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Alternative routes

Nanotechnology opens up the way for bio-compounds that will replace bones and tissues and target medical drugs

marina ladeira/ufmgHigh levels of protein in liver cells, when incubated only with nanotubes, in the first picture (in green and red) drop significantly in the second picture as a result of gene silencingmarina ladeira/ufmg

Nanostructures to carry pharmaceutical substances that fight cancer, infectious and parasite diseases and that can also act as diagnostic agents are examples of Brazilian university research studies into the use of nanotechnology for the production of new drugs. One of the innovative lines of research uses carbon and collagen nanotubes to produce new tissue such as skin, for example, or to help bone regeneration. The carbon nanotubes are cylinder-shaped structures synthesized from carbon, with specific mechanical, thermal and electric properties that are far superior to those of other materials. Collagen is a crucial molecule for any live system and is responsible for the structuring of the skeleton and of the organs. Studies conducted at the Federal University of Minas Gerais/ UFMG, initially led by professors Luiz Orlando Ladeira and Rodrigo Lacerda at the Nanomaterials Laboratory of the Physics Department showed that this is a very promising bio-compound.

The idea of creating the bio-material arose from Ladeira’s observation that the size of collagen and of carbon nanotubes is similar. The carbon nanotubes are produced by the said laboratory in sizes that range in diameter from 1 to 3 nanometers (nm) (1 nanometer equals one millionth of one millimeter) and are up to one thousand nm long (see Pesquisa Fapesp no.118); they are produced for various applications and at the request of several Brazilian research groups. Live beings have more than 20 types of collagen, but human collagen, referred to as Type 1, which is found in cartilage and bones, is comprised of three amino acid chains that form a three-helix, helicoidal arrangement. “The molecule resembles a fiber, with diameter of one nanometer and length of 300 nanometers,” says Ladeira.

The collagen gel is used as a support matrix for the culture of several kinds of cells, because they generally harbor protein receptors. When added to the carbon nanotube, the gel becomes stronger and allows for the structuring of the cells’ three-dimensional growth in the matrixes. “The collagen matrix is a bio-compatible and bio-degradable structure that allows cells to be anchored for tissue engineering, i.e., to create organs, skin growth, parts of the myocardium and cell structures.” In this case, the researchers are studying the interaction for bone production.

The research project began three years, with the participation of doctoral student Edelma Eleto, who also shares the ownership of the patent, filed in Brazil and abroad. “We saw that the bio-compound induces the production of hydroxiapatite, which is responsible for producing bone rigidity,” says Ladeira. This means that the material is bio-degradable, bio-compatible, and promotes ostheogenesis (the process of forming and developing bones). The bio-compound has attracted the interest of the Nanosolutions corporate group, based in Mexico City. The group has started negotiating with the university to conduct the clinical studies required for the product’s certification.

Biological applications
At present, a number of other research groups at UFMG are working to expand the bio-material’s range of applications. Studies on the functionality and bio-compatibility of the product, especially for biological applications, are being conducted by professor Gregory Kitten, coordinator of the Extracellular Matrix and Development Biology Laboratory of the Biological Sciences Institute. Researcher Heloísa Colleta, a member of Kitten’s team that has a grant from the Aid Program for Institutional Projects Involving New PhDs (Prodoc), run by Capes, the Coordinating Office for Training Personnel with Higher Education, is conducting tests with the bio-compound for future use in dental implants. The bone regeneration period in the event of tooth loss caused by periodontal disease is very long; this has led to a search for faster alternatives.

“In vitro experiments will allow us to verify the interference of nanotubes in the proliferation, adhesion, migration, death and maintenance of the differentiated state of the cells in a culture,” says Heloisa. The in vivo analyses will include the implantation of bio-compounds in the dental alveoli (the cavities where the teeth are wedged) of rats after the first molar is extracted. The evaluation’s aim is to find out whether the implantation of the bio-compound will actually speed up the regeneration of the bone without increasing inflammation. “If this model proves to be functional, the bio-compound can be applied in other places that need bone regeneration, such as fractures, for example, or for the regeneration of tissues, as in the case of artificial skin.”

