PAMAM Dendrimers Mediate siRNA Delivery to Target Hsp27 and Produce Potent Antiproliferative Effects on Prostate Cancer Cells

RNA interference (RNAi) holds great promise for the treatment of inherited and acquired diseases, provided that safe and efficient delivery systems are available. Herein we report that structurally flexible triethanolamine (TEA) core PAMAM dendrimers are able to deliver an Hsp27 siRNA effectively into prostate cancer (PC‐3) cells by forming stable nanoparticles with siRNA, protecting the siRNA nanoparticles from enzymatic degradation, and enhancing cellular uptake of siRNA. The Hsp27 siRNA resulted in potent and specific gene silencing of heat‐shock protein 27, an attractive therapeutic target in castrate‐resistant prostate cancer. Silencing of the hsp27 gene led to induction of caspase‐3/7‐dependent apoptosis and inhibition of PC‐3 cell growth in vitro. In addition, the siRNA–dendrimer complexes are non‐cytotoxic under the conditions used for siRNA delivery. Altogether, TEA core PAMAM dendrimer‐mediated siRNA delivery, in combination with RNAi that specifically targets Hsp27, may constitute a promising approach for combating castrate‐resistant prostate cancer, for which there is no efficacious treatment.     

Off‐Target Effects Related to the Phosphorothioate Modification of Nucleic Acids

Phosphorothioate antisense oligonucleotides have been widely used in clinical studies for rational sequence‐specific gene silencing. However, several sequence‐unspecific off‐target effects have been recently described for this compound class. In contrast to siRNA‐mediated knockdown of the same gene, the bcl‐2‐targeted oblimersen (Genasense, G3139) downregulates a number of proteins involved in apoptotic resistance and several glycolytic enzymes in 607B human melanoma cells. Regardless of their target, phosphorothioate‐modified antisense and siRNA compounds, but not oligonucleotides with a phosphodiester backbone, resulted in a similar impact on the proteome. Unspecifically downregulated proteins include cancer markers involved in apoptotic resistance and endoplasmatic reticulum (ER) stress such as the 78 kDa glucose regulated protein (GRP 78), protein disulfide isomerase A3 (PDIA3, GRP 58), calumenin, and galectin‐1, as well as the glycolytic enzymes triose phosphate isomerase, glyceraldehyde phosphodehydrogenase, and phosphoglycerate mutase. The depletion of the glycolytic enzymes is reflected by a decrease in L ‐lactate production, indicating a partial reversal of the Warburg effect. Compared with other phosphorothioate oligonucleotides, oblimersen generally led to a more pronounced effect both in terms of the number of influenced proteins and the extent of downregulation, suggesting a synergistic effect of Bcl‐2 downregulation.     

Structure and Binding of Peptide‐Dendrimer Ligands to Vitamin B12

The third‐generation peptide‐dendrimer B1 (AcES)₈(BEA)₄(K‐Amb‐Y)₂BCD‐NH₂ (B=branching (S)‐2,3‐diaminopropanoic acid, K=branching lysine, Amb=4‐aminomethyl‐benzoic acid) is the first synthetic model for cobalamin‐binding proteins and binds cobalamin strongly (K_a=5.0×10⁶ M ^?1) and rapidly (k₂=346 M ^?1 s^?1) by coordination of cobalt to the cysteine residue at the dendrimer core. A structure–activity relationship study is reported concerning the role of negative charges in binding. Substituting glutamates (E) for glutamines (Q) in the outer branches of B1 to form N3 (AcQS)₈(BQA)₄(B‐Amb‐Y)₂BCD‐NH₂ leads to stronger (K_a=12.0×10⁶ M ^?1) but slower (k₂=67 M ^?1 s^?1) cobalamin binding. CD and FTIR spectra show that the dendrimers and their cobalamin complexes exist as random‐coil structures without aggregation in solution. The hydrodynamic radii of the dendrimers determined by diffusion NMR either remains constant or slightly decreases upon binding to cobalamin; this indicates the formation of compact, presumably hydrophobically collapsed complexes.     

