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International Journal of Nanomedicine Volume 13, 2018 - Issue
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Original Research

Protein delivery nanosystem of six-arm copolymer poly(ε-caprolactone)–poly(ethylene glycol) for long-term sustained release

Jianwei Duan1 Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China
,
Chao Liu1 Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China
,
Xiaoyu Liang1 Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China
,
Xuanling Li1 Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China
,
Youlu Chen1 Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China
,
Zuoguan Chen2 Department of Vascular Surgery, Beijing Hospital, National Center of Gerontology, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China
,
Xiaoli Wang1 Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, ChinaCorrespondence[email protected] [email protected]
,
Deling Kong1 Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, China;3 Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, Tianjin, China
,
Yongjun Li2 Department of Vascular Surgery, Beijing Hospital, National Center of Gerontology, Chinese Academy of Medical Science and Peking Union Medical College, Beijing, China
&
Jing Yang1 Tianjin Key Laboratory of Biomaterial Research, Institute of Biomedical Engineering, Chinese Academy of Medical Science and Peking Union Medical College, Tianjin, ChinaCorrespondence[email protected] [email protected]
 show all
Pages 2743-2754 | Published online: 08 May 2018

Figures & data

Figure 1 Schematic illustration of the synthesis of 6S-PCL-PEG. The hydrophilic model drug OVA was incorporated into the hydrophilic core with a W1/O/W2 solvent evaporation method.

Abbreviations: 6S-PCL, six-arm poly(ε-caprolactone); CL, ε-caprolactone; PEG, poly(ethylene glycol); 6S-PCL-PEG, six-arm poly(ε-caprolactone)–poly(ethylene glycol); OVA, ovalbumin; W1/O/W2, water-in-oil-in-water double emulsion.

Figure 1 Schematic illustration of the synthesis of 6S-PCL-PEG. The hydrophilic model drug OVA was incorporated into the hydrophilic core with a W1/O/W2 solvent evaporation method.Abbreviations: 6S-PCL, six-arm poly(ε-caprolactone); CL, ε-caprolactone; PEG, poly(ethylene glycol); 6S-PCL-PEG, six-arm poly(ε-caprolactone)–poly(ethylene glycol); OVA, ovalbumin; W1/O/W2, water-in-oil-in-water double emulsion.

Figure 2 Synthesis and characterization of 6S-PCL and 6S-PCL-PEG. (A) 1H-NMR spectrum of 6S-PCL. (B) 1H-NMR spectrum of 6S-PCL-PEG. (C) FTIR spectrometry of 6S-PCL and 6S-PCL-PEG. (D) Molecular weights of 6S-PCL and 6S-PCL-PEG investigated by GPC.

Notes: a, (δ=4.05 ppm); b, (δ=1.66 ppm); c, (δ=1.41 ppm); and d, (δ=2.31 ppm). Compared with the 6S-PCL spectrum, the high intensity of peak e (δ=3.62 ppm, −CH2 in PEG segments) in the 6S-PCL-PEG spectrum indicated the existence of PEG.

Abbreviations: 6S-PCL, six-arm poly(ε-caprolactone); 6S-PCL-PEG, six-arm poly(ε-caprolactone)–poly(ethylene glycol); 1H-NMR, hydrogen-1 nuclear magnetic resonance; FTIR, Fourier-transform infrared; GPC, gel-permeation chromatography.

Figure 2 Synthesis and characterization of 6S-PCL and 6S-PCL-PEG. (A) 1H-NMR spectrum of 6S-PCL. (B) 1H-NMR spectrum of 6S-PCL-PEG. (C) FTIR spectrometry of 6S-PCL and 6S-PCL-PEG. (D) Molecular weights of 6S-PCL and 6S-PCL-PEG investigated by GPC.Notes: a, (δ=4.05 ppm); b, (δ=1.66 ppm); c, (δ=1.41 ppm); and d, (δ=2.31 ppm). Compared with the 6S-PCL spectrum, the high intensity of peak e (δ=3.62 ppm, −CH2 in PEG segments) in the 6S-PCL-PEG spectrum indicated the existence of PEG.Abbreviations: 6S-PCL, six-arm poly(ε-caprolactone); 6S-PCL-PEG, six-arm poly(ε-caprolactone)–poly(ethylene glycol); 1H-NMR, hydrogen-1 nuclear magnetic resonance; FTIR, Fourier-transform infrared; GPC, gel-permeation chromatography.

