited. To obtain better anti-tumor effects, in this study, we took advantage of a new nanotechnology to design novel artesunate-loaded bovine serum albumin nanoparticles to achieve the mitochondrial accumulation of artesunate and induce mitochondrial-mediated apoptosis. The results showed that when compared with free artesunate’s reliance on oncotic death, artesunate-loaded bovine serum albumin nanoparticles showed higher cytotoxicity and their significant apoptotic effects were induced through the distribution of artesunate in the mitochondria. This finding indicated that artesunate-loaded bovine serum albumin nanoparticles damaged the mitochondrial integrity and activated mitochondrial-mediated cell apoptosis by upregulating apoptosis-related proteins and facilitating the rapid release of cytochrome C.
Artemisinin and its derivatives have been widely used in the treatment of malaria due to their high anti-malarial activity and low toxicity. Researchers also found that artemisinin and its derivatives demonstrated significant anti-tumor activity in virtue of their few toxic side effects and greater tolerance by patients . It was reported that artesunate (Ats) definitely inhibited tumor cell growth and it further induced significant anti-cancer effects in tumor cells [2–4]. Some experiments indicated that Ats caused different degrees of apoptosis and oncosis in tumor cells after 48 h, and that the degrees of apoptosis and oncosis were dependent on the dose of Ats. At low concentrations, Ats did not induce obvious apoptosis in tumor cells and Ats-induced cell death was accompanied by oncosis-like death [5–8]. In order to obtain greater anti-tumor effects, a higher dosage of Ats was applied, but this further confirmed its serious toxicity and bone marrow suppression. Therefore, it is necessary to find an effective treatment to reduce the effective dosage of Ats to enhance its anti-tumor efficiency [9–11]. It was found that the mitochondria played an important role in regulating the apoptotic and oncotic effects of Ats. The mitochondria was also involved in regulating the transduction process of a wide variety of apoptotic signals [12–17]. When the mitochondria was attacked by drugs, its permeability was enhanced and membrane potential had been decreased, thus leading to endometrial swelling of mitochondrial membrane and the rapid release of cytochrome C from the mitochondria into the cytoplasm [18–20]. Furthermore, some proteins from the caspase family were activated, and the cascade reaction of cell apoptosis was induced.
To enhance the anti-tumor effects of Ats, many new techniques were attempted to increase the drug’s distribution in tumor cells or to improve the targeted delivery of drugs into cell organelles to induce cell death [21–23]. Nanoparticles (NPs) as a key tool in targeted cancer treatment have been widely investigated, and they have shown promising potential. As NPs featured a smaller particle size and a high surface area, they could enter the blood circulation via the capillaries and pass through the endothelial cell gap and migrate to the tumor site, thus achieving a drug-targeted distribution and enhancing the bioavailability of the drug. Moreover, NPs could control the release of the drug through the degradation of biomaterial in a long and smooth pattern, ultimately prolonging the eliminating half-life, improving the effective blood concentration, and reducing the dosing frequency. Most of all, drug-loaded NPs could be delivered to specific locations within the cells, improving the treatment efficacy [24–26].
To enhance the anti-tumor effects of Ats at low concentrations, we tried to design novel Ats-loaded bovine serum albumin (BSA) NPs. Because of the low pH in the tumor cells, the accumulation of a large number of hydrogen proton present on the outer mitochondrial membrane or in the intermembrane space, oppositely, the inter mitochondrial membrane is rich in negative charge due to its chemical composition and mitochondria matrix secretion, that makes a electropositive outside and negative inside transmembrane potential which can make a favoring delivery of BSA. Then, the massive accumulation of Ats in mitochondria could effectively trigger mitochondria-mediated apoptosis. The results showed that when compared with the typical oncotic death induced by free Ats, Ats was specifically transferred into the mitochondria with the mediation of BSA NPs and promoted the mitochondria-mediated activation of apoptosis-related caspase proteins. This ignited significant cell apoptosis, thus highlighting the higher cytotoxicity.