Another line of research developed at UFMG, also stemming from the study started three years ago, uses carbon nanotubes for genes silencing, a line of investigation that might, in the future, lead to new target drugs that aim at the programmed target. The nanotubes are used to transport tiny RNA molecules (siRNA), which, when taken to inside the cell’s cytoplasm (in a process called transfection), silence the command of the synthesis of specific proteins. This approach consists of keeping the gene that is being studied from generating the protein codified by it, in order to analyze the consequences of this inhibition upon the cell. “Whereas the available commercial transfection methods have a very low efficiency rate, ranging from 30% to 40%, the efficiency of the silencing complex formed by the carbon nanotube and siRNA ranges from 80% to 90%,” says professor Silvia Guatimosim, who coordinates the study together with professor Maria de Fátima Leite, both of whom work at the ICB Physiology and Biophysics Department. Another advantage of the carbon nanotube is that it proved to be non-toxic; otherwise, it would cause the cell to die. “The experiments are being conducted with heart, liver and neural cells,” says Marina de Souza Ladeira, a doctoral student who is also working on this study.

Anti-neoplastic agents
 In addition to the research on gene silencing, which is part of a set of biological applications for carbon nanotubes, other studies in the field of nanomedicine in partnership with companies have been conducted at UFMG. One of them focuses on the development of a release system for transporting anti-neoplastic agents; this study is coordinated by professor Monica Cristina de Oliveira, from the Pharmaceutical Products Department of the College of Pharmacy. The technology uses liposomes, a nanostructure comprised of lipids that are similar to human cells, to transport pharmaceuticals to specific parts of the organism. These nanostructures are manufactured in the laboratory from synthetic raw material or raw material extracted from soy beans and eggs. The experiments used a formula that associates liposomes to cisplatin, a synthetic chemotherapy agent used to treat malignant tumors.

“The liposome has a watery cavity that can be used to place soluble chemotherapy agents,” says Monica. As the region around the tumor has a lower pH than healthy tissues, when the liposome sensitive to acid pH reaches the targeted region, it releases the drug into the specific site, in other words, into the tumor’s cell. “Nanotechnology for transporting pharmaceuticals may reduce treatment toxicity and increase therapeutic efficacy,” the professor points out.

Cisplatin is used for chemotherapy treatment of t head, neck, ovary, lung and prostate cancers. The chief problem is that it is highly toxic to the kidneys, which keeps physicians from increasing the dose. Some patients who are given repeat doses begin to show resistance and no longer respond to the treatment. The transportation of the chemotherapy substance by a nanostructured system might circumvent kidney toxicity, because the pathway of this system is not the same as that of a conventional drug. “Resistance to treatment is closely linked to the cell’s incapacity, over the course of time, to allow the entry of the drug,” says Monica.

This study, which began in 2001, has been conducted since 2005 in partnership with Biocancer Clinical Research, a clinical research company from the State of Minas Gerais. The company is involved in all of the development stages of a medical drug and has an agreement with the UFMG Clínicas Hospital, where it maintains a laboratory. Part of the development of the formulation has been completed and now the pre-clinical studies are being conducted. These are at the final stage and are investigating two types of prescription for the nanomedication – one intravenously and the other via the peritoneum (the membrane that covers the abdomen walls and the surface of the digestive organs). The research is being financed thanks to a R$ 180 thousand grant from Fapemig, the Minas Gerais Foundation for Aid to Research, through the Pappe subvention program, which funds research and development projects for small companies; R$ 510 thousand came from Finep, the Studies and Projects Financing Agency, and R$ 695 thousand was granted by the Minas Gerais Nanobiotechnology Research Network, created in 2002 and funded by Fapemig.

Monica, together with professor Valbert Nascimento Cardoso, is also studying the use of liposomes as an agent for the diagnosis of inflammatory and infectious processes. Marked with a radioactive isotope, such as technetium, the liposome emits gamma rays able to generate images that allow for the identification of inflammatory and infectious centers in initial stages without drawing the patient’s blood for the diagnosis, as is the case with the standard procedure, which uses marked leucocytes.

