Cellular Internalization of Human Calcitonin Derived Peptides in MDCK Monolayers: A Comparative Study with Tat(47-57) and Penetratin(43-58)

PURPOSE: The objective of this study was to evaluate key motif requirements of human calcitonin (hCT)-derived peptides for the permeation through the plasma membrane of MDCK monolayers, as epithelial model. METHODS: Truncated and sequence-modified fluorescent-labeled hCT-derived peptides were synthesized through Fmoc chemistry. Peptide uptake by confluent MDCK was observed by confocal laser scanning microscopy. The cytotoxic effect of the peptides on cellular integrity was followed by LDH release. For direct comparison we covered the cellular uptake of established cell penetrating peptides, Tat(47-57) and penetratin(43-58). RESULTS: Truncated sequences of hCT, from hCT(9-32) to hCT(18-32), penetrated the plasma membrane and demonstrated a sectoral, punctuated cytoplasmic distribution. The uptake process appeared to be temperature-, time- and concentration-dependent. Amino acid modifications of hCT(18-32) indicated that both the proline in position 23 and the positive charge of lysine in position 18 are crucial for peptide uptake. The reverse sequence hCT(32-18) did not penetrate the membrane, indicating the importance of sequence orientation. Tat(47-57) and penetratin(43-58) showed a similar punctuated cytoplasmic distribution in MDCK and HeLa cell lines. No relevant toxicity was observed. CONCLUSIONS: Selected hCT-derived peptides have cell penetrating properties. The uptake mechanism seems to involve an endocytic pathway.     

Peptide amphipathy: a new strategy in design of potential insecticides

A 30-residue peptide [YAA(KALA)₆LAA] with an amphipathic helix repeat unit of Lys-Ala-Leu-Ala (KALA) was synthesized as both the l - and the d -isomer. The peptide was shown to form α-helices and lyse lipid vesicles in a pH dependent fashion. The calculated helical amphipathic moment is + 1.19 kcal/ residue and the mean residue hydrophobicity is +0.4 kcal/residue. The formation of α-helices as the pH is increased is similar to poly-lysine, yielding a pK of 10.2. Though not toxic when fed to insects, KALA killed Spodoptera frugiperda cells at low doses and Manduca sexta larvae when injected.     

A modified Tat peptide for selective intracellular delivery of macromolecules

Objectives The Tat peptide has been widely used for the intracellular delivery of macromolecules. The aim of this study was to modify the peptide to enable regulation of cellular uptake through a dependency on activation by proteases present in the local environment. Methods The native Tat peptide sequence was altered to inhibit the initial interaction of the peptide with the cell membrane through the addition of the consensus sequence for urokinase plasminogen activator (uPA). uPA expression was characterised and semi‐quantitatively rated in three cell lines (U251mg, MDA‐MB‐231 and HeLa). The modified peptide was incubated with both recombinant enzyme and with cells varying in uPA activity. Cellular uptake of the modified Tat peptide line was compared with that of the native peptide and rated according to uPA activity measured in each cell line. Key findings uPA activity was observed to be high in U251mg and MDA‐MB‐231 and low in HeLa. In MDA‐MB‐231 and HeLa, uptake of the modified peptide correlated with the level of uPA expression detected (93 and 52%, respectively). In U251mg, however, the uptake of the modified peptide was much less (19% observed reduction) than the native peptide despite a high level of uPA activity detected. Conclusions Proteolytic activation represents an interesting strategy for the targeted delivery of macromolecules using peptide‐based carriers and holds significant potential for further exploitation.     

