Emission filters were set at 535/40, 460/50, and 630/60 nm for GFP, BFP, and RFP channels, respectively. RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (or use, while others are suitable for both. For safety reasons, nonviral delivery methods, such as peptide- and lipid-based systems, have received more attention over the past twenty years than viral methods. Advantages of nonviral systems include ease and flexibility of assembly, minimal toxicity, and low levels of immunogenicity and insertional mutagenesis. Cell-penetrating peptides (CPPs) that can deliver therapeutic and diagnostic molecules into cells in a nontoxic manner have recently received considerable attention as a promising nonviral tool for the delivery of drugs and diagnostic agents [1,2]. The first CPP discovered, transactivator of transcription (Tat)-protein transduction domain (PTD), consists of eleven amino acids (YGRKKRRQRRR) of the HIV-1 Tat. Tat-PTD is rich in basic amino acids, and is required for Tat translocation through the plasma membrane [3]. Subsequently, a variety of amphipathic, Fatostatin hydrophobic, and cationic peptides with less than thirty amino acids in length were identified and found to be able to deliver a wide range of biological cargos into cells [4]. Approximately 1, 700 CPP sequences have been identified and collected in database CPPsite 2.0 [5] (http://crdd.osdd.net/raghava/cppsite/). The CPPpred (http://bioware.ucd.ie/~compass/biowareweb/Server_pages/cpppred.php) and CellPPD (http://crdd.osdd.net/raghava/cellppd/submission.php) websites provided tools that predict CPP effectiveness [6,7]. A quantitative structure-activity relationship (QSAR) model was recently developed that predicts the physiochemical properties of amphipathic CPPs [8]. However, the mechanisms by which CPPs and CPP/cargo complexes traverse cell membranes remain incompletely understood. Lactoferrin (LF), an 80-kDa glycoprotein with iron-binding ability, is present in most biological fluids of mammals, including milk, saliva, tears, and mucous secretions [9]. Hydrolysates prepared from cleavage of LF with pepsin have strong antibacterial activity [10]. The antimicrobial peptide lactoferricin (LFcin) is located in the N-terminal region of LF [11]. The primary structure of bovine LFcin consists of a loop of 25 amino acids (residues 17C41 of the parent Mouse monoclonal to ALCAM LF sequence [12]) formed by a disulfide bond between cysteine residues 19 and 36 [11]. Many LFcin derivatives possess antiviral [13,14], antifungal [15,16], antimicrobial [17C21], antitumoral [22], antiprotozoal [23], anticancer [9,24], and antihypertensive [25] activities (for a review [26]). Recently, the antimicrobial core of bovine LFcin has been narrowed down to only six amino acids (RRWQWR) [24,25]. A 22-amino acid loop form LFcin was the first CPP isolated from the N-terminal domain of human LF [27], which corresponds to amino acid residues 19C40 in bovine LF [28]. This loop structure formed by a disulfide bond between cysteine residues 20 and 37 is strictly conformation-dependent for efficient uptake into cells [27]. Binding of human LFcin to negatively charged heparin sulfates at the cell surface was the driving force for cellular uptake of arginine-rich CPPs [29]. Subsequently, the bLFcin6 Fatostatin sequence (RRWQWR) was identified from bovine as a new CPP that can effectively deliver small interfering RNA (siRNA) [30]. In contrast, the CPP5 (RWQWR), one of the shortest CPPs described [31], has less internalization activity [30]. Recently, a systematical study using human proteomic databases screened amino acid sequences of peptides or protein domains that reside or interact with cellular plasma Fatostatin membranes [32]. Fifty potential CPPs derived from 46 proteins were identified that could deliver siRNA across plasma membranes. Among them, three human CPPs derived from surfactant B, orexin, and LFcin were studied in further detail. It shall be noted that their published sequences of 25-amino acid LFcin and 12-amino acid LFcin (short) [32] are bovine sequences, not human sources. Antimicrobial peptides play an important role in membrane destroying, alternation, or permeation, and some of them may have antibiotic activity [33]. Alternatively, other membrane interacting peptides that do not compromise membrane integrity are very important in modulating the structure and dynamics of the lipid bilayer, and thereby cell membrane function. It has long been appreciated that antimicrobial peptides and CPPs possess similar functional characteristics [33,34]. Thus, we suspected that bovine LFcin derived peptides with antimicrobial activity in prokaryotes could act as CPPs in eukaryotic cells. In the present study, a novel penta-peptide (L5a) from bovine LFcin was examined. This nontoxic L5a peptide was found to noncovalently deliver DNA into human cells. Materials and Methods Cell culture Human bronchoalveolar carcinoma A549 cells (American Type Culture Collection, Manassas, VA, USA; CCL-185) were cultured in Roswell Park Memorial Institute (RPMI).