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International Journal of Nanomedicine Volume 14, 2019 - Issue
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Original Research

D-α-tocopherol polyethylene glycol 1000 succinate-modified liposomes with an siRNA corona confer enhanced cellular uptake and targeted delivery of doxorubicin via tumor priming

Xi Tan1 College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, People’s Republic of China, [email protected]
,
Yan Fang1 College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, People’s Republic of China, [email protected]
,
Yuanyuan Ren1 College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, People’s Republic of China, [email protected]
,
Yinghuan Li2 School of Pharmaceutical Sciences, Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of Endogenous Prophylactic of Ministry of Education of China, Capital Medical University, Beijing 100069, People’s Republic of China
,
Peicheng Wu3 School of Biosciences and Biopharmaceuticals, Institute of Biopharmaceuticals, Guangdong Pharmaceutical University, Guangzhou 510006, People’s Republic of China
,
Xiangliang Yang1 College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, People’s Republic of China, [email protected];4 National Engineering Research Center for Nanomedicine, Huazhong University of Science and Technology, Wuhan 430074, People’s Republic of China, [email protected]
&
Wei Liu1 College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, People’s Republic of China, [email protected];4 National Engineering Research Center for Nanomedicine, Huazhong University of Science and Technology, Wuhan 430074, People’s Republic of China, [email protected]Correspondence[email protected]
 show all
Pages 1255-1268 | Published online: 18 Feb 2019

Figures & data

Table 1 Characterization of Dox-loaded and siBcl-2/Dox co-loaded LPs

Figure 1 TEM images of Dox-LPs, Dox-TPGS-LPs, and siBcl-2/Dox-TPGS-LPs stained with phosphotungstic acid.

Note: Scale bar represents 500 nm.

Abbreviations: Dox, doxorubicin; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TEM, transmission electron microscopy; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure 1 TEM images of Dox-LPs, Dox-TPGS-LPs, and siBcl-2/Dox-TPGS-LPs stained with phosphotungstic acid.Note: Scale bar represents 500 nm.Abbreviations: Dox, doxorubicin; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TEM, transmission electron microscopy; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure 2 Release kinetics of Dox from free Dox, Dox-LPs, Dox-TPGS-LPs, and siBcl-2/Dox-TPGS-LPs incubated in PBS (0.01 M, pH 7.4) containing Tween 80 (1%, w/v) at 37°C.

Notes: Data are presented as mean ± SD (n=3). Some SDs are obscured as they are smaller than the symbols.

Abbreviations: Dox, doxorubicin; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure 2 Release kinetics of Dox from free Dox, Dox-LPs, Dox-TPGS-LPs, and siBcl-2/Dox-TPGS-LPs incubated in PBS (0.01 M, pH 7.4) containing Tween 80 (1%, w/v) at 37°C.Notes: Data are presented as mean ± SD (n=3). Some SDs are obscured as they are smaller than the symbols.Abbreviations: Dox, doxorubicin; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure 3 The cellular uptake of Dox in H22 cells was measured by flow cytometer after treatment with free Dox, Dox-LPs, Dox-TPGS-LPs, or siBcl-2/Dox-TPGS-LPs at a Dox concentration of 5 µg/mL for 4 hours; untreated cells served as a control.

Notes: Data are presented as mean ± SD (n=3). *P<0.05, compared with Dox-TPGS-LPs.

Abbreviations: Dox, doxorubicin; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure 3 The cellular uptake of Dox in H22 cells was measured by flow cytometer after treatment with free Dox, Dox-LPs, Dox-TPGS-LPs, or siBcl-2/Dox-TPGS-LPs at a Dox concentration of 5 µg/mL for 4 hours; untreated cells served as a control.Notes: Data are presented as mean ± SD (n=3). *P<0.05, compared with Dox-TPGS-LPs.Abbreviations: Dox, doxorubicin; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure 4 The cellular uptake and intracellular distribution of Dox and FAM-siRNA in H22 cells after being treated with free Dox, Dox-LPs, Dox-TPGS-LPs, or FAM-siRNA/Dox-TPGS-LPs (Dox, 5 µg/mL) for 4 hours.

Notes: Blue DAPI staining indicates the nucleus, and green FAM indicates siBcl-2 in the images. Scale bars represent 10 µm.

Abbreviations: Dox, doxorubicin; FAM, fluorescein amidite; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure 4 The cellular uptake and intracellular distribution of Dox and FAM-siRNA in H22 cells after being treated with free Dox, Dox-LPs, Dox-TPGS-LPs, or FAM-siRNA/Dox-TPGS-LPs (Dox, 5 µg/mL) for 4 hours.Notes: Blue DAPI staining indicates the nucleus, and green FAM indicates siBcl-2 in the images. Scale bars represent 10 µm.Abbreviations: Dox, doxorubicin; FAM, fluorescein amidite; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure 5 The cytotoxicity of the various formulations against H22 cells at the designed Dox concentrations after 24 hours.