Peptides with indirect in vivo activity against an intracellular pathogen: selective lysis of infected macrophages

Abstract: A collection of natural peptides, simplified analogs of natural peptides, de novo amphipathic peptides and de novo amphipathic peptides composed of 50–80% α,α‐dialkylated glycines (α,α‐Dags) were synthesized on solid‐phase resin as the C‐terminus amides using N‐α‐fluorenylmethyloxycarbonyl protection. The synthesis of the peptides rich in α,α‐Dags used acid fluoride coupling methods. The peptides show antimicrobial activity against Escherichia coli and Staphylococcus aureus but no direct antimicrobial activity against Brucella abortus at 100 µm in vitro. However, in vivo treatment with several of these peptides results in significant reductions of B. abortus in chronically infected immune BALB/c mice relative to infected control animals. The chronically infected mice were susceptible to peptide toxicity at much lower peptide doses than control animals. The highest nonlethal dose for infected mice was only 25 µg for melittin, whereas 500 µg doses were nonlethal for many of the other peptides. Several of the α,α‐Dag‐rich peptides selectively destroy B. abortus‐infected murine macrophages in vitro. Thus, these peptides apparently reduce the bacterial load in vivo by destroying a portion of the infected macrophages and exposing the sequestered bacteria to the immune response in the mice.     

Self-assembled peptide (CADY-1) improved the clinical application of doxorubicin

CADY-1 is an amphipathic peptide that possesses cell-penetrating activity. As an amphipathic peptide, CADY-1 is capable of forming complexes by self-assembly, and they are these complexes that possess cell-penetrating activity. This distinct characteristic of CADY-1 makes it a potent cell-penetrating drug delivery system. Doxorubicin is a widely used cytotoxic anti-cancer drug but is limited by its toxicity. Although the liposomal formulation of doxorubicin ameliorates its toxicity, its complicated synthesis remains an obstacle to its wide clinical use. In this study, our findings revealed that CADY-1 and doxorubicin form a stable complex at optimised molar ratios in a self-assembling manner. Formation of the complex extended the blood residence time of doxorubicin in a similar fashion to that of liposomal doxorubicin. In addition, the complex was capable of carrying doxorubicin across the cell membrane, which increased the therapeutic index of doxorubicin. Experimental animals treated with a CADY-1/doxorubicin complex exhibited better tolerance and anti-tumour activity than animals treated with either liposomal doxorubicin or the free form of doxorubicin. Collectively, the findings in this study support the advantages of using complexes formed by the self-assembled peptide CADY-1 and suggest that CADY-1 is a potent drug delivery system.     

Quantitative assessment of the cell penetrating properties of RI-Tat-9: evidence for a cell type-specific barrier at the plasma membrane of epithelial cells

Penetration of epithelial cells represents the rate-determining step for the absorption of many drugs and pharmaceutical macromolecules such as proteins and nucleic acid therapeutics. While the potential of using cell-penetrating peptides (CPPs) to facilitate absorption has been increasingly recognized, the mechanism of cell penetration and the uptake into certain cells have recently been called into question due to methodological artifacts. Therefore, the objective of this study was to quantitatively assess the ability of RI-Tat-9, a proteolytically stable CPP, to penetrate epithelial cell monolayers. The permeability of RI-Tat-9 with two epithelial cell lines, Madin-Darby canine kidney (MDCK) and Caco-2 cells, was comparable to the leakiness of the respective intact monolayers. Microscopic imaging showed that fluorescence-tagged RI-Tat-9 did not enter these cells, further supporting a paracellular transport mechanism. Although insufficient data were generated in these studies to generalize the observed phenomenon, the entry of RI-Tat-9 into nonepithelial T lymphocytic MT2 cells, possibly by endocytosis, suggested that a cell type-specific barrier might exist that controlled uptake of RI-Tat-9 by cells. Compared to that in MT2 and HeLa cells, the active uptake of the peptide into MDCK monolayers was much slower and showed no dependence of cell energy. Furthermore, the equilibrium binding of RI-Tat-9 to MDCK cells at 0 degrees C was indicative of an interaction with a nonspecific receptor. A correlation between binding density and concentration difference across a leaky separation barrier suggested that repulsion of free peptide molecules by bound peptide molecules at the MDCK monolayer surface may be significant at micromolar concentrations. The results of this study quantitatively show that Tat CPP uptake into two commonly used epithelial cell types is minimal and possibly cell type-specific. Implications for Tat CPP-assisted drug delivery are discussed.     

