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Internalization of Near-Infrared Fluorescently Labeled Activatable Cell-Penetrating Peptide and of Proteins into Human Fibrosarcoma Cell Line HT-1080
Abstract
The internalization of near-infrared fluorescently labeled cargos into living cells and tissues allows a highly sensitive detection without interference from skin, porphins or other fluorescent cell and tissue compounds. In this study, the uptake of labeled bovine serum albumin and an antibody, into fibrosarcoma (HT-1080) cells was triggered by the formation of non-covalent complexes with different cell-penetrating peptides; uptake efficiency and intracellular localization were determined. To improve selectivity of internalization into tumor cells, a fluorescent activatable cell-penetrating peptide (ACPP) was synthesized and functionally characterized. This 25-mer peptide was designed to be activatable by Matrix-Metallo-Proteases (MMPs). Its uptake selectivity was estimated using cells with different MMP activities. This article is protected by copyright. All rights reserved
... These peptides can be categorized as first generation CPPs and newer generation CPPs. In a previous study, 14 we used first and new generation CPPs to form noncovalent complexes and internalize fluorescent-labeled bovine serum albumin and the clinically approved humanized monoclonal antibody trastuzumab into human fibrosarcoma cells. We observed highly efficient uptake of fluorescent BSA into tumor and nontumor cell lines with the amphipathic peptides MPGα, MPGβ, and CAD-2 and histone. ...
... TANSI ET AL. | 6531 2.9 | Preparation of CPP-protein complexes Noncovalent CPP-protein complexes were prepared as reported earlier. 14,39,40 For near-infrared fluorescence imaging of cell pellets and semiquantitative analysis, 10 pmol Tras-DY505, and where applicable, 0.08 μg CPP-DY676 (that is 32 pmol TAT-DY676, 40 pmol mini-mau-DY676 and 32 pmol mini-crot-DY676) or 1 to 2 μg of the respective unconjugated CPP solutions (that is 640 pmol HIV-TAT(rR), 926 pmol mini-mau, 628 pmol mini-crot, 555 pmol MPGα/MPGβ, and 571 pmol CAD-2) were dissolved in 100 μL of Hank's buffered saline solution (HBSS, pH 7.4; PAA, Pasching, Austria). For microscopic analysis on chamber slides, the CPPs and tras-DY652 were diluted in 30 μL HBSS, separately. ...
... While complex formation was ongoing, the cells were prepared for internalization as reported earlier. 14 Cells were washed three times with HBSS. The preformed complex solution (60 μL) was added to the cells. ...
In the last three decades, many new cell‐penetrating peptides (CPPs) were developed that exhibited enhanced cell selectivity. Thus, we aimed to validate the tumor cell selectivity of peptides from this new generation, namely fragments mini‐crotamine and mini‐maurocalcine. Both of these peptides are derived from venoms. Furthermore, we studied an analog of the classical CPP HIV‐TAT(47‐57) with alternating chirality of Arg residues. To allow covalent coupling of cargoes or fluorophores, a cysteine residue was introduced to the N‐terminus of the synthesized peptides. The therapeutic antibody trastuzumab conjugated to different fluorescent dyes was used for internalization studies. Comparison of uptake efficiencies revealed that CPPs of the new generation are in contrast to MPG‐peptides, nearly unable to internalize the noncovalently formed complexes with trastuzumab. Interestingly, the fluorescent derivative of the crotamine fragment was mainly observed in a subpopulation of breast cancer cells, whereas it was homogenously distributed in fibrosarcoma, colon cancer, and noncancerous endothelia cells. Thus, the fluorescent crotamine fragment reported herein is a potent theranostic tool for image‐guided applications. This peptide can be used to pinpoint the level of heterogeneity present within tumors and aid in the generation of therapeutics that target heterogenic subpopulations.
... They found that the cytochrome C sequence is rich of CPP sequences [60,61]. Two peptides, denoted Cyt C [86][87][88][89][90][91][92][93][94][95][96][97][98][99][100][101] and Cyt C , derived from cytochrome C were found to decrease cell viability and also induced DNA fragmentation and caspase-3 activation, which are hallmarks of apoptosis, hence suggesting that the peptides mimic the function of the parent protein. Comprehensive list of protein mimics with CPP properties, bioportides, is presented in [62]. ...
... Activable CPPs were used for the selective delivery of drugs into tumor cells. In this way doxorubicin [52,90], antisense or pDNA [91,92], siRNA [93], and antibodies [94] were delivered. ...
In this introductory chapter, we first define cell-penetrating peptides (CPPs), give short overview of CPP history and discuss several aspects of CPP classification. Next section is devoted to the mechanism of CPP penetration into the cells, where direct and endocytic internalization of CPP is explained. Kinetics of internalization is discussed more extensively, since this topic is not discussed in other chapters of this book. At the end of this section some features of the thermodynamics of CPP interaction with the membrane is also presented. Finally, we present different cargoes that can be transferred into the cells by CPPs and briefly discuss the effect of cargo on the rate and efficiency of penetration into the cells.
... Several NIR fluorescent dyes have been developed and incorporated, for example with antibodies (86,87), nanoparticles (88) or encapsulated within nanomaterials (89,90), to be used as contrast agents for molecular imaging of different tumors (4). Researchers have identified that elevated levels of fibroblast activation protein (FAP) in stromal fibroblasts are associated with aggressive cancer types (91)(92)(93)(94)(95). FAP is a type II salivary glycoprotein with the ability to cleave biological peptides, including collagen and proteolytic enzymes, and serve a central role in the aggressiveness of the solid tumors. ...
Cancer is a leading cause of cases of mortality worldwide. The most effective method to cure solid tumors is surgery. Every year, >50% of cancer patients receive surgery to remove solid tumors. Surgery may increase the cure rate of most solid tumors by 4-11 fold. Surgery has many challenges, including identifying small lesions, locating metastases and confirming complete tumor removal. Fluorescence guidance describes a new approach to improve surgical accuracy. Near-infrared fluorescence imaging allows for real-time early diagnosis and intraoperative imaging of lesion tissue. The results of previous preclinical studies in the field of near-infrared fluorescence imaging are promising. This review provides examples introducing the three kinds of fluorescent dyes: The passive fluorescent dye indocyanine green, which has been approved by the Food and Drug Administration for clinical use in the USA, the fluorescent prodrug 5-aminolevulinic acid, a porphyrin precursor in the heme synthesis, and biomarker-targeted fluorescent dyes, which allow conjugation to different target sites.
... Transactivator of transcription (TAT) peptide, which is derived from HIV-1 TAT protein, has been extensively studied for viral gene infection. The sequence of HIV-1 TAT is 86 amino acids long and comprises of acidic, cysteine rich, core, cationic, glutamic rich and RGD region [40]. Out of the whole sequence of TAT peptide, the cationic part YGRKKRRQRRR (TAT 47-57 ) is a known CPP and has been observed to be responsible for internalization of HIV-1 TAT through the cell membranes as this region comprises of positively charged arginine groups which are capable of binding to the (negatively charged) proteoglycans present on the cell surface through interactions thus promoting the cellular uptake and therapeutic efficacy of this peptide [41]. ...
Gold nanoparticles (AuNPs) functionalized with different biomolecules find extensive application in therapy, clinical diagnosis and biomedical imaging. Herein, two derivatives of TAT peptide with sequences YGRKKRRQRRR and YGRKKRRQRRR-(β-ala)3-Cys-amide were conjugated with tannic acid capped gold nanoparticles which acted as a carrier for cell penetrating peptides (CPPs) into the bacterial cells. The interaction of YGRKKRRQRRR peptide with AuNPs was non-covalent in nature whereas YGRKKRRQRRR-(β-ala)3-Cys-amide interacted covalently with the AuNPs due to presence of thiol group in cysteine which bind strongly to gold nanoparticles surface. Further, tannic acid functionalised AuNPs conjugated CPPs constructs were duly characterized using critical flocculation essay test, UV–visible and TEM. FITC was tagged over AuNPs-CPPs in order to study the intracellular distribution using confocal microscopy. The confocal results revealed that nanoconjugates (AuNP-CPPs) of 5 nm diameter exhibited strong fluorescent signal in Gram positive and Gram negative bacterial strains. The present method can also be used for the killing of bacterial cells using photo-thermal therapy and therefore can be highly useful for targeting multi-drug resistant bacteria.
... Among the undeniable advantages of CPPs there is the possibility of adding specific localization sequences to directly transport the nanosystem into cell organelles, which is not feasible by electroporation, magnetofection, unmodified NCs, or with the use of amphipathic detergents. Development of effective sorting sequences for nucleus and cellular organelles is an extraordinary challenge for cell biologists and biochemists, and in the last years numerous localization sequences have been designed for transport into and staying in the cytosol [96], nucleus [97], mitochondria [96], lysosomes [98] and Golgi apparatus [99]. The direct transport into the nucleus has also been improved through the use of two consecutive sequences linked through a spacer [100] and by the addition of Glu residues to the nuclear localization sequence [101]. ...