BSA was purchased from Sigma-Aldrich Co. (St Louis, MO, USA), and Ats was purchased from the Guilin Pharmaceutical Corporation (Guilin, People’s Republic of China). SMMC-7721 cells and Plc cells were purchased from the Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences (Shanghai, People’s Republic of China). All of the other purchased chemicals were of analytical grade; they were obtained from a variety of vendors.
Preparation and Characterization of Ats-Loaded BSA NPs
According to the previously reported literature , Ats-loaded BSA NPs were prepared via a desolvation method. Briefly, Ats-loaded BSA NPs were prepared by quickly dropping 1.0 mL of anhydrous alcohol containing a certain amount of Ats into 0.5 mL of BSA solution at 37 °C until opalescence. With the removal of ethanol by rotary evaporation, Ats-loaded BSA NPs were further precipitated from the medium, and then 8% glutaraldehyde in water (0.5 μL/mg of BSA) was added to induce particle crosslinking under stirring of the suspension over a period of 24 h. Finally, NPs were collected and washed three times with deionized water to further analyze their physical characterizations, including their hydrodynamic diameter, polydispersity index (PDI), zeta potential, and morphology using a Brookhaven Zetasizer (Brookhaven Instruments Corporation, Holtsville, NY, USA) and a transmission electron microscope (JEM-1200EX; JEOL, Tokyo, Japan). Determination of the encapsulation efficiency of Ats in BSA NPs was estimated using a previously reported method .
Two kinds of tumor cell lines, SMMC-7721 cells and Plc cells, were separately incubated with 20% fetal bovine serum (FBS). The cell growth density was adjusted to l × l06 cells/mL by cell count, and then the cell suspensions were diluted to l × l05 cells/mL. The diluted suspensions were further separately added into a 96-well plate (100 μL per well, about 1 × 104 cells/well) for continuous incubation for 24 h at 37 °C under conditions of 5% CO2 and 95% O2. The medium was replaced by serum-free medium in the presence of either free Ats or Ats-loaded BSA NPs featuring different concentrations of Ats, and it was subsequently incubated for 24 h. A total of 50 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (5 mg/mL) was added to each well and incubated for 4 h for culture termination. When the tetrazolium dye MTT was reduced to its insoluble formazan, 96-well plates were centrifuged at 1000 rpm for 5 min, and the supernatant was decanted from each well, followed by the addition of 150 μL of dimethyl sulfoxide (DMSO), which completely dissolved the crystals. The absorbance of the solution was measured using a microplate reader (Syneray-2; BioTek Instruments, Inc., Winooski, VT, USA) at 490 nm.
Intracellular Distribution of the BSA NP Group in Cells
SMMC-7721 cells and Plc cells at the logarithmic phase were selected and treated with trypsin digestion; the cell concentration was adjusted to l × l06 cells/mL. Next, the cultured cells were added into a 6-well cell culture plate for adherence, and the culture medium was removed followed by the addition of rhodamine B-labeled BSA NPs. The nucleus was stained with Hoechst (blue) for 15 min at 37 °C, and the mitochondria was stained by Mitotracker Green FM. The location of BSA NPs in cells was tracked within the cells using confocal laser scanning microscopy (FluoView FV10i; Olympus Corporation, Tokyo, Japan).
Mitochondrial Membrane Potential Change
JC-1 can be used to determine changes in the mitochondrial membrane potential. When the mitochondrial membrane potential was high, JC-1 was able to freely pass through the cell membrane and formed aggregates within the mitochondria, exhibiting a red fluorescence (excitation wavelength, 525 nm; emission wavelength, 590 nm); when the mitochondrial membrane potential was decreased, JC-1 was transferred from the mitochondrial matrix to the cell cytoplasm to form a green fluorescent monomer (excitation wavelength, 490 nm; emission wavelength, 530 nm). SMMC-7721 cells and Plc cells were respectively seeded in confocal dishes to reach a density of l × l06 cells/mL for continuous incubation for 12 h. Next, the culture medium was discarded and serum-free culture medium containing the dispersion of Ats or Ats-loaded BSA NPs was added into the dish. After 9 h, the medium was discarded and the cells were washed twice with PBS, followed by the addition of 2 mL of JC-1 at a concentration of 2 μmol/L; the cells were then incubated for 30 min at 37 °C under dark conditions. A laser scanning confocal microscope (FluoView FV10i; Olympus Corporation) was used to observe the imaging changes in the mitochondrial membrane.
