Methotrexate-plasmid DNA polyplexes for cancer therapy: Characterization, cancer cell targeting ability and tuned in vitro transfection

Highlights

• We present a novel strategy combining targeting, chemo and gene therapies.

• Methotrexate vectors exhibit cancer-cell targeting ability through folate receptors.

• Confocal microscopy shows intracellular localization of the polyplexes.

• In vitro transfection efficiency can be tuned to enhance protein expression.

• A preliminary study demonstrates synergistic effect potentiating further research.

Abstract

Cell targeting is still the most auspicious task in the design/development of innovative drug and gene delivery systems for advanced biomedical applications. To evolve from conventional delivery vectors, which present several limitations, we report a novel strategy combining a targeting tool associated with both chemical and gene therapies. To accomplish this innovation, the anti-cancer drug methotrexate (MTX) has been loaded into polyplexes based on polyethylenimine (PEI) and a p53 encoding plasmid DNA. The main properties (size, zeta potential, loading and complexation capacity) of the conceived triple-action vector have been determined and revealed to be adequate for intracellular applications. Furthermore, the fine structure of MTX/pDNA based carriers has been analysed by Fourier-transformed infrared (FTIR) spectroscopy. The cancer-cell targeting ability of the developed formulations has been evaluated. Studies on fibroblast and HeLa cells demonstrate the MTX cancer cell targeting through folate receptors, viewed as surface biomarkers and commonly overexpressed in tumors. This achievement supports the evidence for receptor mediated endocytosis. Confocal microscopy studies confirmed intracellular localization of targeting polyplexes, resulting in enhanced sustained pDNA uptake. Furthermore, the efficiency of in vitro transfection can be modulated by MTX concentration with great implications on gene expression and protein production. Preliminary assays on the treatment of cancer cells with the tri-functional PEI/pDNA/MTX vectors show a significant reduction of their viability.

Introduction

In the last decade, researchers have made a considerable effort to design and develop new and advanced gene delivery systems that can be biocompatible, able of cell uptake/internalization and exhibiting targeting specificity, thus, deeply contributing for the success of gene-based therapies from which the most deadly diseases, such as cancer, can deeply benefit [[1], [2], [3], [4]]. Additionally, the co-delivery of different payloads, namely the simultaneous delivery of anticancer drugs and genes, has brought a tremendous progress on cancer therapeutic efficacy, due to synergistic effect, and deserves to be further explored [[5], [6], [7]]. The success of gene therapy relies on the ability of a genetic-based carrier to cross the cell membrane, avoid intracellular degradation and ultimately reach the nucleus where gene expression can occur. Despite the greater transfection efficiency accomplished with viral vectors, their drawbacks, as their immunogenicity, toxicity and possible random mutagenesis limit their application in gene delivery [8]. On contrary, non-viral vectors present many advantages over viral ones and reveal to be best suited for gene therapy purposes. Synthetic carriers are easy to produce and manipulate, can accommodate large amounts of genetic material and have lack of immune response [[9], [10], [11]]. Beyond these characteristics, non-viral vectors can be chemical or physically engineered and/or functionalized to be cell recognized, promote specific targeting and to enhanced overall gene transfection performance [[12], [13], [14]]. In particular, the chemical modification or the combination of delivery systems with ligands, able to specifically bind receptors overexpressed in cancer cells, can significantly increase therapeutic efficacy [15,16]. For instance, MTX, a therapeutic agent commonly used in cancer treatments that binds to the folate receptors, commonly overexpressed in cancer cells, is responsible for the inactivation of the enzyme dihydrofolate reductase that plays a crucial role in cell survival and division processes [17,18]. Although scarcely studied on gene cancer therapy, the functionalization of nanosystems with MTX already demonstrated to be a powerful strategy to ensure both targeting and therapeutic effect leading to relevant achievements in this field [16,19]. The combination of cell targeting, chemotherapy and gene therapy represent an incredible asset in the development of advanced therapeutic interventions greatly contributing for the evolution of cancer treatment [16,20]. Among the wide range of non-viral vectors commonly used for gene delivery, polyplexes formed through the electrostatic binding between a cationic agent (cationic lipids, polypeptides, dendrimers or PEI) and an anionic oligonucleotide conquered special interest [[20], [21], [22]]. PEI, in particular, has been largely applied in the formation of DNA based nanoparticles to therapeutically operate [20,[23], [24], [25]]. PEI presents a high cationic charge density, that favors its interaction with the negatively phosphate groups of DNA originating the condensation of this latter, along with an impressive endosomolytic activity [26,27]. These main PEI assets turn this polymer in a very convenient option to engineer PEI/DNA based nanocarriers to be applied in gene delivery protocols. In a previous report, our research team performed a complete study on PEI/p53 encoding plasmid DNA polyplexes and the most important aspects influencing the properties of these vectors were revealed [28]. Furthermore, preliminary studies on the cellular transfection were conducted and stimulate further investigation [28]. In line with this, in the present work we took advantage of the previous screening study to select the most suitable systems to precede our research focused on in vitro studies mediated by MTX/pDNA based polyplexes. Confocal microscopy analysis demonstrated that these polymeric nanoparticles can be internalized and localized into the nucleus of cancer cells. Moreover, we found that the transfection efficiency can be modulated by MTX concentration. After transfection, MTX can be found in the cytosol of fibroblast and HeLa cells and the encoded protein produced. A comparative study between non-cancer/cancer cells supports the evidence of MTX cancer cell targeting via folate receptors and, therefore, cellular internalization by receptor mediated endocytosis. The treatment of cancer cells with PEI/pDNA/MTX vectors has been evaluated and revealed their ability in decreasing cellular viability. The results are promising and instigate further research.