Comparative analysis of the methods of drug and protein delivery for the treatment of cancer, genetic diseases and diagnostics

Abstract

The methods of protein and drug delivery for the treatment of cancer, genetic diseases and diagnostics were summarized. The potential of protein transduction is discussed and the recent developments in the field are reviewed. An overview is provided of the non-viral delivery methods such as liposomes, polymer-based delivery, cell-penetrating peptides, bacterial secretion, cells, virosomes, physical methods including electroporation, microinjection, osmotic lysis, nanoparticles, sonoporation to locally inject therapeutic molecules. The characteristic properties of non-viral vectors and their use for the delivery of therapeutic molecules for the diagnosis and treatment of disorders and to target tumors are also discussed. The potential of the transduced peptides and proteins was used as new therapeutic compounds against infectious diseases, to complement deficiencies in specific genes, to specifically kill tumour cells, for gene therapy. The protein delivery vectors can enhance the transfection at low concentrations and help to develop future gene delivery systems with reduced toxicity. Vitamin B12, folic acid, biotin, and riboflavin are essential in the treatment of cancer. Ultrasound has a potential in the delivery of therapeutic agents. The new developing technologies of drug delivery and targeting offer the possibility to improve the therapeutic possibilities of the existing drugs and to develop novel therapeutics.

Key words::

• Electroporation
• nanoparticles
• cell-penetrating peptides
• treatment of cancer
• gene therapy
• vitamin-mediated targeting
• ultrasonic drug delivery

Abbreviations:
CPPs	=	cell-penetrating peptides
PTDs	=	protein transduction domains
MTP	=	membrane-transducing peptides
pAntp	=	Antennapedia homeodomain protein
TAT	=	HIV-1 transactivator of transcription
US	=	Ultrasound

The protein delivery, accomplished indirectly by transfecting transcriptionally active DNA into living cells or by viral infection, where the gene is expressed and the protein is produced by the cellular machinery, is time-consuming (1-3 days to express the transfected genes) with the risk of insertional mutagenesis.

New techniques to deliver functional proteins into living cells (protein transduction) were developed that significantly increase the applications to analyze functions of proteins in living cells, for protein reagents and new human therapeutics. Due to the inability of proteins to spontaneously enter cells, exogenous proteins predominantly interact with extracellular targets to enter through the endocytic pathway (Cronican, 2010). Full-length proteins are delivered into a large number of cells (Morris, 2001), including: intracellular, cell surface and toxin proteins, antibodies, synthetic and biological active peptides, protein domains; protein-nucleic acid and protein-nanoparticle conjugates, multi-protein complexes; conjugates between a protein-organic chemical entity, protein-inorganic chemical entity (Biotin, fluorescent dies, silanol and silane derivatives, mass spectrometry tags, low-molecular weight chemicals) (Ye, 2005). A variety of reagents for the delivery of proteins into mammalian cells were developed including lipid-linked compounds (Zelphati, 2001), nanoparticles (Hasadsri, 2009) and fusions to receptor ligands (Gabel, 2006; Rizk, 2009). The direct introduction of purified preparation of protein into cells by microinjection, electroporation, construction of viral fusion proteins, cationic lipids allow to perform the biological functions immediately after the protein entry into the cell and may be used in the cell cycle regulation, control of apoptosis, oncogenesis and transcription regulation studies. The direct delivery of suicide gene proteins to cells may be an alternative approach to the conventional suicide gene therapy strategies (Zheng, 2003).

The challenges for protein delivery are significantly increased in vivo, where cells in the intact tissues and organs are difficult targets for functional protein delivery (Cai, 2006; Caron, 2001). The delivery of therapeutic proteins into tissues and across the blood-brain barrier is limited by the protein size and properties, the poor permeability and selectivity of the cell membrane. Possible disadvantages are the toxicity to the recipient cells, the non-specificity, the low transfection efficiency and substantial variability. Some protein delivery methods can be cytotoxic and physically disruptive to cells, thus compromising the results from the physiological studies.

Liposomes

Lipid-based drug-delivery systems have evolved from micro-scale to nano-scale. The selection of the appropriate method depends on the physicochemical properties of the nanoparticles. The Liposomes and solid-core micelles are lipid-based nanoparticles with surface modifications, long-circulating, tissue-targeted and pH-sensitive to improve their therapeutic effect. The solid lipid nanoparticles were engineered to reduce toxicity to mammalian cells, while multifunctional lipid-based nanoparticles simultaneously perform therapeutic and diagnostic functions (Buse, 2010). The carrier reagent is a liposome, complexed with a protein of interest and promotes the delivery into the cell. The protein encapsulated in the formulation binds to the negatively vehicle for delivery (Zelphati, 2001). A synthetic ligand “Streptaphage” was constructed to deliver streptavidin to mammalian cells by non-covalent interactions with cholesterol and sphingolipid-rich lipid subdomains of cell plasma membranes (Hussey, 2002). The liposomes fuse with the plasma membrane or are endocytosed and fuse with the endosome, releasing the protein into the cytoplasm. Liposomes were used as drug carriers from decades, while currently as gene and protein carriers. The delivery is easy to obtain, but sometimes it is toxic to the cells and the efficiency is not very high.

Cholesterol-based drug-delivery systems

An amphiphile (CholCSper), consisting of cholesterol, linked to carboxy-spermine by a cysteine, and dimerizable upon mild oxidation of thiol to disulfide, was developed and used with DOPE to prepare intracellular protein delivery system for functionally active proteins (De Campos, 2001). The cholesterol affects the density of the complexes formed with proteins and leads to a prolonged protein release in the cytoplasm of cells exposed to protein carrier assemblies (Dalkara, 2006). The protein entry is rapid, concentration-dependent and works with difficult cell types. The protein can be denatured and needs to be refolded upon its entry into the cell to regain its biological activity. The physical properties of the delivered protein such as charge and hydrophobicity can influence the interaction with cationic lipids that are used for protein delivery (Zelphati, 2001).

Commercial Products with lipid nature

Protein delivery products available commercially can be used, depending on the nature of the particular reagent employed, saving time by bypassing the traditional DNA transfection, transcription and protein translation processes associated with gene expression. The reagent forms a complex with the protein, stabilizes the macromolecule and protects it from degradation during delivery. After the internalization in a cell, the complex can dissociate, leaving the macromolecule biologically active and free to proceed to its target organelle (Ye, 2005).

