Synthesis and screening of a small library of proline-based biodendrimers for use as delivery agents

A small library of defined peptide dendrimers based on polyproline sequences was designed to demonstrate the feasibility of generating a new type of polymeric agent for therapeutic use. Structural modifications to dendrimer surfaces further enriched the diversity of the library. Data show that the prolinerich dendrimers can be internalized in human epithelial (HeLa) cells, demonstrating the importance of the dendrimeric motif. The promising results described herein suggest that controlled modification of the dendrimer surface should eventually yield proline dendrimers with therapeutic potential.     

Peptide and glycopeptide dendrimers. Part II

Recent progress in peptide and glycopeptide chemistry make the preparation of peptide and glycopeptide dendrimers of acceptable purity, with designed structural and immunochemical properties reliable. New methodologies using unprotected peptide building blocks have been developed to further increase the possibilities of their design and improve their preparation and separation. The sophisticated design of peptide and glycopeptide dendrimers has led to their use as antigens and immunogens, for serodiagnosis and other biochemical uses including drug delivery. Dendrimers bearing peptide with predetermined secondary structures are useful tools in protein de novo design. This article covers synthesis and applications of multiple antigen peptides (MAPs), multiple antigen glycopeptides (MAGs), multiple antigen peptides based on sequential oligopeptide carriers (MAP‐SOCs), glycodendrimers and template‐assembled synthetic proteins (TASPs). In part II the preparation of MAPs, and the utility of glycodendrimers and TASPs are discussed. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Molecular Determinants of the Cellular Entry of Asymmetric Peptide Dendrimers and Role of Caveolae

Caveolae are flask-shaped plasma membrane subdomains abundant in most cell types that participate in endocytosis. Caveola formation and functions require membrane proteins of the caveolin family, and cytoplasmic proteins of the cavin family. Cationic peptide dendrimers are non-vesicular chemical carriers that can transport pharmacological agents or genetic material across the plasma membrane. We prepared a panel of cationic dendrimers and investigated whether they require caveolae to enter into cells. Cell-based studies were performed using wild type or caveola-deficient i.e. caveolin-1 or PTRF gene-disrupted cells. There was a statistically significant difference in entry of cationic dendrimers between wild type and caveola-deficient cells. We further unveiled differences between dendrimers with varying charge density and head groups. Our results show, using a molecular approach, that (i) expression of caveola-forming proteins promotes cellular entry of cationic dendrimers and (ii) dendrimer structure can be modified to promote endocytosis in caveola-forming cells.     

Applications of dendrimers in bio-organic chemistry

Dendrimers represent a new class of highly branched polymers whose interior cavities and multiple peripheral groups facilitate potential applications in biomedicine and bio-organic chemistry. Major advances in the past year were made in the synthesis and study of new carbohydrate, nucleic acid, and peptide dendrimers, as well as in the use of dendrimers as magnetic resonance imaging contrast agents, as agents for cellular delivery of nucleic acids, and as scaffolds for biomimetic systems.     

Peptide and glycopeptide dendrimers. Part I

Recent progress in peptide and glycopeptide chemistry make the preparation of peptide and glycopeptide dendrimers of acceptable purity, with designed structural and immunochemical properties reliable. New methodologies using unprotected peptide building blocks have been developed to further increase possibilities of their design and improve their preparation and separation. Sophisticated design of peptide and glycopeptide dendrimers has led to their use as antigens and immunogens, for serodiagnosis and other biochemical uses including drug delivery. Dendrimers bearing peptide with predetermined secondary structures are useful tools in protein de novo design. This article covers synthesis and applications of multiple antigen peptides (MAPs), multiple antigen glycopeptides (MAGs), multiple antigen peptides based on sequential oligopeptide carriers (MAP‐SOCs), glycodendrimers and template‐assembled synthetic proteins (TASPs). Part I deals with the development of various structural forms of MAPs as well as their application as antigens, immunogens, and for immunodiagnostic and biochemical purposes. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Photoinduced hydrogen evolution with peptide dendrimer-multi-Zn(II)-porphyrin, viologen, and hydrogenase

