Research ArticleCANCER

Decreased nonspecific adhesivity, receptor-targeted therapeutic nanoparticles for primary and metastatic breast cancer

See allHide authors and affiliations

Science Advances  15 Jan 2020:
Vol. 6, no. 3, eaax3931
DOI: 10.1126/sciadv.aax3931
  • Fig. 1 Analysis of nanoparticle binding to Fn14 using SPR assays.

    Schematic representation and kinetic binding analysis of (A) PLGA-PEG1%-ITEM41%, (B) PLGA-PEG5%-ITEM41%, (C) PLGA-PEG10%-ITEM40.1%, (D) PLGA-PEG10%-ITEM41%, and (E) PLGA-PEG10%-ITEM410% nanoparticles to Fn14-coated Biacore chip showing binding curves at various concentrations using surface plasmon resonance (SPR) technique (R.U., response units). These curves were fit to a first-order process to determine RUeq values at each concentration. The binding isotherm of these nanoparticles showing RUeq values determined from their respective kinetic binding analysis. The data were fit to a single class of binding sites by nonlinear regression analysis using GraphPad Software (A.U., arbitrary units).

  • Fig. 2 Surface properties of nanoparticles alter their systemic circulation time and biodistribution following intravenous injection.

    (A) Fluorescence image of livers from 231-Luc tumor-bearing mice isolated 1 hour after administration of rhodamine-labeled PLGA-PEG-ITEM41% with 1, 5, or 10% PEG density. (B) Analysis of fluorescence intensity from (A). The same area of regions of interest was used to obtain total radiance [photons/second/square centimeter/steradian (p s−1 cm−2 sr−1)] of the fluorescent signals. Values shown are mean ± SD (n = 3). There was a trend toward lower liver accumulation with 10% PEG, but this difference was not statistically significant (Student’s t test). (C) Fluorescence image of 231-Luc tumors isolated from mice 24 hours after administration of rhodamine-labeled PLGA-PEG-ITEM41% with 1, 5, or 10% PEG density. (D) Analysis of fluorescence intensity from (C). Data obtained as in (B). Values shown are mean ± SD (n = 3). Data analyzed for significance using Student’s t test (*P < 0.01). (E) Fluorescence image of livers, spleens, and kidneys isolated from non–tumor-bearing mice 1 hour after administration of rhodamine-labeled PLGA-PEG10%-ITEM4 with 1 or 10% ITEM4 density. (F) Analysis of fluorescence intensity from (E). Data obtained as in (B). Values shown are mean ± SD (n = 3). Data analyzed for significance using Student’s t test (*P < 0.05).

  • Fig. 3 DART nanoparticles preferentially associate with Fn14-positive 231-Luc cells, exhibit cytotoxic activity in vitro, and can diffuse in 231-Luc tumor tissue slices.

    (A) TEM images show well-dispersed, round-shaped nanoparticles (scale bars, 100 nm). (B) PTX release kinetics from PLGA-PEG and PLGA-PEG-ITEM4 particles in PBS at 37°C. (C) Flow cytometry analysis of PLGA-PEG and PLGA-PEG-ITEM4 nanoparticle association with 231-Luc cells and (D) inhibition of nanoparticle uptake/association after preincubation of cells with free ITEM4. Values shown are mean ± SD (n = 3). Data analyzed for significance using Student’s t test (**P < 0.01). (E) Confocal microscopy images of PLGA-PEG or PLGA-PEG-ITEM4 nanoparticle uptake by 231-Luc cells (scale bars, 20 μm). (F) Viability of 231-Luc cells determined by MTS assay after a short exposure to PTX, Abraxane, PLGA-PEG-PTX particles, or PLGA-PEG-ITEM4-PTX DART particles. Cells were treated for 2 hours, and then the culture medium was removed. Cells were incubated for an additional 24 hours in medium without PTX or nanoparticles. Values shown are mean ± SD (n = 3). Data analyzed for significance between PLGA-PEG-ITEM4-PTX and all other groups using Student’s t test (*P < 0.05). (G) Multiple particle tracking (MPT) analysis of nanoparticles in breast tumor slices ex vivo showing ensemble-averaged mean square displacements (MSDs) as a function of time scale at the 1-s time point. Values shown are mean ± SD (n = 5). Data analyzed for significance using one-way ANOVA, followed by Tukey’s post hoc test (*P < 0.05).