Figure 3 Biocompatibility of 6S-PCL-PEG NPs. (A) NIH-3T3 and RAW-264.7 cell viability after co-culture with NPs for 24 h. (B) Cytokine release of RAW-264.7 cells after co-culture with NPs for 24 h. Bars are mean±SD (n=3). The statistical significance of differences were analyzed using Student’s t-test: **p<0.01.

Abbreviations: 6S-PCL-PEG, six-arm poly(ε-caprolactone)–poly(ethylene glycol); NPs, nanoparticles; TNF-α, tumor necrosis factor-α.

Figure 3 Biocompatibility of 6S-PCL-PEG NPs. (A) NIH-3T3 and RAW-264.7 cell viability after co-culture with NPs for 24 h. (B) Cytokine release of RAW-264.7 cells after co-culture with NPs for 24 h. Bars are mean±SD (n=3). The statistical significance of differences were analyzed using Student’s t-test: **p<0.01.Abbreviations: 6S-PCL-PEG, six-arm poly(ε-caprolactone)–poly(ethylene glycol); NPs, nanoparticles; TNF-α, tumor necrosis factor-α.

Figure 4 Characterization of 6S-PCL-PEG NPs in vitro. (A) Dynamic light scattering of 6S-PCL-PEG NPs. (B) The physical stability of 6S-PCL-PEG NPs in PBS was assessed by the change in NP size and PDI for 30 days. (C) TEM image of normal 6S-PCL-PEG NPs. Magnification is 5,000×. (D) OVA cumulative release from 6S-PCL-PEG NPs which were incubated in PBS (0.01 M, pH 7.4) for 28 days; the protein content was determined by a BCA assay. Bars are mean±SD (n=3).

Abbreviations: 6S-PCL-PEG, six-arm poly(ε-caprolactone)–poly(ethylene glycol); NPs, nanoparticles; PDI, polydispersity index; TEM, transmission electron microscopy; OVA, ovalbumin; BCA, bicinchoninic acid.

Figure 4 Characterization of 6S-PCL-PEG NPs in vitro. (A) Dynamic light scattering of 6S-PCL-PEG NPs. (B) The physical stability of 6S-PCL-PEG NPs in PBS was assessed by the change in NP size and PDI for 30 days. (C) TEM image of normal 6S-PCL-PEG NPs. Magnification is 5,000×. (D) OVA cumulative release from 6S-PCL-PEG NPs which were incubated in PBS (0.01 M, pH 7.4) for 28 days; the protein content was determined by a BCA assay. Bars are mean±SD (n=3).Abbreviations: 6S-PCL-PEG, six-arm poly(ε-caprolactone)–poly(ethylene glycol); NPs, nanoparticles; PDI, polydispersity index; TEM, transmission electron microscopy; OVA, ovalbumin; BCA, bicinchoninic acid.

Figure 5 Secondary structure and molecular weight of OVA encapsulated into 6S-PCL-PEG NPs. (A) CD spectra of OVA. (B) Electrophoretogram of OVA. (C) Proliferation of splenocytes stimulated by OVA. Released OVA means the OVA released from NPs. Bars are mean±SD (n=5). The statistical significance of differences were analyzed using Student’s t-test: ***p<0.001.

Abbreviations: OVA, ovalbumin; 6S-PCL-PEG, six-arm poly(ε-caprolactone)–poly(ethylene glycol); CD, circular dichroism; NPs, nanoparticles.

Figure 5 Secondary structure and molecular weight of OVA encapsulated into 6S-PCL-PEG NPs. (A) CD spectra of OVA. (B) Electrophoretogram of OVA. (C) Proliferation of splenocytes stimulated by OVA. Released OVA means the OVA released from NPs. Bars are mean±SD (n=5). The statistical significance of differences were analyzed using Student’s t-test: ***p<0.001.Abbreviations: OVA, ovalbumin; 6S-PCL-PEG, six-arm poly(ε-caprolactone)–poly(ethylene glycol); CD, circular dichroism; NPs, nanoparticles.

