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Tumor-targeting, pH-sensitive nanoparticles for docetaxel delivery to drug-resistant cancer cells.

Tran TH, Ramasamy T, Choi JY, Nguyen HT, Pham TT, Jeong JH, Ku SK, Choi HG, Yong CS, Kim JO - Int J Nanomedicine (2015)

Bottom Line: The negative surface charge and PEG shell of vehicle remarkably enhanced the blood circulation and physiological activity of DTX-LPH nanoparticles compared with that of free DTX.The nanoparticles were also found to reduce the size of tumors in tumor-bearing xenograft mice.The in vivo anticancer effect of DTX-LPH nanoparticles was further confirmed by the elevated levels of caspase-3 and poly ADP ribose polymerase found in the tumors after treatment.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Yeungnam University, Dae-Dong, South Korea.

ABSTRACT
The attachment of polyethylene glycol (PEG) increases the circulation time of drug-containing nanoparticles; however, this also negatively affects cellular uptake. To overcome this problem, unique lipid polymer hybrid (LPH) nanoparticles were developed with a pH-responsive PEG layer that detached prior to cell uptake. Docetaxel (DTX) was incorporated into the lipid core of the nanoparticles, which was then shielded with the pH-responsive block co-polymer polyethylene glycol-b-polyaspartic acid (PEG-b-PAsp) using a modified emulsion method. The optimized LPH nanoparticles were ~200 nm and had a narrow size distribution. Drug release from DTX-loaded LPH (DTX-LPH) nanoparticles was pH-sensitive, which is beneficial for tumor targeting. More importantly, DTX-LPH nanoparticles were able to effectively induce apoptosis in cancer cells. The negative surface charge and PEG shell of vehicle remarkably enhanced the blood circulation and physiological activity of DTX-LPH nanoparticles compared with that of free DTX. The nanoparticles were also found to reduce the size of tumors in tumor-bearing xenograft mice. The in vivo anticancer effect of DTX-LPH nanoparticles was further confirmed by the elevated levels of caspase-3 and poly ADP ribose polymerase found in the tumors after treatment. Thus, the results suggest that this novel LPH system could be an effective new treatment for cancer.

No MeSH data available.


Related in: MedlinePlus

(A) Nuclear apoptosis assay using confocal laser scan microscopy, (B) Cell apoptosis by flow-cytometric analysis after treatment for 24 h with free DTX or DTX-LPH nanoparticles. DTX concentration was 10 μg/mL on SCC-7 and MCF-7 cells and 25 μg/mL on MDA-MB-231 cells.Abbreviations: DTX, docetaxel; LPH, lipid polymer hybrid; DTX-LPH, docetaxel-loaded lipid polymer hybrid; h, hours.
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f5-ijn-10-5249: (A) Nuclear apoptosis assay using confocal laser scan microscopy, (B) Cell apoptosis by flow-cytometric analysis after treatment for 24 h with free DTX or DTX-LPH nanoparticles. DTX concentration was 10 μg/mL on SCC-7 and MCF-7 cells and 25 μg/mL on MDA-MB-231 cells.Abbreviations: DTX, docetaxel; LPH, lipid polymer hybrid; DTX-LPH, docetaxel-loaded lipid polymer hybrid; h, hours.

