The impact of PEGylation patterns on the in vivo biodistribution of mixed shell micelles

Figures & data

Figure 1 Illustration of fabrication of mixed shell micelles.

Abbreviation: PEG, polyethylene glycol.

Table 1 Characteristics of micelles with various PEG2k/PEG550 ratios

Samples	PEG2k/PEG550 (mol/mol)	Zeta potentiala (mv)	Dha (nm)
PEG-0	1:0	−6.52 ± 0.29b	72 ± 2.5
PEG-25	3:1	−7.86 ± 1.54	80 ± 4.6
PEG-50	1:1	−6.09 ± 0.22	74 ± 3.1
PEG-75	1:3	−7.27 ± 0.65	77 ± 4.0

Note:

a Measured in 10 mM PB (pH 7.4) at 37°C.

b the zeta potential results were presented as mean ± standard deviation.

Abbreviations: PB, phosphate buffer; PEG, polyethylene glycol; D_h, the hydrodynamic diameter.

Figure 2 (A) Hydrodynamic diameter (D_h) distribution of different micelles (PEG-0, PEG-25, PEG-50, and PEG-75) in 100 mM phosphate buffer (pH 7.4) determined by dynamic light scattering. (B) Representative TEM image of PEG-25 at 37°C. The scale bar is 200 nm. The TEM images of PEG-0, PEG-50, and PEG-75 are shown in Figure S3.

Abbreviations: PEG, polyethylene glycol; TEM, transmission electron microscopy.

Table 2 Characterization of iodine-125-labeled micelles

Samples	Labeling rate (%)	Radiochemical purity (%)	Stability (72 hours) (%)
PEG-0	90.4	97.9	91.5
PEG-25	59.0	97.0	90.7
PEG-50	87.7	97.9	92.2
PEG-75	92.3	98.6	92.5

Figure 3 Representative thin-layer chromatograms of iodine-125-labeled micelles (PEG-25) (A) before and (B) after purification.

Abbreviation: PEG, polyethylene glycol. Green bars indicate the location of detected radioactive peaks.

Figure 4 Cell viability of the micelles (PEG-0, PEG-25, PEG-50, and PEG-75) with (A) NIH-3T3 and (B) HepG2 cells measured by MTT assay.

Abbreviations: PEG, polyethylene glycol; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; NIH-3T3, mouse embryonic fibroblast cell line; HepG2, human liver hepa tocellular carcinoma cell line.

Figure 5 The size variation of the micelles (PEG-75) in the absence of serum (10 mM phosphate buffer, pH7.4 at 1, 5, and 10 hours) (A) and incubated with serum at the time period of 5 hours (B) and 10 hours (C) determined by dynamic light scattering at 37°C. The other three kinds of micelles are shown in Figure S5.

Abbreviations: PEG, polyethylene glycol; f(D_h), the hydrodynamic diameter.

Figure 6 Protein adsorption efficiency of various micelles with mixed PEG shell on the surface. One-way analysis of variance was utilized for the statistical analysis. All statistical tests were two-sided.

Notes: *P<0.05, **P<0.01 compared with PEG-0; n=3.

Abbreviation: PEG, polyethylene glycol.

Figure 7 Inverted fluorescent microscopy presenting cellular uptake of micelles in RAW 264.7 murine macrophages after 4 hours of incubation with FITC-labeled micelles: (A) PEG-0, (B) PEG-25, (C) PEG-50, and (D) PEG-75 in medium with 10% FBS.

Abbreviations: FBC, fetal bovine serum; FITC, fluorescein isothiocyanate; PEG, polyethylene glycol; RAW 264.7, mouse macrophage-like cell line.

Figure 8 In vivo biodistribution of different micelles at various time points (1, 4, 8, 24, 48 hours) after injection with (A) PEG-0, (B) PEG-25, (C) PEG-50, and (D) PEG-75, respectively.