Modification of the C16Y peptide on nanoparticles is an effective approach to target endothelial and cancer cells via the integrin receptor

Liposomes have been explored as potential drug and gene-delivery particles. In recent years, tumor-targeted liposomes have been developed to improve the efficacy of antitumor treatment. The C16Y peptide is a modified C16 peptide, which is derived from the laminin γ1 chain, and binds to integrins α(v)β3 and α5β1 on endothelial cells. In this study, we prepared integrin-targeted C16Y peptide-modified liposomes (C16Y-L) to enhance the intracellular uptake of drugs and genes specifically into tumor tissues. The selectivity of C16Y-L for endothelial cells and cancer cells, which strongly express integrins α(v)β3 and α5β1, was assessed by flow cytometry and confocal microscopy. The cellular uptake of C16Y-L by both cell types was higher than uptake of the un-labeled and scramble peptide-modified liposomes. Next, to ascertain the involvement of receptor-mediated endocytosis in the process, these cells were treated with C16Y-L for 1h at 37°C or at 4°C. We found that uptake was also dependent on the temperature. Moreover, to evaluate whether the uptake depended on an integrin-ligand interaction, we examined the inhibition of C16Y-L uptake using recombinant integrin αVβ3 and found that the cellular uptake of C16Y-L treated with αVβ3 integrin decreased. This result suggests that C16Y-L can selectively target cells that highly express integrin αVβ3. Thus, the modification of the C16Y peptide on a Drug Delivery System (DDS) carrier may be a feasible approach for drug or gene delivery into tumors.     

Reduction of doxorubicin resistance in P-glycoprotein overexpressing cells by hybrid cell-penetrating and drug-binding peptide

Drug efflux by the membrane transporter P-glycoprotein (P-gp) plays a key role in multidrug resistance (MDR). In order to bypass P-gp, thus overcoming MDR, a hybrid peptide comprising a cell penetrating peptide (Tat) and a drug binding motif (DBM) has been developed to noncovalently bind and deliver doxorubicin (Dox) into MDR cells. The uptake of Dox into the leukemia cell line K562 and its P-gp overexpressing subline KD30 increased in the presence of DBM-Tat peptide. Confocal microscopy indicated that DBM-Tat associated Dox was directed to a perinuclear area of KD30 cells, while this was not observed in parent K562 cells. When KD30 cells were pretreated with the endosomotropic agent chloroquine (CLQ), peptide associated Dox redistributed into the cytosol, indicating that endocytosis was the predominant uptake route. Altered drug uptake kinetics observed by cellular accumulation assay also supported an endocytic uptake. In the presence of CLQ, DBM-Tat was able to enhance the cytotoxicity of Dox by 68.4% at 5?µM peptide concentration in KD30 cells but there were only minor effects on Dox cytotoxicity in K562 cells even in the presence of CLQ. Thus, combining Dox with DBM-Tat reduces P-gp mediated drug efflux, without a requirement for drug modification or inhibiting P-gp function.     

Stabilization of amphipathic α-helical and β-helical conformations in synthetic peptides in the presence and absence of ionic interactions