Mentions: To ascertain the potential of nanoparticles for intracellular drug delivery, Coumarin 6-loaded LPH (C6-LPH) was exposed to three different cells (SCC-7, MCF-7, and MDA-MB-231). The time-dependent cellular uptake of nanoparticles was analyzed using (fluorescence-activated cell sorting [FACS] and CLSM (Figure 3). In SCC-7 and MCF-7 cells, C6-LPH clearly underwent time-dependent cellular uptake as the fluorescence intensity was significantly higher after 90 minutes of incubation than after 45 minutes (Figure 3B). In addition, confocal microscopy was used to examine the mechanism of cellular uptake. LPH penetrated into cells mainly via endocytosis, which led to the delivery of the transported cargo to lysosomes.24 After 45 minutes of incubation, only a small number of complexes entered cells (less fluorescence intensity) and proceeded into lysosomes, but no complexes entered the nuclei (data not shown). However, a dramatic increase in cellular uptake was observed after 90 minutes of incubation as evidenced by an increase in fluorescence intensity (Figure 3A). The high cellular uptake of LPH in all cancer cells was attributed to the electrostatic interactions between the positively charged nanoparticles and the negatively charged cellular membrane. Generally, the tumor extracellular environment is slightly acidic (pH =6.8), and the pH of the bloodstream is ~7.4. Therefore, the surface charge of LPH will change to positive in the tumor extracellular fluid, which will enhance the uptake of the nanoparticles in the cell. In addition, the nanometric size of the particles (~200 nm) might contribute to their cellular internalization. From the result, it can be expected that negatively charged LPH will minimize the undesirable systemic side effects and facilitate the accumulation of the nanoparticles in the tumors. A similar pH influence on zeta potential has been reported previously in the literature.25–27 As a result, the use of LPH nanoparticles may dramatically enhance the concentration of the drug in cells, thereby improving drug cytotoxicity and more effectively overcoming MDR in cancer cells (Figure 4). In all cell lines studied, DTX exhibited a time- and dose-dependent cytotoxic effect. As expected, DTX-LPH nanoparticles were also more effective than free DTX in killing tumor cells, especially in drug-resistant MDA-MB-231 cells. Interestingly, in the case of MDA-MB-231 cells, free DTX did not induce appreciable cytotoxicity at doses up to 50 μg/mL even after 48 hours of incubation. The limited cytotoxicity of free DTX could be attributed to P-glycoprotein–mediated efflux pumps, which can dramatically reduce the intracellular drug concentration and thus limit the cytotoxicity of free drugs in tumors. In the case of DTX-LPH, enhanced cellular DTX retention, intracellular acid-responsive drug release, and minimization of drug loss due to efflux resulted in enhanced cytotoxicity.28 Blank LPH did not cause any significant cytotoxicity in any cell lines across all concentrations tested (up to 50 μg/mL).29 DTX disrupts microtubule formation resulting in cell death via the apoptosis pathway.30 Therefore, the cytotoxic effect of different formulations was further confirmed by Hoechst staining. As shown in Figure 5A, chromatin condensation and apoptotic body formation of nuclei were clearly evident in the DTX and DTX-LPH treatment group, while control cells were homogenous and smooth. These data further suggested that DTX-LPH more effectively induced apoptosis than free DTX.


Tumor-targeting, pH-sensitive nanoparticles for docetaxel delivery to drug-resistant cancer cells.

Tran TH, Ramasamy T, Choi JY, Nguyen HT, Pham TT, Jeong JH, Ku SK, Choi HG, Yong CS, Kim JO - Int J Nanomedicine (2015)

(A) Nuclear apoptosis assay using confocal laser scan microscopy, (B) Cell apoptosis by flow-cytometric analysis after treatment for 24 h with free DTX or DTX-LPH nanoparticles. DTX concentration was 10 μg/mL on SCC-7 and MCF-7 cells and 25 μg/mL on MDA-MB-231 cells.Abbreviations: DTX, docetaxel; LPH, lipid polymer hybrid; DTX-LPH, docetaxel-loaded lipid polymer hybrid; h, hours.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4552257&req=5