Bioporter protein delivery reagent (Genlantis) is non-cytotoxic and transports proteins inside the target cells with retaining structure and function. Bioporter forms a single non-covalent transfection complex, uses non-endosomal delivery mechanism and provides high cell viability. Fluorescent molecules, antibodies, dextran sulfate, phycoerythrin-BSA, β-galactosidase, apoptotic proteins granzyme B, caspases 3 and 8, delivered with Bioporter are functional and effective in multiple cell types such as HeLa, HeLa-S3, BHK-21, HEK 293, CHO-K1, NIH 3T3, CV-1, B16-F0, COS-1, K562, COS 7, Jurkat, Ki-Ras 267 β1, HepG2, MDCK. Depending on the physical properties, the protein in hydration medium is captured by association with the liposomal membrane and attached to negatively charged cell surfaces. Trifluoroacetylated lipopolyamine and dioleoyl phosphatidylethanolamine (BioPorter) were used to form protein-lipid complexes, containing recombinant HSV type-1 thymidine kinase or Drosophila melanogaster multisubstrate deoxyribonucleoside kinase, imported into human osteosarcoma and Chinese hamster ovary cell lines by endocytosis. Delivered into the cytosol and nucleus, the proteins retained their enzymatic activity. The rEGFP and HeLa cells were used as model system to test the efficiency of Bioporter assay, found to be enough for functional studies of mammalian cells and more efficient than electroporation (Todorova, 2009).

Pro-ject protein transfection reagent (Pierce) uses a cationic lipid with non-covalent nature, is non-cytotoxic and capable to deliver a variety of proteins into numerous cell types, maintaining their functional activity. The liposome-protein complex fuses with the cell membrane or is internalized via an endosome and the protein or macromolecule of interest is released into the cytoplasm free of lipids.

Pulsin (Polyplus transfection) allows to study lethal proteins by controlling the level and time course of protein delivery into the cells. Pulsin is non-toxic and contains a cationic amphiphile that forms non-covalent complexes with proteins and antibodies, internalized via anionic cell-adhesion receptors and released into the cytoplasm. Intracellular proteins and antibodies in living cells can be targeted without fixation (Cassinelli, 2006). Proteins, Peptides, Antibodies, Streptococcus TPE B epitope, FITC-labeled anti-alpha-tubulin, R-phycoerythrin (fluorescent protein of 240 kD) were successfully delivered to HeLa cells up to 98%. The delivery of substrate, inhibitor, modulator, or blocking peptides and antibodies into cell allows protein function studies and RNA interference experiments, as well as the development of therapeutic approaches.

Provectin (Imgenex) is a non-cytotoxic lipid-based protein delivery reagent that allows the delivery of proteins, peptides and other bioactive molecules into the cytoplasm of a variety of different adherent and suspension cells.

Electroporation

The efficiency of electroporation is dependent upon the strength of the applied electrical field, the length of the pulses, temperature and the composition of the buffered medium. The cells are grown on a glass slide, half of which is coated with electrically conductive, optically transparent, indium-tin oxide. A control on the strength of the electric field achieved peptide introduction into 100% of the cells, causing no detectable disruption of their division cycle. The method is applied for the fluorescent dye Lucifer yellow, causing its penetration into the cells, growth on the conductive half of a slide (Raptis, 1995). Membrane-impermeant molecules, such as small fluorescent dyes, large carrier-based dyes (fluorescein-labeled dextran), large macromolecules (antibodies), and metabolic precursors (32P-ATP) were introduced by electroporation into adherent cells with high efficiency. The suitable poration medium includes PBS, PBS-buffered 0.25-3.0 M sucrose, Hepes-buffered sucrose, unbuffered sucrose. The rEGFP and HeLa cells were used as model system to test the efficiency of electroporation, giving a sufficient number of uniformly loaded cells for different studies including transcription assays and gene therapy (Todorova, 2009). The ability to load foreign molecules into adherent cells is used in microscopic approaches, such as fluorescence spectroscopic imaging, as well as in conventional biochemical and physiological techniques (Bright, 1996; Potts, 1997). The electroporation was applied to DU145 prostate cancer cells, incubated with GFP-encoded DNA plasmid, either naked or packaged with cationic lipid (Lipofectin), polycationic peptide (salmon protamine) or retroviral vectors (Moloney murine leukemia viruses), and then assayed for gene expression and cell viability. The combination of electroporation with chemicals as cationic lipids, or viral vectors does not improve the gene transfection in vitro (Coulberson, 2003). The proteins cytochrome c, granzyme-B, caspase-8, known to activate caspase-family cell death proteases, were introduced into human leukemia and lymphoma cell lines, freshly isolated lymphocytes and leukemia cells to contrast the status of various caspase activation pathways, related to pathological defects in the regulation of apoptosis that exist in individual patient specimens (Eksioglu-Demiralp, 2003). Fluorochrome-labeled proteins with MW from 15 to 150 kDa were used to evaluate the electroporation conditions and efficiency by flow cytometry. Effect of electroporation in combination with the plant toxin saporin was studied using a human lung cancer cell line (PC9) and a pancreatic cancer cell line (ASPC-1). High degree of inhibition of the proliferation was obtained when ASPC-1 cells were electroporated in the presence of saporin (0.1-1000 ng/ml) in combination with EP (80–90 V, 10 ms, n = 8). The PC9 or ASPC-1 tumor-bearing nude mice were treated with electroporation, following the intratumoral injection of saporin (1 mg), where tumor necrosis was observed 24–48 hr after the combination therapy (Mashiba, 2001). Electroporation was used to introduce antibodies into cells without affecting the physiological integrity of the cells (Nakanishi, 1993; Liao, 1993). Antibodies to pp60c-src were introduced into cultured RASM cells by electroporation, while still attached to tissue culture plates, that had no effect on the platelet-derived and growth factor-stimulated tyrosine phosphorylation of PLC-gamma 1 (Marrero, 1995). The cell cycle activity (transition from the G1 to S phase) was inhibited by the introduction of monoclonal antibodies against G1-specific cyclin D1 into CV-1 and MCF7 cells with 80% efficiency, without affecting the cell viability. The method approaches the efficiency of microinjection and permits treatment of large number of cells which are required for biochemical analyses (Lukas, 1994). Disadvantage of the method is that electroporation can be inefficient and highly disruptive, causing large scale cell death if compare with other protein delivery methods (Todorova, 2009).

Microinjection

Microinjection has the advantage of introducing macromolecules directly into the cell (Graessmann, 1983) such as fertilized eggs, early zygotes, embryonic stem cells and to generate transgenic animals. The techniques of electroporation and microinjection are membrane rupturing.

