The controlled delivery and release of drug molecules at specific targets increases the therapeutic efficacy of treatment. This paper presents a triple complex which is a new potential drug delivery system. Triple complex contains single-walled carbon nanotubes, Congo red, and doxorubicin. Nanotubes are built of a folded graphene layer providing a large surface for binding Congo red via “face-to-face” stacking which markedly increases the binding capacity of the carrier. Congo red is a compound that self-associates to form supramolecular ribbon-like structures, which are able to bind some drugs by intercalation. The nanotube–Congo red complex can bind the model drug doxorubicin. Thus, a new triple carrier system was obtained. The aim of this paper is to present studies on the controlled release of a model anticancer drug from a triple carrier system through pH changes. The specific aim of the study was to model the structure of the obtained experimental systems and to compare the changes in the average energy of interaction between its components induced by pH changes. The studies also aimed to compare the intensity of pH-dependent changes in hydrodynamic diameters of individual components of the triple carrier system. The effect of pH changes on the stability of the analyzed systems was examined using the molecular modeling method and dynamic light scattering. The decrease in pH influenced the structure and stability of the analyzed triple systems and ensured efficient drug release. The changes in hydrodynamic diameters of the obtained fractions were examined with the use of dynamic light scattering and were confirmed by computer simulation methods. The formulation presented in this paper shows potential for a therapeutic application owing to its high drug binding capacity and pH-dependent release. This ensures prolonged local action of the drug. The results reveal that the studied complex fulfills the basic requirements for its potential use as drug carrier, thus reducing side effects and enhancing pharmacological efficacy of drugs.

Designing an effective targeted anticancer drug delivery method is still a big challenge, since chemotherapeutics often cause a variety of undesirable side effects affecting normal tissues. This work presents the research on a novel system consisting of single walled carbon nanotubes (SWNT), dispersed with Congo Red (CR), a compound that forms self-assembled ribbon-like structures (SRLS) and anticancer drug doxorubicin (DOX). SWNT provide a large surface for binding of planar aromatic compounds, including drugs, while CR supramolecular ribbon-like assemblies can be intercalated by drugs, like anthracycline rings containing DOX. The mechanism of interactions in SWNT–CR–DOX triple system was proposed based on electrophoretic, spectral, Dynamic Light Scattering and scanning electron microscopy analyzes. The profile of drug release from the investigated system was evaluated using dialysis and Differential Scanning Calorimetry. The results indicate that ribbon-like supramolecular structures of CR bind to SWNT surface forming SWNT–CR complexes which finally bind DOX. The high amount of nanotube-bound CR greatly increases the capacity of the carrier for the drug. The high capacity for drug binding and possible control of its release (through pH changes) in the analyzed system may result in prolonged and localized drug action. The proposed SWNT–CR–DOX triple system meets the basic criteria that justifies its further research as a potential drug carrier.

The present study attempts to explain the reason for the selective generation of an increase in intensity of Congo red (CR) fluorescence as an effect of the dye interacting with proteins and polysaccharides. This supramolecular dye, which creates ribbon-shaped micelles in aqueous solutions when excited with blue light (470 nm), presents low fluorescence with a maximum within the orange-red light range (approximately 600 nm). In the same conditions, CR-stained preparations of heat-denatured proteins, some native proteins (e.g. cell surface receptors) and cellulose show intense orange-red fluorescence when observed using a fluorescence microscope. The fluormetric measurements showed that the factors that cause the dissociation of the ribbon-shaped CR micelle – ethanol, urea, dimethyl sulfoxide (DMSO) and cholate – all contributed to a significant increase in the fluorescence intensity of the CR solutions. The fluorescence measurements of CR bound to the immunoglobulin light lambda (L λ) chain and soluble carboxymethyl cellulose (CMC) showed a fluorescence intensity which was many times higher. In the case of the denatured (65°C) immunoglobulin L λ chain, the fluorescence intensity significantly exceeded the values observed for the factors which break down the CR micelles. The dissociation of the ribbon-shaped micelles and the complexation of the monomeric CR form with polymers are two of the factors explaining the intense fluorescence of protein and polysaccharide preparations stained with CR.