The role of ionic interactions in stabilizing amphipathic α-helices was studied in the synthetic peptide Ac-NLEELKKKLEELKG-NH₂ (NLEKG14), potentially stabilized by attraction between complementary ions in successive turns of the helix, and in the peptide Ac-NLEELEEELEELEG-NH₂ (NLEG14), in which no side-chain ionic attractions are possible. At a pH below the pK_a of glutamate, NLEG14 had a higher helix content than NLEKG14. At pH 3 to pH 10, the helicity of NLEKG14 did not change, whereas NLEG14 was converted to random coil at pH 7. The role of ionic interactions in stabilizing the conformation of β-structures was studied in the synthetic peptides Ac-KLKLKLELELELG-NH₂ (KLEG13) and Ac-ELELELELELELG-NH₂ (ELG13). At a pH below the pK_a of glutamate, ELG13 had a higher β-content than KLEG13, as judged by their dichroic spectra, but at higher pH, ELG13 was converted to random coil, whereas KLEG13 retained a predominantly β-conformation. At pH 7, high NaCl concentration produced a significant increase in the α-helix content of NLEKG14, converted NLEG14 from random coil to α-helix and converted ELG13 from random coil to β-conformation. Overall, the results demonstrate that ionic attraction between side-chains plays a lesser role than hydrogen bonding and hydrophobic effects in stabilizing the α- and β-conformations exhibited by these amphipathic peptides.     

Synthesis and evaluation of tripodal peptide analogues for cellular delivery of phosphopeptides

Tripodal peptide analogues were designed on the basis of the phosphotyrosine binding pocket of the Src SH2 domain and assayed for their ability to bind to fluorescein-labeled phosphopeptides. Fluorescence polarization assays showed that a number of amphipathic linear peptide analogues (LPAs), such as LPA4, bind to fluorescein-labeled GpYEEI (F-GpYEEI). LPA4 was evaluated for potential application in cellular delivery of phosphopeptides. Fluorescence microimaging cellular uptake studies with fluorescein-attached LPA4 (F-LPA4) alone or with the mixture of LPA4 and F-GpYEEI in BT-20 cells showed dramatic increase of the fluorescence intensity in cytosol of cells, indicating that LPA4 can function as a delivery tool of F-GpYEEI across the cell membrane. Fluorescent flow cytometry studies showed the cellular uptake of F-LPA4 in an energy-independent pathway and confirmed the cellular uptake of F-GpYEEI in the presence of LPA4. These studies suggest that amphipathic tripodal peptide analogues, such as LPA4, can be used for cellular delivery of phosphopeptides.     

Design of a new pH‐activatable cell‐penetrating peptide for drug delivery into tumor cells

Cell‐penetrating peptides (CPPs) have been considered as potential drug delivery vectors due to their remarkable membrane translocation capacity. However, lack of specificity and extreme systemic toxicity hamper their successful application for drug delivery. Here, we designed a new pH‐activatable CPP, LHHLLHHLHHLLHH‐NH₂ (LH), by substitution of all lysines and two leucines of LKKLLKLLKKLLKL‐NH₂ (LK) with histidines. As expected, histidine‐rich LH could be activated and penetrate into cells at pH 6.0, whereas its membrane transduction activity could be shielded at pH 7.4. In contrast, LK showed no obviously different cellular uptake at both pH conditions. Importantly, LH was significantly less cytotoxicity compared with LK at both pH values, suggesting a better safety for further application. In addition, after conjugation of camptothecin (CPT) with LH, this conjugate displayed remarkably pH‐dependent antitumor activity than free CPT and LK‐CPT. This study provides a new tumor pH‐responsive CPP with low toxicity for selective anticancer drug delivery.     

The fd phage and a peptide derived from its p8 coat protein interact with the HIV-1 Tat-NLS and inhibit its biological functions

Filamentous fd bacteriophages are used to construct phage-display peptide libraries, which have been instrumental in selecting peptides that interact with specific domains within target molecules. Here we demonstrate that the fd bacteriophage itself, as well as NTP8 - a synthetic peptide derived from it and bearing amino acids 1-20 of the phage p8 protein - interact with the nuclear localization signal (NLS) of the HIV-1 Tat protein. Accordingly, fd bacteriophage and the NTP8 peptide inhibit binding mediated by the Tat-NLS to the nuclear-import receptor importin beta and Tat-NLS-mediated translocation into cell nuclei. The NTP8 peptide, at 100 microM concentration, also caused about 50% inhibition of HIV-1 propagation in cultured cells. The fd bacteriophage prevents heparan sulfate proteoglycans-mediated uptake of extracellular Tat by target cells and consequently transactivation of a chloramphenicol acetyltransferase (CAT) reporter gene. A BSA-NTP8 conjugate inhibits Tat-NLS-mediated binding to heparin immobilized on a BIAcore surface. BLAST analysis of the NTP8 amino-acid sequence revealed similarity to sequences in several human proteins, including ADA2 and CD53.     

