Differential immunotoxicities of poly(ethylene glycol)- vs. poly(carboxybetaine)-coated nanoparticles

Abstract

Although the careful selection of shell-forming polymers for the construction of nanoparticles is an obvious parameter to consider for shielding of core materials and their payloads, providing for prolonged circulation in vivo by limiting uptake by the immune organs, and thus, allowing accumulation at the target sites, the immunotoxicities that such shielding layers elicit is often overlooked. For instance, we have previously performed rigorous in vitro and in vivo comparisons between two sets of nanoparticles coated with either non-ionic poly(ethylene glycol) (PEG) or zwitterionic poly(carboxybetaine) (PCB), but only now report the immunotoxicity and anti-biofouling properties of both polymers, as homopolymers or nanoparticle-decorating shell, in comparison to the uncoated nanoparticles, and Cremophor-EL, a well-known low molecular weight surfactant used for formulation of several drugs. It was found that both PEG and PCB polymers could induce the expression of cytokines in vitro and in vivo, with PCB being more immunotoxic than PEG, which corroborates the in vivo pharmacokinetics and biodistribution profiles of the two sets of nanoparticles. This is the first study to report on the ability of PEG, the most commonly utilized polymer to coat nanomaterials, and PCB, an emerging zwitterionic anti-biofouling polymer, to induce the secretion of cytokines and be of potential immunotoxicity. Furthermore, we report here on the possible use of immunotoxicity assays to partially predict in vivo pharmacokinetics and biodistribution of nanomaterials.

Keywords: Cremophor-EL; Immunotoxicity; Nanoparticles; Poly(carboxybetaine); Poly(ethylene glycol); Protein adsorption.

Figure 1

(A) Chemical structures of DOTA- and tyramine-functionalized PEG- and PCB-g-PAA-b-PLA copolymers, their self-assembly in water and crosslinking to form SCKs, with PLA degradable cores, PAA crosslinked shells, DOTA and tyramine available functionalities, and a hydrophilic shell of either PEG or PCB. (B) Characterizations of PEG_5k- and PCB_5k-based SCKs in terms of intensity-, volume- and number-averaged hydrodynamic diameter histograms in Tris buffer (10 mM, pH 7.4).

Figure 1

(A) Chemical structures of DOTA- and tyramine-functionalized PEG- and PCB-g-PAA-b-PLA copolymers, their self-assembly in water and crosslinking to form SCKs, with PLA degradable cores, PAA crosslinked shells, DOTA and tyramine available functionalities, and a hydrophilic shell of either PEG or PCB. (B) Characterizations of PEG_5k- and PCB_5k-based SCKs in terms of intensity-, volume- and number-averaged hydrodynamic diameter histograms in Tris buffer (10 mM, pH 7.4).

Figure 2

(A) Heat map of the relative expression of mouse cytokines, interleukin (IL)-lα, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (P40), IL-12 (P70), IL-13, IL-17, Eotaxin, granulocyte-colony-stimulating factor (G-CSF), granulocyte macrophage-colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), keratinocyte-derived chemokine (KC), monocyte chemotactic protein (MCP)-l, macrophage inflammatory protein (MIP)-lα, MIP-1β, regulated upon activation normal T-cell expressed and presumably secreted (RANTES) and tumor necrosis factor-α (TNF-α) following the treatment of RAW 264.7 cells with media (control), PEG- or PCB-based SCKs and Cremophor-EL. (B) The expression of cytokines that were significantly enhanced upon treatment with the two nanoparticle-formulations. (C) The expression of cytokines that were significantly enhanced upon treatment with Cremophor-EL surfactant.

Figure 2

(A) Heat map of the relative expression of mouse cytokines, interleukin (IL)-lα, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (P40), IL-12 (P70), IL-13, IL-17, Eotaxin, granulocyte-colony-stimulating factor (G-CSF), granulocyte macrophage-colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), keratinocyte-derived chemokine (KC), monocyte chemotactic protein (MCP)-l, macrophage inflammatory protein (MIP)-lα, MIP-1β, regulated upon activation normal T-cell expressed and presumably secreted (RANTES) and tumor necrosis factor-α (TNF-α) following the treatment of RAW 264.7 cells with media (control), PEG- or PCB-based SCKs and Cremophor-EL. (B) The expression of cytokines that were significantly enhanced upon treatment with the two nanoparticle-formulations. (C) The expression of cytokines that were significantly enhanced upon treatment with Cremophor-EL surfactant.

Figure 3

The relative expression of mouse cytokines, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (P40), IL-12 (P70), IL-13, IL-17, Eotaxin, G-CSF, GM-CSF, IFN-γ, KC, RANTES and TNF-α following the treatment of RAW 264.7 cells with media (control), PAA₇₅-b-PLA₃₃ SCKs, functionalized or not with DOTA and tyramine.

Figure 4

Adsorption of IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12 (P40), IL-12 (P70), IL-13, IL-17, Eotaxin, G-CSF, GM-CSF, IFN-γ, KC, MIP-lα, MIP-1β and RANTES cytokines by PCB_5k polymer (white bars), PEG_5k polymer (red bars), PCB_5k-SCKs (black bars) and PEG_5k-SCKs (blue bars). The values are presented as the ratio of concentration of the cytokines in the cytokines/nanoparticles mixture (500 µg/mL polymers or nanoparticles) to that in a solution that contains the same amount of the cytokines but without nanoparticles.

Figure 5

(A) Heat map of the relative expression of 23-mouse cytokines following the treatment of RAW 264.7 cells with media (control), PEG_2k- and PCB_2k-based SCKs. (B) The expression of cytokines that were significantly enhanced upon the treatment with the two nanoparticle-formulations.

Figure 5

(A) Heat map of the relative expression of 23-mouse cytokines following the treatment of RAW 264.7 cells with media (control), PEG_2k- and PCB_2k-based SCKs. (B) The expression of cytokines that were significantly enhanced upon the treatment with the two nanoparticle-formulations.

Figure 6

Adsorption of IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-12 (P40), IL-12 (P70), IL-13, IL-17, Eotaxin, G-CSF, GM-CSF, IFN-γ, KC, MCP-1, MIP-1β and RANTES cytokines by PCB_2k-SCKs (black bars) and PEG_2k-SCKs (white bars). The values are presented as the ratio of cytokines in the cytokines/nanoparticles mixture (500 µg/mL polymers or nanoparticles) to the solution that contain the same amount of the cytokines but without the nanoparticles.

Figure 7

(A) Heat map of the relative expression of 23-mouse cytokines in serum of mice treated with PBS (control), PEG_5k- or PCB_5k-based SCKs and Cremophor-EL at 4 mg/kg. (B) The expression of cytokines that were significantly enhanced upon treatment with the two nanoparticle-formulations and Cremophor-EL surfactant.

Figure 7

(A) Heat map of the relative expression of 23-mouse cytokines in serum of mice treated with PBS (control), PEG_5k- or PCB_5k-based SCKs and Cremophor-EL at 4 mg/kg. (B) The expression of cytokines that were significantly enhanced upon treatment with the two nanoparticle-formulations and Cremophor-EL surfactant.