In vivo evaluation of cetuximab-conjugated poly(γ-glutamic acid)-docetaxel nanomedicines in EGFR-overexpressing gastric cancer xenografts

Abstract

Epidermal growth factor receptor (EGFR), upregulated in gastric cancer patients, is an oncogene of interest in the development of targeted cancer nanomedicines. This study demonstrates in silico modeling of monoclonal antibody cetuximab (CET MAb)-conjugated docetaxel (DOCT)-loaded poly(γ-glutamic acid) (γ-PGA) nanoparticles (Nps) and evaluates the in vitro/in vivo effects on EGFR-overexpressing gastric cancer cells (MKN-28). Nontargeted DOCT-γ-PGA Nps (NT Nps: 110±40 nm) and targeted CET MAb-DOCT-γ-PGA Nps (T Nps: 200±20 nm) were prepared using ionic gelation followed by 1-Ethyl-3-(3-dimethyl aminopropyl)carbodiimide-N-Hydoxysuccinimide (EDC-NSH) chemistry. Increased uptake correlated with enhanced cytotoxicity induced by targeted Nps to EGFR +ve MKN-28 compared with nontargeted Nps as evident from MTT and flow cytometric assays. Nanoformulated DOCT showed a superior pharmacokinetic profile to that of free DOCT in Swiss albino mice, indicating the possibility of improved therapeutic effect in the disease model. Qualitative in vivo imaging showed early and enhanced tumor targeted accumulation of CET MAb-DOCT-γ-PGA Nps in EGFR +ve MKN-28-based gastric cancer xenograft, which exhibited efficient arrest of tumor growth compared with nontargeted Nps and free DOCT. Thus, actively targeted CET MAb-DOCT-γ-PGA Nps could be developed as a substitute to conventional nonspecific chemotherapy, and hence could become a feasible strategy for cancer therapy for EGFR-overexpressing gastric tumors.

Keywords: cetuximab; docetaxel; epidermal growth factor receptor; gastric cancer; poly(γ-glutamic acid) nanoparticles; targeted nanoparticles.

Figure 1

In silico modeling studies. Notes: (A) ChemDraw generated structure of γ-PGA Nps formed by the polyionic complexation between anionic γ-PGA cross-linked with cationic chitosan. Lowest binding energy conformation of CET MAb and DOCT within γ-PGA Np assembly obtained by in silico docking calculations for (B) nontargeted Nps (DOCT-γ-PGA Nps) and (C) targeted Nps (CET MAb-DOCT-γ-PGA Nps). Abbreviations: γ-PGA, poly(γ-glutamic acid); CET MAb, cetuximab monoclonal antibody; DOCT, docetaxel; Np, nanoparticle.

Figure 2

Cellular uptake analysis. Notes: (A) EGFR expression of MKN-28 cells. Flow cytometric histograms. Values are presented as mean ± SD of three independent experiments (n=3), showing AF-647 anti-EGFR antibody binding on MKN-28 cells along with a confocal microscopic image. (B) Cellular binding of Nps. Flow cytometry histograms showing the binding of Nps on the surface of MKN-28 cells with and without 1 h of pretreatment with CET MAb. Values are presented as mean ± SD of three independent experiments (n=3). (C) Cellular internalization of CET MAb-FITC-γ-PGA Nps. Confocal microscopic images of CET MAb-FITC-γ-PGA Np-treated MKN-28 cells, where (a–g) show different sections from the z section scanned imaging (scale bars =100 μm; 20× magnification). (D and E) Magnified images of two different frames where single cells can be individually seen indicating the Np localizations; 40× magnification. Abbreviations: γ-PGA, poly(γ-glutamic acid); AF, Alexafluor; CET MAb, cetuximab monoclonal antibody; DOCT, docetaxel; EGFR, epidermal growth factor receptor; FITC, fluorescein isothiocyanate; Nps, nanoparticles.

Figure 3

In vitro cytotoxicity evaluations. Notes: (A) Cell cycle analysis by flow cytometry. Flow cytometric histograms showing the cell cycle profiles of MKN-28 (a) control cells and followed by 24 h treatment of (b) free DOCT, (c) DOCT-γ-PGA Nps and (d) CET MAb-DOCT-γ-PGA Nps. Data shown as mean ± SD. (B) Cytotoxicity profile by 24 and 48 h MTT assay. (C) Flow cytometry analysis and percentage of cells depicting a reduction in the mitochondrial membrane potential and (D) scatter plot indicating the cell populations in early and late apoptotic and necrotic quadrants with the percentage of cancer cell death posttreatment of targeted Nps compared with that of nontargeted Nps. For all the three assays (MTT, mitochondrial membrane potential and apoptosis), Student’s t-test was performed to check the statistical significance between CET MAb-DOCT-γ-PGA and DOCT-γ-PGA Nps. *p<0.05, **p<0.01 and *** p<0.001. Values represent mean ± SD of three independent experiments (n=3). Abbreviations: γ-PGA, poly(γ-glutamic acid); CET MAb, cetuximab monoclonal antibody; DOCT, docetaxel; FITC, fluorescein isothiocyanate; Nps, nanoparticles.