Evaluation of the transfection property of a peptide ligand for the fibroblast growth factor receptor as part of PEGylated polyethylenimine polyplex

PURPOSE: Experiments were conducted to evaluate the utility of a peptide receptor ligand to improve transfection efficiency as part of a polyethylenimine-polyethylene glycol (PEI-PEG) polyplex. The 7-mer peptide (MQLPLAT), targeted toward the fibroblast growth factor 2 (FGF2) receptor, was recently identified using a phage-display library method as possessing a high degree of specificity for the FGF2 receptor without the mutagenicity associated with FGF itself. Two approaches (pre-modification or post-modification) to incorporate the peptide into the PEGylated polyplex were compared in terms of their effect on particle size, surface charge, DNA condensation ability, toxicity, cellular uptake and transfection efficiency. METHODS: The peptide was conjugated to branched PEI (25 kDa) via a PEG spacer either before (pre-modified) or after (post-modified) complexation of PEI with DNA. Polyethyleneimine was conjugated to the PEG spacer (N-hydroxy succinimide (NHS) -PEG-maleimide (Mal)) through the NHS group. The FGF2 peptide was synthesized to contain a cysteine at the carboxyl end (MQLPLATC) and conjugated to the PEG spacer via the Maleimide group. Conjugates were evaluated using (1)H NMR, amino acid analysis, and picrylsulfonic acid assay. DNA condensation was evaluated using agarose gel electrophoresis and cellular toxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cellular uptake was measured using flow cytometry and transfection efficiency was determined using a luciferase reporter gene assay. RESULTS: Both pre- and post-modification approaches led to a decrease in the zeta potential of the resulting polyplexes but did not alter their size. The pre-modification of PEI did not affect its ability to condense DNA. However, polyplexes formed with the pre-conjugated PEI did not improve cell uptake or transfection efficiency. In contrast, polyplexes that were post-modified with the FGF2 peptide resulted in a 3-fold increase in cell uptake and a 6-fold increase in transfection efficiency. Both pre- and post-modified polyplexes resulted in lower toxicity compared with unmodified PEI. CONCLUSIONS: The results indicate that the FGF2 peptide improves transfection efficiency when used as part of post-modified PEI/PEG polyplex. When used with pre-modified PEI/PEG, the beneficial effect of the peptide on transfection is not evident, probably because, in this case, the peptide ligand is not readily accessible to the FGF receptor.     

Can the Cellular Internalization of Cargo Proteins Be Enhanced by Fusing a Tat Peptide in the Center of Proteins? A Fluorescence Study

The aim of this study was to investigate whether the cellular uptake of cargo proteins can be enhanced by fusing a Tat peptide in the center of proteins; glutathione-S-transferase (GST)-Tat-green fluorescent protein (GFP) and GST-GFP-Tat proteins were first constructed and expressed. The cellular internalization of both proteins was then evaluated and compared in HeLa cells using fluorescent microscopy and flow cytometry, as well as the transdermal delivery in human skin using confocal microscopy. Results from in vitro cell experiments showed that GST-Tat-GFP protein efficiently internalized into HeLa cells when a Tat peptide was fused in the center of proteins, whereas its efficiency is lower than that of GST-GFP-Tat protein with a Tat peptide terminal fused. Ex vivo transdermal delivery data also demonstrated that the lower efficiency of GST-Tat-GFP penetrating through human stratum corneum layer when compared with GST-GFP-Tat. Furthermore, both GST-GFP-Tat and GST-Tat-GFP presented a various degree of a mixture of cytoplasmic diffuse staining and punctate surface staining, and the pattern of distribution varied considerably in HeLa cell experiments depending on the concentration of protein used. Therefore, an improved mechanism for Tat-conjugated proteins was proposed, in which Tat-conjugated proteins were first associated with cell membrane, then accumulated on the cell surface, and finally internalized into cells by pore formation mechanism.     

