CPP-loaded devices and local release − Rakyat Biologi

As already discussed, cationic CPPs have the undesired characteristic of entering most of the cells they get in contact with. Therefore, once injected in the body, most of the peptides and indeed the drug attached to them will be internalized in the “wrong” cells. As described in the previous chapters, efforts to overcome these limitations have led to the generation of new chimerical molecules. These molecules are often complex, certainly rather difficult to synthesize and/or fully characterize, and to market. To circumvent these problems, it is also conceivable to design cell-penetrating systems showing a certain form of specificity, by simply allowing a CPP-loaded device to diffuse very close to the environment of the tissues to be targeted. This is indeed viable only in the case of a localized pathology, such as solid tumors, or of directly accessible organs. It may be indeed feasible for the treatment of various cutaneous pathologies, and possibly lung diseases, and eventually for reaching the ocular cavity or the digestive tract, for instance. Moreover, several drug delivery sustained release systems, including gel matrix, nanocapsules, liposomal structures, to name but a few, have been described in the literature (for recent reviews in this field, see volume 59, issues 4 and 5 of the Advanced Drug Delivery Reviews (2007)). Some of them could trigger the release of the encapsulated drug specifically in the region requiring the therapy, however the problem of the rapid and efficient internalization of these drugs into the targeted cells would remain. This could be solved by attaching a CPP to the therapeutic molecule. To our knowledge, such a strategy combining a local delivery device and a CPP internalization system still remains very marginal, although some examples of its application have been described.

Thermal sensitive polymers

There are some alternative means to efficiently direct drugs into a specific cell type with CPPs. For instance, a chemotherapeutic CPP-coupled doxorubicin conjugate has been included in a macromolecular carrier made of an Elastin-like polypeptide (ELP) [105]. This carrier is thermal sensitive with a phase transition occurring between 39°C and 42°C [105, 106]. This temperature range is sufficiently above the normal body temperature to prevent unwanted systemic aggregation. Therefore, following external and focused hyperthermia, which can be induced with different devices such as pulsed-high intensity focused ultrasounds (HIFU) [107], the Tat-drug conjugate can be released in the tumor environment and preferentially taken up by the tumor cells. Additionally, a Gly-Phe-Leu-Gly tetrapeptide spacer (sequence GFLG) was joined to the Tat peptide and the doxorubicin molecule. This sequence corresponds to the target sequence of a protease belonging to the cathepsin family [108]. Since the CPP is internalized mainly by endocytosis [33], the lysosomal proteases are expected to cleave and release active drug moieties within the cell. In this study, a 20-fold enhancement of the effect of the drug was recorded when aggregation of ELP was induced by localized hyperthermia [105].

More recently, Curley and colleagues have used a thermal sensitive device made of carbon nanotubes [109]. By injecting these nanotubes into cancer cells and then zapping them with radio-frequency waves, they created an important increase in temperature that led to the death of cancer cells. Radio-frequency waves can be used in this case because of the particular electronic properties of carbon nanotubes and they are more attractive than the near-infrared laser heating method used in others strategies. Moreover, carbon nanotubes could be derivatized with various proteins to improve their specificity [110]. Indeed, it might be feasible to link these nanotubes to CPPs, to improve their cellular uptake, or to targeting-peptides to concentrate them at the tumor site.

Ultrasound sensitive particles

Ultrasounds can also be used to deliver drugs in a very precise body area. They are currently evaluated for two main applications. The first one relies on focused sonication to deliver drugs or genes into the tissue where the sonication is applied as ultrasounds can reversibly increase the micro-permeation (sonoporation) of the membrane, thus inducing cellular transfer of the extracellular medium [111, 112]. Indeed if the blood stream is loaded with the drug to be transfected into the cells, a more potent delivery of the molecule can be expected locally in the area of sonication. However, it is worth to mention that the sonication effects do not last long. In other words, this strategy mimics an in vitro transfection performed in vivo, thus leading to a higher degree of drug delivery in the cells within the treated area. Sonication has been shown to improve the delivery efficacy of a cytotoxic agent, leading to a significant reduction of the injected dose [113]. However, if applied to small drugs, a rapid clearance from the blood stream can be expected, which will reduce the time window of the sonication effects.

Therefore, to limit this problem, some attempts have been made to deliver in the blood stream stable molecular structures that are exclusively sensitive to ultrasounds [114]. This strategy allows the entrapment of various drugs prior sonication, inducing their release only in the targeted environment [114]. Moreover, the concomitant use of drugs conjugated to CPPs and sonication should further improve the local cellular delivery. However, only hydrophobic molecules, such as doxorubicin for instance [115], can be successfully inserted in ultrasound sensitive structures. Since most of the CPPs are rather hydrophilic because of their high cationic content, their inclusion in theses structures is compromised unless their coupling to doxorubicin reduces significantly their hydrophilicity, or more extended hydrophobization of the CPPs is performed. Additional lipophilic group(s) could also favor the escape of CPPs from the endosomes and, subsequently, the overall efficacy of this “targeted” delivery strategy. This approach is currently evaluated in our group.