Modeling the Interaction between Dendrimers and Nucleic Acids: a Molecular Perspective through Hierarchical Scales

Cationic dendrimers are promising nanocarriers for gene delivery thanks to their ability to establish strong interactions with oppositely charged strands of DNA and siRNA and to promote their aggregation. The binding between dendrimers and nucleic acids is typically a complex process that involves various types of interactions at different scales. To design efficient dendrimer candidates for DNA and siRNA binding it is necessary to have a detailed understanding of their interactions with oligonucleotides in the solvent. Molecular simulation can support experimental work, providing a privileged point of view on the aggregation process. This Minireview discusses recent computational efforts to unravel dendrimer–oligonucleotide binding, and proposes a perspective of the multiscale aggregation process based on hierarchy and on the transformations of the interacting “molecular units” following intermolecular interactions.     

Binding properties of polyamidoamine dendrimers

Dendrimers are globular, hyperbranched polymers possessing a high concentration of surface functional groups and internal cavities. These unique features make them good host molecules for small ligands. To reveal relationships between dendrimer size and its encapsulating properties, the interactions of the fourth and the sixth generations of polyamidoamine dendrimers (PAMAM G4 and PAMAM G6) with a fluorescent dye 1‐anilinonaphthalene‐8‐sulfonate (ANS) were studied. Because ANS is a fluorescent molecule and its fluorescence is very sensitive to changes in its microenvironment, it was possible to use spectrofluorometric methods to evaluate the interactions with dendrimers. A double fluorometric titration method was used to estimate a binding constant and the number of binding centers. There were two types of dendrimer binding centers characterized by different affinity towards ANS. For PAMAM G4, the values of K_b and n for low‐affinity and high‐affinity sites equaled to 2.6 × 10⁵, 0.60 and 3.70 × 10⁶, 0.34, respectively, whereas in the case of PAMAM G6, these values equaled to 1.2 × 10⁵, 76.34 and 1.38 × 10⁶, 22.73. It was observed that the size of the dendrimer had a strong impact on the number of ANS molecules that interacted with dendrimers and their location within the macromolecule. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 2036–2040, 2007     

Generational, skeletal and substitutional diversities in generation one poly(amidoamine) dendrimers

Structural deviations of ethylenediamine core polyamidoamine (PAMAM) dendrimers and derivatives can be defined as skeletal and/or substitutional diversities. Detailed analysis of dendrimer starting materials and derivatives is necessary to understand the intrinsic characteristics of commercial dendrimer materials and their variations related to subsequent surface modifications. In this paper, structural deviations of ethylenediamine core generation 1 PAMAM dendrimers (PAMAM_E1 or E1) are studied and determined in a frame of a systematic investigation using combined characterization techniques. A primary amine-terminated PAMAM dendrimer of generation 1 (E1.NH₂) was used as a starting material to synthesize glycidol (E1.N(Gly)OH) and acetamide-terminated (E1.NHAc) dendrimers. The purity and homogeneity of these dendrimers were extensively characterized by polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis (CE), gel permeation chromatography (GPC), acid-base titration, nuclear magnetic resonance (NMR), matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) and electrospray ionization (ESI) mass spectrometry. PAGE and CE studies showed that electrophoretic mobilities at pH 2.5 are in the order of E1.NH₂>E1.N(Gly)OH>E1.NHAc. Mass spectrometry and NMR investigations (¹H, ¹³C DEPT-135, and ¹³C NMR, COSY, HETCOR, NOESY) suggested that (a) the studied E1 dendrimers were generationally pure, (b) E1.NHAc and E1.N(Gly)OH dendrimers, and essentially had the same defects and skeletal diversity as E1.NH₂ did. The broad distribution of the main peak in the CE electropherogram of E1.N(Gly)OH revealed the incomplete hydroxylation of E1.NH₂ resulting in additional substitutional diversity between the dendrimer molecules. Potentiometric titration studies proved that overall numbers of terminal and tertiary amine groups also deviated from the theoretical values. NMR spectroscopy was applied for both qualitative and quantitative analysis of the structural defects of dendrimers and derivatives. E1.NH₂ and E1.NHAc exhibited only minor deviations from ideal structures and, respectively, displayed a narrow distribution; while E1.N(Gly)OH had a much broader distribution centered around 14±3 glycidol substituents. The study of structural variations in generation 1 PAMAMs provides new insights for the characterization of higher generation PAMAM dendrimers and derivatives both in terms of the skeletal deviations as well as other resulting diversities related to dendrimer surface functionalization.     