To construct an artificial photosynthetic system, multi-Zn(II)-mesoporphyrins in peptide dendrimers were equipped as a photosensitizer of photoinduced hydrogen evolution in a four-component system (electron donor, photosensitizer, electron carrier, and catalyst), so that hydrogen was evolved effectively by the dendrimer architecture, for the first time. The hydrogen evolution activity was correlated to the photoreduction ability of viologen by the Zn-porphyrin-peptide dendrimers. Additionally, using positively charged methyl-viologen as an electron carrier, the photoinduced hydrogen evolution function with the positively charged peptide dendrimer was superior to that with the negatively charged peptide dendrimer, despite that the positive dendrimer did not strongly bind the positively charged methyl-viologen with the electrostatic interaction. By contrast, when zwitterionic propylviologen sulfonate was used, photoreduction and hydrogen evolution properties were identical between the positively and the negatively charged dendrimers. These results demonstrated that the dynamic interaction between the positive dendrimer and methyl-viologen was preferable for the photoreduction and hydrogen evolution, and that the three-dimensional assembly of Zn(II)-mesoporphyrins using the peptide dendrimers was effective as a photosensitizer in the artificial photosynthesis.     

Biological applications of dendrimers

In the past year, significant advances have been made in the synthesis and study of glycodendrimers and peptide dendrimers. Application of these dendrimers to the study of carbohydrate-protein and protein-protein interactions has facilitated the understanding of these processes. In addition, dendrimers show great promise as DNA- and drug-delivery systems.     

Peptide dendrimers.

Dendrimers are branched structures and represent a fast growing field covering many areas of chemistry. Various types of dendrimers differing in composition and structure are mentioned, together with their practical use spanning from catalysis, transport vehicles to synthetic vaccines. The main stress is given to peptide dendrimers, namely, multiple antigenic peptides (MAPs). Their synthesis, physicochemical properties, biological activities, etc. have been described with many examples. MAPs can be used as diagnostics, mimetics, for complexation of different cations, as vaccines against parasites, bacteria, viruses, etc.     

Combinatorial synthesis, selection, and properties of esterase peptide dendrimers

A 65,536-member combinatorial library of peptide dendrimers was prepared by split-and-mix synthesis and screened on solid support for esterolytic activity in aqueous buffer using 8-butyryloxypyrene-1,3,6-trisulfonate (2) as a fluorogenic substrate. Active sequences were identified by analysis of fluorescent beads. The corresponding dendrimers were resynthesized by solid-phase synthesis, cleaved from the resin, and purified by preparative reverse-phase HPLC. The dendrimers showed the expected catalytic activity in aqueous buffer. Catalysis was studied against a pannel of fluorogenic 8-acyloxypyrene-1,3,6-trisulfonate substrates. The catalytic peptide dendrimers display enzyme-like kinetics in aqueous buffer with substrate binding in the range K(M) approximately 0.1 mM, catalytic rate constants k(cat) approximately 0.1 min(-1), and specific rate accelerations over background up to k(cat)/k(uncat) = 10,000.     

The characterization of a novel dendritic system for gene delivery by isothermal titration calorimetry

Understanding the nature of binding of polycationic dendrimers to DNA provides useful information on their role in gene delivery. In the present study, we have characterized the interaction of several peptide-based polycationic dendrimers with salmon sperm DNA using isothermal titration calorimetry. The dendrimers consisted of the cell penetrating peptide TAT, a nuclear localization signal peptide and dendritic polylysine. The binding affinity and thermodynamic parameters were found to increase as the number of positive charges on the dendrimer increased, indicating that ionic interactions were the major binding forces between the two molecules. The effect of acidic pH (3.2) compared to a more neutral pH (7.2) was also examined. The binding affinity was stronger at the lower pH but precipitation of the complex was more prominent at pH 7.2 which was shown by large enthalpies. The results indicate that our dendrimers are forming stable complexes with DNA.     

