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.     

Structural studies of biologically active glycosylated polyamidoamine (PAMAM) dendrimers

The partial modification of carboxylic acid terminated polyamidoamine (PAMAM) dendrimers with glucosamine has been reported to give dendrimer glucosamine conjugates novel immuno-modulatory and anti-angiogenic properties. Experimental analysis of these glycosylated dendrimers showed that, on average, eight glucosamine molecules were covalently bound to each dendrimer. In order to better understand the surface loading and distribution of these glucosamine molecules, molecular reactivity was determined by evaluation of electronic properties using frontier molecular orbital theory (FMOT) and molecular dynamics simulations. It was shown that the surface loading and distribution of zero length amide bond-conjugated glucosamine molecules was determined by both electronic effects and by the different dynamic conformations adopted by the modified dendrimer during the incremental addition of glucosamine. Importantly, the structural features and the dynamic behavior of the partially glycosylated generation 3.5 PAMAM dendrimer showed that its flexibility and polarity changed with the incremental addition of glucosamine. These peripheral glucosamine molecules remained available on the dendrimer’s surface for interaction with the biological target.     

Synthesis of polyamidoamine dendrimers having poly(ethylene glycol) grafts and their ability to encapsulate anticancer drugs

Polyamidoamine dendrimers having poly(ethylene glycol) grafts were designed as a novel drug carrier which possesses an interior for the encapsulation of drugs and a biocompatible surface. Poly(ethylene glycol) monomethyl ether with the average molecular weight of 550 or 2000 was combined to essentially every chain end of the dendrimer of the third or fourth generation via urethane bond. The poly(ethylene glycol)-attached dendrimers encapsulating anticancer drugs, adriamycin and methotrexate, were prepared by extraction with chloroform from mixtures of the poly(ethylene glycol)-attached dendrimers and varying amounts of the drugs. Their ability to encapsulate these drugs increased with increasing dendrimer generation and chain length of poly(ethylene glycol) grafts. Among the poly(ethylene glycol)-attached dendrimers prepared, the highest ability was achieved by the dendrimer of the fourth generation having the poly(ethylene glycol) grafts with the average molecular weight of 2000, which could retain 6.5 adriamycin molecules or 26 methotrexate molecules/dendrimer molecule. The methotrexate-loaded poly(ethylene glycol)-attached dendrimers released the drug slowly in an aqueous solution of low ionic strength. However, in isotonic solutions, methotrexate and adriamycin were readily released from the poly(ethylene glycol)-attached dendrimers.     

Influence of dendrimers on red blood cells

Dendrimers, highly branched macromolecules with a specific size and shape, provide many exciting opportunities for biomedical applications. However, most dendrimers demonstrate toxic and haemolytic activity because of their positively charged surface. Masking the peripheral cationic groups by coating them with biocompatible molecules is a method to reduce it. It was proven that modified dendrimers can even diminish haemolytic activity of encapsulated drugs. Experiments confirmed that anionic dendrimers are less haemotoxic than cationic ones. Due to the high affinity of dendrimers for serum proteins, presence of these components in an incubation buffer might also influence red blood cell (RBC)-dendrimer interactions and decrease the haemolysis level. Generally, haemotoxicity of dendrimers is concentration-, generation-, and time-dependent. Various changes in the RBCs’ shape in response to interactions with dendrimers have been observed, from echinocytic transformations through cell aggregation to cluster formation, depending on the dendrimer’s type and concentration. Understanding the physical and chemical origins of dendrimers’ influences on RBCs might advance scientists’ ability to construct dendrimers more suitable for medical applications.     

Paramagnetic NMR Investigation of Dendrimer-Based Host-Guest Interactions

In this study, the host-guest behavior of poly(amidoamine) (PAMAM) dendrimers bearing amine, hydroxyl, or carboxylate surface functionalities were investigated by paramagnetic NMR studies. 2,2,6,6-Tetramethylpiperidinyloxy (TEMPO) derivatives were used as paramagnetic guest molecules. The results showed that TEMPO-COOH significantly broaden the ¹H NMR peaks of amine- and hydroxyl-terminated PAMAM dendrimers. In comparison, no paramagnetic relaxation enhancement (PRE) was observed between TEMPO-NH₂, TEMPO-OH and the three types of PAMAM dendrimers. The PRE phenomenon observed is correlated with the encapsulation of TEMPO-COOH within dendrimer pockets. Protonation of the tertiary amine groups within PAMAM dendrimers plays an important role during this process. Interestingly, the absence of TEMPO-COOH encapsulation within carboxylate-terminated PAMAM dendrimer is observed due to the repulsion of TEMPO-COO- anion and anionic dendrimer surface. The combination of paramagnetic probes and ¹H NMR linewidth analysis can be used as a powerful tool in the analysis of dendrimer-based host-guest systems.     