Synthesis and characterization of stearyl‐group‐terminated dendrimers and thermosensitivity of their toluene solutions

Stearyl‐group‐terminated poly(ester amide) dendrimers [PEAD (R)₃ and PEAD (R)₈] and a poly(amino amide) dendrimer [PAMAM (R)₄] were synthesized by the amidation of three, eight, and four terminated primary amino groups in poly(ester amine) dendrimers and a poly(amino amide) dendrimer with stearyl chloride. The dendrimer structures were characterized with IR and elemental analysis. The toluene solutions of the stearyl‐group‐terminated dendrimers were thermosensitive. Not only did gels form in PEAD (R)₃–, PEAD (R)₈–, and PAMAM (R)₄–toluene solutions below 57.5, 60, and 49°C, respectively, but the content of toluene in the gels depended on the temperature, and a break existed at about 30°C. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 98: 341–346, 2005     

Preparation of Ag nanoparticles in the presence of low generational poly(ester‐amine) dendrimers

Stable Ag nanoparticles of 10–20 nm were prepared by reduction of AgNO₃ with NaBH₄ in water solution in the presence of low generational hydroxyl‐ terminated poly(ester‐amine) dendrimer G1.0 (OH)₁₆ and amino‐terminated poly(ester‐amine) dendrimer G1.5 (NH₂)₈ by optimizing preparation conditions. UV–vis absorption spectra and transmission electron microscopy were adopted to characterize absorption properties of Ag⁺/dendrimer complex, Ag/dendrimer nanocomposite aqueous solutions, and the morphology of the formed Ag nanoparticles, respectively. The results showed that the size of the Ag particles increased with Ag⁺/dendrimer molar ratio, and the size of Ag nanoparticles in Ag/G1.0 (OH)₁₆ system was larger than that of Ag nanoparticles in Ag/G1.5 (NH₂)₈ system, while the polydispersities of two systems were similar. Moreover, the Ag/G1.5 (NH₂)₈ nanocomposite system was more stable than the Ag/G1.0 (OH)₁₆ one. © 2007 Wiley Periodicals, Inc. J Appl Polym Sci 104: 422–426, 2007     

Antitumor efficacy of doxorubicin encapsulated within PEGylated poly(amidoamine) dendrimers

We report here a general approach to using poly(amidoamine) (PAMAM) dendrimers modified with polyethylene glycol (PEG) as a platform to encapsulate an anticancer drug doxorubicin (DOX) for in vitro cancer therapy applications. In this approach, PEGylated PAMAM dendrimers were synthesized by conjugating monomethoxypolyethylene glycol with carboxylic acid end group (mPEG‐COOH) onto the surface of generation 5 amine‐terminated PAMAM dendrimer (G5.NH₂), followed by acetylation of the remaining dendrimer terminal amines. By varying the molar ratios of mPEG‐COOH/G5.NH₂, G5.NHAc‐mPEG_n (n = 5, 10, 20, and 40, respectively) with different PEGylation degrees were obtained. We show that the PEGylated dendrimers are able to encapsulate DOX with approximately similar loading capacity regardless of the PEGylation degree. The formed dendrimer/DOX complexes are water soluble and stable. In vitro release studies show that DOX complexed with the PEGylated dendrimers can be released in a sustained manner. Further cell viability assay in conjunction with cell morphology observation demonstrates that the G5.NHAc‐mPEG_n/DOX complexes display effective antitumor activity, and the DOX molecules encapsulated within complexes can be internalized into the cell nucleus, similar to the free DOX drug. Findings from this study suggest that PEGylated dendrimers may be used as a general drug carrier to encapsulate various hydrophobic drugs for different therapeutic applications. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40358.     

