Scientists of the Frank Laboratory of Neutron Physics JINR have developed and studied bio-hybrid drug nano-complexes based on natural components with antibacterial and anticancer efficacy. The results of these structural and morphological studies may become the basis for obtaining a new generation of drugs in the future that will fight antibiotic-resistant microbes.

The first results on the creation of the next-generation substances based on phytogenerated Ag/AgCl nanoparticles were presented by an FLNP senior researcher, Ph.D. Yulia Gorshkova at a thematic seminar on nanostructures on 14 February. She said that the FLNP scientific group had been tasked with the development of the nano-complexes and with conducting of structural, spectral, and morphological studies. Based on the results, stable biohybrids with the required parameters should be selected: considering their composition, shape, and size. After characterisation, it was necessary to conduct a series of biological tests of the selected nanocomplexes in order to determine the possibility of their application in the fight against both resistant bacteria and cancer.

Yulia Gorshkova using atomic force microscope at FLNP JINR

At present, the synthesis of a new generation of nanodrugs for targeted drug delivery has become one of the urgent tasks of modern pharmacology. They may help address issues such as antibiotic-resistant bacteria and cancer. Nearly 5 million people died in 2019 due to drug-resistant bacterial infections, according to The Lancet. The most common of them are E. coli, Staphylococcus aureus, Klebsiella pneumonia, pneumococcus, salmonella. According to the World Health Organization, one in six deaths in the world is caused by cancer.

Yulia Gorshkova noted that today synthetic medicines dominate the medication market, but in recent years, developers have been increasingly replacing individual components with completely natural substances or derivatives of them. They have a number of advantages over synthetic ones, such as decreased toxicity. As well as not only a lesser effect on the immune system, but – to a certain extent – even supporting it.

All substances used by JINR scientists to create biocompatible hybrid nanocomplexes are based on natural components. The first such component was “green” nanoparticles of silver and silver chloride. Pomace from turmeric root or a mixture of grape leaves and mint was used for it. Liposomes of soy lecithin and chitosan were also used as components.

“In addition to a membrane protecting a cell, the membrane itself also has a protective shell called glycocalyx. To overcome this protection and deliver our material into the cell, we have used a natural biopolymer – chitosan. It is non-toxic, low-allergic, biocompatible, and biodegradable,” the speaker explained.

As a result, scientists obtained three biohybrid nanocomplexes, which were further studied by various physico-chemical methods, including small-angle and X-ray small-angle scattering, scanning electron microscopy, and atomic force microscopy. UV-visible and infrared spectroscopy allowed scientists to identify functional groups and the interaction between the components of biohybrid systems. X-ray diffraction made it possible to determine the size of the crystallites that make up the nanoparticles. Using atomic force spectroscopy on topological images, scientists studied the structure and shape of the obtained nanoparticles: smooth particles without a chitosan shell and rougher particles coated with chitosan. Moreover, using this method, the dimensions of the developed materials and their components were analysed. The results obtained were further confirmed by the small-angle scattering method, the advantage of which is the possibility for studying biological systems in a native environment. The measurements were carried out at the YuMO small-angle neutron scattering spectrometer at the IBR-2 pulsed reactor.

“Using these objects as an example, the complementarity of neutron and X-ray scattering works very well. Especially it is applicable when the concentrations of substances are low, as in our case, and it is necessary to look at the structure of individual components. Here, neutrons from the IBR-2 reactor helped us to establish the multilamellar structure of liposomes, because they are not sensitive to silver nanoparticles. In addition, we have used X-rays to characterise the nanoparticles themselves,” Yulia Gorshkova said.

Complex of spectrometers of research reactor IBR-2

At the same time, the stability of the developed nanocomplexes was monitored at each stage of the characterization. According to the experiment, the complex in which the liposomes were surrounded by nanoparticles turned out to be the most stable.

The tests for the biological activity of the obtained biocompatible hybrid nanocomplexes showed their good antioxidant activity.

Antibacterial activity was studied at gram-positive and gram-negative bacterial cells. “We have not got a sufficient result after testing our developed nanomaterials against enterococcus. Thus, it has become the reason for further modification of the developed nanosystem. At the same time, we have reached a significant result against Staphylococcus aureus and Koch’s bacillus. It allows us to state that a new generation of drugs that will fight resistant microbes can be developed on biomaterials based on natural components,” Yulia Gorshkova noted.

Scientists also monitored the anti-cancer efficacy by determining the therapeutic index of the nanocomplexes. For some resulting systems, it exceeded one. That means that its activity against cancer cells is greater than against healthy cells.

YuMO spectrometer

In an in-vitro experiment with cell lines of colon cancer and human liver cancer, all complexes showed their activity. The working concentrations of cell treatment with the developed nanomaterials were also determined. Two out of three complexes developed showed hemolytic activity, which means that they can destroy blood cells. However, the third composite, which has bio-similar lipid bilayers in its composition, showed no hemolytic activity and had the highest efficiency of the three composites.

Speaking about the practical application of the research, Yulia Gorshkova noted that the methods and devices used can be actively applied at all stages of the development of pharmacological substances. “We can monitor the structure, stability at every stage of the development of such drugs, including applying these methods in preclinical trials. It is obvious that the quality of the structural measurements carried out determines the amount of time and money that is spent on clinical trials and introduction of these new materials onto the market,” she emphasised. She also noted the advantages of the methods used, as well as the instruments at the Frank Laboratory of Neutron Physics JINR.

As a result of the work, two articles have been published: Gorshkova Yu., Barbinta-Patrascu M.E., et al. Biological Performances of Plasmonic Biohybrids Based on Phyto-Silver/Silver Chloride. 2021. Nanomaterials. 11. 1811 and Barbinta-Patrascu M.E., Gorshkova, Yu., et al. Characterization and antitumoral activity of biohybrids based on turmeric and silver/silver chloride nanoparticles. 2021. Materials, 14, 4726.

The work was carried out in the international collaboration among chemists, biologists, physicists, and physicians from five countries: Russia, Romania, Serbia, Poland, and the Czech Republic with financial support from JINR-Romania grants and programmes.

The report on the relevance of structural and morphological studies for solving modern biomedical problems was presented to the JINR Scientific Council at the session on 25 February.