Ultrasonic Drug Delivery

Sonorelease of an active alkylating agent from the bioconjugate may provide a new method for the selective release of anticancer drugs and thus potentially reduce systemic toxicity (Howard, 1997). Ultrasound has been used to release covalently bound cobalamin from a carrier in a very specific application (Howard, 1997).

Ultrasound (US) is used in the delivery of therapeutic agents including genetic material, proteins, and chemotherapeutic agents. Cavitating gas bodies such as microbubbles are the mediators that permeabilize cell membranes and disrupt the vesicles that carry drugs. To eliminate the non-targeted interaction of free drug with tissues, it is attached to a carrier from which it is released by degradation of a linker via enzymes or pH at the target site. The current theory is that cavitation events open reversible channels in the lipids layers and provide transport for proteins such as insulin (Pitt, 2004).

Regulatory hormones and specifically insulin delivery for diabetes therapy comprises the majority of protein delivery research, and also the growth-related hormones and birth control hormones. All research is focused on insulin transdermal delivery. Ultrasonic delivery is ideal because a small transducer can be placed on the skin surface for a painless, non-invasive delivery route. The transport of tissue plasminogen activator and other lytic proteins such as urokinase into clots is studied. In most studies, the protein is delivered to the clot via intravenous catheter, and the ultrasound is applied transdermally. There are several reports of protein delivery to the lungs via inhalation of ultrasonically aerosolized protein solutions. These additives- stabilizers appear to complex with the proteins and protect them from degradative stresses and free radical attack. The ultrasonically nebulized protein delivery includes interferon, platelet-activating factor, lactate dehydrogenase, superoxide dismutase, alpha1 protease inhibitor, urokinase plasminogen activator, aviscumine (Pitt, 2004).

The US-enhanced transdermal protein delivery is not yet appeared in the clinic. Because large pores and channels are opened through the natural skin barriers, many hormones and proteins could be candidates for transdermal delivery. The effect of the ultrasound on the protein conformation and activity has to be addressed in more detail. Protein delivery is limited by very slow diffusion of proteins through skin and polymeric depots, and by proteinolysis. A polymeric depot could be implanted in subcutaneous, intraperitoneal and intramuscular locations, where timed drug release could by activated by transdermal ultrasound. Polymers in a depot can be degraded by ultrasound in a slow process and thus a technology that will open and close the depot to protein transport is needed. The microbubbles also could be used to assist drug delivery. Small-sized low-frequency transducers (including catheter-based) needs to be developed so that patients can wear them for continuous insulin delivery (Pitt, 2004).

Cell-penetrating peptides (CPPs) and protein transduction domains (PTDs)

Cell-penetrating peptides (CPPs) are short peptides, used as helper molecules to facilitate cellular uptake of molecular cargo (proteins, enzymes, nanosize particles, large fragments of DNA) via covalent bonds or non-covalent interactions to efficiently penetrate cellular lipid bilayers for overcoming cellular membranes. The most commonly used method for protein delivery is genetic fusion to PTDs including the HIV-1 transactivator of transcription (Tat) peptide, oligoarginine, and the Drosophila Antennapedia-derived penetratin peptide (Wadia, 2003; Wadia, 2005; Heitz, 2009). The method is easily titratable, enabling a precise control over time and dosage of incubation; leaves the genome intact in manipulating cellular functions in the absence of genetic interference (Patsch, 2010). CPPs are used as vectors for multiple effectors of gene expression such as oligonucleotides for antisense, siRNA, dsDNA applications, for plasmid delivery transfection agents (Järver, 2007). The advantage of using splice correction for evaluation of CPPs is that the oligonucleotide-based therapeutics induces a biological response in contrast to traditionally used fluorescently labeled peptides (El-Andaloussi, 2007). The toxicity caused by CPPs depends on peptide concentration, cargo molecule, high efficacy and coupling strategy.

CPPs contain polycationic or amphipathic structures, covalently bound to compounds, peptides, antisense peptide nucleic acids, 40-nm iron beads, in-frame fusions with full-length proteins and enter any cell type in a receptor- and transporter-independent fashion. Introduced into mice, they are delivered to all tissues, even crossing the blood–brain barrier, helping the treatment of human disease (Frankel, 1988; Green, 1988). CPPs translocate the plasma membrane to deliver cargoes to the cytoplasm and organelles by direct penetration in the membrane (TAT), endocytosis-mediated entry (TAT), and transitory structure (amphipathic peptides MPG and Pep-1). The transduction across the cellular membrane results in partial or complete unfolding of the protein, and refolding inside the cell to obtain biologically active protein (Schwarze, 2000). CPPs have the ability to enter cells independent of a membrane receptor and are no cell-type specific in vitro and in vivo for use in research and medicine. The efficiency of PTD-mediated protein transduction is probably controlled by the cell surface adsorption and ionic charge interaction between anionic cellular surface and cationic region of proteins (rich in arginine and lysine), followed by internalization through a caveolar endocytotic pathway. Highly cationic proteins (human eosinophile cationic protein, pI = 11.9) and artificially cationized proteins show efficient intracellular delivery. Cationization of proteins with diamines is a powerful strategy to promote their internalization (Futami, 2005). Non-toxic secretory RNases are converted to highly cytotoxic ones by cationization with ethylenediamine (Futami, 2005). A variety of cationic peptides and proteins have been observed to penetrate mammalian cells (Lawrence, 2007; McNaughton, 2009; Frankel, 1988; Green, 1988; Thoren, 2000; Daniels, 2007; Smith, 2008; Fuchs, 2007; Fuchs, 2007). Peptoids are designed of high stability and resistant to proteolysis, transporting macromolecules into cells by offering a highly efficient, non-toxic and non-immunogenic route for cell entry. The mechanism of entry, trafficking route and degradation pathway of CPPs remains elusive (Sebbage, 2010).

Nature of cell penetrating peptides: CPPs originate from full-length proteins of various origin that exhibit cell permeability and can be classified into three groups, including basic transduction peptides, hydrophobic protein leader sequences and artificial peptides (Patsch, 2010). Carriers may comprise a variety of species - a bioactive cell membrane-permeable reagent, PTD (15 to 30 residues) and small sections of these domains (10–16 residues). PTDs mediate protein secretion and are composed of a positively charged amino-terminus, a central hydrophobic core and a carboxy-terminal cleavage site, recognized by a single peptidase. A mixture, containing a protein and a carrier reagent includes a helper reagent to enhance the protein delivery efficiency greater than 90%. The surface-mediated protein delivery technique or “reverse protein delivery” (Ye, 2005) provide greater control over the exact sequence, content, amount and nature by pre-selecting, modifying or checking the proteins prior to their introduction into cells.

