Multivalent Presentation of MPL by Porous Silicon Microparticles Favors T Helper 1 Polarization Enhancing the Anti-Tumor Efficacy of Doxorubicin Nanoliposomes

<div><p>Porous silicon (pSi) microparticles, in diverse sizes and shapes, can be functionalized to present pathogen-associated molecular patterns that activate dendritic cells. Intraperitoneal injection of MPL-adsorbed pSi microparticles, in contrast to free MPL, resulted in the induction of local inflammation, reflected in the recruitment of neutrophils, eosinophils and proinflammatory monocytes, and the depletion of resident macrophages and mast cells at the injection site. Injection of microparticle-bound MPL resulted in enhanced secretion of the T helper 1 associated cytokines IFN-γ and TNF-α by peritoneal exudate and lymph node cells in response to secondary stimuli while decreasing the anti-inflammatory cytokine IL-10. MPL-pSi microparticles independently exhibited anti-tumor effects and enhanced tumor suppression by low dose doxorubicin nanoliposomes. Intravascular injection of the MPL-bound microparticles increased serum IL-1β levels, which was blocked by the IL-1 receptor antagonist Anakinra. The microparticles also potentiated tumor infiltration by dendritic cells, cytotoxic T lymphocytes, and F4/80⁺ macrophages, however, a specific reduction was observed in CD204⁺ macrophages.</p></div

pSi microparticle association with BMDC and biocompatibility.

<p>a) Scanning electron micrographs show pSi microparticles of varying dimensions (bars 500 nm). b) SEM images show cellular association of 2500×400 nm pSi microparticles with BMDC at various stages of uptake (top row) and cellular association with rod-shaped (left, bottom row) and smaller discoidal particles [600 nm (middle) and 1000 nm (right)]. c) BMDCs, treated with five D25.4 microparticles/cell for three hr, were imaged using confocal microscopy. d) TEM images show BMDC with internalized TLR4-ligand (LPS) bound pSi microparticles four hr after microparticle introduction. Seven 1000×400 nm discoidal microparticles are seen in the cell in the upper image (boxed regions; bar 2 µm) and in the magnified regions below (bars 1 and 0.5 µm). e) TEM images show control (left) and alum-treated BMDC four hr after introduction of 2 µg/ml alum. The boxed region showing internalized alum is magnified in the image to the right. f) BMDCs were incubated with LPS (10 ng/ml), alum (25, 12.5 and 6.25 µg/ml), or pSi microparticles (20 particles/cell) for 24 hr. Cellular necrosis was evaluated using the LIVE/DEAD® Aqua dead cell stain.</p

Size and shape characteristics of pSi microparticles.

<p>Size and shape characteristics of pSi microparticles.</p

pSi microparticles are immunomodulatory both <i>in vitro</i> and <i>in vivo</i>.

<p>a) Fluorescent microparticle uptake by human monocytes (THP-1 cells) was measured by flow cytometry at select time points after adding microparticles to the cell culture media (top) or after settling of the microparticles on the cell surface by incubation of cells and microparticles on ice for 60 min (bottom) prior to adding warm media (37°C) and transferring to an incubator (n = 3/group). B-D) Wildtype (WT) (b, c) or NLRP3^−/− (d) BMDCs were primed with 1 ng/ml (b) or 10 ng/ml (c, d) LPS, followed one hr later with 40 particles/cell using pSi microparticles of varying geometries. After 24 hours, supernatants were collected and IL-1β concentrations were determined by ELISA (***p<0.001). E-G) Female C57BL/6 mice were injected i.p. with PBS, Alum or D10.2, D10.4, D25.4 and RS18.4.4 pSi microparticles (0.3 mg/mouse) and the mice were sacrificed after 24 hr. PECs were isolated and stained with various markers to identify cell populations in the peritoneum, specifically the number of neutrophils (e; CD11b⁺ Gr1^high F4/80⁻), eosinophils (f; Gr1⁻ ckit⁻ SiglecF⁺) and resident macrophages (g; CD11b^high F4/80^high). **p<0.01 compared to PBS group, n = 3/group.</p

Combined chemo and MPL-pSi therapy inhibits cellular proliferation and stimulates tumor infiltration by immune cells which is partly IL-1 dependent.