Nanomedicine has opened the way to the design of more efficient diagnostics and therapeutics. Moreover, recent literature has illustrated the use of short cationic and/or amphipathic peptides, known as cell-penetrating peptides (CPPs), for mediating advanced drug delivery. CPPs exploit their ability to enter cells and enhance the uptake of many cargoes ranging from small molecules to proteins. The distinctive properties of nanocarriers (NC) based systems provide unforeseen benefits over pure drugs for biomedical applications and constitute a challenging research field particularly focused on imaging and delivery; nonetheless, several problems have to be overcome to make them a viable option in clinic. The use of CPPs improves significantly their delivery to specific intracellular targets and thus readily contributes to their use both for effective tumor therapy and gene therapy. A key issue is related to their mechanism of uptake, because although classical CPPs enhance NCs’ uptake, the entry mechanism involves the endocytic pathway, which means that the delivered material is sequestered within vesicles and only a small amount will escape from this environment and reach the desired target. In this review, we will summarize recent advances in the use of CPP for enhanced delivery of nanocarriers, nucleic acids, and drugs, we will discuss their uptake mechanisms and we will describe novel approaches to improve endosomal escape of internalized nanosystems.
... Among the undeniable advantages of CPPs there is the possibility of adding specific localization sequences to directly transport the nanosystem into cell organelles, which is not feasible by electroporation, magnetofection, unmodified NCs, or with the use of amphipathic detergents. Development of effective sorting sequences for nucleus and cellular organelles is an extraordinary challenge for cell biologists and biochemists, and in the last years numerous localization sequences have been designed for transport into and staying in the cytosol [96], nucleus [97], mitochondria [96], lysosomes [98] and Golgi apparatus [99]. The direct transport into the nucleus has also been improved through the use of two consecutive sequences linked through a spacer [100] and by the addition of Glu residues to the nuclear localization sequence [101]. ...
Nanomedicine has opened the way to the design of more efficient diagnostics and therapeutics. Moreover,recent literature has illustratedthe use of short cationic and/or amphipathic peptides, known as cell-penetrating peptides (CPPs), for mediating advanceddrug delivery.CPPs exploit their ability to enter cells to enhance the uptake of many cargoes ranging from small molecules to proteins. The distinctiveproperties of NC based systems provide unforeseen benefitsover pure drugsfor biomedical applicationsand constitute a challenging research field particularly focused on imaging and delivery; nonetheless, several problems have tobe overcome to make them a viable option in clinic. The use of CPPs improves significantly their delivery to specific intracellular targets and thus readily contributes to their use bothfor effective tumor therapy and gene therapy.A key issue is related to their mechanism of uptake, because although classical CPPs enhance NCs' uptake, the entry mechanism involves the endocytic pathway, which means that the delivered material is sequestered within vesicles and only a small amountwill escape from this environment and reach the desired target. In this review, we will summarize recent advances in the use of CPP for enhanced delivery of nanocarriers, nucleic acids, and drugs, wewill discuss their uptake mechanisms and we will describe novel approaches to improve endosomal escape of internalized nanosystems.
... For in vivo purposes, suitable fluorescent probes have their absorption and emission properties within the near-infrared (NIR) window [17], where tissue auto-fluorescence based on water and hemoglobin are minimal. With respect to this observation, several nearinfrared fluorescent dyes have been developed and conjugated for example to antibodies [18,19] and nanoparticles [20] or encapsulated within lipidic nanomaterials [21,22] to serve as contrast agents for molecular imaging of different neoplasms. Molecular targets such as the fibroblast activation protein (FAP) are interesting targets for molecular imaging. ...
Despite intensive research and medical advances met, metastatic disease remains the most common cause of death in cancer patients. This results from late diagnosis, poor therapeutic response and undetected micrometastases and tumor margins during surgery. One approach to overcome these challenges involves fluorescence imaging, which exploits the properties of fluorescent probes for diagnostic detection of molecular structures at the onset of transformation and for intraoperative detection of metastases and tumor margins in real time. Considering these benefits, many contrast agents suitable for fluorescence imaging have been reported. However, most reports only demonstrate the detection of primary tumors and not the detection of metastases or their application in models of image-guided surgery.
... However, most of researchers have proposed endocytosis pathway and energy-dependent pathway for these peptide sequences. Indeed, interaction between the negativelycharged plasma membrane and these cationic carriers (CPPs) facilities their cellular internalization along Protein delivery (neuropilin-1 delivery) 100 Imaging probes (fluorescently labeled bovine serum albumin, protease-activated peptides) 101,102 CCP, cell-penetrating peptide BioImpacts, 2015, 5(2), 103-111 108 with cargo. Regardless of several in vitro researches on CPPs, their in vivo applications are still restrained due to the nonspecificity of these peptides and their accumulation both in targeted and non-targeted cells. ...
The impermeability of biological membranes is a major obstacle in drug delivery; however, some peptides have transition capabilities of biomembranes. In recent decades, cell-penetrating peptides (CPPs) have been introduced as novel biocarriers that are able to translocate into the cells. CPPs are biologically potent tools for non-invasive cellular internalization of cargo molecules. Nevertheless, the non-specificity of these peptides presents a restriction for targeting drug delivery; therefore, a peptidic nanocarrier sensitive to matrix metalloproteinase (MMP) has been prepared, called activatable cell-penetrating peptide (ACPP). In addition to the cell-penetrating peptide dendrimer (DCPP), other analogues of CPPs have been synthesized.
In this study, the most recent literature in the field of biomedical application of CPPs and their analogues, ACPP and DCCP, were reviewed.
This review focuses on CPP and its analogues, ACPP and DCPP, as novel nanocarriers for drug delivery. In addition, nanoconjugates and bioconjugates of these peptide sequences are discussed.
DCCP, branched CPPs, compared to linear peptides have advantages such as resistance to rapid biodegradation, high loading capacities and large-scale production capability.
... This peptide is capable of transporting gold nanoparticles and fluorescent cargos into lysosomes. Using costaining with a specific anti-Golgi antibody, we detected a CPP-internalized fluorescent cargo protein at the Golgi apparatus [197]. This finding indicates that possibly more sorting capabilities may yet be discovered. ...
The penetration of polar or badly soluble compounds through a cell membrane into live cells requires mechanical support or chemical helpers. Cell-penetrating peptides (CPPs) are very promising chemical helpers. Because of their low cytotoxicity and final degradation to amino acids, they are particularly favored in in vivo studies and for clinical applications. Clearly, the future of CPP research is bright; however, the required optimization studies for each drug require considerable individualized attention. Thus, CPPs are not the philosopher's stone. As of today, a large number of such transporter peptides with very different sequences have been identified. These have different uptake mechanisms and can transport different cargos. Intracellular concentrations of cargos can reach a low micromole range and are able to influence intracellular reactions. Internalized ribonucleic acids such as small interfering RNA (siRNA) and mimics of RNA such as peptide nucleic acids, morpholino nucleic acids, and triesters of oligonucleotides can influence transcription and translation. Despite the highly efficient internalization of antibodies, enzymes, and other protein factors, as well as siRNA and RNA mimics, the uptake and stabile insertion of DNA into the genome of the host cells remain substantially challenging.
Here, the brief summary of therapeutic developments and current situation with applications of CPPs is presented, hopefully leading to multiple CPP based drugs in near future.
Possibilities for classification of CPPs based on their type of origin, biochemical properties, and physico-chemical properties are presented together with examples. Eight different and arbitrary levels for classification are presented, due to their partly overlapping properties.
Extensive research has been undertaken in the pursuit of anticancer therapeutics. Many anticancer drugs require specificity of delivery to cancer cells, whilst sparing healthy tissue. Cell-penetrating peptides (CPPs), now well established as facilitators of intracellular delivery, have in recent years advanced to incorporate target specificity and thus possess great potential for the targeted delivery of anticancer cargoes. Though none have yet been approved for clinical use, this novel technology has already entered clinical trials. In this review we present CPPs, discuss their classification, mechanisms of cargo internalization and highlight strategies for conjugation to anticancer moieties including their incorporation into therapeutic proteins. As the mainstay of this review, strategies to build specificity into tumor targeting CPP constructs through exploitation of the tumor microenvironment and the use of tumor homing peptides are discussed, whilst acknowledging the extensive contribution made by CPP constructs to target specific protein-protein interactions integral to intracellular signaling pathways associated with tumor cell survival and progression. Finally, antibody/antigen CPP conjugates and their potential roles in cancer immunotherapy and diagnostics are considered. In summary, this review aims to harness the potential of CPP-aided drug delivery for future cancer therapies and diagnostics whilst highlighting some of the most recent achievements in selective delivery of anticancer drugs, including cytostatic drugs, to a range of tumor cells both in vitro and in vivo.