Note: The IC50 of free Dox, Dox-LPs, Dox-TPGS-LPs, and siBcl-2/Dox-TPGS-LPs was 2.73, 0.83, 0.81, and 0.35 µg/mL, respectively.

Abbreviations: Dox, doxorubicin; IC50, half-maximal inhibitory concentration; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure 5 The cytotoxicity of the various formulations against H22 cells at the designed Dox concentrations after 24 hours.Note: The IC50 of free Dox, Dox-LPs, Dox-TPGS-LPs, and siBcl-2/Dox-TPGS-LPs was 2.73, 0.83, 0.81, and 0.35 µg/mL, respectively.Abbreviations: Dox, doxorubicin; IC50, half-maximal inhibitory concentration; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure 6 The images of H22 tumor spheroids treated with free Dox, Dox-LPs, Dox-TPGS-LPs, or siBcl-2/Dox-TPGS-LPs containing 5 µg/mL Dox for 72 hours; untreated tumor spheroids served as a control.

Note: Scale bars represent 50 µm.

Abbreviations: Dox, doxorubicin; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure 6 The images of H22 tumor spheroids treated with free Dox, Dox-LPs, Dox-TPGS-LPs, or siBcl-2/Dox-TPGS-LPs containing 5 µg/mL Dox for 72 hours; untreated tumor spheroids served as a control.Note: Scale bars represent 50 µm.Abbreviations: Dox, doxorubicin; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure 7 In vivo distribution and pharmacokinetics of the different formulations in H22 tumor-bearing BALB/c female mice.

Notes: (A) In vivo pharmacokinetics of siBcl-2/Dox-TPGS-LPs in BALB/c female mice after intravenous injection of free Dox, Dox-LPs, Dox-TPGS-LPs, or siBcl-2/Dox-TPGS-LPs at a Dox dose of 5 mg/kg (n=5). (B) In vivo fluorescence images of BALB/c mice with H22 tumors after the intravenous injection of various DiR-loaded LPs at different time points. (a) Free DiR, (b) DiR-loaded non-coated LPs, (c) DiR-loaded TPGS-coated LPs, and (d) siBcl-2/DiR co-loaded TPGS-coated LPs. Red arrows reprent the tumor sites in BALB/c mice. (C) Ex vivo DiR fluorescence images of excised organs (1: liver, 2: spleen, 3: kidney, 4: lung, 5: heart, and 6: tumor) at 24 hours postinjection. (a) Free DiR, (b) DiR-loaded non-coated LPs, (c) DiR-loaded TPGS-coated LPs, and (d) siBcl-2/Dox co-loaded TPGS-coated LPs. (D) Quantitative analysis of Dox in the excised organs of mice after intravenous injection of free Dox, Dox-LPs, Dox-TPGS-LPs, or siBcl-2/Dox-TPGS-LPs at a Dox dose of 5 mg/kg. Data are presented as mean ± SD (n=5). *P<0.05.

Abbreviations: DiR, 1,1′-dioctadecyltetramethyl indotricarbocyanine iodide; Dox, doxorubicin; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure 7 In vivo distribution and pharmacokinetics of the different formulations in H22 tumor-bearing BALB/c female mice.Notes: (A) In vivo pharmacokinetics of siBcl-2/Dox-TPGS-LPs in BALB/c female mice after intravenous injection of free Dox, Dox-LPs, Dox-TPGS-LPs, or siBcl-2/Dox-TPGS-LPs at a Dox dose of 5 mg/kg (n=5). (B) In vivo fluorescence images of BALB/c mice with H22 tumors after the intravenous injection of various DiR-loaded LPs at different time points. (a) Free DiR, (b) DiR-loaded non-coated LPs, (c) DiR-loaded TPGS-coated LPs, and (d) siBcl-2/DiR co-loaded TPGS-coated LPs. Red arrows reprent the tumor sites in BALB/c mice. (C) Ex vivo DiR fluorescence images of excised organs (1: liver, 2: spleen, 3: kidney, 4: lung, 5: heart, and 6: tumor) at 24 hours postinjection. (a) Free DiR, (b) DiR-loaded non-coated LPs, (c) DiR-loaded TPGS-coated LPs, and (d) siBcl-2/Dox co-loaded TPGS-coated LPs. (D) Quantitative analysis of Dox in the excised organs of mice after intravenous injection of free Dox, Dox-LPs, Dox-TPGS-LPs, or siBcl-2/Dox-TPGS-LPs at a Dox dose of 5 mg/kg. Data are presented as mean ± SD (n=5). *P<0.05.Abbreviations: DiR, 1,1′-dioctadecyltetramethyl indotricarbocyanine iodide; Dox, doxorubicin; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure 8 In vivo antitumor efficacy of the different formulations in H22 tumor-bearing BALB/c female mice.