Regarding the route of intracellular trafficking, evidences exist for both nuclear and cytoplasmic delivery of CPP-internalized cargo. Nuclear delivery is considered to occur as a result of cytoplasmic delivery. Endosome-localized CPPs have been shown to undergo retrograde delivery, through the Golgi apparatus and endoplasmic reticulum (ER), prior to their cytosolic release (ricin and Shigella toxins). CPPs delivery was successfully to different cell types (human Jurkat and HeLa cell lines) and those impermeable to retroviral vectors (osteoclasts). CPPs do not integrate the genetic material they deliver as the viral vectors, do not result in oncogene activation and does not show increased immunogenicity. In contrast to the membrane rupturing techniques of electroporation and microinjection there is no evidence for membrane perturbation caused by CPP cell entry (Sebbage, 2010). The inability of proteins to potently penetrate mammalian cells limits their usefulness as tools and therapeutics (Lawrence, 2007; McNaughton, 2009; Cronican, 2010). The ability to introduce drugs into cells allows the conventional biodistribution of drugs to be altered in order to favorably impact toxicity, patient compliance, and other treatment factors. CPP-conjugated moieties are directed against a growing variety of targets and disease areas, including cancer, cardiology, pain, and stroke. Therapeutics has been tested in humans, including a CPP conjugate for the treatment of acute myocardial infarction. The promising results obtained in a number of these studies indicate that CPPs may have an important role in the development of novel therapeutics (Johnson, 2011).

Carrier reagents include signal sequences and membrane-translocating peptides (MTPs). MTPs were used for translocation of polypeptides, protein domains, antisense peptide nucleic acids, iron beads, full-length proteins and antibodies into cell. The carrier reagent (bioactive peptide or ligand) specifically binds to the cell surface receptors, activates the receptor, and the bound carrier-protein complex undergoes internalization, delivering ligand-protein complexes into cells. The ligand can be complexed, bound by non-covalent interaction (hydrophobic or electrostatic), coupled covalently or by ligand-receptor interaction to the protein. A carrier reagent can be modified with a ligand that can bind specifically to the protein of interest (Ye, 2005). The MTPs (Table 1) include Trojan peptides, (HIV)-1 transcriptional activator (TAT) protein (Frankel, 1988; Green, 1988) or its functional domain peptides, transportan (Rothbard, 2005) and other peptides derived from translocation proteins such as the third helix of Drosophilia homeotic transcription factor Antennapedia (pAntp) (Schwarze, 1999; 2000) or Penetratin (Derossi, 1994), herpes simplex virus DNA-binding protein VP22 (Elliott, 1997), short amphipathic peptide carrier Pep-1 (Morris, 2001), superpositively charged GFP (McNaughton, 2009), cell-permeant Cre and FLP recombinases (Patsch, 2010), Arg-rich peptides (Suzuki, 2002), RGD peptides (Ye, 2005), K-FGF (Lin, 1996), Universal PTK (Clark, 2002), translocator protein TSPO (Rupprecht, 2010). The major cell CPPs penetratin, TAT and transportan 10 (TP10) deliver cargos, including fluoresceinyl moiety, double stranded DNA and proteins (avidin, streptavidin), liposomes and nanoparticles (Langel, 2007).

Table 1.  Cell penetrating peptides and their amino-acid sequences.

CPP	Amino acid sequence	Reference
HIV-1 TAT	YGRKKRRQRRR	(Schwarze, 1999)
Penetratin Antp	RQIKIWFQNRRMKWKK	(Derossi, 1994)
HSV VP22	NAATATRGRSAASRPTQRPRAPARSASRPRRPVQ	(Elliott, 1997)
Universal PTK	AAYANAAVE	(Clark, 2002)
K-FGF	AAVLLPVLLAAP	(Lin, 1996)
Oligoarginine	Rn (n = 4–12)	(Suzuki, 2002)
RGD peptide	RGDS, RGDSPASSKP	(Ye, 2005)
Transportan	GWTLNSAGYLLGKINKALAALAKKIL	(Rothbard, 2005)

The protein transduction domains are used for delivery of therapeutic proteins into animal models of human neurological disorders such as nerve trauma and ischemia (Dietz, 2004). The pAntp was used to introduce peptides into primary lymphocytes and lymphoid cell lines (Frankel, 1988), taken up rapidly into the cytoplasm and nucleus, and retained for at least 48 h. The pAntp peptide is not cytotoxic and the efficiency of delivery is up to 95% (Fenton, 1998).

The HIV-1 TAT delivered proteins from 30 to 120–150 kDa such as horseradish peroxidase and RNase A across cell membrane into the cytoplasm in different cell lines in vitro. TAT protein enters cells when added to culture media and activates transcription of the viral genome (Frankel, 1988; Green, 1988). TAT was found in its normal biological location (nucleus) and could transactivate the viral LTR promoter. A short TAT sequence (residues 37–72), rich in positively charged amino acids, promotes cell permeability when chemically crosslinked to several full-length cargo proteins, such as β-galactosidase and horseradish peroxidase (Patsch, 2010). The intraperitoneal injection of TAT-fused β-galactosidase protein (120-kDa) results in delivery of biologically active fusion protein to all mice tissues (including the brain), thus opening new possibilities for direct delivery of proteins into patients for protein therapy, as well as for epigenetic experimentation with model organisms (Schwarze, 1999). Tat, fused to heterologous proteins traverses the biological membranes and the blood-brain barrier in protein transduction. TAT-eGFP or GST-eGFP proteins were fed to C. elegans worms, which resulted in specific localization of TAT-eGFP to the epithelial intestinal cells. The method is a tool to analyze the mechanisms of protein transduction and to complement RNAi/KO in epithelial intestinal system, combining the bacterial expression system and TAT transduction in living worm (Delom, 2007). The TAT stability, activity and proteolytic cleavage on the surface of TAT-modified micelles and liposomes was investigated, using a phosphatidyl ethanolamine conjugate with a ‘short’ PEG (PEG-PE) in EL-4, HeLa, and B16-F10 cells. A decrease in the cytotoxicity of TAT-modified liposomes, loaded with doxorubicin following trypsin treatment, leaded to the conclusion that steric shielding of TAT is essential to ensure its in vivo therapeutic function (Koren, 2011).

