Nano dump trucks
Nanoparticles have been taught to "unload" a cancer drug only in the presence of disease markers
Natalia Miranda, N+1
German researchers have developed a method of drug delivery in nanoparticles, in which the drug is released inside the cell only in the presence of specific markers of the disease. The basis for the nanoparticles was the glycoprotein mucin with partially complementary DNA molecules attached to each other, playing the role of intermolecular crosslinking. The "key" for breaking DNA cross-links in nanoparticles and releasing the drug in the cell may be microRNA, overexpression of which is observed in some types of cancer. The work was published in the journal ACS Nano (Kimna et al., DNA Strands Trigger the Intracellular Release of Drugs from Mucin-Based Nanocarriers).
Cancer treatment is often complicated by the fact that drugs need to be delivered directly into cancer cells, only there the drug, having reached therapeutic concentrations, will be effective. But having solved this problem, we are faced with the problem of non–specific effects of drugs - for example, not only cancer cells for which they are intended are exposed to cytotoxic effects of chemotherapeutic drugs, but also healthy ones, which leads to undesirable reactions (immunosuppression, gastrointestinal disorders, hair loss, and so on).
Jeren Kimna with colleagues from The Munich Technical University and the Royal Technical Institute in Sweden solved this problem by using intracellular drug delivery in biopolymer nanoparticles, which are revealed only in cancer cells. The researchers chose glycoprotein mucin as the polymer base, since its chemical nature allows it to functionalize in different ways and bind well with both negative and positively charged molecules. In addition, previous studies on the use of mucin as the basis of biomaterials have shown that in mammals it causes either a slight immune response or its complete absence.
Distribution of fluorescently labeled mucin in cancer (HeLa) and healthy (NIH/3T3) cells after treatment with DNA-mucin nanoparticles. Drawings from the article in ACS Nano.
As a crosslinking agent, the authors used partially complementary (8 pairs of nucleotides) short DNA molecules to each other, to the 5’ ends of which thiol groups are attached. Through them, oligonucleotides form disulfide bonds with cysteine residues at the ends of mucin molecules. In the presence of glycerin, mucin molecules reversibly condense, while short DNA is able to get closer and complementarily bind to each other, forming a DNA-mucin polymer nanoparticle, inside which the drug can be delivered.
For the specific release of the drug, the researchers suggest using complementary oligonucleotides with greater affinity for cross-linking short DNA (that is, forming more complementary pairs with it). Thus, due to the competitive interaction, trigger oligonucleotides will be able to break the bonds between the crosslinking DNA, disrupting the structure of the nanoparticle.
A scheme for obtaining DNA-mucin nanoparticles.
To determine the effectiveness of internalization (penetration into cells) of nanoparticles, the researchers used fluorescently labeled mucin molecules and monitored their distribution in HeLa cancer cells using flow cytometry. It turned out, however, that the degree of penetration of DNA-mucin nanoparticles into cells is insignificant, which prompted scientists to think that the surface charge of the particles is to blame. The fact is that they have a negative surface charge in this form, as well as negatively charged components of the cell membrane. It was the electrostatic interactions that probably prevented the penetration of nanoparticles into the cytosol. To overcome this, the authors of the study decided to cover the nanoparticles with polycation, adding another stage to the technique – the second condensation, this time of polylysine molecules (the first option) and chitosan (the second option). At the same time, they note that the polycationic component of the nanoparticle will reduce the minimum required concentration of trigger oligonucleotides, since it itself will attract negatively charged nucleic acid molecules.
Fluorescence intensity of labeled mucin in HeLa cells after treatment (from left to right) DNA-mucin nanoparticles, chitosan-coated nanoparticles and polylysine-coated nanoparticles.
Experiments have shown that nanoparticles in a polylysin shell penetrate the cell better than in a chitosan shell. The authors suggest that an excessively large positive charge of chitosan causes nanoparticles with it to get stuck in a negatively charged cell membrane, interfering with full penetration (the zeta potentials for nanoparticles coated with polylysine and chitosan are +2.3 and +21.7 millivolts, respectively).
The authors called the ultimate goal of the work the creation of nanoparticles capable of autonomous drug release due to cellular, rather than artificial trigger oligonucleotides. To do this, they selected as a crosslinking DNA sequence complementary to miR-21 (oncogenic microRNA, overexpression of which is observed in some tumor cells). The new cross-linking DNA (anti-miR–21) contains 14 base pairs, of which eight are complementary to each other, and 11 nucleotides are complementary to miR-21.
The effectiveness of the created system was tested on the already mentioned HeLa cancer cell line with high miR-21 expression and on NIH/3T3 cells, where miR-21 expression is insignificant. The state of the nanoparticles was monitored by the distribution of fluorescently labeled mucin in the cells. It turned out that in both cells the nanoparticles were capable of internalization, but in healthy cells, where the concentration of trigger oligonucleotides (miR-21) was low, the nanoparticles remained in a condensed state, whereas in tumor cells the nanoparticles opened and fluorescently labeled mucin was evenly distributed in the cytosol.
The researchers note the specificity of drug release in diseased cells, however, they add that the proposed system can be more effective and safer when combined with targeted delivery of nanoparticles – so that they not only specifically release therapeutic agents inside cancer cells, but also specifically penetrate them.
The developments of bioengineers are increasingly encouraging and allow us to hope for the invention of new effective, and sometimes unexpected methods of treating diseases.
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