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.     

Synthesis and evaluation of novel dendrimers with a hydrophilic interior as nanocarriers for drug delivery

Novel polyester-co-polyether dendrimers consisting of a hydrophilic core were synthesized by a combination of convergent and divergent syntheses. The core was synthesized from biocompatible moieties, butanetetracarboxylic acid and aspartic acid, and the dendrons from PEO (poly(ethylene oxide)), dihydroxybenzoic acid or gallic acid, and PEG monomethacrylate. The dendrimers, Den-1-(G 2) (second generation dendrimer-1) and Den-2-(G 2) (second generation dendrimer-2) consisting of 16 and 24 allyl surface groups, respectively, were obtained by coupling the dendrons to the core. The dendrimer (Den-1-(G 2)-OH) with hydroxyl groups at the surface was synthesized by oxidation of the allyl functional groups of Den-1-(G 2), which was divergently coupled to the dendrons to obtain the third generation dendrimer Den-1-(G 3) consisting of 32 surface groups. The modifications in surface groups and generation of dendrimers were shown to influence the shape of dendrimers in the AFM studies. The aggregation as well as self-assembly of dendrimers was observed at high concentration in water by light scattering studies; however, it was reduced on dilution and in the presence of sodium chloride. Dendrimers demonstrated good ability to encapsulate the guest molecule, with loading of 15.80 and 6.47% w/w for rhodamine and beta-carotene, respectively. UV spectroscopy proved the absence of any pi-pi complexation between the dendrimer and encapsulated compounds. (1)H NMR and FTIR studies showed that the physical entrapment and/or hydrogen bonding by PEO in the interior and branch of the dendrimer are the mechanisms of encapsulation. The release of the encapsulated compounds was found to be slow and sustained, suggesting that these dendrimers can serve as potential drug delivery vehicles.     

Synthesis of a new amphiphilic glycodendrimer with antiviral functionality

A new third generation amphiphilic glycodendrimer was synthesized from a stearylamide lysine dendrimer by condensation of the oligosaccharide moiety. By stepwise condensation and deprotection of di-boc lysine from a core of stearyl amide lysine, a third-generation stearylamide lysine dendrimer was constructed. Acetyl cellobiose and glucose units with the carboxylic acid at the end of alkyl chain attached to the reducing end of the sugar moiety was condensed with surface amino groups of the third generation lysine dendrimer, respectively, to give a new stearylamide acetylcellobiose and acetylglucose lysine dendrimers. The structural analysis was carried out using NMR, IR, and matrix-associated laser desorption/ionization time-of-flight (MALDI TOF) mass spectroscopies. After deacetylation to recover hydroxyl groups and subsequent sulfation, the third-generation sulfated cellobiose stearylamide lysine dendrimer was preliminarily found to have high anti-HIV activity at a 50% effective concentration (EC(50)) as low as 6.4μg/ml and low cytotoxicity at a 50% cytotoxic concentration (CC(50)) as high as 1000μg/ml, indicating that the dendrimer gave the enhancement of the functionality of oligosaccharides with low molecular weights. The glycodendrimer with a hydrophobic stearyl chain is immobilized on hydrophobic surfaces by hydrophobic interaction and is expected to provide a new biomedical material with the surface functionality of hydrophilic sulfated oligosaccharides.     

Design of dendritic macromolecules containing folate or methotrexate residues

Polyether dendritic compounds bearing folate residues on their surface were prepared as model drug carriers with potential tumor cell specificity. Starting from ester-terminated polyether dendrimers, hydrazide groups were easily introduced to the surface of the dendrimers by reaction with hydrazine. Folate residues were then conjugated to the hydrazide chain ends of the dendrimers by direct condensation with folic acid in the presence of a condensing agent or by reaction with an active ester derivative of folic acid. Essentially complete functionalization of the terminal hydrazide groups was achieved for both the first and the second generation dendrimers with four and eight hydrazide groups. For the G-2 dendrimer with 16 hydrazide groups, an average number of only 12.6 folate residues were attached to each dendrimer. The conjugates are soluble in aqueous medium above pH 7.4. In addition, a similar conjugation of the antitumor drug methotrexate to the dendrimer was also investigated. Once optimized, these molecules may form the basis for a novel family of multivalent drug carriers.     

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.     

