Right on target

C&I Issue 9, 2014

Most of the drugs we take are swallowed or injected. But how a pharmaceutical is administered can reduce its effect; for example, gastrointestinal acids in our guts can interfere with how well we absorb a drug. So researchers are working to target drugs at specific parts of the body, which will mean lower dosages and fewer side-effects. Here nanotechnology is proving to be one of the most promising platforms.

By using nanoparticles, researchers can target the relevant sites in the body very accurately, so keeping toxicity to a minimum and improving bioavailability. Cancer treatment is a particular research focus. In recent years, scientists have developed nanoparticles that deliver one or two chemotherapy drugs.

The method involves either encapsulating small drug molecules inside the particles or chemically attaching them to the particle. With both of these techniques, as each new drug is added, the reactions required to assemble the particles become increasingly difficult. Combining these two approaches — encapsulating one drug inside a particle and attaching a different one to the surface — has had some success, but is still limited to two drugs.

To make these combination therapies more effective, researchers need to be able to deliver specific, controlled doses of each of the components at specific times. And until recently, it has been difficult to design particles that can carry any more than one or two drugs in a precise ratio.

Now chemists at the Massachusetts Institute of Technology (MIT) in the US have devised a way to build nanoparticles that makes it much easier to include three or more different drugs (J. Am. Chem. Soc., doi: 10.1021/ja502011g). ‘We think it’s the first example of a nanoparticle that carries a precise ratio of three drugs and can release those drugs in response to three distinct triggering mechanisms,’ says MIT’s Jeremiah Johnson.

Instead of building the particle and then attaching drug molecules, Johnson’s team created building blocks that already included a precise amount of drug. In two building blocks, the researchers linked doxorubicin or camptothecin – both ovarian cancer drugs – to a norbornene monomer, and a chain of polyethylene glycol (PEG) that protects the particle from being broken down in the body. In a third unit, they inserted another anti-cancer drug, cisplatin, between two norbornene monomers to form a crosslinking agent.

The researchers then joined these blocks together in a very specific structure using a technique they developed in Johnson’s lab, called [brush-first] ring-opening metathesis polymerisation (ROMP). ‘This is a new way to build the particles from the beginning,’ Johnson says. ‘If I want a particle with five drugs, I just take the five building blocks I want and have those assemble into a particle. In principle, there’s no limitation to how many drugs you can add, and the ratio of drugs carried by the particles just depends on how they are mixed together in the beginning.’

Each particle carries the three drugs in a specific ratio that matches the maximum tolerated dose of each drug, and each drug has its own release mechanism. Cisplatin is freed as soon as the particle enters a cell, as the bonds holding it to the particle break down on exposure to glutathione, an antioxidant present in cells. Camptothecin is also released quickly when it encounters cellular enzymes called esterases. The third drug, doxorubicin, is designed to be released when exposed to ultraviolet light. Once all three drugs are released, all that is left behind is PEG, which biodegrades.

In lab tests, Johnson’s team found that the nanoparticles killed ovarian cancer cells more effectively than particles carrying one or two drugs, and they have begun testing the particles against tumours in animals. They are also working on particles that carry four drugs.

Deadly DNA

Another approach to drug delivery receiving a lot of attention is the use of nanoscale ‘cages’ made from strands of DNA. These cages can encapsulate small-molecule drugs and release them in response to a specific stimulus.

Hanadi Sleiman’s team at McGill University, Canada, works on such DNA nanostructures. Thomas Edwardson, a member of Sleiman’s team, explains that DNA nanostructures have several potential advantages. ‘They can be built very precisely; they are biodegradable; and their size, shape and properties can be easily tuned,’ he says.

However, keeping molecules in a DNA cage is difficult because the pore size is very large. Finding novel ways of getting these cages to carry a cargo and then release it at the right place is therefore an active area of research.

In their experiments, the McGill researchers first created DNA cubes using short DNA strands. Then they added modified DNA chains – dendritic DNA – to each corner of the cube. The chains are ‘amphiphiles’; they are attracted to both water and lipids. And as the chains themselves have lipids on the ends, they are attracted to each other.

‘When four amphiphiles are on one face, the hydrophobic residues [lipid tips] of two neighbouring cubes engage in an intermolecular ‘handshake’, resulting in a dimer,’ the team report (Nature Chemistry, doi:10.1038/nchem.1745). ‘When there are eight amphiphiles – four on the top and bottom cube faces, respectively – they engage in an intramolecular ‘handshake’ inside the cube.’ Essentially, the lipid tips act like sticky patches that come together inside the DNA cube, creating a core that can hold hydrophobic cargo such as small-molecule drugs.

The team tested whether the cages could carry molecules by using the fluorescing molecule Nile Red, with dasatinib, a leukaemia drug. They report that the cage ‘retained elevated amounts’ of the drug compared to controls, the first example of transporting small molecules inside a DNA cage where they not ‘actively attached’ to the DNA.

To release the drug from the cage, the team adds a specific nucleic acid sequence that attaches to the cube and displaces the chain. ‘Many diseased cells, such as cancer cells, overexpress certain genes,’ explains Edwardson. ‘In a future application, one can imagine a DNA cube that carries drug cargo to the diseased cell environment, [and the gene that is over-expressed] will trigger the release of the drug.’

The Sleiman group is now conducting cell and animal studies to assess the viability of this method on chronic lymphocytic leukaemia and prostate cancer. The next hurdle is to ensure that the cages release their cargo in the right places.