A research group at Paulista State University (Unesp), Araraquara campus, coordinated by professors Elson Longo and Maria Valnice Boldrin, is working in partnership with Grupo EMS, a drug manufacturer. Since early 2007, the team has been working on the development of active, anti-hypertension, controlled release nanodrugs. The partnership came into being thanks to CMDMC, the Multidisciplinary Center for Ceramic Materials Development, one of the Research, Innovation and Dissemination Centers maintained by FAPESP. “We are at the beginning stage of the project,” says Longo. “We chose the types of polymers that we will use as the vehicle for the drug and the active agents used to transport it.” Now the study is focusing on the interaction between the polymers and the anti-hypertension agents.

The polymer is a well-known one, but its name cannot be revealed yet because it is still at the patent filing stage. This polymer will encompass the active drug and take it to a specific site. The objective is to produce anti-hypertension drugs able to act only on a specific tissue structure, such as the striated heart muscles. “We are working with five different anti-hypertension agents,” Longo explains. “The technology will allow for the reduction of the quantity of the active drug being used, with more efficacy than that of the drugs that are currently on the market.” The sum of R$ 4.7 million was allocated for the study, 40% of which came from the company and the rest from Finep.

The interest of companies in partnering universities mirrors the importance that the academic community attributes to the fields of nanotechnology and pharmacology. “Across 6,781 publications, crossing the key words nanotechnology and pharmaceuticals, in the database of the Institute for Scientific Information (ISI), we find that Brazil ranks as the 16th country in terms of research, producing knowledge,” says professor Adriana Pohlmann, from the Chemistry Institute of UFRGS, the Federal University of Rio Grande do Sul. Professor Pohlmann, together with professor Sílvia Guterres, from the university’s College of Pharmacy, took part in the development of the first nanotechnological drug produced in Brazil: a local skin anesthetic. This drug is now in the scaling phase for further clinical trials to be conducted by Incrementha PD&I, a company installed in Cietec, the Incubator Center for Technological Companies at the Cidade Universitária (São Paulo city) campus of the University of São Paulo.

Biodegradable polymers
The system being tested uses nanoparticles of biodegradable polymers as the carriers of the pharmacological substance. “The advantage is that this material is naturally metabolized by the body, which means that it is bio-degradable and bio-compatible,” says Henry Suzuki, technical director of Incrementha, an R&D company created by two Brazilian pharmaceutical companies, Biolab and Eurofarma. The basic difference in relation to commercial ointments is that their anesthetic agent is dispersed in oil, while the nanoparticles of the new product are dispersed in water. “As the external layer of the skin absorbs more water, or, in other words, has more affinity with water, the oil-based ointments get lost along the way before they reach the inner layers and as a result, they are less effective,” explains Suzuki. The dispersion of the anesthetic nanoparticles in water allows them to penetrate better not only the outer layer but also the deeper inner layers, with a lower dose of the drug.

“The strength and the duration of the anesthetic make it suitable for use in simple surgical procedures,” says Suzuki. The trials, already conducted on animals, will be validated on patients at the current stage of the study. Once they have been conducted, the anesthetic will be ready to be used safely and efficiently in surgical procedures. The initial schedule, established at the time the new anesthetic was announced in April 2007, was that the product would be launched commercially in 2008. Some problems during the development phase, which usually occur during the transition from the laboratory to the industrial scale, delayed the commercial launch to 2009. “Our objective is to reach the product registration phase, and then pass the product on to the commercial companies, namely, Biolab and Eurofarma,” says Suzuki.

The product development idea came from Biolab, which contacted UFRGS to propose a partnership. For the last 12 years, the university has had two laboratories dedicated to nanotechnology research – the Group for Synthesis and Physical and Chemical Characterization of Microparticles and Nanoparticles Applied to Therapy, coordinated by Adriana Pohlmann, and the Group for Nanostructured Systems for Delivering Pharmaceuticals, coordinated by Silvia Guterres. UFRGS works on the production of knowledge to develop specific research studies upon request. An example is the research study on anti-inflammatory drugs such as diclophenac, which has the same active principle as that of Voltaren and other similar drugs, information on which has been published in leading international scientific journals. “This kind of work is not focused on innovation, but rather on producing knowledge,” says Adriana. The partnership between the university and Biolab resulted in the filing of a co-ownership patent request. The project was funded by CNPq, the National Council for Technological and Scientific Development.