Synthesis and characterization of a triazine dendrimer that sequesters iron(III) using 12 desferrioxamine B groups

The synthesis of a third generation triazine dendrimer, 1, containing multiple, iron-sequestering desferrioxamine B (DFO) groups is described. Benzoylation of the hydroxamic acid groups of DFO and formation of a reactive dichlorotriazine provide the intermediate for reaction with the second generation dendrimer displaying twelve amines. This strategy further generalizes the ‘functional monomer’ approach to generate biologically active triazine dendrimers. Dendrimer 1 is prepared in seven steps in 35% overall yield and displays 12 DFO groups making it 56% drug by weight. Spectrophotometric titrations (UV–vis) show that 1 sequesters iron(III) atoms with neither cooperativity nor significant interference from the dendrimer backbone. Evidence from NMR spectroscopy and mass spectrometry reveals a limitation to this functional monomer approach: trace amounts of O-to-N acyl migration from the protected hydroxamic acids to the amine-terminated dendrimer occurs during the coupling step leading to N-benzoylated dendrimers displaying fewer than 12 DFO groups.     

Acid- and salt-triggered multifunctional poly(propylene imine) dendrimer as a prospective drug delivery system

A novel dendrimeric compound is designed with the objective of simultaneously addressing issues commonly encountered in drug delivery, i.e., stability in biological milieu as well as targeting. For this purpose, a multifunctional dendrimeric system derived from diaminobutane poly(propylene imine) dendrimers (DAB) is prepared bearing at its external surface poly(ethylene glycol) chains and guanidinium moieties. For these moieties, it has been established that they exhibit protective and targeting properties, respectively. The release of encapsulated compounds is triggered by titration with acids followed by the addition of sodium chloride solution. Specifically for pyrene, the solubilization site of which can be clearly traced, protonation leads to a distribution between the core and the poly(ethylene glycol) chains in the periphery of the dendrimer while it is released to the aqueous bulk solution by the addition of sodium chloride. The release of betamethasone valerate is also triggered by the addition of sodium chloride solution.     

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.     

Tumor cell imaging using the intrinsic emission from PAMAM dendrimer: a case study with HeLa cells

HeLa 229 cells were treated with methotrexate (MTX) and doxorubicin (DOX), utilizing fourth generation (G4), amine terminated poly(amidoamine) {PAMAM} dendrimer as the drug carrier. In vitro kinetic studies of the release of both MTX and DOX in presence and absence of G4, amine terminated PAMAM dendrimers suggest that controlled drug release can be achieved in presence of the dendrimers. The cytotoxicity studies indicated improved cell death by dendrimer-drug combination, compared to the control experiments with dendrimer or drug alone at identical experimental conditions. Furthermore, HeLa 229 cells were imaged for the first time utilizing the intrinsic emission from the PAMAM dendrimers and drugs, without incorporating any conventional fluorophores. Experimental results collectively suggest that the decreased rate of drug efflux in presence of relatively large sized PAMAM dendrimers generates high local concentration of the dendrimer-drug combination inside the cell, which renders an easy way to image cell lines utilizing the intrinsic emission properties of PAMAM dendrimer and encapsulated drug molecule.     

Dynamic light scattering and fluorescence study of the interaction between double-stranded DNA and poly(amido amine) dendrimers

The interaction between a cationic poly(amido amine) (PAMAM) dendrimer of generation 4 and double-stranded salmon sperm DNA in 10 mM NaBr solution has been investigated using dynamic light scattering (DLS) and steady-state fluorescence spectroscopy. The structural parameters of the formed aggregates as well as the complex formation process were studied in dilute solutions. When DNA is mixed with PAMAM dendrimers, it undergoes a transition from a semiflexible coil to a more compact conformation due to the electrostatic interaction present between the cationic dendrimer and the anionic polyelectrolyte. The DLS results reveal that one salmon sperm DNA molecule forms a discrete aggregate in dilute solution with several PAMAM dendrimers with a mean apparent hydrodynamic radius of 50 nm. These discrete complexes coexist with free DNA at low molar ratios of dendrimer to DNA, which shows that cooperativity is present in the complex formation. The formation of the complexes was confirmed by agarose gel electrophoresis measurements. DNA in the complexes was also found to be significantly more protected against DNase catalyzed digestion compared to free DNA. The number of dendrimers per DNA chain in the complexes was found to be approximately 35 as determined by steady-state fluorescence spectroscopy.     

Liquid-crystalline dispersions formed by the complexes of linear double-stranded DNA molecules with dendrimers

The specific features of liquid-crystalline dispersions formed by double-stranded DNA molecules interacting with polypropylenimine dendrimers of five generations (G1—G5) in aqueous saline solutions of various ionic strengths were studied. It was demonstrated that the binding of dendrimer molecules to DNA led to the formation of dispersions independently of solution ionic strength and dendrimer structure. By the example of a generation 4 dendrimer, it was shown that the shape of dispersion particles of the (DNA-dendrimer G4) complex were close to a sphere with a diameter of 300–400 nm. The boundary conditions (ionic strength of solution and molecular mass of dendrimer) for the formation of optically active (cholesteric) and optically inactive (DNA-dendrimer) dispersions were determined by circular dichroism spectroscopy. The dispersions formed by dendrimers G1–G3 and G5 were optically inactive. Dendrimers G4 formed liquid-crystalline dispersions of two types. Cholesteric liquid-crystalline dispersions were formed in high ionic strength solutions (μ > 0.4), whereas the dispersions formed in low and intermediate ionic strength solutions (μ < 0.4) lacked an intense negative band in their circular dichroism spectra. The effect of molecular crowding on both the (DNA-dendrimer G4) binding efficiency and the pattern of spatial packing of the (DNA-dendrimer G4) complexes in the liquid-crystalline dispersion particles was demonstrated. The factors determining the structural polymorphism of the liquid-crystalline dispersions of (DNA-dendrimer) complexes are postulated.     

