A monolithic stationary phase with dendritic nanostructures for the separation of PEGylated proteins.

Separation of PEGylated protein mixtures into individual species is a challenging procedure, and many efforts have been focused on creating novel chromatographic supports for this purpose. In this study, a new monolithic stationary phase with hyperbranched nanostructures was chemically synthesized. For this, monoliths with a support matrix of poly (glycidyl methacrylate-co-ethylene dimethacrylate) and ethylenediamine chemistry were modified with third-generation dendrons with butyl-end groups. The new monolith was analyzed by infrared spectroscopy, confirming the dendron with butyl ligands and exhibited low mass transfer resistance as observed by breakthrough frontal analysis. This support was able to separate mono-PEG ribonuclease A from the PEGylation mixture, indicated by a single band (∼30 kDa) in the electrophoretic analysis. Moreover, the separation of mono-PEGylated positional isomers was probably observed, as the protein with ∼30 kDa was found in two separate peaks. Interestingly, the dendronized monolith allowed the separation of the reaction mixture into individual PEGylated species when using high ammonium sulfate concentrations (2 M). A correlation between the PEGylation degree and the strength of the hydrophobic interactions on the monolith was observed. This chromatographic approach combines the natural branched architecture of dendrons and the higher capabilities of the monoliths enhancing the hydrophobic surface area, and therefore the interaction between the PEGylated proteins and ligands. Thus, the novel support represents a novel platform for the purification of PEGylated from non-PEGylated proteins with biotechnological applications.

Self-Assembly Behavior of Amphiphilic Janus Dendrimers in Water: A Combined Experimental and Coarse-Grained Molecular Dynamics Simulation Approach.

Amphiphilic Janus dendrimers (JDs) are repetitively branched molecules with hydrophilic and hydrophobic components that self-assemble in water to form a variety of morphologies, including vesicles analogous to liposomes with potential pharmaceutical and medical application. To date, the self-assembly of JDs has not been fully investigated thus it is important to gain insight into its mechanism and dependence on JDs' molecular structure. In this study, the aggregation behavior in water of a second-generation bis-MPA JD was evaluated using experimental and computational methods. Dispersions of JDs in water were carried out using the thin-film hydration and ethanol injection methods. Resulting assemblies were characterized by dynamic light scattering, confocal microscopy, and atomic force microscopy. Furthermore, a coarse-grained molecular dynamics (CG-MD) simulation was performed to study the mechanism of JDs aggregation. The obtaining of assemblies in water with no interdigitated bilayers was confirmed by the experimental characterization and CG-MD simulation. Assemblies with dendrimersome characteristics were obtained using the ethanol injection method. The results of this study establish a relationship between the molecular structure of the JD and the properties of its aggregates in water. Thus, our findings could be relevant for the design of novel JDs with tailored assemblies suitable for drug delivery systems.

Structural analysis of binding functionality of folic acid-PEG dendrimers against folate receptor.

Dendrimers functionalized with folic acid (FA) are drug delivery systems that can selectively target cancer cells with folate receptors (FR-α) overexpression. Incorporation of polyethylene glycol (PEG) can enhance dendrimers solubility and pharmacokinetics, but ligand-receptor binding must not be affected. In this work we characterized, at atomic level, the binding functionality of conventional site-specific dendrimers conjugated with FA with PEG 750 or PEG 3350 as a linker. After Molecular Dynamics simulation, we observed that both PEG's did not interfere over ligand-receptor binding functionality. Although binding kinetics could be notably affected, the folate fragment from both dendrimers remained exposed to the solvent before approaching selectively to FR-α. PEG 3350 provided better solubility and protection from enzymatic degradation to the dendrimer than PEG 750. Also, FA-PEG3350 dendrimer showed a slightly better interaction with FR-α than FA-PEG750 dendrimer. Therefore, theoretical evidence supports that both dendrimers are suitable as drug delivery systems for cancer therapies.

Self-Assembly of Amphiphilic Dendrimers: The Role of Generation and Alkyl Chain Length in siRNA Interaction.

An ideal nucleic-acid transfection system should combine the physical and chemical characteristics of cationic lipids and linear polymers to decrease cytotoxicity and uptake limitations. Previous research described new types of carriers termed amphiphilic dendrimers (ADs), which are based on polyamidoamine dendrimers (PAMAM). These ADs display the cell membrane affinity advantage of lipids and preserve the high affinity for DNA possessed by cationic dendrimers. These lipid/dendrimer hybrids consist of a low-generation, hydrophilic dendron (G2, G1, or G0) bonded to a hydrophobic tail. The G2-18C AD was reported to be an efficient siRNA vector with significant gene silencing. However, shorter tail ADs (G2-15C and G2-13C) and lower generation (G0 and G1) dendrimers failed as transfection carriers. To date, the self-assembly phenomenon of this class of amphiphilic dendrimers has not been molecularly explored using molecular simulation methods. To gain insight into these systems, the present study used coarse-grained molecular dynamics simulations to describe how ADs are able to self-assemble into an aggregate, and, specifically, how tail length and generation play a key role in this event. Finally, explanations are given for the better efficiency of G2/18-C as gene carrier in terms of binding of siRNA. This knowledge could be relevant for the design of novel, safer ADs with well-optimized affinity for siRNA.

Synthesis of adsorbents with dendronic structures for protein hydrophobic interaction chromatography.

Here, we introduced a new technology based on the incorporation of dendrons-branched chemical structures-onto supports for synthesis of HIC adsorbents. In doing so we studied the synthesis and performance of these novel HIC dendron-based adsorbents. The adsorbents were synthesized in a facile two-step reaction. First, Sepharose 4FF (R) was chemically modified with polyester dendrons of different branching degrees i.e. third (G3) or fifth (G5) generations. Then, butyl-end valeric acid ligands were coupled to dendrons via ester bond formation. UV-vis spectrophotometry and FTIR analyses of the modified resins confirmed the presence of the dendrons and their ligands on them. Inclusion of dendrons allowed the increment of ligand density, 82.5 ± 11 and 175.6 ± 5.7 µmol ligand/mL resin for RG3 and RG5, respectively. Static adsorption capacity of modified resins was found to be ∼ 60 mg BSA/mL resin. Interestingly, dynamic binding capacity was higher at high flow rates, 62.5 ± 0.8 and 58.0 ± 0.5mg/mL for RG3 and RG5, respectively. RG3 was able to separate lipase, ß-lactoglobulin and α-chymotrypsin selectively as well as fractionating of a whole proteome from yeast. This innovative technology will improve the existing HIC resin synthesis methods. It will also allow the reduction of the amount of adsorbent used in a chromatographic procedure and thus permit the use of smaller columns resulting in faster processes. Furthermore, this method could potentially be considered as a green technology since both, dendrons and ligands, are formed by ester bonds that are more biodegradable allowing the disposal of used resin waste in a more ecofriendly manner when compared to other exiting resins.