Drug-encapsulated blend of PLGA-PEG microspheres: in vitro and in vivo study of the effects of localized/targeted drug delivery on the treatment of triple-negative breast cancer

Abstract

Triple-negative breast cancer (TNBC) is more aggressive and difficult to treat using conventional bulk chemotherapy that is often associated with increased toxicity and side effects. In this study, we encapsulated targeted drugs [A bacteria-synthesized anticancer drug (prodigiosin) and paclitaxel] using single solvent evaporation technique with a blend of FDA-approved poly lactic-co-glycolic acid-polyethylene glycol (PLGA_PEG) polymer microspheres. These drugs were functionalized with Luteinizing Hormone-Releasing hormone (LHRH) ligands whose receptors are shown to overexpressed on surfaces of TNBC. The physicochemical, structural, morphological and thermal properties of the drug-loaded microspheres were then characterized using Fourier Transform Infrared Spectroscopy (FTIR), Scanning Electron Microscopy (SEM), Dynamic Light Scattering (DLS), Nuclear Magnetic Resonance Spectroscopy (NMR), Thermogravimetric Analysis (TGA) and Differential Scanning Calorimetry (DSC). Results obtained from in vitro kinetics drug release at human body temperature (37 °C) and hyperthermic temperatures (41 and 44 °C) reveal a non-Fickian sustained drug release that is well-characterized by Korsmeyer-Peppas model with thermodynamically non-spontaneous release of drug. Clearly, the in vitro and in vivo drug release from conjugated drug-loaded microspheres (PLGA-PEG_PGS-LHRH, PLGA-PEG_PTX-LHRH) is shown to result in greater reductions of cell/tissue viability in the treatment of TNBC. The in vivo animal studies also showed that all the drug-loaded PLGA-PEG microspheres for the localized and targeted treatment of TNBC did not caused any noticeable toxicity and thus significantly extended the survival of the treated mice post tumor resection. The implications of this work are discussed for developing targeted drug systems to treat and prevent local recurred triple negative breast tumors after surgical resection.

Figure 1

SEM images of (A) PLGA-PEG_PGS, (B) PLGA-PEG_PGS-LHRH, (C) PLGA-PEG_PTX, (D) PLGA-PEG_PTX-LHRH, (E) PLGA-PEG microspheres. (F) Mean particle size distributions of drug-loaded and control PLGA-PEG microspheres.

Figure 2

(a) FTIR spectra of the synthesized drug-loaded (PLGA-PEG_PGS, PLGA-PEG_PGS-LHRH, PLGA-PEG_PTX, PLGA-PEG_PTX-LHRH) microspheres and control (PLGA-PEG) microspheres. (b) A representative 1HNMR spectrum for drug-loaded PLGA-PEG microspheres.

Figure 3

(a) TGA curves of control PLGA-PEG microspheres and drug-loaded PLGA-PEG microspheres. (b) DSC thermographs of freeze-dried drug-loaded and control PLGA-PEG microspheres, respectively.

Figure 4

In vitro release profile of (a) PLGA-PEG-PGS microspheres, (b) PLGA-PEG-PTX, (c) PLGA-PEG-PGS-LHRH, (d) PLGA-PEG-PTX-LHRH drug-loaded microspheres at 37 °C, 41 °C and 44 °C, respectively. In all cases (n = 3, ^@p > 0.05 vs. control).

Figure 5

A plot of Gibb’s free energy versus Temperature for various drug-loaded PLGA-PEG formulations.

Figure 6

SEM images of surfaces of drug-loaded PLGA-PEG microspheres after 57 days exposure to phosphate buffer saline at pH 7.4: and cross-sections (note the different magnifications/scaling bars). The white arrows show evidence of the progression of material removal and degradation site.

Figure 7

(a) Percentage alamar blue reduction for cells only (MDA-MB-231 cells), drug-loaded and control PLGA-PEG microspheres after 6, 24, 48, 72 and 96 h post-treatment. (b) Percentage cell growth inhibition for drug-loaded and control PLGA-PEG microspheres after 6, 24, 48, 72 and 96 h’ post-treatment [*p < 0.05 (n = 4)].

Figure 8

(a) Cell viability study of MDA-MB-231 cells showing the effect of the treatment time when incubated with drug-loaded and unloaded PLGA-PEG microspheres after for a period of 240 h with MDA-MB-231 cells acting as a control. (b) Representative confocal images of MDA-MB-231 cells after 5 h incubation with respective drug-loaded PLGA-PEG microspheres at 37 °C. Red staining reveals actin-filaments and green staining indicates vinculin. All cells were stained and imaged under the same conditions. White arrows indicate the initiation of cytoskeleton disruption/disintegration (n = 3, ^$p < 0.05 vs. control).

Figure 9

(a) Body weight variation of subcutaneous xenograft tumor-bearing mice treated with drug-loaded microparticles in the presence of control (n = 5, ^p < 0.05) (b) Kaplan Meier survival curves (N = 30) showing the effect of all treatment groups on the survival rate of mice.

Figure 10

(I) Representative photographs showing the steps involved in the treatment of the TNBC tumor with drug-loaded microspheres: (a) subcutaneous xenograft TNBC tumor; (b) surgical tumor removal; (c) residual tumor; (d) stitched residual tumor with implanted drug-loaded microspheres; (e) healing scar 8 weeks after surgery and (f) completely healed mice 18 weeks after surgery and treatment with targeted drug-loaded microspheres (PLGA-PEG_PGSLHRH). (II) (a–c) Representative mice treated with non-drug microparticles (PLGA-PEG) with recurred tumor.

Figure 11

(I) Representative immunofluorescence images of LHRH receptors (green stain) expressed on the (a) tumor, and (b) lungs of mice treated with a control microspheres (PLGA-PEG) and their corresponding H&E stain showing metastasis in the (c) tumor and (d) lungs. (II) Optical images of mice lungs treated with (a) PLGA-PEG_PTX, (b) PLGA-PEG_PGS, (c) PLGA-PEG_PTX-LHRH, (d) PLGA-PEG_PGS-LHRH microspheres.