Curing of DGEBA epoxy resin by low generation amino‐group‐terminated dendrimers

Low generation amino‐group‐terminated poly(ester‐amine) dendrimers PEA1.0 (NH₂)₃ and PEA1.5 (NH₂)₈, and poly(amido‐amine) dendrimer PAMAM1.0 (NH₂)₄ were used as diglycidyl ether of bisphenol A (DGEBA) epoxy resin hardeners. Thermal behavior and curing kinetics of dendrimer/DGEBA systems were investigated by means of differential scanning calorimetry (DSC). Compared with ethylene diamine (EDA)/DGEBA system, the dendrimer/DGEBA systems gradually liberated heat in two stages during the curing process, and the total heat liberated was less. Apparent activation energy and curing reaction rate constants for dendrimer and EDA/DGEBA systems were estimated. Thermal stabilities and mechanical properties of cured thermosetting systems were examined as well. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 3902–3906, 2006     

Synthesis and characterization of triazine‐based dendrimers and their application in metal ion adsorption

This article described the synthesis of triazine‐based dendrimers with poly(ethylene glycol) core by convergent method. Compound 1 was prepared by coupling of amino group of diethanolamine with cyanuric chloride in dry THF (tetrahydrofuran). Reaction of compound 1 with p‐aminobenzylamine resulted in compound 2 . Compound 4 was synthesized using coupling reaction of amino group of compound 2 with cyanuric chloride, then coupling of amine groups of p‐aminobenzylamine with compound 3 in the hybrid solvents. The final dendrimer (den‐OH) were synthesized using reaction of dendron 4 with compound 5. Ethylene diaminetetraacetic acid modified final dendrimer were successfully prepared via coupling ethylene diaminetetraacetic acid dianhydride and den‐OH. The growth of dendrons and their structures were investigated by using usual spectroscopy methods and elemental analysis. The chelating behavior and sorption capacities of triazine dendrimers were determined in relation to pH dependency for some metal ions such as Cu⁺², Ni⁺², and Zn⁺² using atomic absorption methods. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

A study of antimicrobial property of textile fabric treated with modified dendrimers

Dendrimers have been used as a vehicle to develop the antimicrobial properties of textile fabrics. We have taken advantage of the large number of functional groups present in the regular and highly branched three‐dimensional architecture of dendrimers. In this study, the poly(amidoamine) (PAMAM) G‐3 dendrimer was modified to provide antimicrobial properties. Following a procedure similar to what is suggested in the literature, PAMAM (G3) with primary amine end groups was converted into ammonium functionalities. The modification was then confirmed by FTIR and ¹³C‐NMR analysis. Dendrimers have unique properties owing to their globular shape and tunable cavities, this allows them to form complexes with a variety of ions and compounds; and also act as a template to fabricate metal nanoparticles. AgNO₃–PAMAM (G3) complex as well as a MesoSilver–PAMAM (G3) complex were formed and these modified dendrimers were characterized by a UV–Visible spectrophotometer to study the complex formation. Modified dendrimers were applied to the Cotton/Nylon blend fabric. SEM and EDX analysis were performed to study the dispersion of silver nanoparticles onto the fabric. An antimicrobial test of the treated‐fabric against Staphylococcus aureus exhibited significant biocidal activities for each type of modified‐dendrimer. © 2009 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

A structure/function study of polyaminoamide dendrimers as silica scale growth inhibitors

Dendrimers have attracted immense attention during the last decade due to their interesting properties both from a basic and an applied research viewpoint. Encapsulation of metal nanoparticles for catalysis, drug delivery and light harvesting are only some applications of dendrimers that are breaking new ground. A novel application of dendrimer technology is described in the present paper that relates to industrial water treatment. Industrial water systems often suffer from undesirable inorganic deposits. These can form either in the bulk or on metallic surfaces, such as heat exchangers or pipelines. Silica (SiO₂) scale formation and deposition is a major problem in high‐silica‐containing cooling waters. Scale prevention rather than removal is highly desired. In this paper, benchtop screening tests on various silica inhibition chemistries are reported, with emphasis on materials with a dendrimeric structure. Specifically, the inhibition properties of commercially available STARBURST^® polyaminoamide (PAMAM) dendrimers generations 0.5, 1, 1.5, 2, and 2.5 are investigated in detail together with other commonly‐used scale inhibitors. Experimental results show that inhibition efficiency largely depends on structural features of PAMAM dendrimers such as generation number and nature of the end groups. PAMAM dendrimers are effective inhibitors of silica scale growth at 40 ppm dosage levels. PAMAM dendrimers also act as silica nucleators, forming SiO₂–PAMAM composites. This occurs because the SiO₂ formed by incomplete inhibition interacts with cationic PAMAM‐1 and ‐2. The general scope of silica formation and inhibition in industrial waters is also discussed. Copyright © 2005 Society of Chemical Industry     

