Co-delivery nanoparticles with characteristics of intracellular precision release drugs for overcoming multidrug resistance

Abstract

Combination chemotherapy in clinical practice has been generally accepted as a feasible strategy for overcoming multidrug resistance (MDR). Here, we designed and successfully prepared a co-delivery system named S-D1@L-D2 NPs, where denoted some smaller nanoparticles (NPs) carrying a drug doxorubicin (DOX) were loaded into a larger NP containing another drug (vincristine [VCR]) via water-in-oil-in-water double-emulsion solvent diffusion-evaporation method. Chitosan-alginate nanoparticles carrying DOX (CS-ALG-DOX NPs) with a smaller diameter of about 20 nm formed S-D1 NPs; vitamin E D-α-tocopheryl polyethylene glycol 1000 succinate-modified poly(lactic-co-glycolic acid) nanoparticles carrying VCR (TPGS-PLGA-VCR NPs) with a larger diameter of about 200 nm constituted L-D2 NPs. Some CS-ALG-DOX NPs loaded into TPGS-PLGA-VCR NPs formed CS-ALG-DOX@TPGS-PLGA-VCR NPs. Under the acidic environment of cytosol and endosome or lysosome in MDR cell, CS-ALG-DOX@TPGS-PLGA-VCR NPs released VCR and CS-ALG-DOX NPs. VCR could arrest cell cycles at metaphase by inhibiting microtubule polymerization in the cytoplasm. After CS-ALG-DOX NPs escaped from endosome, they entered the nucleus through the nuclear pore and released DOX in the intra-nuclear alkaline environment, which interacted with DNA to stop the replication of MDR cells. These results indicated that S-D1@L-D2 NPs was a co-delivery system of intracellular precision release loaded drugs with pH-sensitive characteristics. S-D1@L-D2 NPs could obviously enhance the in vitro cytotoxicity and the in vivo anticancer efficiency of co-delivery drugs, while reducing their adverse effects. Overall, S-D1@L-D2 NPs can be considered an innovative platform for the co-delivery drugs of clinical combination chemotherapy for the treatment of MDR tumor.

Keywords: co-delivery; combination chemotherapy; intracellular precision release; multidrug resistance; nuclear drug delivery; pH-sensitive nano-particle.

Figure 1

Preparation scheme of co-delivery nanoparticles. Abbreviations: DOX, doxorubicin; VCR, vincristine; TPGS, D-α-tocopheryl polyethylene glycol 1000 succinate; PLGA, poly(lactic-co-glycolic acid); NPs, nanoparticles; CS-ALG-DOX NPs, chitosan-alginate nanoparticles carrying doxorubicin; TPGS-PLGA-VCR NPs, vitamin E D-α-tocopheryl polyethylene glycol 1000 succinate-modified poly(lactic-co-glycolic acid) nanoparticles carrying vincristine; CS-ALG-NPs, chitosan-alginate nanoparticles; TPGS-PLGA NPs, vitamin E d-a-tocopheryl polyethylene glycol 1000 succinate-modified poly(lactic-co-glycolic acid) nanoparticles; CS-ALG-DOX@TPGS-PLGA-VCR NPs, CS-ALG-DOX NPs loaded at TPGS-PLGA-VCR NPs.

Figure 2

Physicochemical characterization of empty nanoparticles and drug-loaded nanoparticles. (A) TEM and SEM images of CS-ALG NPs and CS-ALG@TPGS-PLGA NPs. (B) TEM and SEM images of CS-ALG-DOX NPs and CS-ALG-DOX@TPGS-PLGA-VCR NPs. (C) The fluorescence images of CS-ALG-DOX@TPGS-PLGA-C6 NPs with CLSM. Notes: Green fluorescence originating from C6 shows TPGS-PLGA-C6 NPs; red fluorescence coming from DOX represents CS-ALG-DOX NPs; and the overlapping fluorescence of yellow originating from red plus green indicates CS-ALG-DOX@TPGS-PLGA-C6 NPs. Scale bar, 1 μm. Abbreviations: DOX, doxorubicin; VCR, vincristine; TPGS, D-α-tocopheryl polyethylene glycol 1000 succinate; PLGA, poly(lactic-co-glycolic acid); ALG, alginate; CS, chitosan; NPs, nanoparticles; TEM, transmission electron microscope; SEM, scanning electron microscope; CS-ALG-DOX NPs, chitosan-alginate nanoparticles carrying doxorubicin; TPGS-PLGA-VCR NPs, vitamin E D-α-tocopheryl polyethylene glycol 1000 succinate-modified poly(lactic-co-glycolic acid) nanoparticles carrying vincristine; C6, coumarin-6; CLSM, confocal laser scanning microscopy.