For a DNA nanostructure to treat disease, it must survive the body’s defences long enough to do its job. DNA nanostructures injected into the bloodstream of mice are quickly digested, explains Steve Perrault at Harvard University’s Wyss Institute, US. To protect the nanostructures his team has copied the disguise strategies of viruses (ACS Nano, doi: 10.1021/nn5011914).

In a virus, the genome is enclosed in a solid protein case, which is then further wrapped round with an oily coating identical to that found in the membranes surrounding living cells. This envelope contains a double layer of phospholipid that helps the viruses evade the immune system and delivers them to the cell interior.

Perrault and his team began by folding DNA into a virus-sized octahedron using techniques developed by the Wyss Institute’s William Shih and Peng Yin. Called DNA origami, the technique allows researchers to take a long strand of DNA and program it to fold into specific shapes. Then the researchers built in handles or hooks, on which they hung lipids. This directed the assembly of a single bilayer membrane surrounding the octahedron. Under an electron microscope, the coated nanodevices closely resembled an enveloped virus.

They then loaded the structures with fluorescent dye, injected them into mice, and used whole-body imaging to see which parts of the mouse glowed. Just the bladder glowed in mice that received uncoated structures, which meant that the animals broke them down quickly and were ready to excrete their contents. But the entire body glowed for hours when they received the coated nanodevices, showing that they remained in the bloodstream.

The team also reported that the coated devices evaded the immune system. Levels of two immune-activating molecules were at least 100 times lower in mice treated with coated nanodevices, compared with uncoated nanodevices. The researchers say that this gives them control so that they can now design a device to get the immune response they want.

In the future, ‘cloaked nanorobots’ could activate the immune system to fight cancer or suppress the immune system to help transplanted tissue become established. ‘Activating the immune response could be useful clinically or something to avoid,’ Perrault says. ‘The main point is that we can control it.’

Nanospheres and nanomotors

Other researchers are building nanoscale cages from alternative materials. For example, in the US, Boston College chemists are investigating ‘nanospheres’ made from a metal organic framework.

Chia-Kuang ‘Frank’ Tsung’s team linked tens of thousands of zinc ions using organic chains to create a zeolitic imid–azolate framework (ZIF-8). This series of cage-like structures – a ‘constellation’ – was achieved by carefully controlling temperature during fabrication, they report (ACS Nano, doi: 10.1021/nn406590q).

The size of the constellation is critical; it must be large enough to transport the right dose of drug, yet small enough to penetrate the target cell membrane. Tsung comments: ‘If you have too small a framework, it won’t work. If we stay between 50–100nm, it can penetrate the cancer cell. Our nanosphere is in the 70nm range, which we think is ideal.’

Once inside a cancer cell, the drug is released since the framework reacts to a change in pH. The pH of cancer cells is generally lower than that found in the body (pH7.4). But ZIF-8 is unstable at these low pH levels and disintegrates.

Tsung explains: ‘When the nanosphere enters the cancer cell, the lower pH level destabilises the structure, which begins to break apart and release the drug so it can begin to do its job of attacking and killing cancer cells.’

The team encapsulated several different small molecules, including fluorescein and the anticancer drug camptothecin, inside the ZIF-8 framework. They ran tests on breast cancer cells in the lab. ‘We were very excited to see the results,’ says Tsung, ‘... to see our organic-based drug delivery system attack and kill cancer cells in our lab tests was extremely gratifying. We know there is much work to be done, but we’re excited about the potential in this advance.’

To demonstrate the versatility of their spheres, the team is experimenting with adding iron oxide to the structures. The idea is that external magnetic fields could then be used to direct the spheres to target cells.

Meanwhile, engineers at the University of Texas at Austin, US, claim to have built the smallest, fastest and longest-running synthetic motor to date. One day, they suggest, such devices could move through the body administering insulin or targeting cancer cells. Donglei ‘Emma’ Fan’s three-part nanomotor is small enough to fit inside a human cell; it can rotate for 15 hours continuously at a speed of 18,000 rpm (Nature Communications, doi:10.1038/ncomms4632). Comparable nanomotors run significantly more slowly, and not for this long, the researchers say.

The team assembled the nanomotors using nanowires as rotors, patterned nanomagnets as bearings and quadrupole microelectrodes as stators, the stationary part of a rotary system. The team built and operated the nanomotor using a patent-pending technique that Fan invented while studying at Johns Hopkins University, US. The technique relies on AC and DC electric fields to assemble the nanomotor’s parts one by one.

In lab experiments, the researchers used the technique to turn the nanomotors on and off, and propel the rotation either clockwise or anticlockwise. They found they could arrange the nanomotors in a pattern and move them in a synchronised fashion.

In a ‘non-biological environment’, the motors could move through liquids, and both mix and pump a small molecule drug. To test this, they coated a nanomor with a small molecule drug before setting it spinning. The faster the nanomotor rotated, the faster it released the drug.

‘We were able to establish and control the molecule release rate by mechanical rotation, which means our nanomotor is the first of its kind for controlling the release of drugs from the surface of nanoparticles,’ Fan says. ‘We believe it will help advance the study of drug delivery and cell-to-cell communications.’ The next step is to test the motors in a biological environment, near living cells.

Maria Burke is a freelance science writer based in St Albans, UK

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