Novel Poly(EThyleneAmidoAmine) (PETAA) dendrimers produced through a unique and highly efficient synthesis

Polyamidoamine (PAMAM) dendrimers have unique attributes that have led to their use in a wide variety of biomedical applications. However, the complex synthesis of this polymer leads to variations in the structure and consistency of the final product, and makes scale-up of manufacturing difficult. This has limited the clinical translation of PAMAM-based materials. Here we describe a rapid and highly efficient two-step method for the synthesis of novel Poly(EThyleneAmidoAmine) (PETAA) dendrimers that have many of the favorable characteristics of PAMAM dendrimers. Generation 0 (G0) to 5 (G5) PETAA dendrimers were synthesized using a 3-(bis(2-(2,2,2,-trifluoroacetamido)ethyl)amino)propanoic acid AB₂ (compound 1) building block via a divergent approach. An ethylenediamine core was coupled with the AB₂ building block via O-(7-Azabenzotriazol-1-yl)N,N,N’,N’-tetramethyluronium hexafluorophosphate (HATU) in the presence of diisopropylethyl amine to give a G0 trifluoroacetamide surface dendrimer. The G0 amine surface dendrimer was then obtained by treating the G0 trifluoroacetamide surface dendrimer with potassium carbonate. Repetitions of these two coupling/deprotection reactions were then used to build the dendrimer by coupling the surface amino groups to the carboxyl moiety of the AB₂ building block, followed by the deprotection step with potassium carbonate. The resulting PETAA dendrimers have the same number of surface primary amino groups, the same number of chemical bonds between the dendrimer core and the surface, and the same number of tertiary amino groups throughout the structures as similar generations of PAMAM dendrimers. In contrast, the structure of the PETAA dendrimers is more complete and more uniform than PAMAM dendrimers, especially at higher generations. This unique synthetic process for PETAA dendrimers also offers the potential for large-scale production, therefore providing inherently more uniform and complete structures for exacting biomedical applications.     

Efficient Transfection of siRNA by Peptide Dendrimer–Lipid Conjugates

Efficient delivery of small interfering RNA (siRNA) into cells is the basis of target‐gene‐specific silencing and, ultimately, gene therapy. However, current transfection reagents are relatively inefficient, and very few studies provide the sort of systematic understanding based on structure–activity relationships that would provide rationales for their improvement. This work established peptide dendrimers (administered with cationic lipids) as siRNA transfection reagents and recorded structure–activity relationships that highlighted the importance of positive charge distribution in the two outer layers and a hydrophobic core as key features for efficient performance. These dendrimer‐based transfection reagents work as well as highly optimised commercial reagents, yet show less toxicity and fewer off‐target effects. Additionally, the degrees of freedom in the synthetic procedure will allow the placing of decisive recognition features to enhance and fine‐tune transfection and cell specificity in the future.     

A Versatile and Convenient Synthesis of 34S‐Labeled Phosphorothioate Oligonucleotides

A synthetic protocol for ³⁴S‐labeled phosphorothioate oligonucleotides (PS ONs) was developed to facilitate MS‐based assay analysis. This was enabled by a highly efficient, two‐step, one‐pot synthesis of ³⁴S‐labeled phenylacetyl disulfide (³⁴S‐PADS), starting from ³⁴S‐enriched elemental sulfur (³⁴S₈). ³⁴S‐PADS was subsequently used for stable isotope labeling (SIL) of oligonucleotides containing a phosphorothioate backbone. The ³⁴S‐SIL PS ONs are shown to retain the same melting temperature, antisense activity, and secondary structure as those of the corresponding unlabeled ³²S PS ONs.     

