Synergistic Effect of Antiangiogenic Nanotherapy Combined with Methotrexate in the Treatment of Experimental Inflammatory Arthritis

Hui-fang Zhou; Grace Hu; Samuel A Wickline; Gregory M Lanza; Christine TN Pham

Disclosures

Nanomedicine. 2010;5(7):1065-1074. 

In This Article

Materials & Methods

Synthesis of Nanoparticles

The nanoparticles were directed to the αvβ3-integrin with a peptidomimetic vitronectin antagonist developed by Bristol-Myers Squibb Medical Imaging (Billerica, MA, USA;[101] and related patents), as previously described.[3] In general, nanoparticles were comprised of 20% (v/v) perfluorooctylbromide (PFOB; Exfluor Research Corp., TX, USA) and 2.0% (w/v) of a surfactant comixture, and 1.7% (w/v) glycerin and water for the balance. The surfactant co-mixtures included 97.9 mole% highly purified egg yolk lecithin (Avanti Polar Lipids, Inc., AL, USA), 0.1 mole% peptidomimetic αvβ3-integrin antagonist conjugated to PEG2000-phosphatidylethanolamine (Kereos, Inc., MO, USA) and 2 mole% phosphatidylethanolamine (PE; Avanti Polar Lipids, Inc.). The surfactant components were dissolved in chloroform/methanol and dried in a 50°C vacuum oven overnight. Fumagillin (Fum; producted by the National Cancer Institute) was substituted to the surfactant mixture (1.5 mole%) at the expense of lecithin on an equimolar basis (i.e., molecule for molecule).

Arthritis Induction & Treatment

All animal experiments were performed in strict accordance with the guidelines established by the Division of Comparative Medicine at Washington University (MO, USA). Arthritis was induced using the K/BxN mouse model of inflammatory arthritis as previously described.[3] Male C57BL/6 mice aged 6–8 weeks (The Jackson Laboratory, ME, USA) were injected intraperitoneally with 150 µl of serum from K/BxN mice on day 0 to induce arthritis. Clinical manifestation of arthritis was assessed daily on a scale of 0–3 (0 = no swelling or erythema, 1 = slight swelling or erythema, 2 = moderate erythema and swelling in multiple digits or entire paw, 3 = pronounced erythema and swelling of entire paw, maximum score of 12 per mouse). Change from baseline in paw thickness was determined daily by dial calipers, and an average change in ankle thickness was determined for each mouse from the two hind paw measurements. Mice were also weighed prior to, and every day after, K/BxN serum transfer. Percent weight change was calculated by the formula:

In Figures 1–4, mice were assigned to one of four treatment groups:

Figure 1.

Synergistic effect of fumagillin nanoparticles and methotrexate in inflammatory arthritis. Arthritis was induced by K/BxN serum transfer on day 0. NPs and MTX were provided on the indicated days (arrows). (A) Photograph of a hind paw of a representative mouse from each treatment group taken on day 9 after serum transfer. Arthritic score (B) and change (Δ) in ankle thickness (C) were monitored daily. Values represent mean ± SEM (n = seven to nine mice per treatment group derived from two independent experiments).
*p < 0.05.
**p < 0.001 compared with no Tx group.
Fum-NP: Fumagillin-nanoparticle; MTX: Methotrexate; Tx: Treatment.

Figure 2.

Combination of fumagillin nanoparticles and methotrexate suppresses joint inflammation and damage. On day 9 after K/BxN serum transfer, mice were sacrificed and their paws sectioned and stained with hematoxylin and eosin (A–H) to assess for the number of inflammatory cells (A–D) and erosions (arrows, E–H). Sections were also stained with toluidine blue to assess the proteoglycan content in cartilage (arrowheads, I–L). Scale bars = 0.5 mm (A–D), 0.1 mm (E–H) and 0.05 mm (I–L). The number of inflammatory cells/HPF (×400) (M), erosions (N) and degree of proteoglycan depletion (O) were assessed in a blinded fashion. Values represent mean ± SEM (n = 7–9 mice per treatment group).
*p < 0.01.
**p < 0.001 compared with no Tx group.
Fum-NP: Fumagillin-nanoparticle; MTX: Methotrexate; Tx: Treatment.

Figure 3.

Combination of fumagillin nanoparticles and methotrexate suppresses angiogenesis.
(A) Representative micrograph of blood vessels (V) visualized with an antibody to von Willebrand factor. (B) Number of blood vessels/HPF on day 9 was assessed in a blinded fashion.
Values represent mean ± SEM (n = seven to nine mice per treatment group).
*p < 0.0001 compared with no Tx and Fum-NPs groups.
Fum-NP: Fumagillin-nanoparticle; HPD: High power field; MTX: Methotrexate; Tx: Treatment.