Figure 4

Pharmacokinetics (n=4) and organ distribution analysis (n=4) using HPLC. Notes: (A) Plasma concentration of DOCT versus time profile obtained from blood drawn from the retro-orbital sinuses at different time points from mice treated with free DOCT, DOCT-γ-PGA Nps (nontargeted Nps) and CET MAb-DOCT-γ-PGA Nps (targeted Nps). (B) Concentration of DOCT extracted from organs excised from mice treated with free DOCT, nontargeted DOCT-γ-PGA Nps and targeted CET MAb-DOCT-γ-PGA Nps at (a) 30 min, (b) 2 h, (c) 1 day and (d) 4 days postinjection. Values represent mean ± SD of four independent animals (n=4). Abbreviations: γ-PGA, poly(γ-glutamic acid); CET MAb, cetuximab monoclonal antibody; DOCT, docetaxel; HPLC, high-performance liquid chromatography; Nps, nanoparticles; NT Nps, nontargeted Nps; T Nps, targeted Nps.

Figure 5

Development of gastric cancer xenografts in Balb/c nude mice. Notes: (A) Photographs showing the development and growth of tumor lumps followed by subcutaneous injection of MKN-28 cells. The red circled area indicates the point of tumor induction and further growth. Photographs showing mice with tumors on (B) week 4 (n=4) and (C) week 7. By this time, the mice became morbid due to tumor burden.

Figure 6

Qualitative biodistribution analysis using in vivo imaging in gastric cancer xenografts (n=3). Notes: (A) Biodistribution images of mice treated with IR780-γ-PGA Nps (nontargeted Nps). (B) Biodistribution images of mice treated with CET MAb-IR780-γ-PGA Nps (targeted Nps). The red boxes indicate the time frame during which an enhanced fluorescence intensity was observed in the tumor indicating enhanced tumor accumulation. (C) Mean fluorescence intensity measured from tumors as ROI using the FOBI machine plotted against time of imaging. **p<0.01 indicating the statistical significance of targeted CET MAb-IR780-γ-PGA Nps compared with that of nontargeted IR780-γ-PGA Nps. Values represent mean ± SD of three independent animals (n=3). Abbreviations: γ-PGA, poly(γ-glutamic acid); CET MAb, cetuximab monoclonal antibody; FOBI, fluorescence-labeled organism bioimaging instrument; Nps, nanoparticles; ROI, region of interest.

Figure 7

Quantitative biodistribution analysis using HPLC in gastric cancer xenografts (n=4). Notes: (A) Plasma concentration of DOCT from mice treated with free DOCT, DOCT-γ-PGA Nps (nontargeted Nps) and CET MAb-DOCT-γ-PGA Nps (targeted Nps) on days 1 and 4 postinjection. (B) Concentration of DOCT extracted from tumors excised from mice treated with free DOCT, nontargeted DOCT-γ-PGA Nps (nontargeted Nps) and targeted CET MAb-DOCT-γ-PGA Nps (targeted Nps) on days 1 and 4 postinjection. Similarly, the concentrations of DOCT per milligram of organ are represented in (C) liver, (D) lungs and (E) kidney. Heart, brain and spleen did not have detectable levels of DOCT by HPLC. ^#Represents the p-value indicating the statistical significance of nontargeted DOCT-γ-PGA Nps (nontargeted Nps) and targeted CET MAb-DOCT-γ-PGA Nps (targeted Nps) with that of free DOCT (^##p<0.01 and ^###p<0.001), and *represents the p-value indicating the statistical significance of targeted CET MAb-DOCT-γ-PGA Nps (targeted Nps) with that of nontargeted DOCT-γ-PGA Nps (nontargeted Nps) (**p<0.01). Values represent mean ± SD of four independent animals (n=4). Abbreviations: γ-PGA, poly(γ-glutamic acid); CET MAb, cetuximab monoclonal antibody; DOCT, docetaxel; HPLC, high-performance liquid chromatography; Nps, nanoparticles.

Figure 8

In vivo anticancer efficacy in gastric cancer xenografts (n=4). Notes: (A) Photograph of one representative mouse from each of the four test groups on the day of treatment initiation with tumors of the hind limb. (B) Plot of tumor volume (mm³) versus time, indicating the tumor growth inhibition profile when treated with nontargeted DOCT-γ-PGA Nps (NT Nps) and targeted CET MAb-DOCT-γ-PGA Nps (T Nps) with that of free DOCT along with the saline-treated group. (C) Excised tumors isolated from mice 20 days posttreatment (scale bar =2 mm). (D) Weights of the excised tumors (mg) from mice treated with free DOCT, DOCT-γ-PGA Nps (NT Nps) and CET MAb-DOCT-γ-PGA Nps (T Nps). ^&Represents the p-value indicating the statistical significance of sample treatment compared with that of saline-treated controls (^&&&p<0.001); ^#represents the p-value indicating the statistical significance of DOCT-γ-PGA Nps (NT Nps) and CET MAb-DOCT-γ-PGA Nps (T Nps) with that of free DOCT (^#p<0.05; ^##p<0.01; ^###p<0.001); and *represents the p-value indicating the statistical significance of CET MAb-DOCT-γ-PGA Nps (T Nps) with that of DOCT-γ-PGA Nps (NT Nps) (**p<0.01; ***p<0.001). Scale bar =20 mm. (E) Body weight profile of the mice during the course of treatment. Abbreviations: γ-PGA, poly (γ-glutamic acid); CET MAb, cetuximab; DOCT, docetaxel; Nps, nanoparticles; NT Nps, nontargeted Nps; T Nps, targeted Nps.