ROS Production Measurement and Staining of the Endoplasmic Reticulum (ER)
Cells were incubated with 20% FBS and the cell growth density was adjusted to l × l06 cells/mL by cell count; and then the cell suspensions were diluted to l × l05 cells/mL. The diluted suspensions were further added into 96-well plates (100 μL per well, about 1 × 104 cells/well) for continuous incubation for 24 h at 37 °C under 5% CO2 and 95% O2. Secondly, free Ats and Ats-loaded BSA NPs were incubated with the cells for 6, 12, and 24 h, followed by continuous incubation with 10 μM of 2,7-dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich Co.) for about 30 min. Ice-cold PBS buffer was used to wash the cells three times to remove the uninternalized NPs. The intracellular DCF fluorescence intensity, which is excited at 485 nm and emitted at 530 nm, was detected using a microplate reader (Synergy-2; BioTek Instruments) to investigate the extent of oxidative stress. The test groups were treated with SMMC-7721 cells and Plc cells for 24 h, and the ER-Tracker Blue–White DPX probe (Molecular Probes, Eugene, OR, USA) was added into the cells for incubation for 30 min. After discarding the loading solution and washing the cells with PBS, the morphology change of the ER was observed by confocal laser scanning microscopy.
Cell Oncosis and Apoptosis Evaluation by Flow Cytometry
According to the protocol of our previous study , an Annexin V–fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining assay was used to evaluate the cell oncosis and apoptosis induced by free Ats and Ats-loaded BSA NPs. Cells were lysed with typsin and seeded into six-well plates at a concentration of l × l06 cells/mL for 24 h of continuous incubation. Next, the culture medium was removed and serum-free medium containing free Ats and Ats-loaded BSA NPs was added into the wells. After treatment, the cells were collected and suspended in Nicoletti buffer (Beijing 4A Biotech Co., Ltd., Beijing, People’s Republic of China) containing PI- and FITC-labeled Annexin V (AV-FITC). The morphological change of the cells was observed by confocal laser scanning microscopy. To verify the cell apoptosis and oncosis rates induced by the Ats-loaded NPs, the percentages of early apoptotic (Q4), oncotic (Q2), necrotic (Q1), and live cells (Q3) were quantified by flow cytometry.
Western Blot Analysis of Apoptosis-Related Proteins and Cytochrome C in Cells
A western blot assay was performed to determine the levels of relative proteins when free Ats or Ats-loaded NPs were incubated with SMMC-7721 cells for 24 h. Cells were lysed with ice-cold radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail and phosphatase inhibitors (Roche, Basel, Switzerland). Protein concentrations were determined using a modified BSA assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and normalized before loading on 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE). The levels of the targeted proteins were photographed and analyzed using a UVP gel analysis system (iBox Scientia 600; UVP, LLC., Upland, CA, USA).
Characteristics of Ats-Loaded BSA NPs and Cellular Viability Study
MTT was used to examine the inhibiting effects of free Ats and Ats-loaded BSA NPs in SMMC-7721 cells and Plc cells at different time intervals. The results (Fig. 1d, e) showed that the cytotoxicity of free Ats increased with the increase of the drug concentration, and Ats-loaded BSA NPs showed the gradual enhanced cytotoxicity. This proved that Ats and Ats-loaded BSA NPs inhibited the growth of tumor cells and that the inhibition ratio was dependent on the dose of Ats. Compared with free Ats, Ats-loaded BSA NPs demonstrated higher cytotoxicity and higher sensitivity in both cells, and they resulted in greater cell inhibition. As shown in Fig. 1d, e, treatment of both cells with Ats-loaded BSA NPs caused a significant decrease in cell viability at 24 h when compared with that of free Ats. The 50% maximal inhibitory concentration (IC50) values for the SMMC-7721 cells and Plc cells treated with Ats-loaded BSA NPs were 50.1 and 44.9 μg/mL at 24 h, respectively, which is compared with the values obtained of 69.2 and 74.9 μg/mL at 24 h in cells treated with free Ats. This indicated that when Ats was loaded in BSA NPs, it might change its intracellular location, as mediated by the NPs, and ultimately killed more cells.