Cell‐penetrating peptides (CPPs) can transport various cargoes through membranes of live cells. Since the first generations of CPPs suffered from insufficient cell and tissue selectivity, stability against proteases, and escape from endosomes, a new generation of peptides, with optimized properties, was developed. These are either derived from natural sources or created through the combination of multivalent structures. The second method allows achieving high internalization efficiency, high cell and tissue selectivity, and release from endosomes via hybrid structures, combining sequences for endosomal release, homing sequences, and sequences for activation at the target tissue and for local delivery of cargoes. CPPs with innate tumor selectivity include azurin, crotamine, maurocalcine, lycosin‐I, buffalo cathelicidin, and peptide CB5005. Some of them can penetrate the membranes of live cells and influence intracellular signaling pathways, thereby exerting cytotoxic effects against tumor cells. To obtain multilayer penetration and stabilization against proteolytic degradation, as well as for better handling, CPPs are often conjugated to nanoparticles. A special problem for tumor treatment is the efficiency of drug transport through three‐dimensional cell cultures. Therefore, the capability of CPPs to deliver the drug even to the innermost tissues is of crucial importance. Notably, the ability of certain CPPs to penetrate barriers such as skin, the blood‐brain barrier (BBB), and cornea or conjunctiva of eyes enabled the replacement of dangerous and painful injections with soothing sprays, creams, and drops. However, it is difficult to rank the efficacy of CPPs because transport efficiency and tissue selectivity depend not only on the CPP itself but also on the target tissue or organ, as well as on the cargo and method of CPP‐cargo coupling. Therefore, the present review describes some examples of new‐generation CPPs and aims to provide advice on how to find or create the right CPP for a given task.
It is complicated to classify the cell-penetrating peptides (CPPs) due to the definition obstacles. Considering the definition above, I present here some possibilities for classification of CPPs based on their type of origin, biochemical properties, and physico-chemical properties. Such classification has been the subject in several recent reviews resulting in partly overlapping classes of CPPs.
Cell penetrating peptides as well as their applications have been extensively studied and multiple exciting reports about the topics emerge daily; this book hopefully is the proof for this statement. However, CPPs are not yet on the list of drugs for a patient therapy today and we can talk about the therapeutic potential of CPPs only. I believe that the CPP based drugs will be available in near future, however, several hurdles should be overcome before that. Below, the brief summary of therapeutic developments and current situation with applications of CPPs will be presented.
Modulating signaling pathways for research and therapy requires either suppression or expression of selected genes or internalization of proteins such as enzymes, antibodies, nucleotide binding proteins or substrates including nucleoside phosphates and enzyme inhibitors. Peptides, proteins and nucleotides are transported by fusing or conjugating them to cell penetrating peptides or by formation of non-covalent complexes. The latter is often preferred because of easy handling, uptake efficiency and auto-release of cargo into the live cell. In our studies complexes are formed with labeled or readily detectable cargoes for qualitative and quantitative estimation of their internalization. Properties and behavior of adhesion and suspension vertebrate cells as well as the protozoa Leishmania tarentolae are investigated with respect to proteolytic activity, uptake efficiency, intracellular localization and cytotoxicity. Our results show that peptide stability to membrane-bound, secreted or intracellular proteases varies between different CPPs and that the suitability of individual CPPs for a particular cargo in complex formation by non-covalent interactions requires detailed studies. Cells vary in their sensitivity to increasing concentrations of CPPs. Thus, most cells can be efficiently transduced with peptides, proteins and nucleotides with intracellular concentrations in the low micromole range. For each cargo, cell type and CPP the optimal conditions must be determined separately.
Notch signaling has a pivotal role in numerous cell-fate decisions, and its aberrant activity leads to developmental disorders and cancer. To identify molecules that influence Notch signaling, we screened nearly 17,000 compounds using automated microscopy to monitor the trafficking and processing of a ligand-independent Notch-enhanced GFP (eGFP) reporter. Characterization of hits in vitro by biochemical and cellular assays and in vivo using zebrafish led to five validated compounds, four of which induced accumulation of the reporter at the plasma membrane by inhibiting γ-secretase. One compound, the dihydropyridine FLI-06, disrupted the Golgi apparatus in a manner distinct from that of brefeldin A and golgicide A. FLI-06 inhibited general secretion at a step before exit from the endoplasmic reticulum (ER), which was accompanied by a tubule-to-sheet morphological transition of the ER, rendering FLI-06 the first small molecule acting at such an early stage in secretory traffic. These data highlight the power of phenotypic screening to enable investigations of central cellular signaling pathways.
Management of metastatic disease is integral to cancer treatment. Evaluation of metastases often requires surgical removal of all anatomically susceptible lymph nodes for ex vivo pathological examination. We report a family of novel ratiometric activatable cell penetrating peptides (RACPP), which contain Cy5 as far red fluorescent donor and Cy7 as near-infrared fluorescent acceptor. Cy5 is quenched in favor of Cy7 re-emission until the intervening linker is cut by tumor-associated matrix metalloproteinases-2 & 9 (MMP2,9) or elastases. Such cleavage increases the Cy5:Cy7 emission ratio 40 fold and triggers tissue retention of the Cy5-containing fragment. This ratiometric increase provides an accelerated and quantifiable metric to identify primary tumors and metastases to liver and lymph nodes with increased sensitivity and specificity. This technique represents a significant advance over existing nonratiometric protease sensors and sentinel lymph node detection methods, which give no information regarding cancer invasion.
In real time: Thrombin activation in vivo can be imaged in real time with ratiometric activatable cell penetrating peptides (RACPPs). RACPPs are designed to combine 1) dual-emission ratioing, 2) far red to infrared wavelengths for in vivo mammalian imaging, and 3) cleavage-dependent spatial localization. The most advanced RACPP uses norleucine (Nle)-TPRSFL as a linker that increases sensitivity to thrombin by about 90-fold.
Human matrix metalloproteinases (MMPs) belong to the M10 family of the MA clan of endopeptidases. They are ubiquitarian enzymes, structurally characterized by an active site where a Zn(2+) atom, coordinated by three histidines, plays the catalytic role, assisted by a glutamic acid as a general base. Various MMPs display different domain composition, which is very important for macromolecular substrates recognition. Substrate specificity is very different among MMPs, being often associated to their cellular compartmentalization and/or cellular type where they are expressed. An extensive review of the different MMPs structural and functional features is integrated with their pathological role in several types of diseases, spanning from cancer to cardiovascular diseases and to neurodegeneration. It emerges a very complex and crucial role played by these enzymes in many physiological and pathological processes.
To assess the suitability of asymmetric cyanine dyes for in vivo fluoro-optical molecular imaging, a comprehensive study on the influence of the number of negatively charged sulfonate groups governing the hydrophilicity of the DY-67x family of asymmetric cyanines was performed. Special attention was devoted to the plasma protein binding capacity and related pharmacokinetic properties. Four members of the DY-67x cyanine family composed of the same main chromophore, but substituted with a sequentially increasing number of sulfonate groups (n = 1-4; DY-675, DY-676, DY-677, DY-678, respectively), were incubated with plasma proteins dissolved in phosphate-buffered saline. Protein binding was assessed by absorption spectroscopy, gel electrophoresis, ultrafiltration, and dialysis. Distribution of dye in organs was studied by intraveneous injection of 62 nmol dye/kg body weight into mice (n = 12; up to 180 minutes postinjection) using whole-body near-infrared fluorescence imaging. Spectroscopic studies, gel electrophoresis, and dialysis demonstrated reduced protein binding with increasing number of sulfonate groups. The bovine serum albumin binding constant of the most hydrophobic dye, DY-675, is 18 times higher than that of the most hydrophilic fluorophore, DY-678. In vivo biodistribution analysis underlined a considerable influence of dye hydrophilicity on biodistribution and excretion pathways, with the more hydrophobic dyes, DY-675 and DY-676, accumulating in the liver, followed by strong fluorescence signals in bile and gut owing to accumulation in feces and comparatively hydrophilic DY-678-COOH accumulating in the bladder. Our results demonstrate the possibility of selectively controlling dye-protein interactions and, thus, biodistribution and excretion pathways via proper choice of the fluorophore's substitution pattern. This underlines the importance of structure-property relationships for fluorescent labels. Moreover, our data could provide the basis for the rationalization of future contrast agent developments.
Activatable cell-penetrating peptides (ACPPs) are a new class of promising molecular imaging probes for the visualization of enzymes in vivo. The cell-penetrating function of a polycationic peptide is efficiently blocked by intramolecular electrostatic interactions with a polyanionic peptide. Proteolysis of a cleavable linker present between the polycationic cell-penetrating peptide and polyanionic peptide affords dissociation of both domains and enables the activated cell-penetrating peptide to enter cells. Here, we aimed to develop an ACPP sensitive to matrix metalloproteinase-2 and -9 (MMP-2/9) for nuclear imaging purposes.
MMP-2/9 ACPPs and nonactivatable cell-penetrating peptides (non-ACPP) were prepared by 9-fluorenylmethyloxycarbonyl solid-phase peptide synthesis and labeled with (177)Lu or (177)Lu/(125)I for dual-isotope studies. The in vivo biodistribution of these probes was assessed in MMP-2/9-positive tumor-bearing mice (n = 6) and healthy mice (n = 4) using γ-counting. Furthermore, a radiolabeled cell-penetrating peptide serving as a positive control was evaluated in tumor-bearing mice (n = 6).
