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

Abstract

Development of effective tumor cell-targeted nanodrug formulations has been quite challenging, as many nanocarriers and targeting moieties exhibit nonspecific binding to cellular, extracellular, and intravascular components. We have developed a therapeutic nanoparticle formulation approach that balances cell surface receptor-specific binding affinity while maintaining minimal interactions with blood and tumor tissue components (termed "DART" nanoparticles), thereby improving blood circulation time, biodistribution, and tumor cell-specific uptake. Here, we report that paclitaxel (PTX)-DART nanoparticles directed to the cell surface receptor fibroblast growth factor-inducible 14 (Fn14) outperformed both the corresponding PTX-loaded, nontargeted nanoparticles and Abraxane, an FDA-approved PTX nanoformulation, in both a primary triple-negative breast cancer (TNBC) model and an intracranial model reflecting TNBC growth following metastatic dissemination to the brain. These results provide new insights into methods for effective development of therapeutic nanoparticles as well as support the continued development of the DART platform for primary and metastatic tumors.

Fig. 1. Analysis of nanoparticle binding to Fn14 using SPR assays.

Schematic representation and kinetic binding analysis of (A) PLGA-PEG_1%-ITEM4_1%, (B) PLGA-PEG_5%-ITEM4_1%, (C) PLGA-PEG_10%-ITEM4_0.1%, (D) PLGA-PEG_10%-ITEM4_1%, and (E) PLGA-PEG_10%-ITEM4_10% 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 RU_eq values at each concentration. The binding isotherm of these nanoparticles showing RU_eq 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-ITEM4_1% 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-ITEM4_1% 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-PEG_10%-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.