Effect of size, surface charge, and hydrophobicity of poly(amidoamine) dendrimers on their skin penetration

The barrier functions of the stratum corneum and the epidermal layers present a tremendous challenge in achieving effective transdermal delivery of drug molecules. Although a few reports have shown that poly(amidoamine) (PAMAM) dendrimers are effective skin-penetration enhancers, little is known regarding the fundamental mechanisms behind the dendrimer-skin interactions. In this Article, we have performed a systematic study to better elucidate how dendrimers interact with skin layers depending on their size and surface groups. Franz diffusion cells and confocal microscopy were employed to observe dendrimer interactions with full-thickness porcine skin samples. We have found that smaller PAMAM dendrimers (generation 2 (G2)) penetrate the skin layers more efficiently than the larger ones (G4). We have also found that G2 PAMAM dendrimers that are surface-modified by either acetylation or carboxylation exhibit increased skin permeation and likely diffuse through an extracellular pathway. In contrast, amine-terminated dendrimers show enhanced cell internalization and skin retention but reduced skin permeation. In addition, conjugation of oleic acid to G2 dendrimers increases their 1-octanol/PBS partition coefficient, resulting in increased skin absorption and retention. Here we report that size, surface charge, and hydrophobicity directly dictate the permeation route and efficiency of dendrimer translocation across the skin layers, providing a design guideline for engineering PAMAM dendrimers as a potential transdermal delivery vector.     

Enzymatic activation of hydrophobic self-immolative dendrimers: The effect of reporters with ionizable functional groups

Self-immolative dendrimers are uniquely structured molecules that release multiple tail units through a chain fragmentation initiated by a single cleavage at the dendrimer’s core. Although bioactivation of self-immolative dendritic molecules with only two reporter groups was demonstrated, enzymatic activation failed for self-immolative dendrimers with more reporters. These large and hydrophobic dendrimers aggregated under aqueous conditions and enzyme did not efficiently trigger chain fragmentation. Here we demonstrate a simple solution to the problem of enzymatic activation of hydrophobic self-immolative dendrimers. The reporter units on the dendritic platform were equipped with ionizable functional group. Polar interactions with water significantly decreased hydrophobicity of the dendrimers and prevented aggregate formation. Consequently, hydrophobic self-immolative dendrons were effectively activated.     

树状分子在提高免疫分析性能方面的应用进展

基于抗原-抗体识别的免疫分析技术在小分子化学性污染物监测领域占有重要地位,已成功应用于农药、兽药、生物毒素等的快速检测,为保障食品安全发挥了重要作用.但是,如何提高小分子半抗原的免疫原性及抗体的亲和力仍然是制约该领域发展的关键技术瓶颈.树状分子作为一类新型的高分子化合物,具有分子组成明确、结构规整、高度支化、纳米尺寸、单分散性以及表面呈现高密度功能团等众多优良的结构特性和良好的组织相容性,在小分子免疫分析领域具有潜在的应用优势.本文主要综述了树状分子作为载体在抗体制备及免疫分析方面的应用,重点介绍了树状分子作为载体在免疫原及包被原制备、作为免疫佐剂提高抗原免疫原性以及作为信号放大载体在提高免疫分析灵敏度等方面的研究现状,最后对其在小分子化学性污染物免疫检测领域的应用前景进行了评述,期望能为本领域研究人员提供借鉴.     

