Development and in vivo efficacy of targeted polymeric inflammation-resolving nanoparticles

Abstract

Excessive inflammation and failed resolution of the inflammatory response are underlying components of numerous conditions such as arthritis, cardiovascular disease, and cancer. Hence, therapeutics that dampen inflammation and enhance resolution are of considerable interest. In this study, we demonstrate the proresolving activity of sub-100-nm nanoparticles (NPs) containing the anti-inflammatory peptide Ac2-26, an annexin A1/lipocortin 1-mimetic peptide. These NPs were engineered using biodegradable diblock poly(lactic-co-glycolic acid)-b-polyethyleneglycol and poly(lactic-co-glycolic acid)-b-polyethyleneglycol collagen IV-targeted polymers. Using a self-limited zymosan-induced peritonitis model, we show that the Ac2-26 NPs (100 ng per mouse) were significantly more potent than Ac2-26 native peptide at limiting recruitment of polymononuclear neutrophils (56% vs. 30%) and at decreasing the resolution interval up to 4 h. Moreover, systemic administration of collagen IV targeted Ac2-26 NPs (in as low as 1 µg peptide per mouse) was shown to significantly block tissue damage in hind-limb ischemia-reperfusion injury by up to 30% in comparison with controls. Together, these findings demonstrate that Ac2-26 NPs are proresolving in vivo and raise the prospect of their use in chronic inflammatory diseases such as atherosclerosis.

Fig. 1.

Nanoparticle design and engineering. Nontargeted and targeted (Col IV) NPs encapsulating the Ac2-26 peptide or a randomly generated, isoelectric mismatched scrambled sequence (Scrm Ac2-26) were developed using biodegradable polymers via a single-step nanoprecipitation method. The synthesized polymers and Ac2-26 peptide or Scrm Ac2-26 peptide were dissolved in acetonitrile (total polymer 3 mg/mL), and 2% (wt/wt) of the fluorescent PLGA-Alexa 647 was added to all formulations. All NP samples contained 4% (wt/wt) peptide (either Ac2-26 or scrambled Ac2-26), and targeted NPs contained 5% (wt/wt) of the Col IV peptide-conjugated targeting polymer. The organic mixture containing the polymers and peptide was then added dropwise to nuclease-free water (10 mL). The solution was stirred for 2–4 h, and the particles were filtered, washed, and resuspended in water or PBS.

Fig. 2.

Characterization of polymeric NPs. (A) Dynamic light-scattering measurements of empty, nontargeted (Ac2-26 NP), targeted (Ac2-26 + Col IV NP), scrambled peptide (Scrm Ac2-26 NP), and targeted scrambled peptide (Scrm Ac2-26 + Col IV NP) NPs were measured (mean ± SD, n = 3). (B) ζ-Potential of the NPs in A were also measured. (C) In vitro cumulative release curve of Ac2-26 peptide from targeted and nontargeted NPs incubated at 37 °C is shown (mean ± SD, n = 3). The released peptide at different time points was isolated by filtration, and the absorbance of these samples was measured at 220 nm. (D) Representative TEM images of Ac2-26 Col IV NPs stained with 0.75% uranyl formate at 80 kV (98,000×). (Scale bar, 60 nm.)

Fig. 3.

Ac2-26 NPs are more potent than Ac2-26 and are proresolving in vivo. Zymosan (100 μg per mouse) was administered i.p., followed by i.v. injections of vehicle, empty NPs, or NPs containing Ac2-26 (Ac2-26 NP, 100 ng per mouse), scrambled Ac2-26 (Scrm Ac2-26 NP, 100 ng per mouse), or Ac2-26 native peptide (100 ng per mouse). Peritoneal exudates were harvested 4 h post zymosan initiation, and living cells were quantified using trypan blue exclusion. (A) PMNs were assessed by flow cytometry (n = 3; mean ± SEM). **P < 0.01 for zymosan vs. treatment; ^§P < 0.05 for Ac2-26 NP versus Ac2-26. (B) Representative dot plot of exudate cells. (Upper) Zymosan alone. (Lower) NP-treated group. (C) Peritoneal exudates were harvested 4, 12, and 24 h post zymosan treatment, and PMNs were enumerated (Scrm Ac2-26 NPs, gray; vehicle, black; Ac2-26 NPs, red). *P < 0.05, **P < 0.01 for zymosan vs. Ac2-26 NPs; ^§P < 0.05 for Ac2-26 NP vs. Scrm Ac2-26; ^§§P < 0.01. (D) Resolution indices for zymosan alone (Upper) and Ac2-26 NPs (Lower) (n = 3; mean ± SEM).

Fig. 4.

Col-IV–targeted Ac2-26 NPs limit PMN infiltration into injured tissue. Ischemia was induced by placing a tourniquet around the hind limb for 1 h. After 1 h, the tourniquet was released and vehicle, Scrm Ac2-26 Col-IV NPs, Ac2-26 Col-IV NPs, or Ac2-26 NPs were injected i.v. Reperfusion was carried out for 1 h. The gastroconemius muscle tissue was harvested and (A) sectioned for confocal imaging using a Nikon A1R microscope, 20× magnification. Images are representative of n = 3. (B) Gastroconemius tissue was lysed and homogenized to assess PMNs using an MPO ELISA (n = 3; mean ± SEM). The data are plotted as inhibition of tissue MPO. *P < 0.05 Col-IV for Ac2-26 NPs vs. Ac2-26 NPs or vs. Scrm-Ac2-26 Col-IV–targeted NPs.