<i>In Vivo</i> Pulmonary Delivery and Magnetic-Targeting of Dry Powder Nano-in-Microparticles

This brief communication evaluates the cytotoxicity and targeting capability of a dry powder chemotherapeutic. Nano-in-microparticles (NIMs) are a dry powder drug delivery vehicle containing superparamagnetic iron oxide nanoparticles (SPIONs) and either doxorubicin (w/w solids) or fluorescent nanospheres (w/v during formulation; as a drug surrogate) in a lactose matrix. <i>In vitro</i> cytotoxicity was evaluated in A549 adenocarcinoma cells using MTS and LDH assays to assess viability and toxicity after 48 h of NIMs exposure. <i>In vivo</i> magnetic-field-dependent targeting of inhaled NIMs was evaluated in a healthy mouse model. Mice were endotracheally administered fluorescently labeled NIMs either as a dry powder or a liquid aerosol in the presence of an external magnet placed over the left lung. Quantification of fluorescence and iron showed a significant increase in both fluorescence intensity and iron content to the left magnetized lung. In comparison, we observed decreased targeting of fluorescent nanospheres to the left lung from an aerosolized liquid suspension, due to the dissociation of SPIONs and nanoparticles during pulmonary administration. We conclude that dry powder NIMs maintain the therapeutic cytotoxicity of doxorubicin and can be better targeted to specific regions of the lung in the presence of a magnetic field, compared to a liquid suspension

Binding of serum IgG to intact pneumococci.

<p>Sera from mice immunized with two doses of the indicated formulations were tested for the ability to bind to pneumococcal strains expressing PspA from clades 1 (A), 2 (B), 3 (C), 4 (D) and 5 (E). Results are shown as fluorescence intensity histograms and are representative of two experiments using sera from independent immunizations.</p

Immunophenotyping of cells recovered in BALF collected 12 hours after lethal challenge with ATCC6303.

<p>Mice were immunized with 2 doses of the indicated formulations and challenged with pneumococcal strain ATCC6303. BALF was collected 12 hours after challenge and recovered cells were immunophenotyped by flow cytometry. Percentages of alveolar macrophages (AM—F4/80+ CD11c+ CD11b-) (A), exudate macrophages (EM—F4/80+ CD11c- CD11b+) (B), B cells (F4/80- B220+) (C), CD4+ T cells (F4/80- CD4+) (D), CD8+ T cells (F4/80- CD8+) (E) and neutrophils (F4/80- Ly6G+) (F) are shown. Means and standard errors of each group are shown.</p

Induction of serum anti-PspA4Pro IgG antibodies by mucosal immunization targeting the lungs.

<p>The induction of anti-PspA4Pro IgG antibodies in sera from mice inoculated with the indicated formulations was determined by ELISA. Mice were inoculated with 1 (A) or 2 (B and C) doses of the formulations. Control mice were inoculated sc with saline or PspA4Pro. Log₁₀ anti-PspA4Pro IgG titers (A and B) and log₁₀ anti-PspA4Pro IgG1 titer/log₁₀ anti-PspA4Pro IgG2a titer ratios (C) are shown. * indicates statistically significant difference with saline and # indicates statistically significant difference with PspA4Pro sc (One-way ANOVA, Tukey’s Multicomparison Test for A and B; Unpaired t-test for C). Symbols represent each individual. Means±standard errors are shown. Representative of at least two independent experiments.</p

Induction of anti-PspA4Pro antibodies in BALF by mucosal immunization targeting the lungs.

<p>The induction of anti-PspA4Pro IgG (A) and IgA (B) antibodies in BALF from mice inoculated with the indicated formulations was determined by ELISA. Mice were inoculated with 2 doses of the formulations. Control mice were inoculated sc with saline or PspA4Pro. <i>A</i>_<i>405</i> nm of samples diluted 1:2 is shown. * indicates statistically significant difference with saline (One-way ANOVA, Tukey’s Multicomparison Test). Symbols represent each individual. Means±standard errors are shown.</p

Pneumococcal load in BALF collected 24 hours after non-lethal challenge with EF3030.

<p>Mice were immunized with 2 doses of the indicated formulations and challenged with pneumococcal strain EF3030. Pneumococcal load in BALF was determined 24 hours after challenge. Symbols represent each individual. Means±standard errors are shown.</p

Cytokine/chemokine levels in BALF collected 12 hours after lethal challenge with ATCC6303.

<p>Mice were immunized with 2 doses of the indicated formulations and challenged with pneumococcal strain ATCC6303. Levels of IL-6 (A), TNF-α (B), KC/CXCL1 (C) and MIP-2/CXCL2 (D) were determined in BALF collected 12 hours after challenge by Luminex. Naive mice were not immunized nor challenged. * indicates statistically significant difference (One-way ANOVA, Tukey’s Multicomparison Test). Symbols represent each individual. Means±standard errors are shown. Representative of two independent experiments.</p