Guanidino- and urea-modified dendrimers as potent solubilizers of misfolded prion protein aggregates under non-cytotoxic conditions. dependence on dendrimer generation and surface charge

Amino-terminated dendrimers are well-defined synthetic hyperbranched polymers and have previously been shown to destabilize aggregates of the misfolded, pathogenic, and partially protease-resistant form of the prion protein (PrPSc), transforming it into a partially dissociated, protease-sensitive form with strongly reduced infectivity. The mechanism behind this is not known, but a low pH, creating multiple positively charged primary amines on the dendrimer surface, increases the efficiency of the reaction. In the present study, surface amines of the dendrimers were modified to yield either guanidino surface groups (being positively charged at neutral pH) or urea groups (uncharged). The ability of several generations of modified dendrimers and unmodified amino-terminated dendrimers to deplete PrPSc from persistently PrPSc-infected cells in culture (SMB cells) was studied. It was found that destabilization correlated with both the generation number of the dendrimer, with higher generations being more efficient, and the charge density of the surface groups. Urea-decorated dendrimers having an uncharged surface were less efficient than positively charged unmodified- (amino) and guanidino-modified dendrimers. The most efficient dendrimers (generation 4 (G4) and G5-unmodified and guanidino dendrimers) cleared PrPSc completely by incubation for 4 days at less than 50 nM. In contrast to both unmodified and guanidine-modified dendrimers, the uncharged urea dendrimers showed much lower cytotoxicity toward noninfected SMB cells. Therapeutic uses of modified dendrimers are indicated by the low concentrations of dendrimers needed.     

Carbohydrate-functionalized dendrimers to investigate the predictable tunability of multivalent interactions

Multivalent protein-carbohydrate interactions mediate a wide variety of intercellular recognition processes with high selectivity and specificity. Many synthetic multivalent molecules have been designed to mimic and to inhibit these processes. Using carbohydrate functionalized dendrimers, our goal is to devise a system where the binding activity and the degree of protein clustering induced by the glycopolymer can be readily attenuated. In this paper, dendrimers were functionalized with mixtures of mannose, glucose, and galactose. Their association with concanavalin A was studied using precipitation and hemagglutination assays. With less idealized systems where the association was not optimized, mixtures of low- and high-affinity ligands caused smaller than the theoretically determined differences in binding activity, although linear binding trends were observed. When systems were optimized so that high-affinity binding was achieved, then mixing low- and high-affinity ligands on the dendrimer's surface showed a predictable trend for lectin binding.     

Interactions between PAMAM dendrimers and bovine serum albumin

Dendrimers are a new class of polymeric materials. They are globular, highly branched, monodisperse macromolecules. Due to their structure, dendrimers promise to be new, effective biomedical materials as oligonucleotide transfection agents and drug carriers. More information about biological properties of dendrimers is crucial for further investigation of dendrimers in therapeutic applications.In this study the mechanism of interactions between polyamidoamine (PAMAM) dendrimers and bovine serum albumin (BSA) was examined. PAMAM dendrimers are based on an ethylenediamine core and branched units are constructed from both methyl acrylate and ethylenediamine. We used three types of PAMAM dendrimers with different surface groups (-COOH, -NH(2), -OH). As BSA contains two tryptophan residues we were able to evaluate dendrimers influence on protein molecular conformation by measuring the changes in the fluorescence of BSA in the presence of dendrimers. Additionally experiments with a fluorescent probe 1-anilinonaphthalene-8-sulfonic acid (ANS) were carried out. The differential scanning calorimetry (DSC) was chosen to investigate impact on protein thermal stability upon the dendrimers.Our experiments showed that the extent of the interactions between BSA and dendrimers strongly depends on their surface groups and is the biggest for amino-terminated dendrimers.     

Using starburst dendrimers as linker molecules to radiolabel antibodies

Starburst dendrimers, spherical polymers constructed from methyl acrylate and ethylenediamine, were successfully used to covalently couple synthetic porphyrins to antibody molecules. The dendrimers, as linker molecules, have great potential for increasing the specific activity of radiolabeled antibodies for tumor therapy and diagnosis.     