  • Fig. 4 Effect of systemic delivery of PTX-loaded DART nanoparticles on 231-Luc tumor targeting, tumor growth, and indicators of adverse toxicologic effects.

    (A) Fluorescence images of 231-Luc tumors isolated from mice 24 hours after administration of rhodamine-labeled PLGA-PEG-IgG or PLGA-PEG-ITEM4 nanoparticles. (B) Analysis of fluorescence intensity from (A). The same area of regions of interest was used to obtain total radiance (p s−1 cm−2 sr−1) of the fluorescent tumor signals. Values shown are mean ± SD (n = 3). Data analyzed for significance using Student’s t test (***P < 0.001). (C) Tumor growth curves for mice treated with saline, Abraxane, PLGA-PEG-IgG-PTX nanoparticles, or PLGA-PEG-ITEM4-PTX nanoparticles (n = 9 per group) at 10 mg/kg PTX equivalent by one intravenous injection. Values are means ± SEM. Data analyzed using linear mixed-effects models to compare tumor growth rate between treatment groups. The tumor growth difference between mice receiving PLGA-PEG-ITEM4-PTX nanoparticles versus either saline (**P < 0.0001) or Abraxane (*P < 0.05) was statistically significant. (D) Cumulative Kaplan-Meier survival curve of mice from (C). Arrow indicates injection day. Mice receiving PLGA-PEG-ITEM4-PTX nanoparticles had significantly longer survival compared with saline control (*P < 0.0001). (E) Body weight of individual mice in each treatment group from Fig. 4 (C and D), measured every 2 to 3 days. Blood was collected from each mouse at euthanization, and (F) AST and (G) ALT hepatic enzyme levels were determined. Each point represents an individual mouse. Black horizontal bars represent mean and SEM.

  • Fig. 5 Effect of systemic delivery of PTX-loaded DART nanoparticles on 231-Br-Luc tumor growth in the mouse brain.

    (A) Representative BLI images of 231-Br-Luc intracranial tumor-bearing mice treated with either saline, Abraxane, PLGA-PEG-IgG-PTX nanoparticles, or PLGA-PEG-ITEM4-PTX nanoparticles (n = 9 per group) at 10 mg/kg PTX equivalent by one intravenous injection. (B) Cumulative Kaplan-Meier survival curves of mice treated with saline or PTX nanoformulations. Arrow indicates injection day. Mice receiving PLGA-PEG-ITEM4-PTX nanoparticles had significantly longer survival compared with saline control (*P < 0.0001). In contrast, the median survival times between the saline and PLGA-PEG-IgG-PTX groups and the saline and Abraxane groups were not statistically significant.

  • Table 1 Physicochemical properties of nanoparticles.

    Physicochemical characterization data represent the average of three independent experiments ± SD.

    A
    FormulationSize (nm)*PDIζ Potential
    (mV)
    PEG density
    (no./100 nm2)§
    PEG conformation
    [Γ/Γ*]||
    mAB density
    (no. per particle)
    KD (nM)#
    PLGA-PEG1%-
    ITEM41%
    95.4 ± 5.30.14 ± 0.05−4.2 ± 0.39.22.06.13.2
    PLGA-PEG5%-
    ITEM41%
    93.2 ± 7.20.12 ± 0.02−4.6 ± 0.310.32.36.42.4
    PLGA-PEG10%-
    ITEM40.1%
    92.7 ± 5.40.10 ± 0.03−3.4 ± 0.213.42.11.726.5
    PLGA-PEG10%-
    ITEM41%
    94.8 ± 8.70.09 ± 0.04−3.1 ± 0.114.13.26.31.9
    PLGA-PEG10%-
    ITEM410%
    96.4 ± 5.60.12 ± 0.04−5.1 ± 0.413.72.921.82.6
    B
    FormulationSize (nm)*PDIζ Potential
    (mV)
    DL (%)**PEG density
    (no./100 nm2)§
    PEG
    conformation
    [Γ/Γ*]||
    mAB density
    (no. per
    particle)
    MSDwater/
    MSDtumor††
    PLGA66.9 ± 5.50.16 ± 0.02−4.3 ± 0.45.7 ± 1.2124
    PLGA-PEG75.4 ± 5.10.03 ± 0.01−1.4 ± 0.57.7 ± 1.013.02.917
    PLGA-PEG-IgG99.7 ± 12.80.18 ± 0.05−4.8 ± 0.48.2 ± 0.619.54.44.2
    PLGA-PEG-
    ITEM4
    94.8 ± 8.70.09 ± 0.04−3.1 ± 0.18.7 ± 0.214.13.26.315
    Abraxane143.1 ± 4.60.11 ± 0.01−13.5 ± 1.310

    *Hydrodynamic diameter (number mean) measured by dynamic light scattering (DLS).