TP10 vector was evaluated in different in vitro plasmid delivery assays that can enhance polyethyleneimine mediated transfection at relatively low concentrations to develop future gene delivery systems with reduced toxicity (Kilk, 2005).

The synthetic peptide AAYANAAVE has been used as a universal PTK substrate for rapid detection of PTK activity in recombinant yeast (Clark, 2002).

M918 internalizes via endocytosis and macropinocytosis, independently from cell surface glycosaminoglycans and is non-toxic. In a splice correction assay, using antisense peptide nucleic acid (PNA) conjugated via a disulphide bridge (M918-PNA), was observed a dose-dependent increase in correct splicing. This method possesses the potential of a therapeutic approach for regulating splicing in a variety of diseases (El-Andaloussi, 2006; El-Andaloussi, 2007).

The TSPO (18 kDa) is localized primarily in the outer mitochondrial membrane of steroid-synthesizing cells, including those in the central and peripheral nervous system (Rupprecht, 2010). TSPO is a therapeutic target for neurological and psychiatric disorders.

When fused to superpositively charged GFP (McNaughton, 2009), proteins rapidly (within minutes) entered mammalian cells 100-fold greater than the corresponding fusions of Tat, oligoarginine and penetratin. Ubiquitin-fused supercharged GFP in human cells is partially deubiquitinated and can access the cytosol. Supercharged GFP delivered functional, nonendosomal recombinase enzyme with greater efficiencies than PTDs in vitro to the retinae of mice when injected in vivo (Cronican, 2010). The superpositively charged GFP variants can enter a variety of mammalian cells by binding to anionic cell-surface proteoglycans and undergoing endocytosis in an energy-dependent and clathrin-independent fashion (McNaughton, 2009).

Cell-permeant Cre and FLP recombinases were used to reversibly induce transgenes in embryonic stem cells. FLP protein transduction is a highly efficient and non-toxic method for the removal of undesired selectable marker genes introduced into ES cells by gene targeting. This system is particularly useful for genes whose overexpression has deleterious effects on fundamental cellular processes, such as proliferation, cell death and differentiation. Cell permeant FLP was used for excising prespecified fragments from transgenes, expressed in fibroblasts and mouse embryonic stem cells. The combination of site specific recombination and protein transduction provides a highly efficient methodology for manipulating the mammalian genome (Patsch, 2010).

Several Gram-negative pathogenic bacteria have a conserved and unique protein secretion system type III (T3SS) to deliver bacterial effector proteins into eukaryotic cells to modulate host cellular functions. These bacterial devices are present in both plant and animal pathogenic bacteria and are evolutionarily related to flagellar apparatus. The development of prevention and therapeutic approaches for several infectious diseases can be based on T3SS (Galan, 1999). T3SS are encoded by bacterial species that are symbiotic or pathogenic for humans, other animals (including insects or nematodes) and plants (Galan, 2006). By attaching carrier peptides, efficient protein transduction of p16P^INK4, p27P^Kip1, p53, caspase-3 and Cre recombinase in vitro and in vivo was obtained (Futami, 2005).

Coupled cell-free transcription/translation of DNA templates produces the corresponding proteins immobilized on the surface. An array of DNA templates could be deposited on a substrate surface, performed in situ protein synthesis on it and then treated the resulting protein array with a vehicle reagent to transport the proteins into cells (Ye, 2005). The helper reagent can be polymer (DEAE-dextran, dextran, polylysine, polyethylamine); a cell adherent-enhancing protein (fibronectin and gelatin), a sugar-based gelatin (polyethylene glycol), a synthetic or chemical-based gelatin (acrylamide), a RGD peptide (Arg-Gly-Asp-Ser, Arg-Gly-Asp-Ser-Pro-Ala-Ser-Ser-Lys-Pro), a mixture of a hydrogel and a RGD peptide and combination of molecules (Ye, 2005). By spotting a library of fusion proteins on an appropriate substrate surface, and then plating adherent cell lines on the surface is possible to assess the protein function in vivo in eukaryotic cells. Interactions between the delivered protein and other native cellular proteins may be studied in vivo. Fusing the delivered protein with an auto-fluorescent marker (GFP) may monitor its intercellular localization. Reverse delivery into cells could be accomplished using VP22, HSV protein, or any other protein with similar properties (Ye, 2005).

The pharmacological chaperone is a new concept in the treatment of certain chronic disabling diseases. Proteins that are denatured by any form of proteotoxic stress are cooperatively recognized by heat shock proteins (HSP) and directed for refolding or degradation. Under non-denaturing conditions HSP functions in transmembrane protein transport and in enabling assembly and folding of newly synthesized polypeptides. Besides cellular molecular chaperones, which are stress-induced proteins, pharmacological chaperones demonstrated ability to be effective in preventing misfolding of different disease causing proteins. They are used in the therapeutic management of sight-threatening eye diseases, essentially reducing the severity of several neurodegenerative disorders (age-related macular degeneration), cataract and many other protein-misfolding diseases. The family of imidazole-containing peptidomimetics Carcinine (β-alanylhistamine), N-acetylcarnosine (N-acetyl-β-alanylhistidine) and Carnosine (β-alanyl-L-histidine) are essential constituents possessing diverse biological and pharmacological chaperone properties in human tissues (Babizhayev, 2010).

Commercial peptide products

Some commerically available peptides such as penetratin 1 (Pep-1, Chariot reagent, Active Motif) and HIV GP41 fragment (519–541) can be used to save time. The amphipathic peptide carrier Pep-1 is able to efficiently deliver a variety of peptides and proteins into several cell lines in a fully biologically active form, without the need for prior chemical covalent coupling or denaturation steps. The Pep-1 is stable in physiological buffer, not sensitive to serum and could facilitate rapid cellular uptake of various peptides, proteins, and full-length antibodies with high efficiency and less toxicity. Pep-1 technology should be extremely useful for targeting specific protein–protein interactions in living cells and for screening novel therapeutic proteins (Morris, 2001).

Entrypep (Inbiolabs) contains a cell membrane permeable polypeptide sequence that crosslinks by thioester bond and delivers peptides, proteins, antibodies, protein complexes, chemically modified proteins, all compounds with -SH group, dye-labeled proteins. The method may be used for visualization and co-localization of cell membrane permeable polypeptide sequence-conjugated antibodies and specific ligands, protein-protein interactions in the cell, inhibition or activation of intracellular processes, functional analysis, intracellular effects of chemically modified proteins (Säälik, 2004).