Figure 4.

Analysis of toxicity profile of fumagillin nanoparticles and methotrexate combination therapy. On day 9 following K/BxN serum transfer, blood samples were drawn and (A) liver enzymes (including serum concentration of aspartate amino transferase, alanine amino transferase and alkaline phosphatase) and (B) hematologic parameters (white blood cells, hemoglobin and platelets) were assessed. Values represent mean ± SEM (n = seven to nine mice per treatment group). (C) Mice were weighed daily and weight change from baseline weight (prior to serum transfer) was calculated, as detailed in the Materials & methods section.
*p < 0.01 compared with no treatment group. Minimal (min) and maximal (max) values for liver enzymes in C57BL/6 mice were indicated.
ALT: Alanine amino transferase; AST: Aspartate amino transferase; Fum-NP: Fumagillin-nanoparticle; Hbg: Hemoglobin; MTX: Methotrexate; Tx: Treatment; WBC: White blood cell.

  • No treatment (n = 9)

  • αvβ3-targeted nanoparticles with Fum (2.5 µg/g body weight; n = 9)

  • MTX (1.0 µg/g body weight; n = 7)

  • αvβ3-targeted nanoparticles with Fum (2.5 µg/g body weight) and MTX (1.0 µg/g body weight; n = 9)

In Figure 5, mice were assigned to one of three treatment groups:

  • No treatment (n = 7)

  • αvβ3-targeted nanoparticles without drug (2.5 µg/g body weight) and MTX (1.0 µg/g body weight; n = 9)

  • αvβ3-targeted nanoparticles with Fum (2.5 µg/g body weight) and MTX (1.0 µg/g body weight; n = 10)

αvβ3-targeted nanoparticles (αvβ3-NPs) or αvβ3-targeted Fum nanoparticles (Fum-NPs) were administered intravenously once on day 2 after K/BxN serum transfer. MTX was administered intraperitoneally on days 3, 5 and 7 after serum transfer.

Histological Analysis

Mouse paws were harvested on day 9 after serum transfer, fixed in 10% formalin for 48 h, decalcified in EDTA solution, embedded in paraffin and sectioned at 5 µm. The sections were stained with hematoxylin and eosin (H&E) or toluidine blue. Digital images of five random areas per H&E-stained paw section were acquired at ×400 and the number of exuded inflammatory cells enumerated. The number of bone erosions was enumerated per mm of bone surface using the ImageJ program.[201] Proteoglycan content, an indication of cartilage integrity, was graded on toluidine blue-stained sections on a scale of 0–4 as previously described[8] (0 = fully stained cartilage; 1 = less than 25% unstained; 2 = 25–50% unstained; 3 = 50–75% unstained; and 4 = greater than 75% unstained cartilage). Quantitative scoring was performed by an observer blinded to the treatment. Each value represents the average per animal derived from the cumulative scoring of two hind paws.

Evaluation of Angiogenesis

Angiogenesis was evaluated as previously described.[3] Briefly, mouse paw sections were incubated with rabbit anti-von Willebrand factor antibody (1:200 dilution, Chemicon International, CA, USA) for 2 h at room temperature, followed by biotinylated antirabbit antibody and alkaline phosphatase conjugated streptavidin. Color was visualized using a substrate kit (Vector Laboratories, CA, USA). The slides were counterstained with 1% methyl green. Digital images of five random areas per paw were acquired at ×400 and the number of blood vessels enumerated by an observer blinded to the treatment.

Cytokine Analysis

Plasma was collected in EDTA-containing tubes on day 6 following K/BxN serum transfer and cytokine levels (IL-1β, IL-6, IL-10, IL-12, TNF-α, IFN-γ and MCP-1) were measured by Cytometric Bead Array as recommended by the manufacturers (BD Biosciences, CA, USA).

Blood Cell Count & Serum Chemistry Blood samples were drawn from the inferior vena cava by a single stick after the mice were euthanized on day 9 following K/BxN serum transfer and analyzed by the Washington University Department of Comparative Medicine using routine clinical procedures. Minimal and maximal values for liver enzymes in C57BL/6 male mice were compared with previously published reference ranges.[9]

Statistical Analysis Comparisons between groups were done by two-way ANOVA followed by the Bonferroni post hoc test to compare all groups of data. Comparisons between two groups were performed using student's t-test. p-values of less than 0.05 were considered significant. Numerical values are reported as mean ± SEM.

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