Figure 6 Cellular uptake of nanoparticles by NIH-3T3 cells. (A) NIH-3T3 cells were incubated with OVA-FITC (green) and OVA-FITC-loaded NPs for 3 h; the nucleus was labeled with DAPI (blue). The uptake of NPs was observed by confocal microscopy. (B) Percentage of OVA-FITC-positive cells. (C) Total FI of OVA-FITC; Total FI=% of positive cells × Mean fluorescence intensity. Bars are mean±SD (n=3). The statistical significance of differences were analyzed using Student’s t-test: *p<0.05, **p<0.01, ***p<0.001.

Abbreviations: 6S-PCL, six-arm poly(ε-caprolactone); 6S-PCL-PEG, six-arm poly(ε-caprolactone)–poly(ethylene glycol); OVA, ovalbumin; DAPI, 4′,6-diamidino- 2-phenylindole; BF, bright field; FITC, fluorescein isothiocyanate; FI, fluorescence intensity.

Figure 6 Cellular uptake of nanoparticles by NIH-3T3 cells. (A) NIH-3T3 cells were incubated with OVA-FITC (green) and OVA-FITC-loaded NPs for 3 h; the nucleus was labeled with DAPI (blue). The uptake of NPs was observed by confocal microscopy. (B) Percentage of OVA-FITC-positive cells. (C) Total FI of OVA-FITC; Total FI=% of positive cells × Mean fluorescence intensity. Bars are mean±SD (n=3). The statistical significance of differences were analyzed using Student’s t-test: *p<0.05, **p<0.01, ***p<0.001.Abbreviations: 6S-PCL, six-arm poly(ε-caprolactone); 6S-PCL-PEG, six-arm poly(ε-caprolactone)–poly(ethylene glycol); OVA, ovalbumin; DAPI, 4′,6-diamidino- 2-phenylindole; BF, bright field; FITC, fluorescein isothiocyanate; FI, fluorescence intensity.

Figure S1 Integration areas of NMR spectra of 6S-PCL-PEG.

Abbreviations: NMR, nuclear magnetic resonance; 6S-PCL-PEG, six-arm poly(ε-caprolactone)–poly(ethylene glycol).

Figure S1 Integration areas of NMR spectra of 6S-PCL-PEG.Abbreviations: NMR, nuclear magnetic resonance; 6S-PCL-PEG, six-arm poly(ε-caprolactone)–poly(ethylene glycol).

Figure S2 Size and PDI of 6S-PCL nanoparticles.

Abbreviations: PDI, polydispersity index; 6S-PCL, six-arm poly(ε-caprolactone).

Figure S2 Size and PDI of 6S-PCL nanoparticles.Abbreviations: PDI, polydispersity index; 6S-PCL, six-arm poly(ε-caprolactone).

Figure S3 Representative flow plot of NPs taken up by NIH-3T3 cells.

Abbreviations: NPs, nanoparticles; OVA, ovalbumin; 6S-PCL, six-arm poly(ε-caprolactone); 6S-PCL-PEG, six-arm poly(ε-caprolactone)–poly(ethylene glycol); FITC, fluor-escein isothiocyanate.

Figure S3 Representative flow plot of NPs taken up by NIH-3T3 cells.Abbreviations: NPs, nanoparticles; OVA, ovalbumin; 6S-PCL, six-arm poly(ε-caprolactone); 6S-PCL-PEG, six-arm poly(ε-caprolactone)–poly(ethylene glycol); FITC, fluor-escein isothiocyanate.

Figure S4 Mean fluorescence intensity plot of NPs taken up by NIH-3T3 cells. The statistical significance of differences were analyzed using Student’s t-test: **p<0.01, ***p<0.001.

Abbreviations: NPs, nanoparticles; OVA, ovalbumin; 6S-PCL, six-arm poly(ε-caprolactone); 6S-PCL-PEG, six-arm poly(ε-caprolactone)–poly(ethylene glycol); FI, fluorescence intensity.

Figure S4 Mean fluorescence intensity plot of NPs taken up by NIH-3T3 cells. The statistical significance of differences were analyzed using Student’s t-test: **p<0.01, ***p<0.001.Abbreviations: NPs, nanoparticles; OVA, ovalbumin; 6S-PCL, six-arm poly(ε-caprolactone); 6S-PCL-PEG, six-arm poly(ε-caprolactone)–poly(ethylene glycol); FI, fluorescence intensity.

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