Cell-Penetrating Peptides: design strategies beyond primary structure and amphipathicity

Efficient intracellular drug delivery and target specificity are often hampered by the presence of biological barriers. Thus, compounds that efficiently cross cell membranes are the key to improving the therapeutic value and on-target specificity of non-permeable drugs. The discovery of cell-penetrating peptides (CPPs) and the early design approaches through mimicking the natural penetration domains used by viruses have led to greater efficiency of intracellular delivery. Following these nature-inspired examples, a number of rationally designed CPPs has been developed. In this review, a variety of CPP designs will be described, including linear and flexible, positively charged and often amphipathic CPPs, and more rigid versions comprising cyclic, stapled, or dimeric and/or multivalent, self-assembled peptides or peptido-mimetics. The application of distinct design strategies to known physico-chemical properties of CPPs offers the opportunity to improve their penetration efficiency and/or internalization kinetics. This led to increased design complexity of new CPPs that does not always result in greater CPP activity. Therefore, the transition of CPPs to a clinical setting remains a challenge also due to the concomitant involvement of various internalization routes and heterogeneity of cells used in the in vitro studies

Bottom-Up Design Approach for OBOC Peptide Libraries

One-bead-one-compound peptide libraries, developed following the top-down experimental approach, have attracted great interest in the identification of potential ligands or active peptides. By exploiting a reverse experimental design approach based on the bottom-up strategy, we aimed to develop simplified, maximally diverse peptide libraries that resulted in the successful characterization of mixture components. We show that libraries of 32 and 48 components can be successfully detected in a single run using chromatography coupled to mass spectrometry (UPLC-MS). The proposed libraries were further theoretically evaluated in terms of their composition and physico-chemical properties. By combining the knowledge obtained on single libraries we can cover larger sequence spaces and provide a controlled exploration of the peptide chemical space both theoretically and experimentally. Designing libraries by using the bottom-up approach opens up the possibility of rationally fine-tuning the library complexity based on the available analytical methods

Algorithm-supported, mass and sequence diversity-oriented random peptide library design.

Random peptide libraries that cover large search spaces are often used for the discovery of new binders, even when the target is unknown. To ensure an accurate population representation, there is a tendency to use large libraries. However, parameters such as the synthesis scale, the number of library members, the sequence deconvolution and peptide structure elucidation, are challenging when increasing the library size. To tackle these challenges, we propose an algorithm-supported approach to peptide library design based on molecular mass and amino acid diversity. The aim is to simplify the tedious permutation identification in complex mixtures, when mass spectrometry is used, by avoiding mass redundancy. For this purpose, we applied multi (two- and three-)-objective genetic algorithms to discriminate between library members based on defined parameters. The optimizations led to diverse random libraries by maximizing the number of amino acid permutations and minimizing the mass and/or sequence overlapping. The algorithm-suggested designs offer to the user a choice of appropriate compromise solutions depending on the experimental needs. This implies that diversity rather than library size is the key element when designing peptide libraries for the discovery of potential novel biologically active peptides

MMP-9 triggered self-assembly of doxorubicin nanofiber depots halts tumor growth

A central challenge in cancer care is to ensure that therapeutic compounds reach their targets. One approach is to use enzyme-responsive biomaterials, which reconfigure in response to endogenous enzymes that are overexpressed in diseased tissues, as potential site-specific anti-tumoral therapies. Here we report peptide micelles that upon MMP-9 catalyzed hydrolysis reconfigure to form fibrillar nanostructures. These structures slowly release a doxorubicin payload at the site of action. Using both in vitro and in vivo models we demonstrate that the fibrillar depots are formed at the sites of MMP-9 overexpression giving rise to enhanced efficacy of doxorubicin, resulting in inhibition of tumor growth in an animal model

Ispitivanje fenolnog profila i antioksidacijskog učinka meda od metvice (Mentha spp.)