Chariot (Active Motif) is a peptide delivery vector manufactured by Panomics and advertised as the ‘Revolutionary new transfection agent’ (Sebbage, 2010). Chariot peptide forms a non-covalent bond, is non-cytotoxic; quickly and efficiently (up to 60–95%) transports biologically active proteins, peptides and antibodies directly into cells. The method is used for functional studies, including delivery of inhibitory proteins, label organelles, screen peptide libraries, determine protein half-lives and transient complementation. The complex dissociates, leaving the macromolecule biologically active and free to proceed to its target organelle. Chariot was used to deliver carcinoma proteins, peptides and antibodies in HeLa cells with up to 95% efficiency, determined by Flow cytometry. Protein transduction was performed using Chariot in Rat pheochromocytoma PC12 cell line (RCB009) (Watanabe, 2008).

Nanoparticles

Nanoparticles made of hydrophilic polysaccharides, chitosan (CS) and glucomannan (GM) can be used for association and delivery of proteins. The release of peptide/protein can be modulated by varying the composition of the system. Two different types of glucomannan (non-phosphorylated Konjac GM (KGM) and phosphorylated GM) exhibited a great capacity for association of the model peptide insulin and immunomodulatory protein P1, reaching 89% association efficiency (Alonso-Sandel, 2006; Cuña, 2006). A targeted proapoptotic anticancer drug delivery system (DDS) contains PEG as a carrier, camptothecin (CPT) as anticancer drug/cell death inducer, a synthetic analogue of luteinizing hormone-releasing hormone (LHRH) peptide as targeting moiety/penetration enhancer, and a synthetic analogue of BCL2 homology 3 domain (BH3) peptide as suppressor of cellular antiapoptotic defense. The design of multicomponent DDS allowed conjugation of one or two copies of each active ingredient (CPT, LHRH and BH3) to one molecule of PEG carrier, thus ensuring highest anticancer efficiency in vitro and in vivo. The ligand-targeted DDS preferentially accumulated in the tumor and allowed the delivery of active ingredients into the cellular cytoplasm and nuclei of cancer cells (Chandna, 2007).

Polyethylenimine (PEI)-cationized proteins appear to adhere to the cell surface by ionic charge interaction internalizing into cells in a receptor and transporter-independent fashion. PEI results in endowment of sufficient cationic charge to proteins without causing serious decline in their fundamental functions. A number of PEI-cationized proteins, such as RNase, GFP and IgG, efficiently entered cells and functioned in the cytosol (Futami, 2005). PEIs with molecular masses, ranging from 250 to 70,000 are available. PEI is toxicologically safe and used as a food additive. A number of PEI-cationized proteins were efficiently delivered into living cells both in vitro and in vivo. The covalent conjugation of PEI to proteins is also a possible approach in protein transduction methodology for analyzing protein function in living cells or for specific manipulation of cellular events. PEI-cationized antibody successfully binds to target antigen in living cell, suggesting that this technology may be applicable for intracellular antibody (intrabody) therapy (Futami, 2005). The high efficiency and low toxicity of protein transduction using Polyethylenimine (PEI)-cationized proteins may be used to develop new teurapeutics against cancer and other diseases.

The Influx reagent cell-loading method (Invitrogen) is based on the osmotic lysis of pinocytic vesicles for loading water-soluble and polar compounds into live cells, no altering normal cell function (Okada, 1982). The compounds to be loaded are mixed at high concentration with a hypertonic medium, allowing the material to be carried into the cells via pinocytic vesicles. The cells are then transferred to a hypotonic medium resulting in release of the trapped material into the cytosol. The Influx pinocytic cell-loading reagent contains a mixture of sucrose crystals and PEG that allows loading of compounds into cells, grown on coverslips, in suspension and in flasks.

Virus-like particles (VLPs) consist of viral structural proteins that are capable of self-assembly into a nanoparticle, but are non-infectious because they lack viral nucleic acids. VLPs have been used in viral vaccines, such as those for human papilloma virus and hepatitis B. They have great potential as cancer vaccines, transport of nucleic acids into target cells (gene therapy), functional fusion proteins, transport of biologics and other large molecules into target cells for therapeutic purposes. The chimeric VLP contains a GAG-Cre recombinase fusion protein that retains Cre recombinase activity, and can excise a LOX-flanked gene in a transduced target cell. VLPs containing GAG-protein of interest co-packaged with GAG-protease to deliver protein of interest in target site as a fully-processed protein rather than as a fusion protein (Kaczmarczyk, 2008).

MPG technology uses virus-derived amphipathic peptides that directly interact with nucleic acid cargos to form nanoparticles (150–200 nm), capable of diffusing through plasma membranes and releasing their contents inside the cell. The mechanism of entry is receptor-independent, involves MPG/lipid membrane interactions, avoids the endocytic pathway and prevents endosomal or lysosomal degradation of cargos. Peptide transfection is used for modulation of cell signaling pathways and creation of potential therapeutic agents such as development of siRNAs (Deshayes, 2005; Crombez, 2007).

Nanobiotechnology has extended the limits of molecular diagnosis of cancer, through the use of gold nanoparticles and quantum dots. Nanoparticles enable targeted drug delivery in cancer, increase the efficacy and decrease adverse effects through reducing the dosage of anticancer drugs; cross the biological barriers and achieve therapeutic concentrations in tumor and spare the surrounding normal tissues from toxic effects; facilitate the delivery of various forms of energy for noninvasive thermal destruction of surgically inaccessible malignant tumors; optical imaging of tumors as well as contrast agents to enhance detection of tumors by magnetic resonance imaging; monoclonal antibody nanoparticle complexes for diagnosis and targeted delivery of cancer therapy. These will facilitate the combination of diagnostics with therapeutics, which is an important feature of a personalized medicine approach to cancer (Jain, 2010). Attaching monoclonal antibodies (mAbs) which can recognize a specific cancer cell to gold nanoparticles or nanorods the “heating phenomenon” can be used in cancer detection. Gold nanoparticles conjugated to anti-epidermal growth factor receptor (anti-EGFR) mAbs specifically and homogeneously bind to the surface of the cancer cells with 600% greater affinity than to the noncancerous cells and are not toxic to human cells (Jain, 2010). Due to the large surface area and the controllable surface functionality of mesoporous silica nanoparticles (MSNs), they can be controllably loaded with large amounts of drugs and coupled to homing molecules to facilitate active targeting, simultaneously carrying traceable (fluorescent or magnetically active) modalities. They possess increased relative surface area, small size, flexible surface used in nanomedicine, but also potential risks in the interactions with biological systems (Rosenholm, 2010). The ability to functionalize the surface of mesoporous-silica-based nanocarriers with stimuli-responsive groups, nanoparticles, polymers, and proteins for controlled release of various cargos and intracellular drug delivery is important (Vivero-Escoto, 2010). The drug delivery targeted specifically to defected cells and the development of site-specific drug delivery non-toxic vehicles is promising for use in the medicine.