Biodistribution studies showed a 5-fold-higher retention of ACPP in tumor than in muscle (P < 0.01) and a 6-fold-higher tumor retention relative to non-ACPP (P < 0.01), supporting earlier studies on fluorescently labeled ACPPs proposing activation by tumor-associated MMP-2/9. Surprisingly, however, the uptake of ACPP was significantly higher than that of non-ACPP in almost all tissues (P < 0.01). To unravel the activation process of ACPP in vivo, we developed dual-isotope ACPP analogs (dACPPs) that allowed us to discriminate between uncleaved dACPP and activated dACPP. In vivo biodistribution of dACPP indicated that the tissue-associated counts originated from activated dACPP. Interestingly, dACPP administration to healthy mice, compared with MMP-2/9-positive tumor-bearing mice, resulted in a similar dACPP biodistribution. Furthermore, a radiolabeled cell-penetrating peptide showed tumor-to-tissue ratios equal to those found for ACPP (P > 0.05).
This study demonstrates that the tumor targeting of radiolabeled MMP-2/9 ACPPs is most likely caused by the activation in the vascular compartment rather than tumor-specific activation, as suggested earlier. The results in the present paper indicate that different and more tissue-specific enzyme-ACPP combinations are needed to unleash the full potential of the elegant ACPP concept in living animals.
Abstract Here we review a novel class of delivery vehicles based on pH-sensitive, moderately polar membrane peptides, which we call pH (Low) Insertion Peptides (pHLIPs), that target cells located in the acidic environment found in many diseased tissues, including tumours. Acidity targeting by pHLIPs is achieved as a result of helix formation and transmembrane insertion. In contrast to the earlier technologies based on cell-penetrating peptides, pHLIPs act as monomeric membrane-inserting peptides that translocate one terminus across a membrane into the cytoplasm, while the other terminus remains in the extracellular space, locating the peptide in the membrane lipid bilayer. Therefore pHLIP has a dual delivery capability: it can tether cargo molecules or nanoparticles to the surfaces of cells in diseased tissues and/or it can move a cell-impermeable cargo molecule across the membrane into the cytoplasm. The source of energy for moving polar molecules attached to pHLIP through the hydrophobic layer of a membrane bilayer is the membrane-associated folding of the polypeptide. A drop in pH leads to the protonation of negatively charged residues (Asp or Glu), which enhances peptide hydrophobicity, increasing the affinity of the peptide for the lipid bilayer and triggering peptide folding and subsequent membrane insertion. The process is accompanied by the release of energy that can be utilized to move cell-impermeable cargo across a membrane. That the mechanism is now understood, and that targeting of tumours in mice has been shown, suggest a number of future applications of the pHLIP technology in the diagnosis and treatment of disease.
What are the molecular events that occur when a peptide inserts across a membrane or exits from it? Using the pH-triggered insertion of the pH low insertion peptide to enable kinetic analysis, we show that insertion occurs in several steps, with rapid (0.1 sec) interfacial helix formation, followed by a much slower (100 sec) insertion pathway to give a transmembrane helix. The reverse process of unfolding and peptide exit from the bilayer core, which can be induced by a rapid rise of the pH from acidic to basic, proceeds approximately 400 times faster than folding/insertion and through different intermediate states. In the exit pathway, the helix-coil transition is initiated while the polypeptide is still inside the membrane. The peptide starts to exit when about 30% of the helix is unfolded, and continues a rapid exit as it unfolds inside the membrane. These insights may guide understanding of membrane protein folding/unfolding and the design of medically useful peptides for imaging and drug delivery.
The completeness of tumor removal during surgery is dependent on the surgeon's ability to differentiate tumor from normal tissue using subjective criteria that are not easily quantifiable. A way to objectively assess tumor margins during surgery in patients would be of great value. We have developed a method to visualize tumors during surgery using activatable cell-penetrating peptides (ACPPs), in which the fluorescently labeled, polycationic cell-penetrating peptide (CPP) is coupled via a cleavable linker to a neutralizing peptide. Upon exposure to proteases characteristic of tumor tissue, the linker is cleaved, dissociating the inhibitory peptide and allowing the CPP to bind to and enter tumor cells. In mice, xenografts stably transfected with green fluorescent protein show colocalization with the Cy5-labeled ACPPs. In the same mouse models, Cy5-labeled free ACPPs and ACPPs conjugated to dendrimers (ACPPDs) delineate the margin between tumor and adjacent tissue, resulting in improved precision of tumor resection. Surgery guided by ACPPD resulted in fewer residual cancer cells left in the animal after surgery as measured by Alu PCR. A single injection of ACPPD dually labeled with Cy5 and gadolinium chelates enabled preoperative whole-body tumor detection by MRI, intraoperative guidance by real-time fluorescence, intraoperative histological analysis of margin status by fluorescence, and postoperative MRI tumor quantification. Animals whose tumors were resected with ACPPD guidance had better long-term tumor-free survival and overall survival than animals whose tumors were resected with traditional bright-field illumination only.
High-resolution imaging of molecules intrinsically involved in malignancy and metastasis would be of great value for clinical detection and staging of tumors. We now report in vivo visualization of matrix metalloproteinase activities by MRI and fluorescence of dendrimeric nanoparticles coated with activatable cell penetrating peptides (ACPPs), labeled with Cy5, gadolinium, or both. Uptake of such nanoparticles in tumors is 4- to 15-fold higher than for unconjugated ACPPs. With fluorescent molecules, we are able to detect residual tumor and metastases as small as 200 microm, which can be resected under fluorescence guidance and analyzed histopathologically with fluorescence microscopy. We show that uptake via this mechanism is comparable to that of other near infrared protease sensors, with the added advantage that the approach is translatable to MRI. Once activated, the Gd-labeled nanoparticles deposit high levels (30-50 microM) of Gd in tumor parenchyma with even higher amounts deposited in regions of infiltrative tumor, resulting in useful T(1) contrast lasting several days after injection. These results should improve MRI-guided clinical staging, presurgical planning, and intraoperative fluorescence-guided surgery. The approach may be generalizable to deliver radiation-sensitizing and chemotherapeutic agents.
Activatable cell penetrating peptides (ACPPs) are novel in vivo targeting agents comprised of a polycationic cell penetrating peptide (CPP) connected via a cleavable linker to a neutralizing polyanion (). Adsorption and uptake into cells are inhibited until the linker is proteolyzed. An ACPP cleavable by matrix metalloproteinase-2 (MMP-2) in vitro was the first one demonstrated to work in a tumor model in vivo, but only HT-1080 xenografts and resected human squamous cell carcinomas were tested. Generality to other cancer types, in vivo selectivity of ACPPs for MMPs, and spatial resolution require further characterization. We now show that ACPPs can target many xenograft tumor models from different cancer sites, as well as a thoroughly studied transgenic model of spontaneous breast cancer (mouse mammary tumor virus promoter driving polyoma middle T antigen, MMTV-PyMT). Pharmacological inhibitors and genetic knockouts indicate that current ACPPs are selective for MMP-2 and MMP-9 in the above in vivo models. In accord with the known local distribution of MMP activity, accumulation is strongest at the tumor-stromal interface in primary tumors and associated metastases, indicating better spatial resolution (<50 mum) than other currently available MMP-cleavable probes. We also find that background uptake of ACPPs into normal tissues such as cartilage can be decreased by appending inert macromolecules of 30-50 KDa to the polyanionic inhibitory domain. Our results validate an approach that should generally deliver imaging agents and chemotherapeutics to sites of invasion, tumor-promoting inflammation, and metastasis.
Cell penetrating peptides (CPPs) have been developed as vehicles for payload delivery into cells in culture and in animals. However several biologic features limit their usefulness in living animals. Activatable cell penetrating peptides (ACPPs) are polycationic CPPs whose adsorption and cellular uptake are minimized by a covalently attached polyanionic inhibitory domain. Cleavage of the linker connecting the polyanionic and polycationic domains by specific proteases (tumor associated matrix metalloproteases discussed herein) dissociates the polyanion and enables the cleaved ACPP to enter cells. In contrast to their CPP counterpart, ACPPs are relatively nonadherent and distributed uniformly to normal tissues. While nonaarginine (r(9)) CPP administered intravenously into mice initially bind to the local vasculature and redistribute to the liver, where >90% of the injected dose accumulates 30 min after injection. Regardless of the presence of the polyanionic inhibitory domain, confocal imaging of live tissues reveals that the majority of the ACPP and CPP remain in punctate organelles, presumably endosomes. Therefore further improvements in the efficiency of delivery to the cytosol and nucleus are necessary. In addition to improved target specificity, a major advantage of ACPPs over CPPs for potential clinical applications is reduced toxicity. Systemically administered r(9) CPP causes acute toxicity in mice at a dose 4-fold lower than the MMP cleavable ACPP, a complication not observed with an uncleavable ACPP presumably because the polycationic charge remains masked systemically. These data suggest that ACPPs have greater potential than CPPs for systemic delivery of imaging and therapeutic agents.
