Trastuzumab Targeted Neratinib Loaded Poly-Amidoamine Dendrimer Nanocapsules for Breast Cancer Therapy

Abstract

Background: Human epidermal growth factor receptor2 (Her2) positive breast cancer represents 25% of breast cancer cases. Targeted therapy with Her2 monoclonal antibody, trastuzumab (TZ), represents the first-line treatment for this type of breast cancer. In addition, neratinib, an irreversible inhibitor of the HER-2 receptor tyrosine kinase, has recently been approved as adjuvant therapy to TZ. This study aims to formulate (TZ)-grafted dendrimers loaded with neratinib, allowing a dual treatment alongside reducing the associated resistance as well as targeted therapy.

Methods: TZ was conjugated on the surface of dendrimer using hetero-cross linker, MAL-PEG-NHS, and the zeta potential, and in vitro release of neratinib from dendrimers was characterized. Formulated dendrimers were also fluorescently conjugated with fluorescein isothiocyanate to visualize and quantify their SKBR-3 cellular uptake.

Results: The G4 PAMAM dendrimer showed successful encapsulation of neratinib and a sustained release profile. Comparative in vitro studies revealed that these TZ-targeted dendrimers loaded with neratinib were more selective and have higher antiproliferation activity against SKBR-3 cells compared to neratinib alone and neratinib loaded dendrimer.

Conclusion: In the current study, neratinib loaded in plain and trastuzumab-grafted dendrimer were successfully prepared. Enhanced cellular uptake of trastuzumab conjugated dendrimers was shown, together with a higher cytotoxic effect than plain neratinib dendrimers. These findings suggest the potential of TZ-conjugated dendrimers as targeting carrier for cytotoxic drugs, including neratinib.

Keywords: Her2-positive; breast cancer; neratinib; targeted dendrimer; trastuzumab.

Figure 1

Schematic illustrating the five-step reaction to prepare a formula of neratinib loaded into G4 PAMAM dendrimers then conjugated to trastuzumab as targeted therapy for breast cancer. Step 1: loading of neratinib into G4 PAMAM dendrimers, step 2: synthesis of FITC-labeled dendrimers, step 3: conjugation of heterocross-linker maleimide-poly(ethylene) glycol-N-hydroxysuccinimide (NHS-PEG-MAL) to FITC-labeled dendrimers, step 4: TZ thiolation, step 5: bioconjugation of thiolated TZ with loaded dendrimers.

Figure 2

TEM images at different magnification to study the morphology of (A) blank PAMAM dendrimers and (B) neratinib loaded PAMAM dendrimers verifying encapsulation of neratinib inside the dendrimer.

Figure 3

Efficiency of bioconjugation of thiolated TZ with dendrimers (A) SDS-PAGE analysis of TZ (lane 2), thiolated TZ (Lane 3) and marker (lane 1) [left side]. Tests run at a constant voltage of 200 V in Tris/glycine/SDS buffer. The activity of TZ was checked after thiolation (B) Bradford assay of the formula after each step checking the activity of TZ within the formula [right side].

Figure 4

In vitro release of neratinib calculated as percentage of cumulative release from dendrimers and TZ-conjugated dendrimers showing similar sustained release pattern with decreased amount from TZ-conjugated dendrimers as 10% of loaded drug lost through the process. No significance difference between the two formulas.

Figure 5

Percentage of cell viability of human breast cancer cell lines SKBR-3 (HER2-positive) treated with neratinib, neratinib-loaded-dendrimers, and neratinib-loaded-dendrimers trastuzumab grafted. Untreated cells were used as control, data are presented as the mean ± SD of triplicate experiments. Cytotoxicity assay was performed using alamar blue assay. Then cells were incubated with neratinib, neratinib-dendrimer or neratinib-dendrimer-TZ for 48 h then 50 μL of alamar blue reagent for 2–3 h. The fluorescence intensity of the reagent was measured at 560 nm excitation wavelength and 590 nm emission wavelength.

Figure 6

Cellular uptake studies of fluorescently labeled neratinib-loaded-dendrimers. (A) Fluorescent microscopic images of neratinib-dendrimer-FITC, and neratinib-dendrimer-FITC-trastuzumab after 5 h of incubations, and control untreated cells. Scale bars = 10 µm. (B) Quantitative fluorescence intensity of prepared dendrimers. Data are presented as the mean ± SD of triplicate experiments. *p < 0.05 in Student’s t-test relative to non-targeted neratinib-dendrimer formula.

Figure 7

HPLC assessment of neratinib. (A) Calibration curve of standard neratinib. A stock solution containing 10 mg of drug in mobile phase was used. A daily standard calibration curve (n=9) of 10, 20, 40, 60, 80, and 100 µg/mL was prepared. The standards were transferred to glass autosampler vials with pre-slit septum (Waters, USA), where 20 µL was injected into the HPLC system for analysis. (B) Representative HPLC chromatogram of neratinib at 220 nm using Waters HPLC system, utilizing Waters 2707 autosampler delivery system and using symmetry C18 column (4.6 x 1.0 cm). The total run time was 5.0 min and the autosampler temperature was ambient temperature. The flow rate was 1 mL/min and the injection volume was 20 μL and detection was performed at 220 nm. Data were analyzed with an Empower Pro Chromatography Manager Data Collection System. All experiments were performed three times in triplicate.

Figure 8

Stability of released neratinib from formula in autosampler and after freeze– thaw cycles for 6 months. Data are presented as the mean ± SD of triplicate of three different experiments (n = 9). *p < 0.05 in Student’s t-test relative to neratinib loss by 23% after 6 months.