Notes: 5 mg micelles/kg mice body weight; data are expressed as %ID/g ± standard deviation; n=3.

Abbreviations: %ID/g, percent injected dose per gram; PEG, polyethylene glycol.

Figure 9 Representative Gamma camera images of the BALB/c mice. (A) PEG-0, (B) PEG-25, (C) PEG-50, and (D) PEG-75 at 4 hours after intravenous administration. (The y axis means the relative radioactive intensity).

Abbreviation: PEG, polyethylene glycol.

Figure 10 Blood clearance curves of micelles after intravenous administration.

Notes: Data are expressed as percent injected dose (%ID) ± standard deviation; n=3.

Scheme S1 Synthesis of (A) PAsp₄₂, (B) PLys₁₈, (C) PEG₄₅-b-PLys₃₂, (D) PEG-b-PAsp (PEG₄₅-b-PAsp₃₆ and PEG₁₂-b-PAsp₁₆), (E) PEG₄₅-b-P(Lys_31.7-co-LysTyr_0.3) and (F) PEG_45-b-P(Lys₂₄-co-LysFITC₈).

Abbreviations: PEG, polyethylene glycol; PLys, poly(L-lysine); PAsp, poly(aspartic acid); FITC, fluorescein isothiocyanate; EDC, 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide; NHS, N-hydroxysuccinimide; DMSO, dimethyl sulfoxide; DMF, N,N-Dimethylformamide; TFA, trifluoroacetic acid; Tyr, tyrosine.

Figure S1 ¹H NMR spectra of (A) PAsp₄₂, (B) PLys₁₈, (C) PEG₄₅-b-PLys₃₂, (D) PEG₄₅-b-PAsp₃₆, and (E) PEG₁₂-b-PAsp₁₆ in D₂O.

Abbreviations: NMR, nuclear magnetic resonance; PEG, polyethylene glycol; PLys, poly(L-lysine); PAsp, poly(aspartic acid).

Figure S2 ¹H NMR spectra of (A) PEG₄₅-b-P(Lys_31.7-co-LysTyr_0.3) and (B) PEG₄₅-b-P(Lys₂₄-co-LysFITC₈) in D₂O.

Abbreviations: NMR, nuclear magnetic resonance; PEG, polyethylene glycol; PLys, poly(L-lysine); PAsp, poly(aspartic acid); FITC, fluorescein isothiocyanate; Tyr, tyrosine.

Figure S3 TEM images of micelles in the absence (left) and presence (right) of serum (stained with uranyl acetate) at 37°C (scale bar: 0.2 μm).

Abbreviation: TEM, transmission electron microscopy.

Figure S4 Thin-layer chromatograms of iodine-125-labeled micelles (A) before and (B) after purification. Green bars indicate the location of detected radioactive peaks.

Figure S5 Size distributions of (A) PEG-0, (B) PEG-25, (C) PEG-50, and (D) PEG-75 in phosphate buffer (pH 7.4, 10 mM) at 37°C (a) without serum (1, 5, and 10 hours), and in the presence of serum at different time points, (b) 5 hours and (c) 10 hours.

Abbreviation: PEG, polyethylene glycol; D_h, hydrodynamic diameter.

Table S1 Formulation of mixed shell micellesa

Samples	PEG2k-b-PLys32/mg	PEG2k-b-PAsp16/mg	PEG550-b-PAsp16/mg	PAsp/mg	PLys/mg
PEG-0	1.1	1	0	0	0
PEG-25	1.5	0	0.2	0.8	0
PEG-50	1	0	0.4	0.35	0
PEG-75	0.9	0	1.08	0	0.4

Notes:

a The micelles were prepared in 4 mL water. After mixing relative polymer solutions together for 0.5 hours, about 5 mg EDC was added in each mixture and the solution was stirred for another 24 hours at room temperature.

Abbreviations: PEG, polyethylene glycol; PLys, poly(L-lysine); PAsp, poly(aspartic acid).