Cells

A major limitation of cancer gene therapy is the difficulty of delivering a therapeutic gene to distant sites of metastatic disease. A promising strategy to overcome this difficulty is to use expanded ex vivo cells to produce a therapeutic protein when it is unstable ex vivo or has a short circulating half life in vivo. The initial step is the identification of a cell type that migrates to the tumor site, is readily available for harvesting, and is manipulated easily ex vivo. The endothelial progenitor, precursor, and blood outgrowth endothelial cells are attracted to the tumor vasculature by its angiogenic drive. The systemic delivery of gene therapy to distant metastases is the most attractive feature of using ELCs as gene delivery vehicles. Preclinical development is necessary to demonstrate the selectivity of ELC-mediated gene delivery to the tumor site and to enhance the various strategies used to impede tumor growth (Dudek, 2010).

Applications of protein transduction technology for diagnostics and treatment of cancer and genetic diseases

Vitamin-mediated targeting for cancer treatment

The research was focused on targeting Vitamin B12, folic acid, biotin, and analogs of these substances. Not all cancer cells have increased demand for vitamin analogs. The most desirable cancer treatment is that in which the anti-cancer agent targets only tumour cells. The antibody–drug conjugates used are specific for only a small number of tumour types, may be highly immunogenic and lead to an antibody response against the conjugate. Vitamin B12, folic acid, biotin, and riboflavin are essential vitamins for the cell division, but particularly for the growth of tumour cells. Vitamin B12 depletion kills cancer cells that are resistant to conventional chemotherapeutic drugs.

The supply route of vitamin B12 was exploited to deliver Co⁵⁷ and In¹¹¹. Transcobalamin I accumulates on tumors as a possible new receptor for preferential accumulation of vitamin-mediated targeting. The cobalamin is water soluble and has no known toxicity. Vitamin B12–mediated targeting is dependent on undisturbed interaction of cobalamin with the main transport proteins IF, TCI, and TCII (Waibel, 2008). Vitamin B12, coupled sterically, stabilized liposomes (SL-VB12) and could be used as targeted carrier to facilitate the delivery of the encapsulated anticancer drugs into tumor cells (Gupta, 2011).

The receptor for the vitamin folic acid is overexpressed in cancers of the ovary, kidney, uterus, testis, brain, colon, lung and myelocytic blood cells. The affinity of folate conjugates for cell surface folate receptors is high (K_D~10⁻¹⁰ M), folic acid derivatization allows the selective delivery of diagnostic and therapeutic agents to cancer cells in the presence of normal cells (Wang, 1998). The degree of over-expression correlates with the stage of tumour growth, with the highest levels found on stage IV carcinomas. Binding of the targeted fluorochrome leads to rapid internalization in the cells to levels that are two to thirty times higher than with non-targeted polymers. The folate receptor for tumour diagnosis was applied using the MOv18 and MOv19 monoclonal antibodies to identify the over-expressing cells in tumour sections (Russell-Jones, 2004). Folate-mitomycin C conjugates, EC72 and EC118, are highly cytotoxic and selective for FR-positive M109 cells, thus in vitro and in vivo enhancing drug specificity and reducing lethal toxicity (Reddy, 2006; Leamon, 2005). There are existing anti-folate chemotherapeutic drugs such as Methotrexate and 5-Fluoraracil, used in cancer treatment, but they are significantly toxic and inducing drug resistance. Folate and its drug conjugates enter cancer cells by receptor-mediated endocytosis, thus allowing very hydrophilic drugs and macromolecules to enter folate receptor-bearing cells as their hydrophobic counterparts. Bovine IgG and bovine ribonuclease were labeled with fluorescent probes and conjugated to folic acid, where only the folate-conjugated fluorescent proteins were taken up by folate receptor-positive KB cancer cells (Leamon, 1991). Internalization of the folate-conjugated proteins was found to be time- and concentration-dependent, and could be quantitatively blocked by addition of excess free folic acid to the culture medium (Leamon, 1991). The site of folate attachment has to be at the vitamin’s γ-carboxyl group. The folic acid is internalized for consumption and folate conjugates remain stable and functional for hours following uptake by cancer cells, allowing that proteins and nucleic acids remain undigested, enzymes retain their activities (Leamon, 1991), and liposomes resist disruption (unless engineered to disintegrate at endosomal pH). Thus, if sensitivity to hydrolytic enzymes (nucleases, proteases, glycosidases and lipases) is a concern, vitamin-mediated delivery may constitute a more protected pathway for intracellular delivery (Wang, 1998). Drugs linked to folic acid for tumor-selective drug delivery include protein toxins, chemotherapeutic agents, gene therapy vectors, oligonucleotides (including siRNA), radioimaging agents, MRI contrast agents, liposomes with entrapped drugs, radiotherapeutic agents, immunotherapeutic agents, enzyme constructs for prodrug therapy. Folate receptor-targeting holds great promise for High tumor selectivity in vitro and in vivo with folate-targeted imaging agents, antineoplastic drugs, protein toxins, and antisense oligonucleotides (Low, 2008). Folate-targeted liposomes are effective vehicles for gene therapy: to incorporate a fusogenic oligopeptide whose conformation changes upon acidification and initiates membrane fusion; to develop a pH-sensitive lipid composition stable at neutral pH but lysogenic or fusogenic upon entry into acidic endosomes (Wang, 1998).

The advantages of the vitamin folic acid compared to the tumor-specific monoclonal antibodies as a tumor targeting agent are its low cost, high chemical and biological stability, compatibility with organic solvents, non-immunogenicity, stronger affinity for its receptor, and small size for fast tumor extravasation and thorough intercellular infiltration. The folic acid method has significant clinical potential, since overexpression of folate receptors has been found in a large fraction of human cancers but rarely in normal tissues, allowing high tumor specificity in the diagnosis and treatment of cancer.