Visibly fluorescent proteins (FPs) from jellyfish and corals have revolutionized many areas of molecular and cell biology,
but the use of FPs in intact animals, such as mice, has been handicapped by poor penetration of excitation light. We now show
that a bacteriophytochrome from Deinococcus radiodurans, incorporating biliverdin as the chromophore, can be engineered into monomeric, infrared-fluorescent proteins (IFPs), with
excitation and emission maxima of 684 and 708 nm, respectively; extinction coefficient >90,000 M−1 cm−1; and quantum yield of 0.07. IFPs express well in mammalian cells and mice and spontaneously incorporate biliverdin, which
is ubiquitous as the initial intermediate in heme catabolism but has negligible fluorescence by itself. Because their wavelengths
penetrate tissue well, IFPs are suitable for whole-body imaging. The IFPs developed here provide a scaffold for further engineering.
RNA interference constitutes a powerful tool for biological studies, but has also become one of the most challenging therapeutic strategies. However, small interfering RNA (siRNA)-based strategies suffer from their poor delivery and biodistribution. Cell-penetrating peptides (CPPs) have been shown to improve the intracellular delivery of various biologically active molecules into living cells and have more recently been applied to siRNA delivery. To improve cellular uptake of siRNA into challenging cell lines, we have designed a secondary amphipathic peptide (CADY) of 20 residues combining aromatic tryptophan and cationic arginine residues. CADY adopts a helical conformation within cell membranes, thereby exposing charged residues on one side, and Trp groups that favor cellular uptake on the other. We show that CADY forms stable complexes with siRNA, thereby increasing their stability and improving their delivery into a wide variety of cell lines, including suspension and primary cell lines. CADY-mediated delivery of subnanomolar concentrations of siRNA leads to significant knockdown of the target gene at both the mRNA and protein levels. Moreover, we demonstrate that CADY is not toxic and enters cells through a mechanism which is independent of the major endosomal pathway. Given its biological properties, we propose that CADY-based technology will have a significant effect on the development of fundamental and therapeutic siRNA-based applications.
The pH low-insertion peptide (pHLIP) serves as a model system for peptide insertion and folding across a lipid bilayer. It has three general states: (I) soluble in water or (II) bound to the surface of a lipid bilayer as an unstructured monomer, and (III) inserted across the bilayer as a monomeric α-helix. We used fluorescence spectroscopy and isothermal titration calorimetry to study the interactions of pHLIP with a palmitoyloleoylphosphatidylcholine (POPC) lipid bilayer and to calculate the transition energies between states. We found that the Gibbs free energy of binding to a POPC surface at low pHLIP concentration (state I–state II transition) at 37°C is approximately −7 kcal/mol near neutral pH and that the free energy of insertion and folding across a lipid bilayer at low pH (state II–state III transition) is nearly −2 kcal/mol. We discuss a number of related thermodynamic parameters from our measurements. Besides its fundamental interest as a model system for the study of membrane protein folding, pHLIP has utility as an agent to target diseased tissues and translocate molecules through the membrane into the cytoplasm of cells in environments with elevated levels of extracellular acidity, as in cancer and inflammation. The results give the amount of energy that might be used to move cargo molecules across a membrane.
• thermodynamics
• drug delivery
• imaging
• membrane protein
Tat is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1). Several studies have shown that exogenous Tat protein was able to translocate through the plasma membrane and to reach the nucleus to transactivate the viral genome. A region of the Tat protein centered on a cluster of basic amino acids has been assigned to this translocation activity. Recent data have demonstrated that chemical coupling of a Tat-derived peptide (extending from residues 37 to 72) to several proteins allowed their functional internalization into several cell lines or tissues. A part of this same domain can be folded in an alpha-helix structure with amphipathic characteristics. Such helical structures have been considered as key determinants for the uptake of several enveloped viruses by fusion or endocytosis. In the present study, we have delineated the main determinants required for Tat translocation within this sequence by synthesizing several peptides covering the Tat domain from residues 37 to 60. Unexpectedly, the domain extending from amino acid 37 to 47, which corresponds to the alpha-helix structure, is not required for cellular uptake and for nuclear translocation. Peptide internalization was assessed by direct labeling with fluorescein or by indirect immunofluorescence using a monoclonal antibody directed against the Tat basic cluster. Both approaches established that all peptides containing the basic domain are taken up by cells within less than 5 min at concentrations as low as 100 nM. In contrast, a peptide with a full alpha-helix but with a truncated basic amino acid cluster is not taken up by cells. The internalization process does not involve an endocytic pathway, as no inhibition of the uptake was observed at 4 degrees C. Similar observations have been reported for a basic amino acid-rich peptide derived from the Antennapedia homeodomain (1). Short peptides allowing efficient translocation through the plasma membrane could be useful vectors for the intracellular delivery of various non-permeant drugs including antisense oligonucleotides and peptides of pharmacological interest.
Arginine-rich peptides, penetratins, as part of a number of cellular and viral proteins, can penetrate across plasma membrane directly, without participation of endocytosis. We show that one of penetratins, the basic domain 47-57 of human immunodeficiency virus, type 1, transcription factor Tat (Tat peptide), is able to interact with plasmid DNA electrostatically. These interactions result in formation of polyelectrolytic complexes at various negative/positive charge ratios of plasmid DNA and Tat peptide. Plasmid DNA is capable of binding to Tat peptide up to 1.7-fold excess of the complex positive charge. The DNA-Tat complexes can be used for delivery of plasmid DNA into mammalian cells. Transfection efficacy of cultured cells by DNA-Tat complexes is stimulated by free Tat peptide, most likely because it protects DNA-Tat complexes from disruption by anionic proteoglycans of cellular surface. Our data strongly argue in favor of the endocytosis-dependent mechanism of DNA-Tat complex uptake by mammalian cells similarly to internalization of complexes of plasmid DNA with other polycationic carriers. Moreover, different cell lines use different endocytosis-mediated pathways for DNA-Tat complex internalization. Intravenous injections to mice of DNA-Tat complexes in comparison with injections of naked DNA showed an inhibitory effect of DNA-Tat complex positive charge on expression of transferred gene. A low level of foreign gene expression in the liver of mice injected intravenously with positively charged DNA-Tat complexes is accounted for by inactivation of DNA-Tat complexes in the bloodstream due to their interactions with serum albumin. These data should be taken into account in an attempt to develop versatile gene delivery systems based on penetratin application for human disease therapy.
Fluorescent labels are commonly used to investigate the mechanisms of cellular uptake and intracellular distribution of cell-penetrating peptides. However, labels such as fluorescein and rhodamine are relatively large and very lipophilic and may significantly alter physicochemical properties of small peptides. To minimize the impact of the fluorescent probe on a tetrapeptide, we substituted one of the amino acids (Lys4) in a tetrapeptide ([Dmt1]DALDA, Dmt-D-Arg-Phe-Lys-NH2 where Dmt = 2',6'-dimethyltyrosine) with two different fluorescent amino acids (beta-dansyl-L-alpha,beta-diaminopropionic acid (dnsDap4) or beta-anthraniloyl-L-alpha,beta-diaminopropionic acid (atnDap4)). Initial studies with confocal laser scanning microscopy (CLSM) showed very different localization patterns for the two fluorescent analogs, with [Dmt1,atnDap4]DALDA showing mitochondrial localization and [Dmt1,dnsDap4]DALDA showing diffuse cytoplasmic localization. Studies with isolated mouse liver mitochondria suggested that [Dmt1,dnsDap4]DALDA targeted the mitochondrial matrix resulting in mitochondrial depolarization, opening of the permeability transition pore, mitochondrial swelling, and rapid release of the peptide into the cytoplasm. In contrast, [Dmt1,atnDap4]DALDA was retained in the inner mitochondrial membrane and did not induce mitochondrial swelling. Furthermore, [Dmt1,atnDap4]DALDA protected mitochondria against Ca2+-induced swelling. Importantly, the unlabeled parent peptide [Dmt1]DALDA behaved like [Dmt1,atnDap4]DALDA and was mitoprotective. These findings suggest that experimental results obtained with fluorescent labels must be interpreted with caution, and the use of multiple fluorophores, together with confirmation using the original or radiolabeled molecule, is recommended.
Objectives:
The use of fluorescent compounds and techniques in neurosurgery has aided in delimitating tumor margins and visualizing blood dynamics in vascular malformations. However, the use of these techniques for the treatment of spinal lesions has not been well established.
Methods:
In this manuscript, we conduct a literature review to assess the role of fluorescent compounds in the treatment of vascular malformations and tumors of the spine. Other potential technologies such as activatable cell-penetrating peptides (ACPPs) and nanoparticles are also explored.
Results:
Several case reports and case series have described successful visualization and treatment of these lesions mainly with the aid of indocyanine green (ICG), with no complications reported. Nonetheless, no large-scale studies or prospective studies have been conducted.
Discussion:
Spinal lesions need to be adequately visualized intraoperatively to attain surgical success, and the use of fluorescent techniques may become a cornerstone in treating these lesions in the near future. Wide-scale applications and the potential use of other fluorophores such as 5-ALA and fluorescein should be further investigated.
