Date Published: February 6, 2017
Publisher: Public Library of Science
Author(s): Angelika Kwiatkowska, Ludomira H. Granicka, Anna Grzeczkowicz, Radosław Stachowiak, Michał Kamiński, Zuzanna Grubek, Jacek Bielecki, Marcin Strawski, Marek Szklarczyk, Bing Xu.
The inadequate efficiency of existing therapeutic anti-cancer regiments and the increase in the multidrug resistance of cancer cells underscore the need to investigate novel anticancer strategies. The induction of apoptosis in tumors by cytotoxic agents produced by pathogenic microorganisms is an example of such an approach. Nevertheless, even the most effective drug should be delivered directly to targeted sites to reduce any negative impact on other cells. Accordingly, the stabilized nanosystem (SNS) for active agent delivery to cancer cells was designed for further application in local anti-tumor therapy. A product of genetically modified Escherichia coli, listeriolysin O (LLO), was immobilized within the polyelectrolyte membrane (poly(ethylenimine)|hyaluronic acid) shells of ‘LLO nanocarriers’ coupled with the stabilizing element of natural origin.
The impact of LLO was evaluated in human leukemia cell lines in vitro. Correspondingly, the influence of the SNS and its elements was assessed in vitro. The viability of targeted cells was evaluated by flow cytometry. Visualization of the system structure was performed using confocal microscopy. The membrane shell applied to the nanocarriers was analyzed using atomic force microscopy and Fourier transform infrared spectroscopy techniques. Furthermore, the presence of a polyelectrolyte layer on the nanocarrier surface and/or in the cell was confirmed by flow cytometry. Finally, the structural integrity of the SNS and the corresponding release of the fluorescent solute listeriolysin were investigated.
The construction of a stabilized system offers LLO release with a lethal impact on model eukaryotic cells. The applied platform design may be recommended for local anti-tumor treatment purposes.
Modern medicine undoubtedly can boast great successes in the control of tumor development and progression. Nevertheless, an increase in cancer-related mortality is still observed in developed countries [1–4]. This increase is caused by both side effects associated with the conventional treatment (e.g., surgery, radiotherapy and chemotherapy) and low efficiency of used drugs, which are increasingly proven to be inadequate . Furthermore, only a small amount of an active factor is delivered directly to the targeted cells due to significant losses during transport. Accordingly, it is often necessary to use an excess of applied medicines. Unfortunately, the vast majority of cytostatic agents are dissipated through the body, causing significant emaciation of patient organisms. As a consequence, the benefits obtained from the application of antitumor factors are overshadowed by numerous excessive side effects. The most frequently occurring afflictions recorded during clinical use of typical chemotherapeutic agents include bone marrow suppression, heart failure (as a consequence of cardiomyopathy) typhlitis and dyspigmentation [6–9]. In recent decades, considerable efforts have been made to change this state of affairs and reduce the undesirable effects that are connected with classic anti-tumor therapies.
After examining three different platforms, we conclude that the SNS is the most effective platform. Based on biologically derived elements, the unique design of the developed system supplies LLO to the targeted cells. The constructed system provides immediate cytotoxic effects on tumor cells while simultaneously ensuring the protection of the active agent from potential destruction during the experiment. Comparing the performance of constructed platforms in targeted cells, the increased lethal effects of systems bearing transferrin is observed, indicating that the ligand increases the platform affinity towards the tumor cells. Finally, the ‘cell core’ with transferrin ligands ensures system stability and enhances the titer of the LLO nanocarriers that are delivered to the targeted cells. Of note, the macromolecular substances involved because of phagocytosis induced visible intracellular effects after a few dozen minutes. This process cannot be observed in cases of macrophages because of their phagocytic function. The unique advantage of the system is its possible application in local listeriolysin O anti-tumor therapies. These experiments provided the basis for the further development of LLO delivery systems as an alternative to targeting chemotherapeutic drugs in local anti-tumor therapy.