PEGylated dendrimer polystyrene support: synthesis, characterisation and evaluation of biologically active peptides

Poly(N,N-bisethylamine) dendrimers with high content of poly(ethylene glycol) were synthesized on 3-(Acryloyloxy)-2-hydroxypropylmethacrylate-crosslinked polystyrene (PS-AHMA) resin and tested in various conditions of solid phase peptide synthesis. The dendritic templates were generated to the second generation on cross-linker active site of 3-(Acryloyloxy)-2-hydroxypropylmethacrylate (AHMA). First generation dendrimer was designed by series of four-stage reactions, such as Schiff base incorporation, acidolytic cleavage, diazotization and thionyl chloride treatment and same synthetic routes were followed for second generation also. Poly(ethylene glycol) (PEG1000) has been grafted to second-generation dendrimer and used to check various physico-chemical parameters in Fmoc/Boc peptide synthetic conditions. The utility of PEGylated dendrimer support was demonstrated by synthesizing biologically potent linear as well as disulfide-bonded peptide by Fmoc method.     

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.     

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.     

Interaction of poly(amidoamine) dendrimers with supported lipid bilayers and cells: hole formation and the relation to transport

We have investigated poly(amidoamine) (PAMAM) dendrimer interactions with supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers and KB and Rat2 cell membranes using atomic force microscopy (AFM), enzyme assays, flow cell cytometry, and fluorescence microscopy. Amine-terminated generation 7 (G7) PAMAM dendrimers (10-100 nM) were observed to form holes of 15-40 nm in diameter in aqueous, supported lipid bilayers. G5 amine-terminated dendrimers did not initiate hole formation but expanded holes at existing defects. Acetamide-terminated G5 PAMAM dendrimers did not cause hole formation in this concentration range. The interactions between PAMAM dendrimers and cell membranes were studied in vitro using KB and Rat 2 cell lines. Neither G5 amine- nor acetamide-terminated PAMAM dendrimers were cytotoxic up to a 500 nM concentration. However, the dose dependent release of the cytoplasmic proteins lactate dehydrogenase (LDH) and luciferase (Luc) indicated that the presence of the amine-terminated G5 PAMAM dendrimer decreased the integrity of the cell membrane. In contrast, the presence of acetamide-terminated G5 PAMAM dendrimer had little effect on membrane integrity up to a 500 nM concentration. The induction of permeability caused by the amine-terminated dendrimers was not permanent, and leaking of cytosolic enzymes returned to normal levels upon removal of the dendrimers. The mechanism of how PAMAM dendrimers altered cells was investigated using fluorescence microscopy, LDH and Luc assays, and flow cytometry. This study revealed that (1) a hole formation mechanism is consistent with the observations of dendrimer internalization, (2) cytosolic proteins can diffuse out of the cell via these holes, and (3) dye molecules can be detected diffusing into the cell or out of the cell through the same membrane holes. Diffusion of dendrimers through holes is sufficient to explain the uptake of G5 amine-terminated PAMAM dendrimers into cells and is consistent with the lack of uptake of G5 acetamide-terminated PAMAM dendrimers.     

Optimization of transfection properties of DNA-lysine dendrimer complexes

We studied the possibility of optimizing the DNA transfection properties of carriers based on lysine dendrimers of the third and the fifth generation, including those containing a chloroacetyl or a lipophilic palmitoyl moiety at C-end. The use of lysosome-destroying antibiotic chloroquine and an amphipathic polycationic nonadecapeptide JTS-1 was found to enhance the DNA transfecting properties of the lysine dendrimers. The triple complex including DNA, a lysine dendrimer of the third generation modified with lipophylic moieties of palmitic acid at its C-end, and JTS-1 was shown to be comparable in its transfecting activity to a complex containing Escort, a commercial cationic liposome carrier.     