    †PDI indicates the distribution of individual molecular masses in a batch of nanoparticles, measured by DLS.

    ‡Surface charge measured at 25°C in 15× diluted PBS with ~10 mM NaCl (pH 7.4).

    §PEG surface density determined by NMR.

    ||PEG surface coverage/total surface area [a value <1 indicates mushroom coverage (low density), whereas a value >1 indicates brush regime (high density)].

    ¶Surface density reported from the LavaPep fluorescent protein assay.

    #Equilibrium binding affinity (KD) values determined on a per-nanoparticle basis from fit of Fn14-bindng Biacore data.

    **DL is the percentage of PTX encapsulated into nanoparticles (% w/w).

    ††Ratio indicates the extent to which diffusion of nanoparticles in breast tumor tissues is reduced compared to their diffusion in water.

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/3/eaax3931/DC1

      Fig. S1. Comparison of Fn14 mRNA expression levels in normal breast versus breast cancer subtypes and Fn14 expression/breast cancer patient survival analysis.

      Fig. S2. Analysis of Fn14-specific binding of free targeting ligands and corresponding ligand-conjugated nanoparticles using SPR assays.

      Fig. S3. Surface properties of nanoparticles affect blood clearance time and liver accumulation following intravenous injection.

      Fig. S4. Analysis of Fn14 expression using Western blot and fluorescence-activated cell sorting analyses.

      Fig. S5. SPR analysis reveals Fn14-targeted nanoparticles maintain specific binding interactions with Fn14 even after incubation with mouse blood serum.

      Fig. S6. Effect of PTX and Abraxane on MB-231-Luc cell viability.

      Fig. S7. Nontargeted and Fn14-targeted DART nanoparticles do not exhibit nonspecific interaction with tumor ECM proteins as determined using SPR analysis.

      Fig. S8. PTX-loaded DART nanoparticles inhibit 231-Luc tumor growth after intratumoral injection.

      Fig. S9. PTX-loaded, Fn14-targeted DART nanoparticles do not induce histologic evidence of inflammatory or cytotoxic damage to healthy tissues.

      Fig. S10. Effect of PTX on MB-231-Br-Luc cell viability and distribution of PLGA-PEG-IgG and PLGA-PEG-ITEM4 nanoparticles after systemic administration into mice bearing TNBC tumors in the brain.

    • Supplementary Materials

      This PDF file includes:

      • Fig. S1. Comparison of Fn14 mRNA expression levels in normal breast versus breast cancer subtypes and Fn14 expression/breast cancer patient survival analysis.
      • Fig. S2. Analysis of Fn14-specific binding of free targeting ligands and corresponding ligand-conjugated nanoparticles using SPR assays.
      • Fig. S3. Surface properties of nanoparticles affect blood clearance time and liver accumulation following intravenous injection.
      • Fig. S4. Analysis of Fn14 expression using Western blot and fluorescence-activated cell sorting analyses.
      • Fig. S5. SPR analysis reveals Fn14-targeted nanoparticles maintain specific binding interactions with Fn14 even after incubation with mouse blood serum.
      • Fig. S6. Effect of PTX and Abraxane on MB-231-Luc cell viability.
      • Fig. S7. Nontargeted and Fn14-targeted DART nanoparticles do not exhibit nonspecific interaction with tumor ECM proteins as determined using SPR analysis.
      • Fig. S8. PTX-loaded DART nanoparticles inhibit 231-Luc tumor growth after intratumoral injection.
      • Fig. S9. PTX-loaded, Fn14-targeted DART nanoparticles do not induce histologic evidence of inflammatory or cytotoxic damage to healthy tissues.
      • Fig. S10. Effect of PTX on MB-231-Br-Luc cell viability and distribution of PLGA-PEG-IgG and PLGA-PEG-ITEM4 nanoparticles after systemic administration into mice bearing TNBC tumors in the brain.

      Download PDF

      Files in this Data Supplement:

    Stay Connected to Science Advances

    Navigate This Article