In Vitro Cellular Uptake of BSA NPs
Mitochondrial Membrane Potential Analysis
ROS Production Measurement and Staining of the ER
It was widely confirmed that the generation of a large number of ROS can cause phospholipid peroxidation in the inner mitochondrial membrane and that it can also induce a decrease in the mitochondrial membrane potential, thus resulting in the rapid release of cytochrome C. We used DCFH-DA as a fluorescent probe to detect the change of ROS. DCFH-DA passed freely through the cell membrane into the cell and was transformed into DCFH by esterase hydrolysis. The generated DCFH cannot pass through the cell membrane, and it can be easily loaded into the cells. Intracellular ROS oxidized non-fluorescent DCFH to DCF with a green fluorescent color. Therefore, DCF fluorescence detection can indicate the level of intracellular ROS.
Evaluation of Cell Apoptosis and Necrosis
The percentages of early apoptotic (Q4), oncotic (Q2), necrotic (Q1), and live cells (Q3) were shown in Fig. 5b. This finding demonstrated that when cells were treated with free Ats, the oncotic rates were gradually increased to 24.4 and 4.6%, and the apoptotic rate remained at 4.9 and 7.1% in SMMC-7721 cells and Plc cells, respectively, suggesting that free Ats triggered the occurrence of oncosis and apoptosis to lead to cell death. On the contrary, Ats-loaded BSA NPs significantly improved the rate of cell apoptosis and oncosis. The apoptotic ratios were significantly increased to 10.9% in SMMC-7721 cells and to 11.5% in Plc cells. The oncotic ratios were increased to 29.0% in SMMC-7721 cells and to 21.6% in Plc cells. This indicated that the mitochondrial delivery of Ats with the mediation of BSA NPs accelerated the death of tumor cells by enhancing the oncotic and apoptotic effects. Ats-loaded BSA NPs triggered the apoptotic signal transduction process and promoted the mitochondrial-mediated cascade reaction of cellular apoptosis.
Western Blot Analysis
Oncosis and apoptosis represent the two different ways in which cells undergo death. Apoptosis is an active process of programmed cell death that occurs in multicellular organisms. Oncosis, on the other hand, describes a caspase-independent cell death that is characterized by swelling, increased permeability, and membrane rupture, which is often referred to as necrosis. This form of cell death is believed to be accidental and uncontrolled. Based on our investigation, we found that Ats inhibited the growth of tumor cells and that the inhibition ratio was dependent on the dose of Ats. Ats primarily depended on the degree of oncosis and led to cell death; it also activated caspase-independent cell death in the form of oncosis. Conversely, and separately from the occurrence of obvious oncosis-like death, when tumor cells were treated with Ats-loaded BSA NPs, Ats-loaded BSA NPs were internalized into the cytoplasm and were quickly located within the mitochondria to release Ats, as mediated by the NPs. Ats in the mitochondria generated ROS and triggered ER stress; it further activated the mitochondria-meditated caspase-dependent cell apoptotic pathway by reducing the mitochondrial membrane potential, releasing cytochrome C, and promoting the protein expressions of Bax, cleaved caspase 3, and caspase 9. Taken together, Ats-loaded BSA NPs increased the mitochondrial delivery of Ats and enhanced the degree of oncosis and apoptosis to induce cell death, thus increasing the cytotoxicity of the drug and inducing significant cell death.
Briefly, we clarified that free Ats in the tumor cells was strongly dependent on the degree of oncosis to inhibit the proliferation of tumor cells in the form of an oncosis-like death; thus, the cytotoxicity of the drug was limited and undesirable. In contrast, Ats-loaded BSA NPs activated the mitochondrial apoptotic pathway and simultaneously triggered oncotic effects; together, they enhanced the synergistic anti-tumor efficacy of Ats. The results of this study highlighted the significance of Ats-loaded BSA NPs in the enhancement of the cytotoxic and apoptotic effects of Ats, and they further signify the role of BSA NPs in diversifying the pathways of cell death induced by Ats. Compared with free Ats, Ats-loaded BSA NPs induced greater cytotoxicity and significant cell apoptosis effects in tumor cells.