A fluorescence study of the interactions between sodium alginate and surfactants

The interactions between the polysaccharide alginate with charged ionic surfactants (anionic and cationic) in aqueous solution have been investigated using pyrene as a photophysical probe. Static fluorescence determinations have been used to obtain information about the new microenvironments arising by these interactions. Micropolarity studies using the I(1)/I(3) ratio of the vibronic bands and I(E)/I(M) ratio between the excimer and monomer emissions of pyrene shows the formation of hydrophobic domains. The interactions between the natural polyelectrolytes and the oppositely charged surfactants lead to the formation of pre-micelles at surfactant concentrations lower than the CMC of the surfactants. The aggregation process is assumed to be due to electrostatic attraction. On the other side, systems containing an anionic surfactant do not show the same behaviour at low concentrations.     

Influence of dendrimer's structure on its activity against amyloid fibril formation

Inhibition of fibril assembly is a potential therapeutic strategy in neurodegenerative disorders such as prion and Alzheimer's diseases. Highly branched, globular polymers-dendrimers-are novel promising inhibitors of fibril formation. In this study, the effect of polyamidoamine (PAMAM) dendrimers (generations 3rd, 4th, and 5th) on amyloid aggregation of the prion peptide PrP 185-208 and the Alzheimer's peptide Abeta 1-28 was examined. Amyloid fibrils were produced in vitro and their formation was monitored using the dye thioflavin T (ThT). Fluorescence studies were complemented with electron microscopy. The results show that the higher the dendrimer generation, the larger the degree of inhibition of the amyloid aggregation process and the more effective are dendrimers in disrupting the already existing fibrils. A hypothesis on dendrimer-peptide interaction mechanism is presented based on the dendrimers' molecular structure.     

Complex formation between endogenous toxin bilirubin and polyamidoamine dendrimers: a spectroscopic study

The effects of 4th and 5th generation cationic, neutral and anionic polyamidoamine (PAMAM) dendrimers on bilirubin absorbance and fluorescence were studied. Cationic and neutral PAMAM dendrimers shifted the bilirubin absorption maximum from 435 to 442-455 nm, increased the peak absorbance 1.5-fold, shifted the bilirubin fluorescence excitation and emission maxima, increased the fluorescence emission several-fold and significantly protected bilirubin against photodestruction. Using double fluorescence titration technique allowed to receive such constant of binding and the number of binding centers at 20 degrees C: for PAMAM G4 dendrimer, (2.4+/-1.4) x 10(6) (mol/l)(-1) and 0.07+/-0.012; for PAMAM G4-OH dendrimer, (3.1+/-1.3) x 10(6) (mol/l)(-1) and 0.08+/-0.014; for PAMAM G5 dendrimer, (7.6+/-3.6) x 10(6) (mol/l)(-1) and 0.09+/-0.02; and for PAMAM G5-OH dendrimer, (8.5+/-3.2) x 10(6) (mol/l)(-1) and 0.09+/-0.02. These effects can be explained by the formation of bilirubin-PAMAM dendrimer complexes and the formation of bilirubin monomers from tetramers. The formation of complexes sharply increased bilirubin solubility. We conclude that cationic and neutral PAMAM dendrimers bind bilirubin effectively and suggest that such dendrimers may serve as detoxication agents for hydrophobic endogenous toxins.     

Synthesis of new polymer-bound adenine nucleotides using starburst PAMAM dendrimers

Two types of new polymer-bound adenine nucleotides were synthesized by coupling adenine nucleotides (ATP and ADP) with starburst polyamidoamine (PAMAM) dendrimers. The first type was obtained by coupling native adenine nucleotides directly with a carboxy-terminated PAMAM dendrimer. In the second type, the nucleotides were modified by introducing a spacer arm containing a carboxylic end group (N(6)-R-ATP and N(6)-R-ADP) and coupled with an amine-terminated PAMAM dendrimer. Both types of the dendrimers were coupled with native or the modified nucleotides using the well-known carbodiimide activation technique. The optimum coupling pH and temperature were 4 and 30 degrees C, respectively, for preparing the carboxy-terminated PAMAM-bound ATP or ADP, and were 9 and 50 degrees C, respectively, for preparing the amine-terminated PAMAM-bound N(6)-R-ATP or N(6)-R-ADP. The ATP or ADP contents in the synthesized polymers were found to be 4 mol of ATP or of ADP/mol of carboxy-terminated PAMAM-bound ATP or ADP and 25 mol of ATP or of ADP/mol of amine-terminated PAMAM-bound N(6)-R-ATP or N(6)-R-ADP. The coenzymatic activities relative to the native ATP of the carboxy-terminated PAMAM-bound ATP against glucokinase and hexokinase were 16 and 7%, respectively, and those of the amine-terminated PAMAM-bound N(6)-R-ATP 2 and 1%, respectively. The coenzymatic activities relative to the native ADP of the carboxy-terminated PAMAM-bound ADP and the amine-terminated PAMAM-bound N(6)-R-ADP against acetate kinase were 24 and 3.5%, respectively.