Figure 3

In vitro release characteristics of the nanoparticles with pH sensitivity. (A) TEM images of CS-ALG@TPGS-PLGA NPs incubated with PBS at pH 7.4 for 6 h. (B) TEM images of CS-ALG@TPGS-PLGA NPs incubated with PBS at pH 5.0 for 6 h. (C) The release curve of VCR from CS-ALG@TPGS-PLGA-VCR NPs in PBS (pH 7.4 and pH 5.8). (D) The release curve of DOX from CS-ALG-DOX NPs in PBS (pH 7.4, pH 5.8, and pH 5.0). Notes: The data of (C) and (D) are presented as the mean values ± SD (n=3). Black arrows indicate that some smaller NPs released from the larger NPs. Abbreviations: TEM, transmission electron microscope; CS-ALG-DOX NPs, chitosan-alginate nanoparticles carrying doxorubicin; TPGS-PLGA-VCR NPs, vitamin E D-α-tocopheryl polyethylene glycol 1000 succinate-modified poly(lactic-co-glycolic acid) nanoparticles carrying vincristine; PBS, phosphate-buffered saline; SD, standard deviation; VCR, vincristine; NPs, nanoparticles; DOX, doxorubicin.

Figure 4

The evaluation of in vitro pharmacological effects of free drug VCR and drug-loaded nanoparticles TPGS-PLGA-VCR NPs incubated for 12 h. The effect on the microtubule in A549 cell (A) and A549/taxol cell (B) with tubulin-tracker red under CLSM. The effect on the microfilament in A549 cell (C) and A549/taxol cell (D) with actin-tracker green under CLMC. (E) Cell cycle of A549 cell and A549/taxol cell using a flow cytometer. Notes: Red fluorescence indicates the microtubules labeled with tubulin-tracker, and blue fluorescence represents nuclear staining by Hoechst 33342. Green fluorescence indicates microfilament labeled with actin-tracker and blue fluorescence represents nuclear staining by Hoechst 33342. Scale bar, 10 μm. Abbreviations: TPGS-PLGA-VCR NPs, vitamin E D-α-tocopheryl polyethylene glycol 1000 succinate-modified poly(lactic-co-glycolic acid) nanoparticles carrying vincristine; VCR, vincristine; NPs, nanoparticles; CLSM, confocal laser scanning microscopy.

Figure 4

The evaluation of in vitro pharmacological effects of free drug VCR and drug-loaded nanoparticles TPGS-PLGA-VCR NPs incubated for 12 h. The effect on the microtubule in A549 cell (A) and A549/taxol cell (B) with tubulin-tracker red under CLSM. The effect on the microfilament in A549 cell (C) and A549/taxol cell (D) with actin-tracker green under CLMC. (E) Cell cycle of A549 cell and A549/taxol cell using a flow cytometer. Notes: Red fluorescence indicates the microtubules labeled with tubulin-tracker, and blue fluorescence represents nuclear staining by Hoechst 33342. Green fluorescence indicates microfilament labeled with actin-tracker and blue fluorescence represents nuclear staining by Hoechst 33342. Scale bar, 10 μm. Abbreviations: TPGS-PLGA-VCR NPs, vitamin E D-α-tocopheryl polyethylene glycol 1000 succinate-modified poly(lactic-co-glycolic acid) nanoparticles carrying vincristine; VCR, vincristine; NPs, nanoparticles; CLSM, confocal laser scanning microscopy.