Synthesis and Microscopic Characterization of Dendrimer-Derived Ru/Al2O3 Catalysts

Fourth generation poly(amidoamine) dendrimers have been used to template and stabilize Ru nanoparticles in solution. UV-visible spectroscopic results indicate that Ru³⁺ ions from a RuCl₃ precursor can complex with functional groups within the dendrimer structure. Subsequent reduction of the Ru³⁺ ions yields finely dispersed Ru nanoparticles with a narrow particle size distribution. These dendrimer-stabilized nanoparticles were deposited onto an alumina support and thermally activated to remove the dendrimer “shell”, as indicated by in situ Fourier transform infrared (FTIR) spectroscopic measurements. High resolution transmission electron microscopy (HRTEM) measurements indicate that the resulting Ru/Al₂O₃ catalyst has a smaller mean metal particle size and a narrower particle size distribution than a similar catalyst prepared by a traditional wet impregnation from the same RuCl₃ precursor.     

siRNA Modified with 2′‐Deoxy‐2′‐C‐methylpyrimidine Nucleosides

(2′S)‐2′‐Deoxy‐2′‐C‐methyluridine and (2′R)‐2′‐deoxy‐2′‐C‐methyluridine were incorporated in the 3′‐overhang region of the sense and antisense strands and in positions 2 and 5 of the seed region of siRNA duplexes directed against Renilla luciferase, whereas (2′S)‐2′‐deoxy‐2′‐C‐methylcytidine was incorporated in the 6‐position of the seed region of the same constructions. A dual luciferase reporter assay in transfected HeLa cells was used as a model system to measure the IC₅₀ values of 24 different modified duplexes. The best results were obtained by the substitution of one thymidine unit in the antisense 3′‐overhang region by (2′S)‐ or (2′R)‐2′‐deoxy‐2′‐C‐methyluridine, reducing IC₅₀ to half of the value observed for the natural control. The selectivity of the modified siRNA was measured, it being found that modifications in positions 5 and 6 of the seed region had a positive effect on the ON/OFF activity.     

CO Oxidation and Toluene Hydrogenation by Pt/TiO2 Catalysts Prepared from Dendrimer Encapsulated Nanoparticle Precursors

Generation 4 hydroxyl terminated polyamidoamine (PAMAM) dendrimer encapsulated nanoparticles (DENs) were examined as precursors for Pt/TiO₂ catalysts. In this preparation method, the dendrimers were initially used to template and stabilize Pt nanoparticles in solution. DENs were then deposited onto titania, and activation conditions for dendrimer thermolysis were examined. The interactions between PAMAM dendrimers and the titania were found to differ from previous reports of dendrimer-support interactions with silica, alumina, and zirconia. In the case of titania, the amide bonds were found to shift 100 cm^?1, indicating adsorption occurs primarily through amide–titania interactions. Infrared spectroscopy, CO oxidation catalysis, and toluene hydrogenation catalysis were used to evaluate protocols for removing the dendrimer. Thermal decomposition of the DENs in O₂ or CO/O₂ atmospheres led to the formation of surface isocyanates that were preferentially bound to the metal nanoparticles. CO oxidation catalysis was insensitive to the activation protocol used, and infrared spectroscopy of adsorbed CO showed only small differences in the basic surface properties of the resulting Pt catalysts. Toluene hydrogenation catalysis was more sensitive to different activation pretreatments. The most active hydrogenation catalysts resulted from short, low temperature (150 °C) hydrogen treatments while longer treatments at higher temperature (300 °C) resulted in slightly less active catalysts.     

Fabrication of Ag nanoparticle–encapsulating multilayer films based on PAMAM dendrimers with covalent interlayer linkages

Ag‐dendrimer nanoclusters were prepared with Ag⁺ and carboxyl shell (G4.5) poly(amino amine) dendrimers. Self‐assembly photosensitive ultrathin films were then fabricated with these Ag‐dendrimer nanoclusters as polyanions and diazoresin (DR) as polycation. With UV irradiating the films became stable because of the formation of covalent linkages between the layers. Compared to similar films containing no Ag nanoclusters, the obtained films showed greatly enhanced electric conductivity. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 89: 1515–1519, 2003