Object:
Bone invasion is a major concern in meningioma surgery, since it is predictive of the recurrence of cranial involvement, morbidity, and mortality. Bone invasion has been reported in 20%-68% of studies with histopathologically confirmed data. Unfortunately, radical resection of bone invasion remains challenging. The aim of this study was to assess the role of 5-aminolevulinic acid (5-ALA) fluorescence in guiding the resection of bone-invading meningiomas. To this purpose, the sensitivity, specificity, and positive and negative predictive values of 5-ALA in detecting meningioma bone invasion were evaluated.
Methods:
Data from 12 patients affected by bone-invading meningiomas (7 with skull base and 5 with convexity meningiomas) who had undergone surgery with the assistance of 5-ALA fluorescence and neuronavigation between July 2012 and March 2013 at the Department of Neurosurgery of Padua were retrospectively analyzed. To evaluate the sensitivity and specificity of 5-ALA fluorescence in detecting meningioma tissue, a pathologist analyzed 98 surgical bone samples under blue light, according to different fluorescence patterns. Magnetic resonance images and CT scans were obtained pre- and postoperatively to determine the extent of bone invasion resection.
Results:
The rate of 5-ALA-induced fluorescence of both tumor and bone invasion was 100%. Based on the pathological examination of bone specimens, 5-ALA presented a sensitivity of 89.06% (95% CI 81.41%-96.71%) and a specificity of 100% in detecting meningioma bone invasion, while the positive and negative predictive values were 100% and 82.93% (95% CI 71.41%-94.45%), respectively. At the postoperative stage, MRI did not detect cases of meningioma bone invasion, whereas CT scans revealed residual hyperostosis in 2 cases.
Conclusions:
In summary, 5-ALA fluorescence represents a suitable and reliable technique for identifying and removing bone infiltration by meningiomas. However, further studies are needed to prove the clinical consequences of this promising technique in a larger population.
Cell-penetrating peptides (CPPs) are used to transport peptides, proteins, different types of ribonucleic acids (or mimics of these molecules) and DNA into live cells, both plant and mammalian. Leishmania belongs to the class of protozoa having, in comparison to mammalian cells, a different lipid composition of the membrane, proteoglycans on the surface, and signal pathways. We investigated the uptake of two different and easily detectable proteins into the non-pathogenic strain Leishmania tarentolae. From the large number of CPPs available, six and a histone were chosen specifically for their ability to form non-covalent complexes. For Leishmania we used the enzyme β-galactosidase and fluorescent labeled bovine serum albumin as cargoes. The results are compared to similar internalization studies using mammalian cells [Mussbach et al. 2011a]. Leishmania cells can degrade CPPs by a secreted and membrane-bound chymotrypsin-like protease. Both cargo proteins were internalized with sufficient efficiency and achieved intramolecular concentrations similar to mammalian cells. The transport efficiencies of the CPPs differed from each other, and showed a different rank order for both cargoes. The intracellular distribution of fluorescent-labeled bovine serum albumin showed highest concentrations in the nucleus and kinetoplast. Leishmania are susceptible to high concentrations of some CPPs, although comparably dissimilar to mammalian cells. MPG-peptides are more cytotoxic in Leishmania than in mammalian cells, acting as antimicrobial peptides. Our results contribute to a better understanding of molecular interactions in Leishmania cells and possibly to new treatments of leishmaniasis. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.
The intraoperative identification and resection of glioma is a significant and important challenge in neurosurgery. Complete resection of the enhancing tumour increases the median survival time in glioblastoma compared to partial glioma resection; however, it is achieved in fewer than half of eligible patients when conventional tumour identification methods are used. Increasing the incidence of complete resection, without causing excess morbidity, requires new methods to accurately identify neoplastic tissue intraoperatively, such as use of the drug 5-amino-levulinic acid (ALA). After ALA ingestion, the fluorescent molecule protoporphyrin IX (PpIX) accumulates in high grade glioma, allowing the neurosurgeon to more easily detect and accurately resect tumour. The utility of ALA has been demonstrated in a large, multicentre phase III randomised control trial of 243 patients with high grade glioma. ALA use led to a significant increase in the incidence of complete resection (65% compared to 36%), improved progression-free survival at 6months (41% compared to 21%), fewer reinterventions, and delayed onset of neurological deterioration. This review provides a broad assessment of ALA-PpIX fluorescence-guided resection, with Part 1 focusing on its clinical efficacy, and correlations with imaging and histology. The theoretical, biochemical and practical aspects of ALA use are reviewed in Part 2.
There is considerable interest in the sub-cellular targeting and delivery of biomolecules, therapeutic and imaging agents, and nanoparticles and nanoparticle conjugates into organelles for therapeutic and imaging purposes. To date, a number of studies have used sorting peptides for targeted delivery of cargo into different cell organelles but not into lysosomes. In this study, the delivery of 13-nm gold nanoparticles across the cell membrane followed by targeted localisation into the lysosomes of a mammalian cell line was examined using novel combinations of cell-penetrating peptides and lysosomal sorting peptides conjugated to the nanoparticles. Using a combination of fluorescence spectroscopy, fluorescence microscopy and transmission electron microscopy techniques, we show that these nanoconjugates were efficiently and selectively delivered into the lysosomes with minimal cytotoxic effects. This novel targeted delivery system may underpin the development of a new strategy for the treatment of lysosomal storage diseases by exploiting the large surface area of nanoparticles to deliver drugs or replacement enzymes directly to the lysosomes.
How to target cancer cells with high specificity and kill cancer cells with high efficiency remains an urgent demand for anticancer drugs. Temporin-La, which belongs to the family of temporins, presents antitumor activity against many cancer cell lines. We first used a whole bioinformatic analysis method as a platform to identify new anticancer antimicrobial peptides (AMPs). On the basis of these results, we designed a temporin-La analog (temporin-Las) and related constructs containing the Arg-Gly-Asp (RGD) tripeptide, the integrin αvβ3 homing domain (RGD-La and RGD-Las). We detected a link between the net charges and integrin αvβ3 expression of cancer cell lines and the antitumor activities of these peptides. Temporin-La and its synthetic analogs inhibited cancer cell proliferation in a dose-dependent manner. Evidence was provided that the affinity between RGD-Las and tumor cell membranes was stronger than other tested peptides using a pull-down assay. Morphological changes on the cell membrane induced by temporin-La and RDG-Las, respectively, were examined by scanning electron microscopy. Additionally, time-dependent morphological changes were detected by confocal microscopy, where the binding process of RGD-Las to the cell membrane could be monitored. The results indicate that the electrostatic interaction between these cationic peptides and the anionic cell membrane is a major determinant of selective cell killing. Thus, the RGD tripeptide is a valuable ligand motif for tumor targeting, which leads to an increased anticancer efficiency by RGD-Las. These AMP-derived peptides have clinical potential as specifically targeting agents for the treatment of αvβ3 positive tumors.
Thrombin and other coagulation enzymes have been shown to be important during atherosclerotic disease development. Study of these proteases is currently limited because of lack of robust molecular imaging agents for imaging protease activity in vivo. Activatable cell penetrating peptides (ACPPs) have been used to monitor MMP activity in tumors and, in principle, can be modified to detect other proteases. We have developed a probe that incorporates the peptide sequence DPRSFL from the proteinase activated receptor 1 (PAR-1) into an ACPP and shown that it is preferentially cleaved by purified thrombin. Active thrombin in serum cleaves DPRSFL-ACPP with >90% inhibition by lepirudin or argatroban. The DPRSFL-ACPP cleavage product accumulated in advanced atherosclerotic lesions in living mice, with 85% reduction in retention upon pre-injection of mice with hirudin. Uptake of the ACPP cleavage product was highest in plaques with histological features associated with more severe disease. Freshly resected human atheromas bathed in DPRSFL-ACPP retained 63% greater cleavage product compared to control ACPP. In conclusion, DPRSFL-ACPP can be used to study thrombin activity in coagulation and atherosclerosis with good spatial and temporal resolution. Thrombin-sensitive ACPPs may be developed into probes for early detection and intraoperative imaging of high risk atherosclerotic plaques.
Cell-penetrating peptides (CPPs) are short cationic peptides that penetrate cells by interacting with the negatively charged plasma membrane; however, the detailed uptake mechanism is not clear. In contrary to the conventional mode of action of CPPs, we show here that a CPP, PepFect14 (PF14), forms negatively charged nanocomplexes with oligonucleotides and their uptake is mediated by class-A scavenger receptors (SCARAs). Specific inhibitory ligands of SCARAs, such as fucoidin, polyinosinic acid, and dextran sulfate, totally inhibit the activity of PF14-oligonucleotide nanocomplexes in the HeLa pLuc705 splice-correction cell model, while nonspecific, chemically related molecules do not. Furthermore, RNA interference (RNAi) knockdown of SCARA subtypes (SCARA3 and SCARA5) that are expressed in this cell line led to a significant reduction of the activity to <50%. In line with this, immunostaining shows prevalent colocalization of the nanocomplexes with the receptors, and electron microscopy images show no binding or internalization of the nanocomplexes in the presence of the inhibitory ligands. Interestingly, naked oligonucleotides also colocalize with SCARAs when used at high concentrations. These results demonstrate the involvement of SCARA3 and SCARA5 in the uptake of PF14-oligonucleotide nanocomplexes and suggest for the first time that some CPP-based systems function through scavenger receptors, which could yield novel possibilities to understand and improve the transfection by CPPs.