Potent antioxidant dendrimers lacking pro-oxidant activity

It is well known that antioxidants have protective effects against oxidative stress. Unfortunately, in the presence of transition metals, antioxidants, including polyphenols with potent antioxidant activities, may also exhibit pro-oxidant effects, which may irreversibly damage DNA. Therefore, antioxidants with strong free radical-scavenging abilities and devoid of pro-oxidant effects would be of immense biological importance. We report two antioxidant dendrimers with a surface rich in multiple phenolic hydroxyl groups, benzylic hydrogens, and electron-donating ring substituents that contribute to their potent free radical-quenching properties. To minimize their pro-oxidant effects, the dendrimers were designed with a metal-chelating tris(2-aminoethyl)amine (TREN) core. The dendritic antioxidants were prepared by attachment of six syringaldehyde or vanillin molecules to TREN by reductive amination. They exhibited potent radical-scavenging properties: 5 times stronger than quercetin and 15 times more potent than Trolox according to the 1,1-diphenyl-2-picrylhydrazyl assay. The antioxidant dendrimers also protected low-density lipoprotein, lysozyme, and DNA against 2,2'-azobis(2-amidinopropane) dihydrochloride-induced free radical damage. More importantly, unlike quercetin and Trolox, the two TREN antioxidant dendrimers did not damage DNA via their pro-oxidant effects when incubated with physiological amounts of copper ions. The dendrimers also showed no cytotoxicity toward Chinese hamster ovary cells.     

Interaction of multicomponent anionic liposomes with cationic pyridylphenylene dendrimer: Does the complex behavior depend on the liposome composition?

Dendrimers are individual macromolecular compounds having a great potential for biomedical application. The key step of the cell penetration by dendrimers is the interaction with lipid bilayer. Here, the interaction between cationic pyridylphenylene dendrimer of third generation (D₃⁵⁰⁺) and multicomponent liquid (CL/POPC), solid (CL/DPPC) and cholesterol-containing (CL/POPC/30% Chol) anionic liposomes was investigated by dynamic light scattering, fluorescence spectroscopy, conductometry, calorimetric studies and molecular dynamic (MD) simulations. Microelectrophoresis and MD simulations revealed the interaction is electrostatic and reversible with only part of pyridinium groups of dendrimers involved in binding with liposomes. The ability of dendrimer molecules to migrate between liposomes was discovered by the labeling liposomes with Rhodamine B. The phase state of the lipid membrane and the incorporation of cholesterol into the lipid bilayer were found to not affect the mechanism of the dendrimer - liposome complex formation. Rigid dendrimer adsorption on liposomal surface does not induce the formation of significant defects in the lipid membrane pave the way for possible biological application of pyridylphenylene dendrimers.     

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.     

Surface acetylation of polyamidoamine (PAMAM) dendrimers decreases cytotoxicity while maintaining membrane permeability

Improving the oral bioavailability of therapeutic compounds remains a challenging area of research. Polyamidoamine (PAMAM) dendrimers are promising candidates for oral drug delivery due to their well-defined compact structure, versatility of surface functionalities, low polydispersity, and ability to enhance transepithelial transport. However, potential cytotoxicity has hampered the development of PAMAM dendrimers for in vivo applications. In this article, we have systematically modified the surface groups of amine-terminated PAMAM dendrimers with acetyl groups. The effect of this modification on cytotoxicity, permeability, and cellular uptake was investigated on Caco-2 cell monolayers. Cytotoxicity was reduced by more than 10-fold as the number of surface acetyl groups increased while maintaining permeability across the cell monolayers. Furthermore, a decrease in nonspecific binding was evident for surface-modified dendrimers compared to their unmodified counterparts. These studies point to novel strategies for minimizing PAMAM dendrimer toxicity while maximizing their transepithelial permeability.     

Preparation of poly(ethylene glycol)-attached dendrimers encapsulating photosensitizers for application to photodynamic therapy

Photodynamic therapy (PDT) is a noninvasive treatment of some diseases including cancer. We have developed poly(ethylene glycol) (PEG)-attached dendrimers as a drug-carrier candidate. In this study, we prepared nanocapsules of photosensitizers using PEG-attached dendrimers for application to PDT. Two PEG-attached dendrimers derived from poly(amido amine) (PAMAM) and poly(propylene imine) (PPI) dendrimers (PEG-PAMAM and PEG-PPI) were synthesized, and rose bengal (RB) and protoporphyrin IX (PpIX) were used as photosensitizers. Results showed that fewer PpIX molecules were encapsulated by both PEG-attached dendrimers than RB, but the complexes were more stable under physiological conditions. Furthermore, we demonstrated that PEG-PPI held photosensitizers in a more stable manner than PEG-PAMAM because of their inner hydrophobicity. We described the cytotoxicity of the complexes of photosensitizers induced by light irradiation in vitro. The complex of PpIX with PEG-PPI exhibited efficient cytotoxicity, compared with free PpIX. It was suggested that the cytotoxicity was caused by the high level of singlet oxygen production and the efficient delivery to mitochondria. Our results suggest that these PEG-attached dendrimers are a promising vehicle for PDT.     

