The fields of molecular imaging, drug targeting, bioinformatics and biomedical research are currently merging in to the expanding field of theranostics 1-3
. The theranostic trends change increasingly from systemic towards patient-specific strategies 4-6
. Instead of native nucleic acids whose use as a drug is hampered by their nuclease sensitivity, the use of amine-polymers functionalized with the potential of Watson-Crick binding lacking nuclease sensitivity can produce relief 7-10
. The cell membrane however, forms an almost impassable obstacle. This basic problem of nucleic acid-based active molecules, responsible for their poor uptake into living cells and tissues, remains to be solved 11-14
. Great efforts resulted in multifaceted methods of viral 15-17
and non-viral carrier 18-22
solutions. Peptide-based molecules which harbour cell membrane penetrating properties are documented as CPP (cell penetrating peptides) 23-27
. In our hands, one approach to circumvent this hurdle and enter cells is based on functional peptides which are modularly composed depending on their conceptual formulation, hereafter called “BioShuttle” 11
: BioShuttle-based formulations employ amphiphilic peptides, ligated to their functional groups via disulfide bridge formation, for transport across the cell membrane. By the use of different cell immanent mechanisms, based on reductive conditions located in the cytoplasm, the disulfide-bridges can be cleaved. As a consequence, the CPP-part of the BioShuttle is separated after passage across the cell membrane from a nuclear localization sequence (NLS) -based address moiety 28-30
, enabling the delivery of imaging molecules and/or pharmacologically active components into the nucleus 11, 31
To provide an experimental proof-of-principle for these features, three BioShuttle constructs were tested, which are illustrated schematically in Figure :
illustrates schematically the investigated Bioshuttle-conjugates.
A. The CPP-Rd110 consists of the pAntp peptide fragment which is labeled with Rhodamine 110 at the N-terminus. The pAntp's (CPP) amino acid sequence is KKWKMRRNQFWIKIQRC which facilitates the passage of molecules across the cell membrane. All constructs are able to pass through cell membranes into the cytosol via the CPP.
B. The double-labeled BioShuttle construct (Atto647N)-CPP-S∩S-NLS-(Atto488) comprises pAntp (CPP) (KKWKMRRNQFWIKIQRC) conjugated to a NLS (VKRKKP) module by a disulfide linker. Both modules are conjugated to fluorescent dyes (coupled by a peptide bond to the N-terminus or to lysine, respectively): the CPP is labeled with Atto647N and the NLS sequence is labeled with Atto488. After crossing the cell membrane, the linker between the modules should be cleaved and the NLS should be transported into the nucleus. Hence, cytoplasm and nucleus will be labeled differently. After cleavage, the residual fluorescently labeled NLS can diffuse freely and can actively enter the cell nucleus.
The CtsB-PNA construct's structure is more complex (Rd110-NLS
: The CPP is coupled to a peptide nucleic acid (PNA) sequence by a disulfide linker which can be cleaved by intracellular disulfide reductases. The PNA sequence (in italic
) acts as an antisense sequence molecule and should hybridize specifically with the mRNA transcript of cathepsin B, which is a peptidase overexpressed in many (metastatic) cancer cell lines. A short peptide sequence (GFGRK) inserted between the complementary PNA and the nuclear localization sequence (NLS) module acts as a substrate, enabling specific cleavage by cathepsin B 33
. Nuclei of cells with normal Cathepsin B expression without an active gene product should be fluorescence-identifiable barely by this construct. This experiment should demonstrate the potential of FFM for imaging of gene expression in diagnostics. Here we used as an example the CtsB
gene encoding the matrix metal proteinase CtsB whose activity is associated with tumor malignancy 34, 35
Whereas functional modules associated with transmembrane-transport facilitating properties, like the cell penetrating peptides12, 15, 23, 36, 36-39
, nuclear localization sequences 28, 40-46
and the antisense molecules 47-53
have been broadly characterized, the intra- and extracellular dynamics and the subcellular distribution of modular carrier materials containing such moieties have not been evaluated in great detail. Here, the mobility of the three abovementioned BioShuttle constructs was investigated by fluorescence correlation spectroscopy (FCS). In FCS, dynamic parameters of a fluorescent sample are derived from statistical fluctuations in fluorescence intensity in a small detection volume of a confocal microscope 54
. These fluctuations are mainly induced by the diffusion of the fluorescent probe through the observation volume. The high spatial resolution and the applicability of FCS also in vivo
are powerful tools for measuring the local concentration and the diffusion coefficient in different subcellular compartments 55
. In addition, BioShuttle-facilitated transmembrane transport, nuclear targeting and intracellular localization were investigated using an adapted confocal laser scanning microscopy (CLSM) protocol.