The development of cell penetrating peptides (CPPs) for the cellular delivery of attached cargo is an area of growing interest. Many CPPs, however, are found trapped within endosomes, thereby limiting their use as drug delivery agents with sub-cellular applications. Herein, we detail the properties of a highly efficient class of CPPs, cationic amphiphilic polyproline helices (CAPHs), that are found localized to the mitochondria by direct transport into cells.
Internalization of peptides and proteins into live cells is an essential prerequisite for studies on intracellular signal pathways, for treatment of certain microbial diseases and for signal transduction therapy, especially for cancer treatment. Cell penetrating peptides (CPPs) facilitate the transport of cargo-proteins through the cell membrane into live cells. CPPs which allow formation of non-covalent complexes with the cargo are used primarily in this study due to the relatively easy handling procedure. Efficiency of the protein uptake is estimated qualitatively by fluorescence microscopy and quantitatively by SDS-PAGE. Using the CPP cocktail JBS-Proteoducin, the intracellular concentrations of a secondary antibody and bovine serum albumin can reach the micromolar range. Internalization of antibodies allows mediation of intracellular pathways including knock down of signal transduction. The high specificity and affinity of antibodies makes them potentially more powerful than siRNA. Thus, CPPs represent a significant new possibility to study signal transduction processes in competition or in comparison to the commonly used other techniques. To estimate the highest attainable intracellular concentrations of cargo proteins, the CPPs are tested for cytotoxicity. Cell viability and membrane integrity relative to concentration of CPPs are investigated. Viability as estimated by the reductive activity of mitochondria (MTT-test) is more sensitive to higher concentrations of CPPs versus membrane integrity, as measured by the release of dead cell protease. Distinct differences in uptake efficiency and cytotoxic effects are found using six different CPPs and six different adhesion and suspension cell lines.
Cell-penetrating peptides (CPPs) constitute a family of peptides with the characteristic ability to cross biological membranes and deliver cargo into the intracellular milieu. Several CPPs have been proposed for delivery of polypeptides and proteins into cells through either of two strategies: covalent or complexed in a non-covalent fashion. Members of the PEP family are primary amphipathic peptides which have been shown to deliver peptides and proteins into a wide variety of cells through formation of non-covalent complexes. CADY is a secondary amphipathic peptide which has been demonstrated to deliver short nucleic acids, in particular siRNA with high efficiency. Here we review the characteristics of the PEP and CADY carriers and describe a novel derivative of CADY termed CADY2, which also presents sequence similarities to Pep1. We have compared Pep1, CADY and CADY2 in their efficiency to interact with and internalize short fluorogenic peptides and proteins into cultured cells, and provide evidence that CADY2 can interact with proteins and peptides and deliver them efficiently into living cells, similar to Pep1, but in contrast to CADY which is unable to deliver any peptide, even short negatively charged peptides. This is the first study to investigate the influence of the cargo on the interactions between PEP and CADY carriers, thereby providing novel insights into the physicochemical parameters underlying interactions and cellular uptake of peptides and proteins by these non-covalent CPPs.
Cell-penetrating peptides (CPPs) are short peptides capable of translocating across the plasma membrane of live cells and transporting conjugated compounds intracellularly. Fifteen years after discovering the first model cationic CPPs, penetratin and TAT, CPP internalization is still challenging many questions. Particularly it has been unknown whether CPPs enter the cells with or without mediation of a specific surface receptor. Here we report that syndecan-4, the universally expressed isoform of the syndecan family of transmembrane proteoglycans, binds and mediates transport of the three most frequently utilized cationic CPPs (penetratin, octaarginine and TAT) into the cells. Quantitative uptake studies and mutational analyses demonstrate that attachment of the cationic CPPs is mediated by specific interactions between the heparan sulfate chains of syndecan-4 and the CPPs. Protein kinase C alpha is also heavily involved in the uptake mechanism. The collected data give the first direct evidence on the receptor-mediated uptake of cationic CPPs and may replace the long-thought, but already contradicted membrane penetration hypothesis. Thus our study might give an answer for a decade long debate and foster the development of rationalized, syndecan-4 targeted novel delivery technologies.
The pH (low) insertion peptide (pHLIP) has exceptional characteristics: at neutral pH it is an unstructured monomer in solution or when bound to lipid bilayer surfaces, and it inserts across a lipid bilayer as a monomeric alpha-helix at acidic pH. The peptide targets acidic tissues in vivo and may be useful in cancer biology for delivery of imaging or therapeutic molecules to acidic tumors. To find ways to vary its useful properties, we have designed and analyzed pHLIP sequence variants. We find that each of the Asp residues in the transmembrane segment is critical for solubility and pH-dependent membrane insertion of the peptide. Changing both of the Asp residues in the transmembrane segment to Glu, inserting an additional Asp into the transmembrane segment, or replacing either of the Asp residues with Ala leads to aggregation and/or loss of pH-dependent membrane insertion of the peptide. However, variants with either of the Asp residues changed to Glu remained soluble in an aqueous environment and inserted into the membrane at acidic pH with a higher pK(app) of membrane insertion.
A class of mitochondria-penetrating peptides (MPPs) was studied in an effort to optimize their applications in the delivery of bioactive cargo to this therapeutically important organelle. The sequence requirements for mitochondrial entry were monitored, and it was discovered that while an alternating cationic/hydrophobic residue motif is not required, the inclusion of a stretch of adjacent cationic amino acids can impede access to the organelle. In addition, a variety of N- and C-terminal cargo were tested to determine if there are limitations to the lipophilicity, charge, or polarity of compounds that can be transported to mitochondria by MPPs. The results reported demonstrate that these peptide sequences are versatile transporters that will have a range of biological applications.
MMP-2 and MMP-9 secretion is elevated in several types of human cancers and their elevated expression has been associated with poor prognosis. Expression of MMPs is highly regulated by cytokines and signal transducation pathways, including those activated by phorbol 12-myristate 13-acetate (PMA). The aim of this study was to examine the effect of PMA on MMP-2 and MMP-9 secretion in 42 different human cancer cell lines, selected on the basis of their organ malignancies. They were cultured in the recommended media supplemented with 10% FBS and antibiotics in 24-well tissue culture plates. At near confluence, the cells were washed with PBS, 0.5 ml of medium was added, and the cultures were incubated. Parallel sets of cultures were also treated with PMA for induction of enzymes. After 24 h the media were collected and MMP-2 and MMP-9 levels were assayed by gelatinase zymography. Based on MMP-2 and MMP-9 secretion without and with PMA treatment, the various human cancer cell lines fell into one of two major groups. The first group characterized by low basal MMP-9 secretion fell into three different categories of susceptibility to PMA induction of MMP-9 expression: resistant, moderately susceptible and highly susceptible. High basal MMP-9 levels responsive to PMA induction characterized the second group. Most cancer cell lines examined exhibited basal levels of MMP-2, MMP-9 or both. MMP-2 secretion was not induced by PMA in any of the cancer cells examined.
Structural simplification of a 42-residue venom peptideby N-to-C-terminal splicing led to two sequences [YKQCHKKGGXKKGSG, where X = nil (1) or 6-aminohexanoyl (2)], both efficiently uptaken by HeLa cells and, most interestingly, specifically localized at the nucleolus. Retro-2 was uptaken less efficiently, but a single (His --> Ile) replacement recovered the translocation ability. None of the peptides were cytotoxic up to 100 microM. Enantio-1 did not translocate, suggesting that peptide uptake was receptor-mediated.
Two peptides designed for drug delivery were generated by the combination of a signal peptide with a nuclear localization sequence and are shown to facilitate the cellular internalization of small molecules which are covalently linked to these peptides. In order to understand the mechanism of internalization, the conformations of the peptides were investigated through different approaches both in solution and in membrane-mimicking environments. These peptides are highly versatile and adopt different conformational states depending on their environment. While in a disordered form in water, they adopt an alpha-helical structure in TFE and in the presence of micelles of SDS or DPC. The structured domain encompasses the hydrophobic part of the peptides, whereas the charged C-termini remain unstructured. In contrast, in the presence of lipids and whatever the nature of the phosphate headgroup, the two peptides mainly adopt an antiparallel beta-sheet form and embed in the lipidic cores. This result suggests that the beta-sheet is responsible for the translocation through the cellular membranes but also questions the conformational state of signal peptides when associated to hydrophilic sequences.