Enzymatic activation of second-generation dendritic prodrugs: Conjugation of self-immolative dendrimers with poly(ethylene glycol) via click chemistry

Single-triggered disassemble dendrimers were recently developed and introduced as a potential platform for a multi-prodrug. These unique structural dendrimers can release all of their tail units through a self-immolative chain fragmentation initiated by a single cleavage at the dendrimer's core. There are several examples for the bioactivation of first-generation self-immolative dendritic prodrugs. However, enzymatic activation failed for second-generation self-immolative dendrimers. The hydrophobic large molecular structure of the dendritic prodrugs results in aggregation under aqueous conditions and prevented the enzyme from reaching the triggering substrate. Here we show a simple solution for the enzymatic activation of second-generation self-immolative dendrimers. Poly(ethylene glycol) (PEG) was conjugated to the dendritic platform via click chemistry. The poly(ethylene glycol) tails significantly decreased the hydrophobic properties of the dendrimers and thereby prevented aggregate formation. We designed and synthesized a dendritic prodrug with four molecules of the anticancer agent camptothecin and a trigger that can be activated by penicillin-G-amidase. The PEG5000-conjugated, self-immolative dendritic prodrug was effectively activated by penicillin-G-amidase under physiological conditions and free camptothecin was released to the reaction media. Cell-growth inhibition assays demonstrated increased toxicity of the dendritic prodrug upon incubation with the enzyme.     

Heavy metal-free 19F NMR probes for quantitative measurements of glutathione reductase activity using silica nanoparticles as a signal quencher

For the quantitative assessment of the glutathione reductase (GR) activity with a (19)F NMR spectroscopy, we developed the heavy metal-free probes based on silica nanoparticles modified with water-soluble perfluorinated dendrimers via the disulfide linkers. Before enzymatic reaction, the molecular rotation of the perfluorinated dendrimers is highly restricted, and the magnitude of (19)F NMR signals from the perfluorinated dendrimers can be suppressed. By the reductive cleavage of the disulfide linkers with the reduced glutathione-mediated enzymatic reaction of GR, perfluorinated dendrimers can be released from the surfaces of the nanoparticles. Consequently, the (19)F NMR signals of perfluorinated dendrimers were recovered. The enzymatic activity of GR was determined from the increase of the magnitude of (19)F NMR signals. Finally, to demonstrate the feasibility of the probe in the presence of miscellaneous molecules under bio-mimetic conditions, the comparison study was executed with the cancer cell lysate. The value determined from our method showed a good agreement with that from the conventional method.     

Partitioning of model toxins to hydrophobically terminated DAB dendrimers

Dendrimers are attractive in biological and biomedical applications due to the similarity in their molecular size to biologically relevant molecules and the large number of chain ends available functionalization. In the current work, we examined the potential of diamino butane (DAB) dendrimers functionalized with long alkyl chains as partitioning agents for hydrophobic toxins for use as a prefiltering stage in a bioartiticial liver. DAB dendrimers of various generations that had been previously fully modified with palmitoyl chloride were obtained. A study of the kinetics of partitioning of acetylsalicylic acid (ASA) suggested that while significant toxin removal occurred in 30 s, although a slight time dependent increase in removal was noted up to 60 minutes. The partitioning of 6 hydrophobic toxins from aqueous solution to the modified dendrimers in 30 minutes was examined. The results demonstrated that a number of factors, including the pKa of the toxin, its octanol water partitioning coefficient and molecular size contributed to the level of toxin removal. Toxin removal on a molar basis increased with increasing dendrimer generation for all toxins, with the modified G5 dendrimers partitioning 50-100 toxin molecules in most cases. Dendrimer modification with C4 alkyl chains rather than Cl5 chains significantly decreased toxin removal, although chains longer than C10 seemed to partition equal amounts of toxins. The results of the study demonstrate that water-soluble dendrimers modified with hydrophobic end groups may be useful for the removal of toxins from the blood in a prereaction step for a bioartificial liver, but that a better understanding of the molecular mechanisms of removal may be necessary before it is possible to predict the levels of toxin removal.