Have you ever taken medicines that didn't really work? Wouldn't it be great if they worked exactly where they were supposed to and were as effective as possible? This has been the goal since the birth of medicine, and it seems that the infamous ‘Trojan' approach feared by even the bravest soldiers in the past could be effectively applied to achieve this dream.
The major focus in therapeutic drug development today is delivering an effective drug exactly where it is needed. First, the drug must be sent to the right place so it can produce a beneficial effect; for example in anti-cancer treatment the drug needs to be delivered to the cancer, where it can destroy harmful cells while leaving the surrounding healthy tissue undamaged.
Second, for any therapeutic drug to be effective, it needs to enter the right cells, and then bind to the target site or molecule within them. Inefficient delivery of medicines into the cell has been the limiting step in their efficient use, and extensive research is being carried out to find new technologies to improve this.
One of the most promising strategies for delivering biologically active compounds into the cells is the use of cell-penetrating peptides (CPPs), in other words short sequences from either naturally occurring proteins or produced by chemical engineering. Since their discovery about 15 years ago, the properties of CPPs—often called ‘Trojan' peptides because they can enter cells while carrying a vast range of bioactive molecules—has been closely studied by several research groups worldwide.
Substantial progress has been made in the effective delivery of various bioactive ‘cargos', and this is a fascinating and innovative field of study. Our own research carried out at the Institute of Molecular and Cell Biology at the University of Tartu (Estonia) under the supervision of Professor Margus Pooga is currently concentrating on elucidating the mechanisms of effective uptake, and so the focus of this article is on the delivery of the bioactive drugs to the cell interiorO.
One of the Least Toxic Transporters
A common characteristic of CPPs is their net positive electrical charge, which enables them to bind to the negatively charged cell membrane and be taken into the cell. The uptake of the cargo attached to CPPs is achieved by budding and subsequent formation of vesicular structures, almost like the forming of a soap bubble into the cell interior from the cell membrane containing the CPP-cargo complexes, a process called endocytosis.
Since endocytosis is a normal way for a cell to communicate with and acquire information from the surrounding environment, this process is neither harmful nor invasive to the cell or the organism as a whole. Consequently CPPs are unlikely to cause an immune response, and are one of the least toxic transporters available today. This gives them a clear advantage over other transport techniques currently being explored, for example the systems that use viruses.
Different Ways of Getting Into Cells
Once inside the cells, the cargo molecules attached to CPPs are mostly captured inside endocytic vesicles, much like the air is captured inside the soap bubble, and sent to the cellular ‘stomach', where digestive enzymes break them down. However, we have shown that a fraction of the CPP-cargo complexes are able to escape from this fate and are found in vesicles following a different path. Also, at least some CPPs are able to cause their cargo to leak from the entrapping vesicles, making them available for transportation to the necessary target within the cell by the cell's own molecular machinery.
Because only the intact cargo that has reached its target inside the cell can exert its activity, much effort has been put into developing strategies to enhance this step. It has also been shown that different CPPs as well as different cargo molecules attached to the CPPs can alter the entry, the intracellular targeting and processing of the drug cargo. This is why it is important to define and understand the processes involved in the way different CPPs and CPP-cargo complexes enter the cell, and their fate within it, so that even more effective carrier systems can be developed.
The Need For Further Research
Patients do not have the chance to benefit from many new and highly potent therapeutic drugs due to their poor delivery into the cell, low bioactivity and lack of specific targeting. Improving the delivery of these drugs is the key to future therapeutic development. We believe that the innovative approach of using CPPs as transporters could bypass the problems mentioned here and offer an alternative either for scientific research or in patient care.
However, it is important to understand that further research has to be done to clarify the selectivity, cellular uptake, intracellular targeting, processing and bioactivity of the CPP-cargo constructs. Understanding these mechanisms more precisely can help us to make a rational and systematic selection and to use delivery vehicles with a better rate of success and less waste of resources.