Probing of the assembly structure and dynamics within nanoparticles during interaction with blood proteins - PubMed (original) (raw)

. 2012 Nov 27;6(11):9485-95.

doi: 10.1021/nn302317j. Epub 2012 Oct 30.

Affiliations

Probing of the assembly structure and dynamics within nanoparticles during interaction with blood proteins

Yuanpei Li et al. ACS Nano. 2012.

Abstract

Fully understanding the influence of blood proteins on the assembly structure and dynamics within nanoparticles is difficult because of the complexity of the system and the difficulty in probing the diverse elements and milieus involved. Here we show the use of site-specific labeling with spin probes and fluorophores combined with electron paramagnetic resonance (EPR) spectroscopy and fluorescence resonance energy transfer (FRET) measurements to provide insights into the molecular architecture and dynamics within nanoparticles. These tools are especially useful for determining nanoparticle stability in the context of blood proteins and lipoproteins and have allowed us to quantitatively analyze the dynamic changes in assembly structure, local stability, and cargo diffusion of a class of novel telodendrimer-based micellar nanoparticles. When combined with human plasma and individual plasma components, we find that non-cross-linked nanoparticles immediately lose their original assembly structure and release their payload upon interaction with lipoproteins. In contrast, serum albumins and immunoglobulin gamma have moderate affects on the integrity of the nanoparticles. Disulfide cross-linked nanoparticles show minimal interaction with lipoproteins and can better retain their assembly structure and payload in vitro and in vivo. We further demonstrate how the enhanced stability and release property of disulfide cross-linked nanoparticles can be reversed in reductive conditions. These findings identify factors that are crucial to the performance of nanomedicines and provide design modes to control their interplay with blood factors.

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Conflict of interest statement

Conflict of Interest: K.S. Lam is the founding scientist of LamnoTherapeutics which plan to develop the nanotherapeutic described in the manuscript. J. Luo and K.S. Lam are the inventors of pending patent on telodendrimers.

Figures

Figure 1

Figure 1

Representative EPR spectra of the S-NCMN1 (a), S-NCMN2 (b) and S-DCMN2 (c) in the presence of 1x PBS and 2.5 mg/mL SDS for 1 min, respectively. TEM images of S-NCMN1 (d), S-NCMN2 (e) and S-DCMN2 (f) in the presence of 2.5 mg/mL SDS for 1 min (scale bar: 100 nm).

Figure 2

Figure 2

Representative time-resolved EPR spectra S-NCMN1 (a), S-NCMN2 (b) and S-DCMN2 (c) in the presence of CM (1.0 mg/mL), HDL (2.0 mg/mL), LDL (2.0 mg/mL), PBS and VLDL (1.0 mg/mL). The percentage changes in rotational correlation time ((τ1 − τ0)/τ0) of the three types of spin labelled nanoparticles in human plasma at different time points (d), where τ0 is the rotational correlation time of the sample in PBS while τ1 is the rotational correlation time of the corresponding peak in a different media under identical instruments conditions, values reported are the mean diameter ± SD for duplicate samples; The percentage of EPR intensity changes ((I1−I0)/I0) of the three types of spin labelled nanoparticles in human plasma at different time points (e), where I0 is the intensity of the highest peak in the EPR spectrum of the sample in PBS while I1 is the intensity of the corresponding peak in a different media under identical instruments conditions, values reported are the mean diameter ± SD for duplicate samples; The ((τ1 − τ0)/τ0) value of the three types of spin labelled nanoparticles in PBS and HSA at different time points (f).