Synthetic peptides in the form of dendrimers become resistant to protease activity

In previous papers, we observed that dendrimers of peptide mimotopes of the nicotinic receptor ligand site are strong antidotes against the lethality of the nicotinic receptor ligand alpha-bungarotoxin. Although their in vitro activity is identical to that of dendrimers, the corresponding monomeric peptide mimotopes are not effective in vivo. Because the higher in vivo efficiency of dendrimers could not in this case be related to polyvalent interaction, the stability to blood protease activity of monomeric versus tetrabranched dendrimeric mimotope peptides was compared here by incubating three different mimotopes with human plasma and serum. Unmodified peptides and cleaved sequences were followed by high pressure liquid chromatography and mass spectrometry. Tetrabranched peptides were shown to be much more stable in plasma and also in serum. To assess the notable stability of multimeric peptides, different bioactive neuropeptides, including enkephalins, neurotensin and nociceptin, were synthesized in monomeric and tetrabranched forms and incubated with human plasma and serum and with rat brain membrane extracts. All the tetrabranched neuropeptides fully retained biological activity and generally showed much greater stability to blood and brain protease activity. Some tetrabranched peptides were also resistant to trypsin and chymotrypsin. Our findings provide new insights into the possible therapeutic use of bioactive peptides.     

Temperature‐sensitive elastin‐mimetic dendrimers: Effect of peptide length and dendrimer generation to temperature sensitivity

Dendrimers are synthetic macromolecules with unique structure, which are a potential scaffold for peptides. Elastin is one of the main components of extracellular matrix and a temperature‐sensitive biomacromolecule. Previously, Val‐Pro‐Gly‐Val‐Gly peptides have been conjugated to a dendrimer for designing an elastin‐mimetic dendrimer. In this study, various elastin‐mimetic dendrimers using different length peptides and different dendrimer generations were synthesized to control the temperature dependency. The elastin‐mimetic dendrimers formed β‐turn structure by heating, which was similar to the elastin‐like peptides. The elastin‐mimetic dendrimers exhibited an inverse phase transition, largely depending on the peptide length and slightly depending on the dendrimer generation. The elastin‐mimetic dendrimers formed aggregates after the phase transition. The endothermal peak was observed in elastin‐mimetic dendrimers with long peptides, but not with short ones. The peptide length and the dendrimer generation are important factors to tune the temperature dependency on the elastin‐mimetic dendrimer. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 603–612, 2014.     

Diffusion and conformation of peptide-functionalized polyphenylene dendrimers studied by fluorescence correlation and 13C NMR spectroscopy

We report on the combined use of fluorescence correlation spectroscopy (FCS) and 1H and 13C NMR spectroscopy to detect the size and type of peptide secondary structures in a series of poly-Z-L-lysine functionalized polyphenylene dendrimers bearing the fluorescent perylenediimide core in solution. In dilute solution, the size of the molecule as detected from FCS and 1H NMR diffusion measurements matches nicely. We show that FCS is a sensitive probe of the core size as well as of the change in the peptide secondary structure. However, FCS is less sensitive to functionality. A change in the peptide secondary conformation from beta-sheets to alpha-helices detected by 13C NMR spectroscopy gives rise to a steep increase in the hydrodynamic radii for number of residues n > or = 16. Nevertheless, helices are objects of low persistence.     

Peptide and glycopeptide dendrimers and analogous dendrimeric structures and their biomedical applications

The size of information that can be stored in nucleic acids, proteins, and carbohydrates was calculated. The number of hexamers for peptides is 64,000,000 (20⁶) and seems to be impressive in comparison with 4,096 (4⁶) hexanucleotides, but the number of isomers of hexasaccharides is 1.44 × 10¹⁵. Carbohydrates are therefore the best high-density coding system. This language has been named glycocode resp. sugar code. In comparison with peptide dendrimers, the amount of information carried by glycopeptide dendrimers or glycodendrimers is therefore much higher. This is reflected by the variability of structures and functions (activities). This review is about the broad area of peptide and glycopeptide dendrimers. The dendrimeric state and physicochemical properties and general consequences are described, together with a cluster effect. The impact of cluster effect to biological, chemical, and physical properties is discussed. Synthesis of dendrimers by convergent and divergent approaches, “Lego” chemistry, ligation strategies, and click chemistry is given with many examples. Purification and characterization of dendrimers by chromatographic methods, electromigration methods, and mass spectrometry are briefly mentioned. Different types of dendrimers with cyclic core, i.e. RAFTs, TASPs and analogous cyclic structures, carbopeptides, carboproteins, octopus glycosides, inositol-based dendrimers, cyclodextrins, calix[4]arenes, resorcarenes, cavitands, and porphyrins are given. Dendrimers can be used for creation of libraries, catalysts, and solubilizing agents. Biocompatibility and toxicity of dendrimers is discussed, as well as their applications in nanoscience, nanotechnology, drug delivery, and gene delivery. Carbohydrate interactions of glycopeptide dendrimers (bacteria, viruses, and cancer) are described. Examples of dendrimers as anti-prion agents are given. Dendrimers represent a fast developing area which partly overlaps with nanoparticles and nanotechnologies.     

