Alg-g-PEG nanosphere-mediated intracellular growth factor delivery inhibits cancer cell proliferation

摘要

Introduction: Recent reports suggest intracrine high molecular weight (hi) fibroblast growth factor 2 (FGF-2) signaling may play a role in inhibiting cell proliferation and promoting cell death. Herein we report on the use of alginate-graft-poly (ethylene glycol) (Alg-g-PEG) nanospheres to intracellularly deliver hi FGF-2 via a stealth mechanism, without interacting with FGF-2 cell surface receptors. We hypothesize that the intracellular delivery of FGF-2 may be a potential strategy for cancer therapy. Materials and Methods: The preparation of Alg-g-PEG nanospheres was based on the literature by Miao et al. (2014) Briefly, Alg-g-PEG copolymers were synthesized via carbodiimide chemistry. Alg-g-PEG solution was mixed with hi FGF-2 and formed into nanospheres using a water/oil emulsion and calcium crosslinking. Carbodiimide chemistry was also performed between the Alg-g-PEG nanosphere surface and Alexa Fluor® 647 to form fluorescent nanospheres. Dynamic light scattering (DLS) was used to measure diameter and zeta-potential of FGF-2-encapsulaled nanospheres. A human bFGF ELISA was used to determine FGF-2 encapsulation efficiency and in vitro release profile. For flow cytometry assays, human lung cancer cells (A549 ATTC®) were co-cultured with fluorescent Alg-g-PEG nanospheres for 24 h. For cell proliferation assays, an equal number of A549 cells were seeded into 12-well plates. Experimental groups included: cells without any treatment (control), cells with 200 μg/mL FGF-2-encapsulated nanospheres, cells with empty nanospheres, and cells with 10 ng/ml FGF-2 (extracellular). Cell viability was measured using an MTT-based assay (Sigma-Aldrich). Resultsand Discussion: Figure 1 illustrates the intracellular delivery of FGF-2 into cell nuclei to shut down proliferation. DLS determined that the nanospheres were 84 nm in diameter with a zeta-potential = -7.82 mV (see Figure 2 above). The FGF-2 release profile demonstrated a burst release within the first few days (Figure 2, bottom). Flow cytometry showed that over 90% of the cells had internalized Alg-g-PEG nanospheres (Figure 3, Left). Cells treated with FGF-2-encapsulated nanospheres exhibited significantly lower viability (p = 0.01), whereas cells treated with extracellular FGF-2 had significantly higher viability compared with control cells (p = 0.036). The empty nanospheres had no significant effect, indicating that the Alg-g-PEG nanospheres were not cytotoxic (Figure 3, right). Conclusion: As a potential cancer therapy, intracellular delivery of hi FGF-2 may provide an alternative treatment to chemotherapeutics or an adjunct treatment (in combination with chemotherapeutics).