Influence of preformed α-helix and α-helix induction on the activity of cationic antimicrobial peptides

One prominent class of cationic antibacterial peptides comprises the α-helical class, which is unstructured in free solution but folds into an amphipathic α-helix upon insertion into the membranes of target cells. To investigate the importance of α-helicity and its induction on interaction with membranes, a series of peptides was constructed based on a hybrid of moth cecropin (amino acids 1-8) and bee melittin (amino acids 1-18) peptides. The new peptides were predicted to have a high tendency to form α-helices or to have preformed α-helices by virtue of construction of a lactam bridge between glutamate and lysine side-chains at positions i and i+ 4 at various locations along the primary sequence. In two examples where the use of lactam bridge constraints induced and stabilized α-helical structure in benign (aqueous buffer) and/or hydrophobic medium, there was a decrease in antibacterial activity relative to the linear counterparts. Thus the preformation of α-helix in solution was not necessarily beneficial to antimicrobial activity. In the one case where the lactam bridge did result in increased antibacterial activity (lower minimal inhibitory concentration values) it did not increase α-helical content in benign or hydrophobic medium. Broadly speaking, good activity of the peptides against Pseudomonas aeruginosa correlated best (r²= 0.88) with a helican parameter which was calculated as the induction of α-helix in α membrane-mimicking environment divided by the α-helix formation under benign conditions. Interestingly, the activity of the lactam bridge peptide constructs correlated in part with alterations in bacterial outer or cytoplasmic membrane permeability.     

Analysis of in vitro toxicity of five cell-penetrating peptides by metabolic profiling

Cell-penetrating peptides (CPPs) are promising candidates for safe and efficient delivery vectors for a wide range of cargoes. However, any compound that is internalized into cells may affect the cell homeostasis. The plethora of possible biological responses makes large scale “omics” studies appealing approaches for hunting any unsuspected side-effects and evaluate the toxicity of drug candidates. Here we have compared the alterations in cytosolic metabolome of CHO cells caused by five representatives of the most common CPPs: transportan (TP), penetratin (pAntp), HIV Tat derived peptide (pTat), nonaarginine (R₉) and model amphipathic peptide (MAP). Analysis was done by liquid chromatography–mass spectrometry techniques, principal component analysis and heatmap displays. Results showed that the intracellular metabolome was the most affected by TP followed by pTat and MAP. Only minor changes could be associated with pAntp or R₉ treatment. The cells could recover from a treatment with 5 μM TP, but no recovery was observed at higher concentration. Both metabolomic and control experiments showed that TP affected cellular redox potential, depleted energy and the pools of purines and pyrimidines. In conclusion, we have performed a metabolomic analysis comparing the safety of cell-penetrating peptides and demonstrate the toxicity of one of them.     

The delivery of biologically active (therapeutic) peptides and proteins into cells

Biologically active peptides and proteins have a great potential to act as targeted drug therapies in the treatment of a variety of diseases, including cancer. However, their use in vivo is limited by their low stability and cell permeability. Thus, it is necessary to develop efficient and safe peptide/protein delivery systems that can overcome these problems and increase a therapy's bioavailability. The search for promising vectors has led to the use of compounds called cell-penetrating peptides or protein transduction domains. The cell-penetrating peptides, as effective transporter, are utilized to enhance uptake of various biologically active peptide/protein cargos upon fusion or attachment to its sequences. Cell-penetrating peptides have been the subject of investigation of many researchers, however this review only focuses on the arginine-rich and amphipathic carriers and their potential therapeutic use.