A direct method for the formation of peptide and carbohydrate dendrimers.

Two new methods for the modification of PAMAM dendrimers have been developed which allow the covergent synthesis of either peptide or carbohydrate-bearing dendrimer molecules. Both methods involve condensation between hydroxylamino nucleophiles and appropriate carbonyl-bearing reaction partners.     

Polyphenylene dendrimers as scaffolds for shape-persistent multiple peptide conjugates

The present work describes synthetic concepts for the coupling of peptides to polyphenylene dendrimers (PPDs). Novel functionalized cyclopentadienones have been synthesized whose Diels-Alder cycloaddition with various core molecules leads to polyphenylene dendrimers possessing (protected) amino or carboxyl groups. In addition, the resulting functionalized molecules exhibit the characteristic shape-persistence and monodispersity of PPDs. Their functions have been used for the attachment of polylysine to the dendritic scaffold. Three different methods for the decoration of dendrimers with polypeptides are presented. First, polylysine segments are grafted from the surface of the dendrimers employing alpha-amino acid N-carboxyanhydride (NCA) polymerization. Second, the C-terminal carboxyl groups of protected polypeptides are activated and then coupled to the amino groups on the surface of the PPD. Finally, cysteine terminated, unprotected peptide sequences are attached to polyphenylene dendrimers utilizing the addition of the sulfhydryl group of a cysteine to the maleimide functions on the dendrimer surface. Moreover, Diels-Alder cycloaddition of suitably functionalized cyclopentadienons to a desymmetized core molecule allows the design of a dendritic scaffold with a specific number of different anchor groups on its periphery. These approaches are important for the tailoring of new, shape-persistent, polyfunctional multiple antigen conjugates.     

Efficient orthogonal bioconjugation of dendrimers for synthesis of bioactive nanoparticles

Nanoparticles carrying biologically active functional sets (e.g., targeting moiety, payload, tracer) have potential use in a wide range of clinical applications. Though complex, such constructions should, as far as possible, have a defined molecular architecture and be monodisperse. However, the existing methods to achieve this goal are unsuitable for the incorporation of peptides and proteins, and those that provide for orthogonal introduction of two different types of functional element are incompatible with the use of commercially available materials. In this study, we have developed approaches for the production of nanoparticles based on commercially available polyamidoamine (PAMAM) dendrimers. First, we identified an optimized oxime conjugation strategy under which complex dendrimers can be fully decorated not only with model peptides, but also with recombinant proteins (insulin was taken as an example). Second, we developed a strategy based on a two-chain covalent heterodendrimer (a "diblock") based on cystamine core PAMAM dendrimers and used it to generate heterodendrimers, into which a peptide array and a mannose array were orthogonally introduced. Finally, by incorporating a functionalized linker into the diblock architecture we were able to site-specifically introduce a third functional element into the nanoparticle. We exemplified this approach using fluorescein, a mannose array, and a peptide array as the three functionalities. We showed that incorporation of a mannose array into a nanoparticle strongly and specifically enhances uptake by sentinel cells of the immune system, an important property for vaccine delivery applications. These PAMAM dendrimer-based approaches represent a robust and versatile platform for the development of bioactive nanoparticles.     