f5-ijn-10-5249: (A) Nuclear apoptosis assay using confocal laser scan microscopy, (B) Cell apoptosis by flow-cytometric analysis after treatment for 24 h with free DTX or DTX-LPH nanoparticles. DTX concentration was 10 μg/mL on SCC-7 and MCF-7 cells and 25 μg/mL on MDA-MB-231 cells.Abbreviations: DTX, docetaxel; LPH, lipid polymer hybrid; DTX-LPH, docetaxel-loaded lipid polymer hybrid; h, hours.
Mentions: To ascertain the potential of nanoparticles for intracellular drug delivery, Coumarin 6-loaded LPH (C6-LPH) was exposed to three different cells (SCC-7, MCF-7, and MDA-MB-231). The time-dependent cellular uptake of nanoparticles was analyzed using (fluorescence-activated cell sorting [FACS] and CLSM (Figure 3). In SCC-7 and MCF-7 cells, C6-LPH clearly underwent time-dependent cellular uptake as the fluorescence intensity was significantly higher after 90 minutes of incubation than after 45 minutes (Figure 3B). In addition, confocal microscopy was used to examine the mechanism of cellular uptake. LPH penetrated into cells mainly via endocytosis, which led to the delivery of the transported cargo to lysosomes.24 After 45 minutes of incubation, only a small number of complexes entered cells (less fluorescence intensity) and proceeded into lysosomes, but no complexes entered the nuclei (data not shown). However, a dramatic increase in cellular uptake was observed after 90 minutes of incubation as evidenced by an increase in fluorescence intensity (Figure 3A). The high cellular uptake of LPH in all cancer cells was attributed to the electrostatic interactions between the positively charged nanoparticles and the negatively charged cellular membrane. Generally, the tumor extracellular environment is slightly acidic (pH =6.8), and the pH of the bloodstream is ~7.4. Therefore, the surface charge of LPH will change to positive in the tumor extracellular fluid, which will enhance the uptake of the nanoparticles in the cell. In addition, the nanometric size of the particles (~200 nm) might contribute to their cellular internalization. From the result, it can be expected that negatively charged LPH will minimize the undesirable systemic side effects and facilitate the accumulation of the nanoparticles in the tumors. A similar pH influence on zeta potential has been reported previously in the literature.25–27 As a result, the use of LPH nanoparticles may dramatically enhance the concentration of the drug in cells, thereby improving drug cytotoxicity and more effectively overcoming MDR in cancer cells (Figure 4). In all cell lines studied, DTX exhibited a time- and dose-dependent cytotoxic effect. As expected, DTX-LPH nanoparticles were also more effective than free DTX in killing tumor cells, especially in drug-resistant MDA-MB-231 cells. Interestingly, in the case of MDA-MB-231 cells, free DTX did not induce appreciable cytotoxicity at doses up to 50 μg/mL even after 48 hours of incubation. The limited cytotoxicity of free DTX could be attributed to P-glycoprotein–mediated efflux pumps, which can dramatically reduce the intracellular drug concentration and thus limit the cytotoxicity of free drugs in tumors. In the case of DTX-LPH, enhanced cellular DTX retention, intracellular acid-responsive drug release, and minimization of drug loss due to efflux resulted in enhanced cytotoxicity.28 Blank LPH did not cause any significant cytotoxicity in any cell lines across all concentrations tested (up to 50 μg/mL).29 DTX disrupts microtubule formation resulting in cell death via the apoptosis pathway.30 Therefore, the cytotoxic effect of different formulations was further confirmed by Hoechst staining. As shown in Figure 5A, chromatin condensation and apoptotic body formation of nuclei were clearly evident in the DTX and DTX-LPH treatment group, while control cells were homogenous and smooth. These data further suggested that DTX-LPH more effectively induced apoptosis than free DTX.

Bottom Line: The negative surface charge and PEG shell of vehicle remarkably enhanced the blood circulation and physiological activity of DTX-LPH nanoparticles compared with that of free DTX.The nanoparticles were also found to reduce the size of tumors in tumor-bearing xenograft mice.The in vivo anticancer effect of DTX-LPH nanoparticles was further confirmed by the elevated levels of caspase-3 and poly ADP ribose polymerase found in the tumors after treatment.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Yeungnam University, Dae-Dong, South Korea.

ABSTRACT
The attachment of polyethylene glycol (PEG) increases the circulation time of drug-containing nanoparticles; however, this also negatively affects cellular uptake. To overcome this problem, unique lipid polymer hybrid (LPH) nanoparticles were developed with a pH-responsive PEG layer that detached prior to cell uptake. Docetaxel (DTX) was incorporated into the lipid core of the nanoparticles, which was then shielded with the pH-responsive block co-polymer polyethylene glycol-b-polyaspartic acid (PEG-b-PAsp) using a modified emulsion method. The optimized LPH nanoparticles were ~200 nm and had a narrow size distribution. Drug release from DTX-loaded LPH (DTX-LPH) nanoparticles was pH-sensitive, which is beneficial for tumor targeting. More importantly, DTX-LPH nanoparticles were able to effectively induce apoptosis in cancer cells. The negative surface charge and PEG shell of vehicle remarkably enhanced the blood circulation and physiological activity of DTX-LPH nanoparticles compared with that of free DTX. The nanoparticles were also found to reduce the size of tumors in tumor-bearing xenograft mice. The in vivo anticancer effect of DTX-LPH nanoparticles was further confirmed by the elevated levels of caspase-3 and poly ADP ribose polymerase found in the tumors after treatment. Thus, the results suggest that this novel LPH system could be an effective new treatment for cancer.

No MeSH data available.


Related in: MedlinePlus