Notes: (A) Tumor volume and (B) body weight changes during the treatment period. The black arrows on the x-axis represent the time points of intravenous injections (ie, Days 1, 3, 5, 8, and 11). The injected dose of Dox and siBcl-2 per administration was 5 mg/kg and 1.2 mg/kg, respectively (n=5). (C) The weight and (D) image of tumors separated from the mice at the end of the experiment. (E) H&E staining of the heart and tumor tissues, and immunofluorescence analysis of the tumor tissues. The red dotted circles represent damaged cardiomyocytes after free Dox treatment. Data are presented as mean ± SD (n=5). *P<0.05 and **P<0.01.

Abbreviations: Dox, doxorubicin; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure 8 In vivo antitumor efficacy of the different formulations in H22 tumor-bearing BALB/c female mice.Notes: (A) Tumor volume and (B) body weight changes during the treatment period. The black arrows on the x-axis represent the time points of intravenous injections (ie, Days 1, 3, 5, 8, and 11). The injected dose of Dox and siBcl-2 per administration was 5 mg/kg and 1.2 mg/kg, respectively (n=5). (C) The weight and (D) image of tumors separated from the mice at the end of the experiment. (E) H&E staining of the heart and tumor tissues, and immunofluorescence analysis of the tumor tissues. The red dotted circles represent damaged cardiomyocytes after free Dox treatment. Data are presented as mean ± SD (n=5). *P<0.05 and **P<0.01.Abbreviations: Dox, doxorubicin; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.
Figure 8 In vivo antitumor efficacy of the different formulations in H22 tumor-bearing BALB/c female mice.Notes: (A) Tumor volume and (B) body weight changes during the treatment period. The black arrows on the x-axis represent the time points of intravenous injections (ie, Days 1, 3, 5, 8, and 11). The injected dose of Dox and siBcl-2 per administration was 5 mg/kg and 1.2 mg/kg, respectively (n=5). (C) The weight and (D) image of tumors separated from the mice at the end of the experiment. (E) H&E staining of the heart and tumor tissues, and immunofluorescence analysis of the tumor tissues. The red dotted circles represent damaged cardiomyocytes after free Dox treatment. Data are presented as mean ± SD (n=5). *P<0.05 and **P<0.01.Abbreviations: Dox, doxorubicin; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure S1 Entrapment efficiency, particle sizes, and zeta potentials of different formulations.

Notes: (A) The entrapment efficiency of siRNA in FAM-siRNA/Dox-TPGS-LPs with varied N/P ratios. (B) The particle sizes and zeta potentials of Dox-TPGS-LPs and FAM-siRNA/Dox-TPGS-LPs with varied N/P ratios.

Abbreviations: Dox, doxorubicin; FAM, fluorescein amidite; LPs, liposomes; N/P, nitrogen to phosphorus; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure S1 Entrapment efficiency, particle sizes, and zeta potentials of different formulations.Notes: (A) The entrapment efficiency of siRNA in FAM-siRNA/Dox-TPGS-LPs with varied N/P ratios. (B) The particle sizes and zeta potentials of Dox-TPGS-LPs and FAM-siRNA/Dox-TPGS-LPs with varied N/P ratios.Abbreviations: Dox, doxorubicin; FAM, fluorescein amidite; LPs, liposomes; N/P, nitrogen to phosphorus; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure S2 The cytotoxicity of blank liposomes (LPs and TPGS-LPs) against H22 cells at the designed total lipids concentrations from 1 to 200 µg/mL after 24 hours.

Abbreviations: LPs, liposomes; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure S2 The cytotoxicity of blank liposomes (LPs and TPGS-LPs) against H22 cells at the designed total lipids concentrations from 1 to 200 µg/mL after 24 hours.Abbreviations: LPs, liposomes; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure S3 Blood biochemical indicators, including liver-related ALT and AST, kidney-related BUN and CRE, and heart-related LDH and CK-MB, in the blood samples of mice were analyzed on Day 15 after treatment with different formulations. *P<0.05.

Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CK-MB, creatinine kinase-MB; CRE, creatinine; Dox, doxorubicin; LDH, lactate dehydrogenase; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure S3 Blood biochemical indicators, including liver-related ALT and AST, kidney-related BUN and CRE, and heart-related LDH and CK-MB, in the blood samples of mice were analyzed on Day 15 after treatment with different formulations. *P<0.05.Abbreviations: ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen; CK-MB, creatinine kinase-MB; CRE, creatinine; Dox, doxorubicin; LDH, lactate dehydrogenase; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure S4 H&E-staining of liver, spleen, lung, and kidney extracted from the mice on Day 15 after treatment with various formulations.

Abbreviations: Dox, doxorubicin; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

Figure S4 H&E-staining of liver, spleen, lung, and kidney extracted from the mice on Day 15 after treatment with various formulations.Abbreviations: Dox, doxorubicin; LPs, liposomes; siBcl-2, Bcl-2 siRNA; TPGS, D-α-tocopherol polyethylene glycol 1000 succinate.

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