Antibacterial activities of poly(amidoamine) dendrimers terminated with amino and poly(ethylene glycol) groups

Poly(amidoamine) (PAMAM) dendrimer derivatives have been investigated for their biological applications, especially for delivery of drugs, including antimicrobial drugs to eukaryotic cells, but their effects on bacterial cells are largely unexplored. Herein we report that amino-terminated PAMAM dendrimers are highly toxic to the common Gram-negative pathogen Pseudomonas aeruginosa. The concentration that kills 50% of the bacteria (EC50) was in the range of approximately 0.9-1.5 microg/mL for the generation 5, amino-terminated dendrimers with or without partial (43%) coating of poly(ethylene glycol) (PEG). These EC50 values were lower than that ( approximately 1.9-2.8 microg/mL) for LL-37, a potent antimicrobial peptide expressed in a variety of epithelia. On the contrary, the dendrimers were far less toxic (EC50 > 21 microg/mL) to the Gram-positive pathogen Staphylococcus aureus than LL-37 (EC50 = approximately 1.9 microg/mL). In agreement with the previous studies on other cell types, the dendrimers were not cytotoxic to human corneal epithelial cells at the concentrations that were toxic to P. aeruginosa. Our findings indicate that amino-terminated PAMAM dendrimers and their partially PEG-coated derivatives possess attractive antimicrobial properties, particularly against Gram-negative bacteria, thus expanding the potential biological application of the dendrimers.     

Synthesis, isolation and characterization of Plasmodium falciparum antigenic tetrabranched peptide dendrimers obtained by thiazolidine linkages.

Different chemical alternatives were evaluated for obtaining immunogenic polypeptidic macromolecules which could then be used as vaccines. These were based on the ligation reaction between an unprotected immunogenic peptide and an unprotected multifunctional core peptide; polyantigens, designated dendrimers because their form resembles that of dendritic cells, were thus obtained. The antigen-core ligation alternatives, studied by indirect synthesis, were the formation of oxime, hydrazone and thiazolidine linkages, making use of the reaction between a weak base (acting as nucleophile) and an alkyl aldehyde. The other alternative was the formation of a thioether linkage between a sulfydryl and an alkyl halide. Finally, a multiple antigen peptide (MAP) was synthesized by direct synthesis. All reactions were monitored by SEC-HPLC and SDS-PAGE. Dendrimer molecular mass obtained was confirmed by MS MALDI-TOF. Dendrimer purification was first carried out by concentrating crude reaction products with CP-5000 centricons and (using SEC-HPLC) pure tetramers were then obtained. A 20-residue 9376 immunogenic sequence, from Plasmodium falciparum apical merozoite antigen protein (AMA-1), was used to study the best alternative for chemical ligation. It was observed that thiazolidine formation proceeded with greater yield and in less time than the others. A tetramer has been simultaneously synthesized via thiazolidine with the SPf-66 antimalarial vaccine 45-residue monomer, proving the technique's versatility. The 9376 peptide disulfide bound polymer and SPf-66 (as well as their tetrameric thiazolidine dendrimers) were inoculated in rabbits to evaluate their antibody response. It was observed that titers for tetrameric thiazolidine dendrimers were not just greater but were also sustained over time. Western blot for pre-immune and immune sera showed that dendrimer sera recognized specific Plasmodium falciparum proteins as well as disulfide-bound polymers.     

Low‐generation asymmetric dendrimers exhibit minimal toxicity and effectively complex DNA

Conventional dendrimers are spherical symmetrically branched polymers ending with active surface functional groups. Polyamidoamine (PAMAM) dendrimers have been widely studied as gene delivery vectors and have proven effective at delivering DNA to cells in vitro. However, higher‐generation (G4‐G8) PAMAM dendrimers exhibit toxicity due to their high cationic charge density and this has limited their application in vitro and in vivo. Another limitation arises when attempts are made to functionalize spherical dendrimers as targeting moieties cannot be site‐specifically attached. Therefore, we propose that lower‐generation asymmetric dendrimers, which are likely devoid of toxicity and to which site‐specific attachment of targeting ligands can be achieved, would be a viable alternative to currently available dendrimers. We synthesized and characterized a series of peptide‐based asymmetric dendrimers and compared their toxicity profile and ability to condense DNA to spherical PAMAM G1 dendrimers. We show that asymmetric dendrimers are minimally toxic and condense DNA into stable toroids which have been reported necessary for efficient cell transfection. This paves the way for these systems to be conjugated with targeting ligands for gene delivery in vitro and in vivo. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.