Nanoparticles are modified to selectively combat cancer, viruses and bacteria

A Brazilian research group tested the effects of different chemical groups added to the surface of nanoparticles with the aim of making them ‘invisible’ to the immune system, safe for human cells, and effective against pathogens and tumors.

By Sidnei Santos de Oliveira  |  Agência FAPESP – Researchers at the National Energy and Materials Research Center (CNPEM) in Campinas, São Paulo State, Brazil, are developing nanoparticles for use in the treatment of tumors, infections and inflammation. Their work is supported by FAPESP. The proposal is to deliver drugs in ideal doses directly to sick cells, avoiding unnecessary damage to the organism.

However, two obstacles must be surmounted to achieve this goal. The first entails finding a way to prevent proteins from sticking to the surface of the nanoparticle when it comes into contact with the patient’s blood, forming a structure known as a protein corona; this could merge with another nanoparticle’s protein corona to form a tightly bound monolayer that reduces the nanoparticles’ capacity to act and could lead to blood vessel blockage. The other challenge is how to ensure nanoparticle stability in fluids such as blood plasma.

Novel strategies to address these problems are described in articles that the group at CNPEM has recently published in ACS Applied Materials Interfaces and the Journal of Colloid and Interface Science. The study is featured on the cover of the latter journal.

The two articles report the action of particles with dual functionalization, meaning that their surface is modified to avoid protein corona formation and, at the same time, guarantee colloidal stability in the bloodstream.

“Now that we know about the possibility of working with dual-functionalized structures, we can identify the proportions of different chemical groups that favor nanoparticle stability while avoiding toxicity and protein corona formation,” Mateus Borba Cardoso, leader of the CNPEM group, told Agência FAPESP.

The article “Dual functionalization of nanoparticles for generating corona-free and noncytotoxic silica nanoparticles,” published in ACS Applied Materials Interfaces, describes the search for the ideal proportion between the two chemical groups used in dual functionalization: zwitterions and amino acids.

“Zwitterions have separate positively and negatively charged structures that all but cancel each other out [so that the net charge is almost neutral]. These structures prevent protein corona formation and maintain the system’s colloidal stability. The amino group potentially serves to anchor antibodies that direct the particles to the targeted cells, but amino acids are known to induce protein corona formation, as well as destabilize the particles and be highly toxic. We therefore set out to identify the ideal proportion between these two components,” Cardoso said.

The experiment was initially performed using mammalian cells (murine fibroblasts). The next step consisted of a hemolysis assay with human blood supplied by the Blood Center of the University of Campinas’s Medical School (FCM-UNICAMP).

“In this case, the aim was to see whether the particles ruptured red blood cells, which would make them contraindicated for therapeutic use,” Cardoso said.

“The results showed that nanoparticles containing a significant fraction of surface zwitterions did not induce hemolysis and are potentially safe for intravenous administration.”

The second study, “Shielding and stealth effects of zwitterion moieties in double-functionalized silica nanoparticles,” published in the Journal of Colloid and Interface Science, describes the use of zwitterionic structures to retain corona-forming compounds.

“This zwitterionic layer forms a sort of water membrane around the nanoparticle, so that it isn’t identified as an invader by the organism’s defense mechanisms,” Cardoso explained.

Some of the proteins that tend to stick to the surface of the particles when they come into contact with blood, he added, act as immune system signalers, attracting defense cells such as macrophages to the site, where these immune cells attempt to eliminate the foreign body.

“Avoiding protein corona formation is therefore fundamental for the particle to go unnoticed by the organism’s defense mechanisms. We’re trying to obtain these ‘invisible’ particles. But if they’re ‘invisible,’ will they be able to interact with any biological structure?”

To answer this question, the group conducted dual functionalization experiments with compounds containing zwitterionic groups and another biologically active group. “We tested the hemolytic properties [risk of causing hemolysis] of these ‘invisible’ nanoparticles, their protein corona formation capacity, and whether they could interact with different biological structures,” Cardoso said.

The effect was assessed in vitro in cultured mammalian cells, bacteria of the species Escherichia coli, and zika viruses. The results showed that the water membrane kept the protein corona-forming compounds hidden and simultaneously prevented the interaction between the nanoparticles and the animal cells, bacteria or viruses, thereby blocking any therapeutic action.

The next step, according to Cardoso, will be to try to solve the impasse by finding a way to have the biologically active groups leave this hydrated layer so that they can be selectively recognized.

“We’ll try to insert the biologically active groups or other structures with proven biological activity outside the zwitterionic group’s zone of influence. To this end, the amino group will be essential,” he said.

Advances in nanomedicine

Research in nanomedicine has been underway since the early 1980s, based on the insertion of drugs into nanoparticles that carry them in the bloodstream and deliver them to a targeted site or organ. The nanoparticles may be made of proteins, solid lipidic structures or other substances. Currently, the pharmaceutical industry mainly uses liposomes, a kind of membrane that closely resembles the membranes surrounding cells in the human body.

“Nanoparticles in general can increase the time during which a drug moves through the organism from six hours to eight or even 12 hours, depending on the drug and on the nanoparticle. This may lead to better treatment outcomes,” Cardoso said.

Liposomes are not toxic to humans, but they cannot deliver drugs to a specific site and may have undesired side effects, such as hair loss in cancer treatment, for example.

The nanoparticles developed by the researchers at CNPEM have a rigid structure, unlike liposomes. This structure consists of a nuclear layer made primarily of silica. The strategy is different, as the core is coated with chemicals that only react at specific sites and their action is therefore selective. “Think of a tennis ball in which the center is made of silica and the surrounding tissue contains the functionalizations,” Cardoso said.

The CNPEM group had previously demonstrated the feasibility of this strategy in cancer treatment, using nanoparticles to target tumor cells with chemotherapeutic compounds and avoid the interaction with healthy cells. They also showed the efficacy of the strategy to inactivate HIV in vitro.

The article “Dual functionalization of nanoparticles for generating corona-free and noncytotoxic silica nanoparticles” by Jessica Fernanda Affonso de Oliveira, Francine Ramos Scheffer, Ryan F. Landis, Érico Teixeira Neto, Vincent M. Rotello and Mateus Borba Cardoso can be retrieved from: https://pubs.acs.org/doi/10.1021/acsami.8b12351.

The article “Shielding and stealth effects of zwitterion moieties in double-functionalized silica nanoparticles” by Lívia M. D. Loiola, Marina Batista, Larissa B. Capelettia, Gabriela B. Mondo, Rhubia S. M. Rosa, Rafael E. Marques, Marcio C. Bajgelman and Mateus B. Cardoso can be retrieved from: www.sciencedirect.com/science/article/pii/S0021979719307179?via%3Dihub.