Leukemia Inhibitory Factor-Loaded Nanoparticles with Enhanced Cytokine Metabolic Stability and Anti-Inflammatory Activity - PubMed (original) (raw)

Leukemia Inhibitory Factor-Loaded Nanoparticles with Enhanced Cytokine Metabolic Stability and Anti-Inflammatory Activity

Stephanie M Davis et al. Pharm Res. 2018.

Abstract

Purpose: To synthesize and assess the in vitro biological activity of nanoparticles containing leukemia inhibitory factor (LIF). These NanoLIF particles are designed to prolong the neuroprotective and anti-inflammatory actions of LIF in future preclinical studies of ischemic stroke.

Methods: LIF was packaged in nanoparticles made of poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) polymer to form LIF-loaded nanoparticles (NanoLIF). The surface of NanoLIF was also modified with the CD11b antibody (CD11b-NanoLIF) targeting activated peripheral macrophages to increase cytokine delivery to inflammatory macrophages. ELISA was used to quantify bioactive cytokine inside and releasing from NanoLIF. NanoLIF biological activity was measured using the M1 murine leukemia cell proliferation assay.

Results: NanoLIF and CD11b-NanoLIF had diameters of approximately 30 nm, neutral surface charge, and physicochemical stability retaining biological activity of the cytokine during incubation at 25°C for 12 h. NanoLIF particles released LIF relatively fast from 0 to 6 h after incubation at 37°C followed by slow release from 24 to 72 h according to a two-phase exponential decay model. NanoLIF and CD11b-NanoLIF significantly decreased M1 cell proliferation over 72 h compared to free LIF.

Conclusions: NanoLIF and CD11b-NanoLIF preserved the metabolic stability and biological activity of LIF in vitro. These results are promising to improve the therapeutic potential of LIF in treating neurodegenerative and inflammatory diseases.

Keywords: cytokine delivery; inflammation; macrophages; neurodegeneration; stroke.

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Figures

Fig. 1

Fig. 1

Post-stroke pathological events in the brain and novel therapeutic approach using NanoLIF (a) Neurodegeneration involves acute brain damage in the early stage after stroke followed by secondary immune response due to macrophage activation. (b) NanoLIF releasing LIF in the blood and damaged brain sites for a prolonged time is expected to increase concentrations and therapeutic efficacy of the cytokine. (c) CD11b-NanoLIF targeting inflammatory macrophages hold promise to suppress secondary immune response and thus improve stroke treatment and patient recovery

Fig. 2

Fig. 2

Synthesis of NanoLIF and CD11b-NanoLIF

Fig. 3

Fig. 3

Seventy-seven percent of the LIF that was stored at 4°C was recognized as biologically active LIF but none of the denatured LIF was detected by the ELISA. All measurements are reported as mean ± standard error of the mean (n = 3 measurements per sample).

Fig. 4

Fig. 4

The purified NanoLIF samples that were lyophilized after processing had significantly higher concentration of encapsulated LIF compared to the purified PGLA nanoemulsions that were preserved via lyophilization (*p < 0.05; n = 2 measurements per sample).

Fig. 5

Fig. 5

The unlabeled NanoLIF particles contained approximately 222 ng/ml of biologically active LIF and the CD11b-NanoLIF particles contained approximately 33 1 ng/ml (n = 3 measurements per sample).

Fig. 6

Fig. 6

There was a significant change in the quantity of LIF released from NanoLIF particles over the 72 h period (*p < 0.0001). However, the concentration of LIF in the supernatant was not significantly altered over this period (n = 3 measurements per time point).

Fig. 7

Fig. 7

The pattern of LIF release from the NanoLIF particles over a 72 h time period was closely resembled a second order exponential decay pattern (n = 3 measurements per time point; p < 0.05).

Fig. 8

Fig. 8

NanoLIF reduced degradation of biologically active LIF after incubation at 25°C for 12 h as measured by (a) ELISA (*p < 0.001; n = 3 measurements per group) and (b) the M1 cell proliferation assay (*p < 0.05; n = 4 independent experiments).

Fig. 9

Fig. 9

(a) NanoLIF did not significantly reduce the degradation of biologically active LIF after one freeze/thaw cycle as measured by the ELISA (n = 3 measurements per group). (b) Although there was a trend towards decreased M1 proliferation after treatment with NanoLIF this decrease was not statistically significant (p > 0.05; n = 5 independent experiments).

Fig. 10

Fig. 10

(a) LIF, NanoLIF (*p < 0.05), and CD11b-NanoLIF (**p < 0.01) significantly reduced the proliferation of M1 cells compared to PBS, while NanoLIF (#p < 0.05) and CD11b-NanoLIF (^p < 0.001) significantly reduced proliferation compared to their empty counterparts. (b) When reduction in M I proliferation was normalized to ng/LIF (*p < 0.05) NanoLIF and CD11b-NanoLIF were significantly more efficient than LIF (*p < 0.01) while CD11b-NanoLIF was significantly more efficient than NanoLIF (#p < 0.05; n = 3 independent experiments).

References

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