was introduced and commonly used [5, 6]. An increasing number
of CPPs have been subsequently identified and extensively studied
over the past three decades. Currently, about 1850 CPP sequences
have been deposited in the CPPsite 2.0 database [7].
CPPs are capable of penetrating the cell membrane due to
cationic, amphipathic, or hydrophobic domains [1, 8–11]. They
are taken up in almost all cell types [1]. The CPP family hosts a wide
array of variously structured proteins which differ in charge and
polarity [3]. Although most are linear, some have α-helix and
β-sheet secondary structure [3]. CPP uptake is often mediated by
endocytosis [4], and it is common for CPPs to have stretches of
polyarginine [3, 12]. In fact, a simple polyarginine sequence (such
as SR9, RRRRRRRRR) can act as a CPP [11].
Transfection and gene expression can be controlled by the
delivery of macromolecules for gene therapy. CPP carriers can
bring transcription factors [13], RNA interference (RNAi)
[1, 11], plasmid DNA [1, 11], functional proteins [1, 11], and
other nanomaterials [11] into cells. Some CPPs are specifically
designed to naturally enter the nucleus to deliver cargo with nuclear
localization sequences and other motifs [4]. Delivery of these
compounds can directly alter or influence the transcription of
genes, or add new genes into cells.
Recently, several CPPs have been identified from bovine lactoferricin [8]. Among them, the novel pentapeptide L5a (RRWQW)
has demonstrated the ability to form stable complexes with plasmid
DNA and to transfer plasmid DNA into cells with no cytotoxicity
[8]. L5a represents one of the shortest CPPs characterized to date
and has helical structure [9]. Mechanistic studies have revealed that
L5a appears to enter A549 cells by direct membrane translocation
[9], while L6 (RRWQWR) and HL6 (CHHHHHRRWQWRHH
HHHC), also derived from bovine lactoferricin, are internalized by
endocytosis [10] and direct membrane translocation [14], respectively. Lactoferricin-derived CPPs should not be preferential to cells
and will enter most cells effectively given their ability to pass
through cell membranes. All three lactoferricin-derived CPPs
(L5a, L6, and HL6) are positively charged, allowing electrostatic
complexing with negatively charged macromolecules, like DNA
and carboxyl-functionalized nanoparticles [8, 14].
The use of a small peptide derived from lactoferricin has several
advantages. Derivation from mammalian proteins decreases the
chance for immunogenicity compared to peptides from the transduction domain of viral proteins as our bodies have defenses in
place for disease-causing proteins [8]. Although the size of CPPs
does not affect transduction efficiency when complexed with DNA
[9], a smaller delivery vehicle is advantageous. Small peptides have
uniform and rapid biodistribution which reaches deep into tissues
[15]. The short length of L5a also makes it easy to synthesize
artificially [9, 15].
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Natalie J. Holl et al.
of CPPs have been subsequently identified and extensively studied
over the past three decades. Currently, about 1850 CPP sequences
have been deposited in the CPPsite 2.0 database [7].
CPPs are capable of penetrating the cell membrane due to
cationic, amphipathic, or hydrophobic domains [1, 8–11]. They
are taken up in almost all cell types [1]. The CPP family hosts a wide
array of variously structured proteins which differ in charge and
polarity [3]. Although most are linear, some have α-helix and
β-sheet secondary structure [3]. CPP uptake is often mediated by
endocytosis [4], and it is common for CPPs to have stretches of
polyarginine [3, 12]. In fact, a simple polyarginine sequence (such
as SR9, RRRRRRRRR) can act as a CPP [11].
Transfection and gene expression can be controlled by the
delivery of macromolecules for gene therapy. CPP carriers can
bring transcription factors [13], RNA interference (RNAi)
[1, 11], plasmid DNA [1, 11], functional proteins [1, 11], and
other nanomaterials [11] into cells. Some CPPs are specifically
designed to naturally enter the nucleus to deliver cargo with nuclear
localization sequences and other motifs [4]. Delivery of these
compounds can directly alter or influence the transcription of
genes, or add new genes into cells.
Recently, several CPPs have been identified from bovine lactoferricin [8]. Among them, the novel pentapeptide L5a (RRWQW)
has demonstrated the ability to form stable complexes with plasmid
DNA and to transfer plasmid DNA into cells with no cytotoxicity
[8]. L5a represents one of the shortest CPPs characterized to date
and has helical structure [9]. Mechanistic studies have revealed that
L5a appears to enter A549 cells by direct membrane translocation
[9], while L6 (RRWQWR) and HL6 (CHHHHHRRWQWRHH
HHHC), also derived from bovine lactoferricin, are internalized by
endocytosis [10] and direct membrane translocation [14], respectively. Lactoferricin-derived CPPs should not be preferential to cells
and will enter most cells effectively given their ability to pass
through cell membranes. All three lactoferricin-derived CPPs
(L5a, L6, and HL6) are positively charged, allowing electrostatic
complexing with negatively charged macromolecules, like DNA
and carboxyl-functionalized nanoparticles [8, 14].
The use of a small peptide derived from lactoferricin has several
advantages. Derivation from mammalian proteins decreases the
chance for immunogenicity compared to peptides from the transduction domain of viral proteins as our bodies have defenses in
place for disease-causing proteins [8]. Although the size of CPPs
does not affect transduction efficiency when complexed with DNA
[9], a smaller delivery vehicle is advantageous. Small peptides have
uniform and rapid biodistribution which reaches deep into tissues
[15]. The short length of L5a also makes it easy to synthesize
artificially [9, 15].
114
Natalie J. Holl et al.