Figure 3

Figure 3

Schematic illustration of non-crosslinked FRET-based nanoparticles FRET-NCMN1 (DiO and rhodamine B pair) (a), FRET-NCMN2 (DiO and DiI pair) (b), FRET-NCMN3 (FITC and rhodamine B pair) in PBS (c) and FRET-NCMN1 (DiO and rhodamine B pair) in human plasma (d); representative fluorescence spectra of FRET-NCMN1 (DiO loading: 2.5%, rhodamine B conjugated PEG5k-CA8: 5.0 mg) in PBS (red line) and in acetonitrile (black line), NCMN with rhodamine B alone (rhodamine B conjugated PEG5k-CA8: 5.0 mg) (blue line) with 480 nm excitation (e); representative fluorescence spectra of FRET-NCMN2 (DiO loading: 2.5%, DiI loading: 2.5%) in PBS (red line) and in acetonitrile (black line), NCMN with DiI alone (DiI loading: 2.5%) (blue line) with 480 nm excitation (f); representative fluorescence spectra of FRET-NCMN3 (FITC conjugated PEG5k-CA8: 5.0 mg, rhodamine B conjugated PEG5k-CA8: 5.0 mg) in PBS (red line) and in acetonitrile (black line), NCMN with rhodamine B alone (rhodamine B conjugated PEG5k-CA8: 5.0 mg) with 480 nm excitation (g); representative fluorescence spectra of FRET-DCMN1 (DiO loading: 2.5%, rhodamine B conjugated PEG5k-Cys4-CA8: 5.0 mg) in PBS (red line) and in acetonitrile (black line), DCMN with rhodamine B alone (rhodamine B conjugated PEG5k-Cys4-CA8: 5.0 mg) (blue line) with 480 nm excitation (h).

Figure 4

Figure 4

Representative fluorescence emission spectra of non-crosslinked FRET based nanoparticles (FRET-NCMN1, DiO and rhodamine B pair) (a) and disulfide crosslinked FRET nanoparticles (FRET-DCMN1, DiO and rhodamine B pair) (b) in the presence of human plasma, respectively. The final concentrations of the nanoparticles were 0.1 mg/mL; the time-resolved apparent FRET efficiency (Eapp) change of FRET-NCMN1 (DiO and rhodamine B pair), FRET-NCMN2 (DiO and DiI pair), FRET-NCMN3 (FITC and rhodamine B pair), FRET-DCMN1 (DiO and rhodamine B pair) in human plasma and PBS (c), the apparent FRET efficiency is calculated as Eapp = _I_A/(_I_A + _I_D), where _I_A and _I_D represent acceptor and donor intensities, respectively; the time-resolved Eapp change of FRET-DCMN1 (DiO and rhodamine B pair) in the presence of human plasma and reducing agents (glutathione (GSH) and N-acetylcysteine (NAC)) (d); the time-resolved Eapp change of FRET-NCMN1 (DiO and rhodamine B pair) (e) and FRET-DCMN1 (DiO and rhodamine B pair) (f) in the presence of HSA (50 mg/mL), LDL (2.0 mg/mL), HDL (2.0 mg/mL), VLDL (1.0 mg/mL), CM (1.0 mg/mL) and IgG (10 mg/mL). Excitation: 480 nm. Values reported are the mean diameter ± SD for triplicate samples.

Figure 5

Figure 5

(a) The time-resolved apparent FRET efficiency (Eapp) change in blood of nude mice (n=3) over time after intravenous injection of 100 μL FRET-NCMN1 (DiO and rhodamine B pair) and FRET-DCMN1 (DiO and rhodamine B pair) (2.0 mg/mL). Excitation: 480 nm. (b) The fluorescence signal changes of rhodamine B conjugated NCMN and DCMN in the blood collected at different time points after intravenous injection in the nude mice (n=3). Excitation: 540 nm. Values reported are the mean diameter ± SD for triplicate samples.

Scheme 1

Scheme 1

Schematic illustration of the spin labelled non-crosslinked nanoparticles by the self-assembly of (a) the PEG5k-CA8 telodendrimer with a spin label attached to the end of the hydrophilic PEG chain and (b) the PEG5k-CA8 telodendrimer with a spin label attached to the lysine side chain at the junction between the linear PEG chain and the dendritic core. (c) Schematic illustration of the spin labelled disulfide crosslinked nanoparticles (S-DCMN2) by the self-assembly of the cysteine containing PEG5k-Cys4-CA8 telodendrimer with a spin label attached to the lysine side chain at the junction between the linear PEG chain and the dendritic core followed by oxidation to form disulfide crosslinks.

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