Research background. The composition of honey is influenced by the botanical source and geographical area of the nectar from which it is derived. Unifloral honeys reach higher market value than multifloral honeys due to their specific aromas, which result from volatile and phenolic compounds. Experimental approach. The aim of our study is to characterize the phenolic composition of a rare unifloral variety of honey – mint (Mentha spp.) honey. For this purpose, we performed standard physicochemical analyses, pollen analysis, determined total phenolic and flavonoid content, analysed antioxidant activity and performed qualitative and quantitative analyses of phenolic compounds for five mint honeys. Results and conclusions. Our results indicate that mint honey samples have high phenolic content, expressed in gallic acid equivalents, from (76.7±0.6) to (90.1±1.1) mg/100 g, and flavonoid content, expressed as quercetin equivalents, from (6.7±0.6) to (12.5±0.8) mg/100 g. These honey samples also exhibit strong antioxidant activity, expressed as Trolox equivalents, from (33.6±2.8) to (51.3±1.2) mg/100 g and from (14.4±0.8) to (55.1±2.4) mg/100 g when analysed with DPPH and ABTS assays, respectively. Quantitative LC-MS/MS analysis revealed that the most abundant phenols in all samples were chrysin, apigenin and p-coumaric acid. Qualitative LC-MS/MS analysis identified the presence of kaempferide, diosmetin, acacetin and several caffeic acid derivatives. Novelty and scientific contribution. Our study indicates that mint honey contains unique phenolic profiles, which likely contribute to its distinctive aroma and strong antioxidant activity. A detailed description of the rare honey varieties gives beekeepers greater visibility and easier access to the demanding natural product market.Pozadina istraživanja. Na sastav meda utječu botanički izvor i geografsko podrijetlo nektara iz kojeg je dobiven. Uniflorni medovi postižu veću tržišnu vrijednost od multiflornih zbog svoje specifične arome, koja je rezultat prisustva hlapljivih i fenolnih spojeva. Eksperimentalni pristup. Svrha je našeg rada bila okarakterizirati fenolni sastav rijetke uniflorne sorte meda – meda od metvice (Mentha spp.). U tu svrhu smo proveli standardne fizikalno-kemijske analize i analizu peludi, odredili ukupne udjele fenola i flavonoida, ispitali antioksidacijsku aktivnost te kvalitativno i kvantitativno ispitali fenolne spojeve u pet uzoraka meda od metvice. Rezultati i zaključci. Rezultati pokazuju da uzorci meda od metvice imaju velik udjel fenola, izražen u ekvivalentima galne kiseline, od (76,7±0,6) do (90,1±1,1) mg/100 g, te udjel flavonoida, izražen u ekvivalentima kvercetina, od (6,7±0,6) do (12,5±0,8) mg/100 g. Uzorci su također imali snažnu antioksidacijsku aktivnost, izraženu u Trolox ekvivalentima, i to prema metodi DPPH od (33,6±2,8) do (51,3±1,2) mg/100 g, a prema metodi ABTS od (14,4±0,8) do (55,1±2,4) mg/100 g. Kvantitativnim ispitivanjem pomoću spregnutog sustava tekućinske kromatografije i tandemske spektrometrije masa utvrđeno je da su najzastupljeniji fenoli u svim uzorcima bili krizin, apigenin i p-kumarinska kiselina, dok je kvalitativnom analizom pomoću istog sustava utvrđena prisutnost kemferida, diosmetina, akacetina i nekoliko derivata kafeinske kiseline. Novost i znanstveni doprinos. U istraživanju smo pokazali da med od metvice ima jedinstven fenolni profil, koji pridonosi njegovoj prepoznatljivoj aromi i snažnom antioksidativnom djelovanju. Detaljan opis rijetkih sorti meda omogućava pčelarima veću vidljivost i lakši pristup zahtjevnom tržištu prirodnih proizvoda

Alignment of nanostructured tripeptide gels by directional ultrasonication

We demonstrate an in-situ ultrasonic approach to influence self-assembly across the supramolecular to micron length scales, showing enhancement of supramolecular interactions, chirality and orientation, which depends on the peptide sequence and solvent environment. This is the first successful demonstration of using oscillating pressure waves to generate anisotropic organo- and hydro- gels consisting of oriented tripeptides structures

Enzyme responsive nanomaterials for cancer applications

Expression levels of enzymes dictate the difference between health and disease in many cases, including cancer. This leads scientists to explore strategies to incorporate enzyme sensitivity in materials where the goal is to achieve dynamic and targeted changes in material properties to influence cancer cells. Peptide amphiphiles were designed (PhAc-FFAGLDD (1a) and GFFLGLDD (2a) and their expected products of enzyme cleavage PhAc-FFAG (1b) and GFFLG (2b)) such that, upon cleavage by a disease-associated enzyme, reconfigure from micellar aggregates to fibres. After the designed peptides (1a, 1b, 2a, 2b) were shown to be suitable for controlling the morphology of the supramolecular aggregates based on peptide length, hydrophobicity and charge, the enzyme triggered micelle to fibre transition was explored. Following this it was investigated whether the micelles were capable to perform as mobile vehicles for encapsulation and release of hydrophobic drugs. It was observed that the assembled fibres provide a scaffold for prolonged drug delivery due to the partial entrapment (localised depots) of the drug and the intrinsic biodegradable nature of peptide carriers themselves. The capacity of retention of doxorubicin in the hydrophobic core of the micelles followed by its entrapment in the fibres was exploited in the development of a method for visualisation of fibre formation around cancer cells. In vitro studies were performed on human cancer cell lines using different types of microscopy. MMP-9 activity was quantified in the mentioned cancer cell lines. In addition, preliminary toxicity studies of the designed peptides to cancer cells were performed. Being purely peptidic (compared to conventional aromatic or aliphatic peptide amphiphiles), these systems have the advantage of being non toxic to cells and can be used as carriers for doxorubicin in vivo. They are currently tested on animal models where the cancer growth is slowed down by administration of doxorubicin loaded peptides compared to doxorubicin only. Another, complementary system was investigated based on crosslinked polymer particles- microgels as a possible way to obtain enzyme responsive materials. Amine rich microgels, poly(VAM-co-BEVAME) were synthesised and functionalised post-polymerisation with peptides. Due to aggregation issues these systems were not further explored for biomedical applications.Expression levels of enzymes dictate the difference between health and disease in many cases, including cancer. This leads scientists to explore strategies to incorporate enzyme sensitivity in materials where the goal is to achieve dynamic and targeted changes in material properties to influence cancer cells. Peptide amphiphiles were designed (PhAc-FFAGLDD (1a) and GFFLGLDD (2a) and their expected products of enzyme cleavage PhAc-FFAG (1b) and GFFLG (2b)) such that, upon cleavage by a disease-associated enzyme, reconfigure from micellar aggregates to fibres. After the designed peptides (1a, 1b, 2a, 2b) were shown to be suitable for controlling the morphology of the supramolecular aggregates based on peptide length, hydrophobicity and charge, the enzyme triggered micelle to fibre transition was explored. Following this it was investigated whether the micelles were capable to perform as mobile vehicles for encapsulation and release of hydrophobic drugs. It was observed that the assembled fibres provide a scaffold for prolonged drug delivery due to the partial entrapment (localised depots) of the drug and the intrinsic biodegradable nature of peptide carriers themselves. The capacity of retention of doxorubicin in the hydrophobic core of the micelles followed by its entrapment in the fibres was exploited in the development of a method for visualisation of fibre formation around cancer cells. In vitro studies were performed on human cancer cell lines using different types of microscopy. MMP-9 activity was quantified in the mentioned cancer cell lines. In addition, preliminary toxicity studies of the designed peptides to cancer cells were performed. Being purely peptidic (compared to conventional aromatic or aliphatic peptide amphiphiles), these systems have the advantage of being non toxic to cells and can be used as carriers for doxorubicin in vivo. They are currently tested on animal models where the cancer growth is slowed down by administration of doxorubicin loaded peptides compared to doxorubicin only. Another, complementary system was investigated based on crosslinked polymer particles- microgels as a possible way to obtain enzyme responsive materials. Amine rich microgels, poly(VAM-co-BEVAME) were synthesised and functionalised post-polymerisation with peptides. Due to aggregation issues these systems were not further explored for biomedical applications