Targeted immunotherapy has expanded to simultaneous delivery of drugs, including chemotherapeutics. The aim of the presented research is to design a new drug carrier system. Systems based on the use of proteins as natural components of the body offer the chance to boost safety and efficacy of targeted drug delivery and excess drug removal. Congo red (CR) type supramolecular, self-assembled ribbon-like structures (SRLS) were previously shown to interact with some proteins, including albumin and antibodies complexed with antigen. CR can intercalate some chemotherapeutics including doxorubicin (Dox). The goal of this work was to describe the CR-Dox complexes, to analyze their interaction with some proteins, and to explain the mechanism of this interaction. In the present experiments, a model system composed of heated immunoglobulin light chain Lλ capable of CR binding was used. Heat aggregated immunoglobulins (HAI) and albumin were chosen as another model system. The results of experiments employing methods such as gel filtration chromatography and dynamic light scattering confirmed the formation of the CR-Dox complex of large size and properties different from the free CR structures. Electrophoresis and chromatography experiments have shown the binding of free CR to heated Lλ while CR-Dox mixed structures were not capable of forming such complexes. HAI was able to bind both free CR and CR-Dox complexes. Albumin also bound both CR and its complex with Dox. Additionally, we observed that albumin-bound CR-Dox complexes were transferred from albumin to HAI upon addition of HAI. DLS analyses showed that interaction of CR with Dox distinctly increased the hydrodynamic diameter of CR-Dox compared with a free CR supramolecular structure. To our knowledge, individual small proteins such as Lλ may bind upon heating a few molecules of Congo red tape penetrating protein body due to the relatively low cohesion of the dye micelle. If, however, the compactness is high (in the case of, e.g., CR-Dox) large ribbon-like, micellar structures appear. They do not divide easily into smaller portions and cannot attach to proteins where there is no room for binding large ligands. Such binding is, however, possible by albumin which is biologically adapted to form complexes with different large ligands and by tightly packed immune complexes and heat aggregated immunoglobulin-specific protein complex structures of even higher affinity for Congo red than albumin. The CR clouds formed around them also bind the CR-Dox complexes. The presented research is essential in the search for optimum solutions for SRLS application in immuno-targeting therapeutic strategies, especially with the use of chemotherapeutics.

Congo red (CR) type self–assembled ribbon–like structures (SRLS) were previously shown to interact with some proteins, including albumin. SRLS also complex with some drugs with a flat, ring–shaped structure with aromatic characteristics, intercalating them into their ribbon structure. The combination of interaction with proteins and drug binding by SRLS enables the use of such systems for immunotargeting. It is especially interesting in the case of chemotherapeutic agents. The present experiments aimed to show that the model carrier system composed of supramolecular albumin and Congo red efficiently binds doxorubicin (Dox) and that the drug can be released at reduced pH. The presented results come from the studies on such complexes differing in the molar ratio of CR to Dox. The following methods were used for the analysis: electrophoresis, dialysis, gel filtration, spectral analysis, and analysis of the size of the hydrodynamic radius using the dynamic light scattering method (DLS). The applied methods confirmed the formation of the CR–Dox complex, with large dimensions and changed properties compared with free CR. The presented results show that albumin binds both CR and its complex with Dox. Various CR–Dox molar ratios, 5:1, 2:1, and 1:1, were analyzed. The confirmation of the possibility of releasing the drug from the carriers thus formed was also obtained. The presented research is important due to the search for optimal solutions for the use of SRLS in drug immunotargeting, with particular emphasis on chemotherapeutic agents.

The balance between apoptosis and autophagy plays a key role in cancer biology and treatment strategies. The aim of this study was to assess the effect of the mTOR kinase inhibitor everolimus and chloroquine on the regulation of proliferation, caspase-3 activation, and apoptosis in melanoma cells. We studied the activity of caspase-3 and the levels of caspase-3 and -9 using the Western blot technique. Cellular apoptosis was examined using a DNA fragmentation assay, and changes in the cell nucleus and cytoskeleton were examined using fluorescence microscopy DAPI, OA/IP. We also studied the rearrangement of lipid structures using fluorescent dyes: Nile Red and Nile Blue. A low nanomolar concentration of the mTOR kinase inhibitor everolimus in combination with chloroquine activated the apoptosis process and decreased cell proliferation. These changes were accompanied by an obvious change in cell morphology and rearrangement of lipid structures. Alterations in lipid redistribution accompanying the process of apoptosis and autophagy are among the first to occur in the cell and can be easily monitored in in vitro studies. The combination of mTOR inhibitors and chloroquine represents a promising area of research in cancer therapy. It has the potential to enhance treatment efficacy through complementary mechanisms.