Production of matrix-degrading proteases, particularly matrix metalloproteinases (MMPs), by endothelial cells is a critical event during angiogenesis, the process of vessel neoformation that occurs in normal and pathological conditions. MMPs are known to be highly regulated at the level of synthesis and activation, however, little is known about the regulation of MMP secretion by endothelial cells. We found that cultured human umbilical vein endothelial cells shed vesicles (300 to 600 nm) originating from localized areas of the cell plasma membrane, as revealed by ultrastructural analysis. Normal and reverse zymography, Western blot, and immunogold analyses of the vesicles showed two gelatinases, MMP-2 and MMP-9, in both the active and proenzyme forms, the MT1-MMP proenzyme located on the external side of the vesicle membrane and the two inhibitors TIMP-1 and TIMP-2. Serum and the angiogenic factors, fibroblast growth factor-2 and vascular endothelial growth factor, stimulated the shedding of MMPs as vesicle components. Shedding the vesicle was rapid, as it was already completed after 4 hours. Addition of shed vesicles to human umbilical vein endothelial cells resulted in autocrine stimulation of invasion through a layer of reconstituted basement membrane (Matrigel) and cord formation on Matrigel. We conclude that endothelial cells shed MMP-containing vesicles and this may be a mechanism for regulating focalized proteolytic activity vital to invasive and morphogenic events during angiogenesis.
Much progress has been made in recent years in the understanding of angiogenesis, yet signalling pathways involved remain poorly defined. Here we report that small RhoA GTPase is implicated in the invasion of human microvascular endothelial cells (HMEC-1). Ectopic expression of active-RhoA GTPase induced the expression of MMP-9 metalloproteinase, a key proteinase of the basement membrane, and promoted migration of endothelial cells through a 3D-matrix protein gel. MMP-9 was either directed as vesicular-like patches to the apical side of cells, or addressed to specific membrane sites at the cell surface. Confocal microscopy analyses indeed revealed clustering of MMP-9 in advancing lamellipodia at the forefront of endothelial cells, where this proteinase colocalized with RhoA and CD44, a transmembrane receptor known to be proteolysed in tumor cell progression. In addition, TIMP-1, a natural MMP inhibitor, significantly reduced the invasion of RhoAV14 expressing cells, suggesting that MMP-9 was a critical metalloproteinase responsible, at least partly, for the RhoAV14-induced endothelial cell invasion. We propose that RhoA triggers signalling pathways that, upregulating expression of a proteinase at specific membrane localizations, may confer an highly invasive phenotype to endothelial cells.
To identify rules for the design of efficient cell-penetrating peptides that deliver therapeutic agents into subcellular compartments, we compared the properties of two closely related primary amphipathic peptides that mainly differ by their conformational state. On the basis of a peptide Pbeta that is nonstructured in water and that promotes efficient cellular uptake of nucleic acids through noncovalent association, we have designed a peptide [Palpha] that is predicted to adopt a helical conformation. We show that [Pbeta] undergoes a lipid-induced conformational transition into a sheet structure, while [Palpha] remains helical. Penetration experiments show that both peptides can spontaneously insert into phospholipid membranes. Analysis of compression isotherms indicates that both peptides interact with phospholipids in the liquid expanded and liquid condensed states. AFM observations reveal that the peptides strongly disrupt the lipid organization of the monolayers and that the conformational state can influence the uptake by model membranes.
Protein transduction domains (PTDs), such as the TAT PTD, have been shown to deliver a wide variety of cargo in cell culture and to treat preclinical models of cancer and cerebral ischemia. The TAT PTD enters cells by a lipid raft-dependent macropinocytosis mechanism that all cells perform. Consequently, PTDs resemble small-molecule therapeutics in their lack of pharmacologic tissue specificity in vivo. However, several human malignancies overexpress specific receptors, including HER2 in breast cancer, GnRH in ovarian carcinomas, and CXC chemokine receptor 4 (CXCR4) in multiple malignancies. To target tumor cells that overexpress the CXCR4 receptor, we linked the CXCR4 DV3 ligand to two transducible anticancer peptides: a p53-activating peptide (DV3-TATp53C') and a cyclin-dependent kinase 2 antagonist peptide (DV3-TAT-RxL). Treatment of tumor cells expressing the CXCR4 receptor with either the DV3-TATp53C' or DV3-TAT-RxL targeted peptides resulted in an enhancement of tumor cell killing compared with treatment with nontargeted parental peptides. In contrast, there was no difference between DV3 targeted peptide and nontargeted, parental peptide treatment of non-CXCR4-expressing tumor cells. These observations show that a multidomain approach can be used to further refine and enhance the tumor selectivity of biologically active, transducible macromolecules for treating cancer.
CPPs (cell-penetrating peptides), including Tatp (transactivator of transcription peptide), have been successfully used for intracellular delivery of a wide variety of cargoes including various nanoparticulate pharmaceutical carriers such as liposomes, micelles and nanoparticles. Here, we will consider the major results obtained in this area with emphasis on Tatp-mediated delivery of liposomes and various transfection vectors. We will also address the development of 'smart' stimuli-sensitive nanocarriers, where the cell-penetrating function can only be activated when the nanocarrier is inside the biological target, thus minimizing the interaction with non-target cells.
Cell-penetrating peptides (CPPs) including TAT peptide (TATp) have been successfully used for intracellular delivery of a broad variety of cargoes including various nanoparticulate pharmaceutical carriers (liposomes, micelles, nanoparticles). Here, we will consider the main results in this area, with a special emphasis on TATp-mediated delivery of liposomes and DNA. We will also address the development of "smart" stimuli-sensitive nanocarriers, where cell-penetrating function can be activated by the decreased pH only inside the biological target minimizing thus the interaction of drug-loaded nanocarriers with non-target cells.
Matrix metalloproteinases (MMPs) are now acknowledged as key players in the regulation of both cell-cell and cell-extracellular matrix interactions. They are involved in modifying matrix structure, growth factor availability and the function of cell surface signalling systems, with consequent effects on cellular differentiation, proliferation and apoptosis. They play central roles in morphogenesis, wound healing, tissue repair and remodelling in response to injury and in the progression of diseases such as arthritis, cancer and cardiovascular disease. Because of their wide spectrum of activities and expression sites, the elucidation of their potential as drug targets in disease or as important features of the repair process will be dependent upon careful analysis of their role in different cellular locations and at different disease stages. Novel approaches to the specific regulation of individual MMPs in different contexts are also being developed.
PEP and CADY-mediated delivery of fluorescent peptides and proteins into living cells Accepted Article This article is protected by copyright. All rights reserved Cell-penetrating peptide exploited syndecans Review: Progress in matrix metalloproteinase research
- L Kurzawa
- M Pellerano
- Morris
- T Letoha
- Keller
- A Pinter
- E Kusz
- C Koloszi
- Z Bozso
- G Toth
- C Vizier
- Z Olah
- Szilak
Kurzawa L, Pellerano M, Morris MC. 2010. PEP and CADY-mediated delivery of fluorescent peptides and proteins into living cells. Biochim Biophys Acta 1798:2274-2285. Accepted Article This article is protected by copyright. All rights reserved Letoha T, Keller-Pinter A, Kusz E, Koloszi C, Bozso Z, Toth G, Vizier C, Olah Z, Szilak L. 2010. Cell-penetrating peptide exploited syndecans. Biochim Biophys Acta (Biomembranes) 1798:2258-2263. Doi:10.1016/j.bbamem.2010.01.022. Murphy G, Nagase H. 2008. Review: Progress in matrix metalloproteinase research. Molecular Aspects of Medicine 29:290-308.
Real-time in vivo molecular detection of primary tumors and metastases with ratiometric activatable cell-penetrating peptides
- E N Savanier
- C N Felsen
- N Nashi
- T Jiang
- L G Ellies
- P Steinbach
- R Y Tsien
- Nguen Qut
Savanier EN, Felsen CN, Nashi N, Jiang T, Ellies LG, Steinbach P, Tsien RY, Nguen
QuT. 2013. Real-time in vivo molecular detection of primary tumors and metastases with
ratiometric activatable cell-penetrating peptides. Cancer Research 73:855-864.
Review: Human matrix metalloproteinases: An ubiquitarian class of enzymes involved in several pathological processes
- D Sbardella
- G F Fasciglione
- M Gioia
- M Ciaccio
- G R Tundo
- S Marini
Sbardella D, Fasciglione GF, Gioia M, Ciaccio M, Tundo GR, Marini S, Coletta M. 2012.
Review: Human matrix metalloproteinases: An ubiquitarian class of enzymes involved in
several pathological processes. Molecular Aspects of Medicine 33:119-208.
Shedding of the matrix metalloproteinases MMP-2, MMP-9, and MT1-MMP as membrane vesicleassociated compomemts by endothelial cells
- G Taraboletti
- Ascenzo
- P Borsotti
- R Giavazzi
- A Pavan
- V Dolo
Taraboletti G, D`Ascenzo, Borsotti P, Giavazzi R, Pavan A, Dolo V. 2002. Shedding of
the matrix metalloproteinases MMP-2, MMP-9, and MT1-MMP as membrane vesicleassociated compomemts by endothelial cells. Amer J Pathol 160:673-680.
Real-time in vivo molecular detection of primary tumors and metastases with ratiometric activatable cell-penetrating peptides
- Savanier
Review: Human matrix metalloproteinases: An ubiquitarian class of enzymes involved in several pathological processes
- Sbardella