This work was supported by the Liao’ning Educational Committee (No. L2014339) and the Natural Science Foundation of Liaoning Province (No. 2014022039, No. 2015020692, and No. 201602337). English-language editing of this manuscript was provided by Journal Prep.
RL and XY performed the preparation and characteristics of the NPs, and RL wrote the paper. CS and YS helped with the biological study. YS and LZ helped in the analysis of biological data.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
- Yang X, Wu XZ (2015) Main anti-tumor angiogenesis agents isolated from Chinese herbal medicines. Mini Rev Med Chem 15:1011–1023View ArticleGoogle Scholar
- Kim C, Lee JH, Kim SH, Sethi G, Ahn KS (2015) Artesunate suppresses tumor growth and induces apoptosis through the modulation of multiple oncogenic cascades in a chronic myeloid leukemia xenograft mouse model. Oncotarget 6:4020–4035View ArticleGoogle Scholar
- Liu L, Zuo LF, Zuo J, Wang J (2015) Artesunate induces apoptosis and inhibits growth of Eca109 and Ec9706 human esophageal cancer cell lines in vitro and in vivo. Mol Med Rep 12:1465–1472Google Scholar
- Zhou X, Sun WJ, Wang WM, Chen K, Zheng JH, Lu MD et al (2013) Artesunate inhibits the growth of gastric cancer cells through the mechanism of promoting oncosis both in vitro and in vivo. Anticancer Drugs 24:920–927View ArticleGoogle Scholar
- Zhang P, Luo HS, Li M, Tan SY (2015) Artesunate inhibits the growth and induces apoptosis of human gastric cancer cells by downregulating COX-2. Oncol Targets Ther 8:845–854View ArticleGoogle Scholar
- Jin M, Shen X, Zhao C, Qin X, Liu H, Huang L et al (2013) In vivo study of effects of artesunate nanoliposomes on human hepatocellular carcinoma xenografts in nude mice. Drug Deliv 20:127–133View ArticleGoogle Scholar
- Du JH, Zhang HD, Ma ZJ, Ji KM (2010) Artesunate induces oncosis-like cell death in vitro and has antitumor activity against pancreatic cancer xenografts in vivo. Cancer Chemother Pharmacol 65:895–902View ArticleGoogle Scholar
- da Jeong E, Song HJ, Lim S, Lee SJ, Lim JE, Nam DH et al (2015) Repurposing the anti-malarial drug artesunate as a novel therapeutic agent for metastatic renal cell carcinoma due to its attenuation of tumor growth, metastasis, and angiogenesis. Oncotarget 6:33046–33064Google Scholar
- Xu Q, Li ZX, Peng HQ, Sun ZW, Cheng RL, Ye ZM et al (2011) Artesunate inhibits growth and induces apoptosis in human osteosarcoma HOS cell line in vitro and in vivo. J Zhejiang Univ Sci B 12:247–255View ArticleGoogle Scholar
- Yin JY, Wang HM, Wang QJ, Dong YS, Han G, Guan YB et al (2014) Subchronic toxicological study of two artemisinin derivatives in dogs. PLoS One 9:e94034View ArticleGoogle Scholar
- Aquino I, Perazzo FF, Maistro EL (2011) Genotoxicity assessment of the antimalarial compound artesunate in somatic cells of mice. Food Chem Toxicol 49:1335–1339View ArticleGoogle Scholar
- Wang Y, Yang J, Chen L, Wang J, Wang Y, Luo J et al (2014) Artesunate induces apoptosis through caspase-dependent and -independent mitochondrial pathways in human myelodysplastic syndrome SKM-1 cells. Chem Biol Interact 219:28–36View ArticleGoogle Scholar
