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. 2016 Dec 19:6:39119.
doi: 10.1038/srep39119.

Using anti-poly(ethylene glycol) bioparticles for the quantitation of PEGylated nanoparticles

Yuan-Chin Hsieh  1 , Ta-Chun Cheng  2 , Hsin-Ell Wang  3 , Jia-Je Li  3 , Wen-Wei Lin  4 , Chien-Chiao Huang  2 , Chih-Hung Chuang  5 , Yeng-Tseng Wang  6 , Jaw-Yuan Wang  2   7 , Steve R Roffler  8 , Kuo-Hsiang Chuang  9   10 , Tian-Lu Cheng  1   2   11
Affiliations

Using anti-poly(ethylene glycol) bioparticles for the quantitation of PEGylated nanoparticles

Yuan-Chin Hsieh et al. Sci Rep. .

Abstract

Attachment of polyethylene glycol (PEG) molecules to nanoparticles (PEGylation) is a widely-used method to improve the stability, biocompatibility and half-life of nanomedicines. However, the evaluation of the PEGylated nanomedicine pharmacokinetics (PK) requires the decomposition of particles and purification of lead compounds before analysis by high performance liquid chromatography (HPLC), mass spectrometry, etc. Therefore, a method to directly quantify un-decomposed PEGylated nanoparticles is needed. In this study, we developed anti-PEG bioparticles and combined them with anti-PEG antibodies to generate a quantitative enzyme-linked immunosorbent assay (ELISA) for direct measurement of PEGylated nanoparticles without compound purification. The anti-PEG bioparticles quantitative ELISA directly quantify PEG-quantum dots (PEG-QD), PEG-stabilizing super-paramagnetic iron oxide (PEG-SPIO), Lipo-Dox and PEGASYS and the detection limits were 0.01 nM, 0.1 nM, 15.63 ng/mL and 0.48 ng/mL, respectively. Furthermore, this anti-PEG bioparticle-based ELISA tolerated samples containing up to 10% mouse or human serum. There was no significant difference in pharmacokinetic studies of radiolabeled PEG-nanoparticles (Nano-X-111In) through anti-PEG bioparticle-based ELISA and a traditional gamma counter. These results suggest that the anti-PEG bioparticle-based ELISA may provide a direct and effective method for the quantitation of any whole PEGylated nanoparticles without sample preparation.

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Figures

Figure 1
Figure 1. Anti-PEG bioparticle-based ELISA systems.
Schematic representation of the anti-PEG-bioparticles. The anti-PEG bioparticles are derived from the BALB/c 3T3 cells stably expressing anti-PEG antibody Fab on the cell membrane. The sandwich ELISA is generated by PEG-bioparticle and the detective biotinylated anti-PEG antibody, which can estimate the PEGylated nanoparticles (NPs).
Figure 2
Figure 2. Characterization of anti-PEG bioparticles.
Anti-PEG bioparticles (top row) and anti-DNS control bioparticles (bottom row) were stained with α-HA antibody (red fluorescence) and PEG-QD (green fluorescence). Bioparticles were observed with a digital confocal microscope. Merged images are shown. Scale bars in this figure correspond to 10 μm.
Figure 3
Figure 3. Measurement of the concentration of PEG-QD nanoparticles in fixed anti-PEG bioparticle-based sandwich ELISA.
Sandwich ELISA in which anti-PEG bioparticle/AGP4-biotin was employed as the capture/detection reagents to measure PEG-QD in the paraformaldehyde fixation (♢) and non-fixation (●) anti-PEG bioparticle. Representative data from three independent experiments are shown. Data represents mean ± SD. Statistical analysis was performed by independent t-test (P = 0.9044). ns, no significant difference.
Figure 4
Figure 4. Quantification of PEGylated nanomolecules by anti-PEG bioparticle-based sandwich ELISA.
Anti-PEG bioparticles acted as the capture reagents and AGP4-biotin as the detection antibody to measure the concentration of (A) PEG-QD (B) Lipodox, (C) PEG5K-SPIO and (D) PEGASYS. Representative data from three independent experiments are shown. Data represents mean ± SD.
Figure 5
Figure 5. Effect of serum in the anti-PEG bioparticle-based sandwich ELISA.
Sandwich ELISA in which anti-PEG bioparticle/AGP4-biotin were employed as the capture/detection reagents to measure Lipo-Dox and PEG-QD in the presence of saline (○), buffer: saline containing 2% (wt/vol) skim milk (●) or 2% (wt/vol) skim milk containing 2.5% (v/v) serum (△), 5% (v/v) serum (▲), or 10% (v/v) serum (□), respectively. Human serum (A) (D) and mouse serum (B) (C) were investigated. Representative data from three independent experiments are shown. Data represents mean ± SD. Statistical analysis was performed by multiple t-test. *P value < 0.05; **P value < 0.0001 as compared to buffer group (●).
Figure 6
Figure 6. Pharmacokinetics of Nano-X-111In in mice.
BALB/c mice (n = 8) were intravenously injected with 20 μCi of Nano-X-111In. The concentration of Nano-X-111In was measured by the anti-PEG bioparticle-based ELISA (○). The radioactivity of Nano-X-111In in the serum sample was directly measured by a gamma counter (●). Data represents mean ± SD. Statistical analysis was performed by independent t-test (P = 0.9994). ns, no significant difference.
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