Bottom-Up Design Approach for OBOC Peptide Libraries

One-bead-one-compound peptide libraries, developed following the top-down experimental approach, have attracted great interest in the identification of potential ligands or active peptides. By exploiting a reverse experimental design approach based on the bottom-up strategy, we aimed to develop simplified, maximally diverse peptide libraries that resulted in the successful characterization of mixture components. We show that libraries of 32 and 48 components can be successfully detected in a single run using chromatography coupled to mass spectrometry (UPLC-MS). The proposed libraries were further theoretically evaluated in terms of their composition and physico-chemical properties. By combining the knowledge obtained on single libraries we can cover larger sequence spaces and provide a controlled exploration of the peptide chemical space both theoretically and experimentally. Designing libraries by using the bottom-up approach opens up the possibility of rationally fine-tuning the library complexity based on the available analytical methods

Cell-Penetrating Peptides: Design Strategies beyond Primary Structure and Amphipathicity

Efficient intracellular drug delivery and target specificity are often hampered by the presence of biological barriers. Thus, compounds that efficiently cross cell membranes are the key to improving the therapeutic value and on-target specificity of non-permeable drugs. The discovery of cell-penetrating peptides (CPPs) and the early design approaches through mimicking the natural penetration domains used by viruses have led to greater efficiency of intracellular delivery. Following these nature-inspired examples, a number of rationally designed CPPs has been developed. In this review, a variety of CPP designs will be described, including linear and flexible, positively charged and often amphipathic CPPs, and more rigid versions comprising cyclic, stapled, or dimeric and/or multivalent, self-assembled peptides or peptido-mimetics. The application of distinct design strategies to known physico-chemical properties of CPPs offers the opportunity to improve their penetration efficiency and/or internalization kinetics. This led to increased design complexity of new CPPs that does not always result in greater CPP activity. Therefore, the transition of CPPs to a clinical setting remains a challenge also due to the concomitant involvement of various internalization routes and heterogeneity of cells used in the in vitro studies

Cell-Penetrating Peptides: design strategies beyond primary structure and amphipathicity

Efficient intracellular drug delivery and target specificity are often hampered by the presence of biological barriers. Thus, compounds that efficiently cross cell membranes are the key to improving the therapeutic value and on-target specificity of non-permeable drugs. The discovery of cell-penetrating peptides (CPPs) and the early design approaches through mimicking the natural penetration domains used by viruses have led to greater efficiency of intracellular delivery. Following these nature-inspired examples, a number of rationally designed CPPs has been developed. In this review, a variety of CPP designs will be described, including linear and flexible, positively charged and often amphipathic CPPs, and more rigid versions comprising cyclic, stapled, or dimeric and/or multivalent, self-assembled peptides or peptido-mimetics. The application of distinct design strategies to known physico-chemical properties of CPPs offers the opportunity to improve their penetration efficiency and/or internalization kinetics. This led to increased design complexity of new CPPs that does not always result in greater CPP activity. Therefore, the transition of CPPs to a clinical setting remains a challenge also due to the concomitant involvement of various internalization routes and heterogeneity of cells used in the in vitro studies