The disadvantages are that some tumour types do not over-express receptors involved in vitamin uptake (Russell-Jones, 2004). Radiolabeled vitamin derivatives labeled the tumor tissue but also accumulated radioactivity in normal tissue. Limitations of the use of folate or vitamin B12 are that the dose deliverable is small (one molecule of drug for each molecule of targeting agent), and the majority of the complexes are small, thus leading to their undesirable accumulation in the kidney.

Commercial drug delivery systems based on vitamins. By coupling drugs to CobaCyte™ - Cobalamin™ (Access Pharmaceuticals) (an analog of vitamin B12), more drug is taken up by diseased cells. This effect can be amplified by attaching Cobalamin™ and several molecules of the drug to a polymer or by encapsulating the drug in a nanoparticle coated with Cobalamin™. The targeting approach could be useful in cancer, rheumatoid arthritis, psoriasis, acute leukemia, lymphomas, Crohn’s disease, ulcerative colitis, and multiple sclerosis.

The biology of the cancer could be modulated at protein level by direct cellular introduction of peptides, full-length proteins and functional domains into tumor cells, allowing to treat diseases such as cancer. The barrier of the cell plasma membrane restricts the intracellular uptake of macromolecules to non-polar and less than 500 Da in size (Wadia, 2005). Non-viral delivery methods were utilized to enhance the tumor-selective delivery of therapeutic molecules, including proteins, synthetic oligonucleotides, small compounds and genes. By combining therapeutic molecules with drug delivery systems, having antitumor activities, enhancement of the multiple therapeutic pathways may be achieved (Kaneda, 2010). Manipulation of the cell cycle (p53 and p27), inflammation (NF-κb), apoptosis (Bcl-xL), stem cell differentiation (transcription factors HoxB4, Pdx1, Scl, Oct4, Sox2 and Nkx2.2), engineer genomes via Cre recombinase in murine and human ES cells and post-mitotic neurons were accomplished (Patsch, 2010). One trial concerns the drug cyclosporine A (CsA) used in ointment for the topical treatment of psoriasis (Price, 2003). A polyarginine heptamer with a pH-sensitive linker permits skin penetration allowing the release of CsA at functional pH within the skin (Sebbage, 2010). CPPs have found numerous applications in medicine as drug delivery agents in the treatment of different diseases including cancer and virus inhibitors, as well as contrast agents for cell labeling (carriers for GFP; MRI using different metal chelates or quantum dots as fluorescent probes) (Stewart, 2008). Metal chelates increase the contrast signal between normal and diseased tissues (Gd3+ low-molecular-weight chelates, superparamagnetic iron oxide (SPIO)) for disease diagnosis of cancer metastasis and inflammation.

The possibility of targeting to repair or degrade protein misfolding in cancer therapy is important (Nagaraj, 2010), because an effective drug must be active only in the diseased cell. Chemotherapy agents must be able to kill tumour cells selectively, while leaving normal cells unharmed: fusing the Antp PTD to docking site of cyclin–Cdk complexes resulted in specific apoptosis of the tumorigenic cells (Chen, 1999); the p53 tumour-suppressor protein guarded the genome and induced apoptosis in cells, undergoing significant levels of DNA damage (Levine, 1997); the VP22–p53 fusion expression vector resulted in transduction to the surrounding tumour cells and subsequent induction of apoptosis (Phelan, 1998); differentiation between normal and HIV-1 infected cells by including a virus-specific proteolytic cleavage site in TAT protein (‘Trojan horse’ strategy) selectively induced apoptosis in HIV-infected cells by exploiting the HIV protease (Vocero-Akbani, 1999); a caspase-3 proapoptotic TAT-PTD fusion zymogen was engineered that substituted HIV proteolytic-cleavage sites for endogenous caspase cleavage sites (Schwarze, 2000). TAT–caspase-3 was processed into an active form by the HIV protease, initiating the apoptotic cascade in the infected cells, while the uninfected cells were spared. The preclinical approaches harness the therapeutic potential of protein transduction (Schwarze, 2000). The TSPO expression may constitute a biomarker of brain inflammation and reactive gliosis that could be monitored by using TSPO ligands as neuroimaging agents in the treatment of neurological and psychiatric disorders (Rupprecht, 2010).

Peptides, targeted by autoimmune responses, can be synthesised with defined chemical modifications to mimic natural epitopes and deliberately introduce protease resistant peptide bonds to regulate their processing independent of tissue specific proteolysis and to stabilize these compounds in vivo (Dudek, 2010). The potential of peptide-based vaccines for the treatment of chronic viral diseases, cancer and epitope discovery is evident.

Basic pharmacological questions of tissue distribution, protein half-life, immunogenicity, increasing the refolding rates of transduced proteins and domains, existing restrictions on the structures of therapeutic agents, modes of delivery are also important. The ability of the TAT, Antp and VP22 proteins to transduce into cells does not appear to affect their biological functions, namely interacting with nucleic acids. In vitro, the refolding is the rate-limiting step, and not transduction, whereas in vivo, the animal models need improvements in both areas. If protein transduction can be adapted for humans with the efficacy observed in vitro and in vivo, the potential of therapeutic compounds, peptides and proteins to combat infectious diseases, to complement deficiencies in specific genes and specifically kill tumour cells can be realized (Schwarze, 2000). The intracellular concentration may be controlled by varying the protein amount added to culture medium. A transiently internalized protein is likely to be metabolized in biological process (Futami, 2005). For selective targeted delivery is necessary to identify generic markers that are over-expressed on the surface of tumour cells but are not over-expressed on normal tissue.

The delivery to specific tissues will be targeted by decorating the carriers or microbubbles with antibodies or other site-specific adhesive molecules. Small-sized low-frequency transducers including catheter-based transducers need to be developed so that patients can wear them for continuous insulin delivery. Target-specific antibodies could be mixed with generic microbubbles to create drug delivery systems with the drug attached to the bubbles (Pitt, 2004). The response of cells and their membranes to US should not produce collateral damage to the target or adjacent tissues.

Conclusions

The protein transduction technology provides several advantages over DNA transfection, the current standard method of intracellular protein expression. The potential of the transduced peptides and proteins was used as new therapeutic compounds against infectious diseases, to complement deficiencies in specific genes, to specifically kill tumour cells, for gene therapy. The protein delivery vectors can enhance the transfection at low concentrations and help to develop future gene delivery systems with reduced toxicity. CPPs may have an important role in the development of novel therapeutics. The new developing technologies of drug delivery and targeting offer the possibility to improve the therapeutic possibilities of the existing drugs with low risk and low cost.