Alphavbeta3-targeted nanotherapy suppresses inflammatory arthritis in mice

Abstract

The purpose of this study was to assess whether an alternative treatment approach that targets angiogenesis, delivered through ligand-targeted nanotherapy, would ameliorate inflammatory arthritis. Arthritis was induced using the K/BxN mouse model of inflammatory arthritis. After arthritis was clearly established, mice received three consecutive daily doses of alpha(v)beta(3)-targeted fumagillin nanoparticles. Control groups received no treatment or alpha(v)beta(3)-targeted nanoparticles without drugs. Disease score and paw thickness were measured daily. Mice that received alpha(v)beta(3)-targeted fumagillin nanoparticles showed a significantly lower disease activity score (mean score of 1.4+/-0.4; P<0.001) and change in ankle thickness (mean increase of 0.17+/-0.05 mm; P<0.001) 7 d after arthritis induction, whereas the group that received alpha(v)beta(3)-targeted nanoparticles without drugs exhibited a mean arthritic score of 9.0 +/- 0.3 and mean change in ankle thickness of 1.01 +/- 0.09 mm. Meanwhile, the group that received no treatment showed a mean arthritic score of 9.8 +/- 0.5 and mean change in ankle thickness of 1.05 +/- 0.10 mm. Synovial tissues from animals treated with targeted fumagillin nanoparticles also showed significant decrease in inflammation and angiogenesis and preserved proteoglycan integrity. Ligand-targeted nanotherapy to deliver antiangiogenic agents may represent an effective way to treat inflammatory arthritis.

Figure 1.

Rhodamine-conjugated α_vβ₃-targeted nanoparticles (nps) accumulate specifically in arthritic paws. Arthritis was induced by intraperitoneal injection of K/BxN serum as described in Materials and Methods. On d 7 after serum transfer, at the peak of disease, arthritic mice were injected intravenously with the indicated rhodamine-conjugated nps. Normal mice served as controls. Concomitant injection of FITC-conjugated lectin outlined blood vessels (v) in green, while nps fluoresced in red. Arrowheads indicate colocalization (yellow) of nps to areas of blood vessels. Scale bar = 0.1 mm.

Figure 2.

Targeted nanotherapy suppresses K/ BxN serum-induced arthritis. Arthritis was induced by intraperitoneal injection of K/BxN serum, and arthritis development (A) and changes in ankle thickness (B) were monitored daily by two independent observers. Starting on d 2 after serum transfer, when arthritis was clearly established in all animals, mice were given 3 daily intravenous doses of either α_vβ₃-targeted nps without drugs or α_vβ₃-targeted nps with 1.5 mol% fumagillin. Another control group received no treatment. Values are means ± se; n = 6–7 mice/group. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3.

Targeted nanotherapy reduces inflammatory cell influx, proteoglycan depletion, and angiogenesis. On d 7 after K/BxN serum transfer, groups of mice that received α_vβ₃-targeted nps without drugs or α_vβ₃-targeted nps with fumagillin were sacrificed, their paws were harvested, and sections were stained with H&E (A, B, E, F) to assess degree of inflammation and inflammatory cell influx, with toluidine blue (C, G) to assess the proteoglycan content in cartilage, or with an antibody to vWF to visualize blood vessels (arrows, D, H). Insets from A and E are shown at higher magnification in B and F, respectively. Scale bars = 0.5 mm (A, E); 0.05 mm (B, F); 0.1 mm (C, G); 0.02 mm (E, H). Micrographs represent 10 paws/group. Number of leukocytes (I) per HPF, degree of proteoglycan depletion by toluidine blue staining (J), and number of vWf⁺ blood vessels (K) per HPF were assessed in a masked fashion. Values are means ± se; 5 random fields/paw, 10 paws/group. *P < 0.0001. B, bone; JS, joint space; S, subsynovial tissue.

Figure 4.

α_vβ₃-Targeted nanoparticles accumulate in the reticuloendothelial system. A) Cryosections from different organs were examined for nanoparticle accumulation (red). Blood vessels were visualized with FITC-lectin (green). On d 5 after the initiation of nanoparticle injection, blood samples were drawn, and liver enzymes, including serum concentrations of aspartate aminotransferase (AST; B), alanine aminotransferase (ALT; C), and alkaline phosphatase (AP; D) were assessed. Increase in liver enzymes was transient, with levels falling toward normal on d 11 after initiation of nanotherapy (E). Values are means ± se; 3–4 animals/group. *P < 0.05; **P < 0.01.