- Hamacher-Brady A, Stein HA, Turschner S, Toegel I, Mora R, Jennewein N et al (2011) Artesunate activates mitochondrial apoptosis in breast cancer cells via iron-catalyzed lysosomal reactive oxygen species production. J Biol Chem 286:6587–6601View ArticleGoogle Scholar
- Kvansakul M, Hinds MG (2015) The Bcl-2 family, structures, interactions and targets for drug discovery. Apoptosis 20:136–150View ArticleGoogle Scholar
- Williams MM, Cook RS (2015) Bcl-2 family proteins in breast development and cancer, could Mcl-1 targeting overcome therapeutic resistance? Oncotarget 6:3519–3530View ArticleGoogle Scholar
- Besbes S, Mirshahi M, Pocard M, Billard C (2015) Strategies targeting apoptosis proteins to improve therapy of chronic lymphocytic leukemia. Blood Rev 29:345–350View ArticleGoogle Scholar
- Smoot RL, Blechacz BR, Werneburg NW, Bronk SF, Sinicrope FA, Sirica AE et al (2010) A Bax-mediated mechanism for obatoclax-induced apoptosis of cholangiocarcin- oma cells. Cancer Res 70:1960–1969View ArticleGoogle Scholar
- Modica-Napolitano JS, Weissig V (2015) Treatment strategies that enhance the efficacy and selectivity of mitochondria-targeted anticancer agents. Int J Mol Sci 16:17394–17421View ArticleGoogle Scholar
- Brahmbhatt H, Oppermann S, Osterlund EJ, Leber B, Andrews DW (2015) Molecular pathways, leveraging the BCL-2 interactome to kill cancer cells—mitochondrial outer membrane permeabilization and beyond. Clin Cancer Res 21:2671–2676View ArticleGoogle Scholar
- Pang Y, Qin G, Wu L, Wang X, Chen T (2016) Artesunate induces ROS-dependent apoptosis via a Bax-mediated intrinsic pathway in Huh-7 and Hep3B cells. Exp Cell Res 347:251–260View ArticleGoogle Scholar
- Ibrahim N, Ibrahim H, Dormoi J, Briolant S, Pradines B, Moreno A et al (2014) Albumin-bound nanoparticles of practically water-insoluble antimalarial lead greatly enhance its efficacy. Int J Pharm 464:214–224View ArticleGoogle Scholar
- Shen S, Liu SZ, Zhang YS, Du MB, Liang AH, Song LH et al (2015) Compound antimalarial ethosomal cataplasm, preparation, evaluation, and mechanism of penetration enhancement. Int J Nanomedicine 10:4239–4253View ArticleGoogle Scholar
- Agnihotri J, Saraf S, Singh S, Bigoniya P (2015) Development and evaluation of anti-malarial bio-conjugates, artesunate-loaded nanoerythrosomes. Drug Deliv Transl Res 5:489–497View ArticleGoogle Scholar
- Ikoba U, Peng H, Li H, Miller C, Yu C, Wang Q (2015) Nanocarriers in therapy of infectious and inflammatory diseases. Nanoscale 7:4291–4305View ArticleGoogle Scholar
- Yang Y, Wang S, Wang Y, Wang X, Wang Q, Chen M (2014) Advances in self-assembled chitosan nanomaterials for drug delivery. Biotechnol Adv 32:1301–1316View ArticleGoogle Scholar
- Song F, Li X, Wang Q, Liao L, Zhang C (2015) Nanocomposite hydrogels and their applications in drug delivery and tissue engineering. J Biomed Nanotechnol 11:40–52View ArticleGoogle Scholar
- Zhao L, Su R, Cui W, Shi Y, Liu L, Su C (2014) Preparation of biocompatible heat-labile enterotoxin subunit B-bovine serum albumin nanoparticles for improving tumor-targeted drug delivery via heat-labile enterotoxin subunit B mediation. Int J Nanomedicine 9:2149–2156View ArticleGoogle Scholar
- Yu X, Yang G, Shi Y, Su C, Liu M, Feng B, Zhao L (2015) Intracellular targeted co-delivery of shMDR1 and gefitinib with chitosan nanoparticles for overcoming multidrug resistance. Int J Nanomedicine 10:7045–7056Google Scholar