DNA nanopyramids slip through the brain’s protective barrier and release cancer drugs only when they detect the acidic environment surrounding a tumor.
(Nanowerk Spotlight) Brain tumors are exceptionally difficult to treat. The blood-brain barrier, an intricate network of tightly packed endothelial cells lining cerebral blood vessels, shields the central nervous system from pathogens and toxins circulating in the bloodstream. This evolutionary advantage becomes a devastating liability when cancer develops in brain tissue.
Chemotherapy drugs that decimate tumors elsewhere in the body cannot reach gliomas, the most common and aggressive form of brain cancer. Patients with malignant gliomas face median survival times measured in months rather than years, not because effective drugs do not exist, but because those drugs cannot cross this biological checkpoint.
Researchers have engineered nanoparticles, liposomes, and polymer micelles to ferry chemotherapy agents across the barrier. Yet these approaches suffer from limited penetration efficiency, accumulation in unintended organs, and toxic side effects. The barrier demands carriers small enough to slip through, stable enough to survive the journey, and capable of releasing their payload only at the tumor site.
DNA nanotechnology offers a promising alternative. The technique exploits the predictable base-pairing rules of DNA molecules to fold single strands into precise three-dimensional shapes, a method known as DNA origami. These structures are inherently biocompatible and can respond to specific biological signals.
Tetrahedral DNA nanostructures have shown particular promise, with multiple studies demonstrating they cross the blood-brain barrier more effectively than other geometric configurations. The challenge has been engineering these structures to remain stable during circulation yet disassemble on command to release their therapeutic cargo.
A study published in Advanced Functional Materials (“Allosteric DNA Nanorobots for Targeted Glioma Therapy: Acid‐Triggered Doxorubicin Delivery”) presents a tetrahedral DNA nanostructure (TDN) equipped with pH-sensitive switches that trigger controlled drug release in the acidic environment characteristic of tumor tissue. The nanorobot carries doxorubicin, a chemotherapy agent that kills cancer cells by intercalating into their DNA and costs substantially less than temozolomide, the current standard treatment for gliomas. In mice with brain tumors, the system achieved significant tumor suppression and extended survival.
Schematic of the allosteric DNA nanorobot for targeted drug delivery. (A) Assembly process of the pH-responsive tetrahedral DNA nanorobot. (B) Allosteric mechanism of the DNA nanorobot in response to acidic conditions. (C) The pH-responsive DNA nanorobots serves as a drug carrier for inhibiting brain tumor growth. (Image: Reproduced with permission from Wiley-VCH Verlag) (click on image to enlarge)
The design begins with a planar DNA origami sheet measuring 15 nm × 15 nm × 2 nm. Cytosine-rich pH sensors then fold this sheet into a stable tetrahedral configuration with a hydrodynamic diameter of 13 nm, small enough to traverse the blood-brain barrier. At normal physiological pH around 7.0, these sensors maintain the locked tetrahedral shape. When the nanorobot encounters the acidic microenvironment surrounding tumor cells, typically around pH 5.0, the cytosine bases become protonated and form an alternative triplex structure. This destabilizes the tetrahedron, causing it to unfold and release its drug payload.
Atomic force microscopy revealed distinct morphological changes in the nanorobots when shifted from neutral to acidic conditions. Molecular dynamics simulations confirmed that the tetrahedral configuration maintains structural stability under physiological conditions while remaining responsive to pH changes. Fluorescence measurements showed maximum sensor response at pH 5.0, with weaker signals at pH 6.0 and pH 4.5.
To enhance tumor targeting, the investigators conjugated AS1411 aptamers, short DNA sequences that bind specifically to nucleolin proteins overexpressed on glioma cells. This variant, designated TDN-AS, achieved approximately twice the cellular uptake compared to the unmodified structure.
The critical test came with an in vitro blood-brain barrier model consisting of a monolayer of brain endothelial cells positioned above glioma cell spheroids. TDN-AS nanorobots successfully crossed the endothelial barrier and penetrated into the tumor spheroids at significantly higher levels than control structures. Experiments using methyl-β-cyclodextrin, which inhibits caveolin-dependent endocytosis, confirmed that barrier penetration relies on this specific cellular uptake pathway.
The investigators then tracked Cy5-fluorescent-tagged nanorobots in living mice following intravenous injection. While substantial accumulation occurred in the liver, as is typical for nanoparticle therapies, TDN-AS achieved significantly higher brain penetration than control structures. Serum levels of inflammatory cytokines remained unchanged compared to saline-treated mice, and liver function tests revealed no hepatotoxicity.
Therapeutic efficacy experiments used mice bearing bioluminescent human glioblastoma cells implanted in the brain. The investigators administered intravenous injections of doxorubicin-loaded nanorobots on days 8, 11, and 14 after tumor implantation. By day 17, mice treated with drug-loaded TDN-AS showed the smallest increase in tumor bioluminescence.
Confocal microscopy of brain tissue slices revealed co-localized fluorescence from the nanorobot framework and released doxorubicin in tumor regions. No doxorubicin signals appeared in other organs, ruling out significant drug leakage during circulation.
One-third of mice receiving doxorubicin-loaded TDN-AS survived beyond 40 days, while all animals in other treatment groups died within 36 days. Mice receiving empty TDN-AS nanorobots showed no survival benefit compared to saline-treated controls, confirming the therapeutic effect stems from delivered chemotherapy rather than any intrinsic property of the DNA structures.
The study clarifies how pH-responsive behavior governs drug release. Doxorubicin loading efficiency was nearly identical between the planar DNA origami precursor and the folded tetrahedron. However, the tetrahedral form released far less drug during incubation in serum, consistent with its greater resistance to nuclease degradation. Acidic conditions dramatically increased drug release, reaching 42.4% at pH 5.0 compared to just 2.9% at pH 7.0.
This approach differs from earlier DNA nanorobot designs, which typically relied on mechanical opening to expose therapeutic cargo. By modulating release efficiency through pH-triggered destabilization, the system maintains structural integrity during circulation while achieving controlled delivery at tumor sites. The tetrahedral architecture provides blood-brain barrier permeability, the pH sensors enable tumor-specific release, and the aptamer conjugation enhances targeting.
While organ retention and the heterogeneity of clinical gliomas require further investigation before human trials, the combination of efficient barrier crossing, controlled drug delivery, and extended survival positions this platform as a promising step toward precision chemotherapy for brain cancer.
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