<p>a) Excised tumors from mice treated with low dose DOX-NPs with or without pSi-MPL microparticles were stained with antibodies specific for Ki67 (red), CD8 (red), F4/80 (green), CD204 (red), or 33D1 (red). Nuclei were stained with Prolong Gold Antifade Reagent with DAPI (blue). b) The percentage of the population comprised of each cell type is shown graphically as the mean of 3–4 regions in randomly selected tissues representing at least two animals per group. *p<0.05; **p<0.01. c) Plasma IL-1β level six hr after intravenous injection of free MPL or 5×10⁸ control or MPL-pSi microparticles (10 µg MPL equivalents; n = 3/group; ***p<0.001; *p<0.05). d) Effect of anakinra on MPL-pSi microparticle-induced changes in plasma IL-1β six hr after injection (30 mg, 3×/week; *p<0.05; n = 3/group). e) Tumor growth of mice injected with MPL-pSi microparticles (weekly as indicated by arrows) with and without anakinra (30 mg, 3×/week; n = 3/group). Control verses MPL-pSi, *p<0.05; **p<0.01; ***p<0.001.</p

Injection of pSi microparticles with the TLR4 agonist MPL induces a synergistic recruitment of inflammatory cells.

<p>Female wild-type C57BL/6 mice were injected with PBS, MPL (50 µg/mouse), pSi microparticles (5×10⁸) or MPL plus pSi microparticles. The mice were sacrificed 24 hr later and PECs were isolated. PECs were stained with various markers to identify cell populations in the peritoneum at this time point, specifically the number of neutrophils (a; CD11b⁺ SiglecF⁻ Gr-1⁺), M1-like macrophages (b; CD11b⁺ SiglecF⁻ F4/80⁺ ), M2-like macrophages (c; CD11b^high F4/80^high), eosinophils (d; Gr-1⁻ ckit⁻ SiglecF⁻) and mast cells (e; ckit⁺ SiglecF⁻). Indicated variable versus MPL+pSi, *p<0.05, ***p<0.001, n = 3/group.</p

Injection of pSi microparticles with the TLR4 agonist MPL induces a synergistic production of proinflammatory cytokines.

<p>PECs were restimulated with CpG (5 µg/ml) or HK E. coli (10 E. coli : 1 PEC)(F). After 24 hr, supernatants were collected and analyzed for the cytokines IFN-γ, TNF-α, IL-12p40 and IL-10 by ELISA. MPL versus MPL-pSi, **p<0.01, ***p<0.001; pSi versus MPL-pSi, •p<0.05, •••p<0.001, n = 3/group.</p

Combination therapy is superior to single agent therapy in a mouse model of breast cancer.

<p>a) 4T1 tumor growth in BALB/c mice was monitored using calipers. Mice were treated intravenously with MPL-pSi microparticles (5×10⁸) with and without high dose DOX-NPs weekly as indicated by arrows. Control verses MPL-pSi, *p<0.05, n = 3–5/group. b) Final mass of excised tumor. c) Images showing the relative size of extracted tumors and spleens. d) Mice were imaged weekly using the IVIS imaging system five min following injection with luciferin. e) Caliper-derived tumor volume measurements over time for BALB/c mice treated intravenously with low dose DOX-NPs in the presence or absence of MPL-pSi microparticles (5×10⁸) (time of microparticle injections indicated by arrows; DOX-NPs verses DOX-NPs plus MPL-pSi, *p<0.05, n = 3–4/group). f) Final weights of excised tumors, *p<0.05. g) Relative size of excised tumors and spleens. h) Weekly IVIS imaging of mice injected with luciferin. i) Quantitation of the bioluminescence data is presented as total flux in protons/sec. j) Increase in tumor growth blockade due to MPL-pSi microparticles (red) over that induced by DOX-NPs (blue) shown graphically across time. k) Similar to A & E, 4T1 tumor growth was monitored in BALB/c mice using calipers. Mice were injected with PBS (control), MPL-liposomes, or free MPL as indicated by the arrows (control vs MPL liposome, *p<0.05, n = 3/group).</p