Research ArticleHEALTH AND MEDICINE

High-capacity poly(2-oxazoline) formulation of TLR 7/8 agonist extends survival in a chemo-insensitive, metastatic model of lung adenocarcinoma

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Science Advances  17 Jun 2020:
Vol. 6, no. 25, eaba5542
DOI: 10.1126/sciadv.aba5542
  • Fig. 1 Physical characterization of Resiquimod PM.

    (A) Particle size distribution of Resiquimod PM at different feeding ratios as a function of intensity (percentage), measured by DLS. (B and C) Transmission electron micrographs at different magnifications show the spherical morphology of Resiquimod PM particles (4/10 g/liter) and illustrate the uniformity of particle shape and size. (D) Characterization of POx micelles loaded with TLR 7/8 agonist (Resiquimod).

  • Fig. 2 Cytotoxicity assay of POx formulations.

    In vitro cytotoxicity of anticancer agent and chemosensitizers in the 344SQ LUAD cell line (A, B, D, and E). Dose-response curves of free and micelle incorporated drugs and drug combinations in the 344SQ cell line after 72 hours of treatment. Cell viability as a function of individual drug concentrations after treatment with a combination of the drugs AZD8055 and C6CP (A and B) and VE-822 and PTX (D and E). The data were fit into sigmoidal curve using nonlinear regression. Data represent mean ± CV. n = 6. (C and F) Fa-CI plots of the C6CP/AZD7762 and C6CP/VE-822 combinations. Data represent means. n = 6. (G) Comparison of the IC50 values of POx formulations and free drugs in the 344SQ cell line.

  • Fig. 3 In vitro activation of BMDM.

    (A) Relative mRNA expression of classically activated (M1-like) macrophage markers (TNFα, IL-1β, IL-6, and NOS2) normalized to 18S. (B) Relative mRNA expression of M2-like macrophage markers (c-myc and IL-10) normalized to 18S. Data represent means ± SEM. n = 3. **P < 0.01 and ***P < 0.001 computed by unpaired Student’s t test with Welch’s correction. Significance level (α) was set at 0.05. (C) Cell morphology of resting macrophages and M1-polarized macrophages following Resiquimod and Resiquimod PM (2/10 g/liter) treatments (bottom). Scale bars: 50 μm.

  • Fig. 4 Tumor inhibition in 344SQ lung adenocarcinoma–bearing mice.

    Kaplan-Meier survival plots of (A) tumor-bearing mice treated with four intravenous injections of saline, C6CP/AZD7762 PM, and C6CP/VE-822 PM. (B) Tumor-bearing mice treated with four intravenous injections of saline, Resiquimod PM, eight intraperitoneal injections of anti–PD-1 antibody, and a combination of Resiquimod PM (four intravenous injections) anti–PD-1 antibody (eight intraperitoneal injections). P values were computed by log-rank (Mantel-Cox) test. Significance level (α) was set at 0.05. (C) Quantification of BLI signal; data represent means ± SEM. n = 13. (D) Representative IVIS images of mice from each treatment group on the days of the treatment.

  • Fig. 5 Resiquimod PM induces TH1 polarization of immune cells in the TME.

    (A) Representative fluorescence-activated cell sorting (FACS) plots of CD11b+/CD11c/Ly6C+, CD45+/CD3+/CD4+, and CD45+/CD3+/CD8+ cell populations from the tumors of mice treated with saline and Resiquimod PM. FSC-A: forward scatter area. (B to E) Quantification of the indicated population of cells. Data represent means ± SEM. n = 4. *P < 0.05 computed by unpaired Student’s t test with Welch’s correction. Significance level (α) was set at 0.05. ns, not significant.

  • Table 1 Characterization of POx micelles coloaded with anticancer agent and chemosensitizers.

    Characterization of POx micelles coloaded with anticancer agent and chemosensitizers.. LE, loading efficiency; LC, loading capacity; PDI, polydispersity index.

    Checkpoint kinase inhibitor (AZD7762) and anticancer agent (C6CP)
    Feeding ratio
    (g/liter)
    LE (%)LC (%)Drug concentration in
    solution (g/liter)
    Deff (nm)PDI
    C6CP/
    AZD7762/
    POx
    C6CPAZD7762C6CPAZD7762TotC6CPAZD7762
    8/0/1071.336.35.7124 ± 0.50.05 ± 0.05
    0/8/1073.837.15.964 ± 3.60.64 ± 0.02
    4/8/1052.577.511.533.945.42.16.225 ± 0.80.42 ± 0.02
    6/6/1040.070.014.525.339.82.44.282 ± 4.70.45 ± 0.07
    8/4/1038.880.019.019.638.63.13.2128 ± 0.70.09 ± 0.02
    9/3/1058.990.029.415.044.45.32.7112 ± 1.10.09 ± 0.02
    ATR inhibitor (VE-822) and anticancer agent (C6CP)
    Feeding ratio
    (g/liter)
    LE (%)LC (%)Drug concentration in
    solution (g/liter)
    Deff (nm)PDI
    C6CP/VE-822/
    POx
    C6CPVE-822C6CPVE-822TotC6CPVE-822
    8/0/1098.844.07.9122 ± 0.90.10 ± 0.03
    0/8/1083.840.06.7351 ± 6.70.40 ± 0.08
    4/8/1092.575.018.830.549.33.76.0354 ± 4.70.50 ± 0.02
    6/6/1091.783.326.824.451.25.55.0240 ± 1.90.30 ± 0.01
    8/4/1096.385.036.516.152.67.73.4211 ± 3.80.30 ± 0.02
    9/3/109190.039.213.052.28.22.7160 ± 2.40.20 ± 0.01

Supplementary Materials

  • Supplementary Materials

    High-capacity poly(2-oxazoline) formulation of TLR 7/8 agonist extends survival in a chemo-insensitive, metastatic model of lung adenocarcinoma

    Natasha Vinod, Duhyeong Hwang, Salma H. Azam, Amanda E. D. Van Swearingen, Elizabeth Wayne, Sloane Christian Fussell, Marina Sokolsky-Papkov, Chad V. Pecot, Alexander V. Kabanov

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