Figure 5

The intracellular migration fluorescence images of CS-ALG-DOX@TPGS-PLGA-C6 NPs. Notes: Red fluorescence indicates inner smaller CS-ALG NPs labeled with free DOX, green fluorescence indicates outer larger TPGS-PLGA-C6 NPs, and blue fluorescence indicates the region of the nucleus stained with Hoechst 33342. Yellow fluorescence (red overlapping green) represents the co-localization of CS-ALG NPs and TPGS-PLGA NPs, and purple fluorescence (red overlapping blue) represents the co-localization of CS-ALG NPs and the nucleus. Scale bar, 10 μm. Abbreviations: DOX, doxorubicin; C6, coumarin-6; CS-ALG NPs, chitosan-alginate nanoparticles carrying doxorubicin; TPGS-PLGA-C6 NPs, vitamin Ed-a-tocopheryl polyethylene glycol 1000 succinate-modified poly(lactic-co-glycolic acid) nanoparticles carrying C6; CS-ALG-DOX@TPGS-PLGA-C6 NPs, CS-ALG NPs located at TPGS-PLGA-C6 NPs.

Figure 6

The fluorescence image of inner smaller CS-ALG-DOX NPs escaped from the endosome. Notes: CS-ALG-DOX NPs are used to trace the intracellular localization of the nanoparticles to evaluate the correlation between the nanoparticles and lysosomes. Red fluorescence indicates CS-ALG-DOX NPs, green fluorescence from lyso-tracker green DND-26 indicates the lysosomes, and blue fluorescence represents the nuclear staining with Hoechst 33342. Scale bars, 10 μm. Abbreviations: CS-ALG-DOX NPs, chitosan-alginate nanoparticles carrying doxorubicin; NPs, nanoparticles; DOX, doxorubicin.

Figure 7

General state of tumor-bearing nude mice with A549 xenograft model. (A) Schematic plan of in vivo experiments. (B) The growth curve of mice during 19-day course of therapy. (C) Organic coefficient of mice. Notes: Data are presented as mean ± SD (n=5). *P≤0.05; **P≤0.01, compared with control. Abbreviations: CS-ALG-DOX NPs, chitosan-alginate nanoparticles carrying doxorubicin; TPGS-PLGA-VCR NPs, vitamin E D-α-tocopheryl polyethylene glycol 1000 succinate-modified poly(lactic-co-glycolic acid) nanoparticles carrying vincristine; SD, standard deviation; VCR, vincristine; NPs, nanoparticles; DOX, doxorubicin; CS-ALG-DOX@TPGS-PLGA-VCR NPs, CS-ALG-DOX NPs located at TPGS-PLGA-VCR NPs.

Figure 8

The comparison of in vivo antitumor efficiency between DOX and VCR and CS-ALG-DOX@TPGS-PLGA-VCR NPs. (A) The photographs of tumors tissue. (B) The weight of tumors tissue. (C) Pathological sections of the tumor tissues with H&E stain and TUNEL stain. Notes: Data are presented as mean ± SD (n=5). *P<0.05. **P≤0.01, compared with control. ^≤P≤0.05, compared between co-delivery nanoparticles with free DOX and VCR. Abbreviations: CS-ALG-DOX NPs, chitosan-alginate nanoparticles carrying doxorubicin; TPGS-PLGA-VCR NPs, vitamin E D-α-tocopheryl polyethylene glycol 1000 succinate-modified poly(lactic-co-glycolic acid) nanoparticles carrying vincristine; SD, standard deviation; H&E, hematoxylin and eosin; TUNEL, transferase-mediated deoxyuridine triphosphate-biotin nick end labeling; VCR, vincristine; NPs, nanoparticles; DOX, doxorubicin; CS-ALG-DOX@TPGS-PLGA-VCR NPs, CS-ALG-DOX NPs located at TPGS-PLGA-VCR NPs.

Figure 9

Schematic illustration of the co-delivery nanoparticles reversing MDR. Abbreviations: MDR, multidrug resistance; P-gp, P-glycoprotein.