A Molecular Dynamics Simulation of Complexes of Fullerenes and Lysine-Based Peptide Dendrimers with and without Glycine Spacers.

The development of new nanocontainers for hydrophobic drugs is one of the most important tasks of drug delivery. Dendrimers with hydrophobic interiors and soluble terminal groups have already been used as drug carriers. However, the most convenient candidates for this purpose are peptide dendrimers since their interiors could be modified by hydrophobic amino acid residues with a greater affinity for the transported molecules. The goal of this work is to perform the first molecular dynamics study of the complex formation of fullerenes C60 and C70 with Lys-2Gly, Lys G2, and Lys G3 peptide dendrimers in water. We carried out such simulations for six different systems and demonstrated that both fullerenes penetrate all these dendrimers and form stable complexes with them. The density and hydrophobicity inside the complex are greater than in dendrimers without fullerene, especially for complexes with Lys-2Gly dendrimers. It makes the internal regions of complexes less accessible to water and counterions and increases electrostatic and zeta potential compared to single dendrimers. The results for complexes based on Lys G2 and Lys G3 dendrimers are similar but less pronounced. Thus, all considered peptide dendrimers and especially the Lys-2Gly dendrimer could be used as nanocontainers for the delivery of fullerenes.

Influence of the Chemical Structure on the Mechanical Relaxation of Dendrimers.

The rheological properties of macromolecules represent one of the fundamental features of polymer systems which expand the possibilities of using and developing new materials based on them. In this work, we studied the shear-stress relaxation of the second generation PAMAM and PPI dendrimer melts by atomistic molecular dynamics simulation. The time dependences of relaxation modulus G(t) and the frequency dependences of the storage G'(ω) and loss G″(ω) moduli were obtained. The results were compared with the similar dependences for the polycarbosilane (PCS) dendrimer of the same generation. The chemical structure of the dendrimer segments has been found to strongly influence their mechanical relaxation. In particular, it has been shown that hydrogen bonding in PAMAM dendrimers leads to an entanglement of macromolecules and the region is observed where G'(ω) > G″(ω). This slows down the mechanical relaxation and rotational diffusion of macromolecules. We believe that our comprehensive research contributes to the systematization of knowledge about the rheological properties of dendrimers.

NMR Studies of Two Lysine Based Dendrimers with Insertion of Similar Histidine-Arginine and Arginine-Histidine Spacers Having Different Properties for Application in Drug Delivery.

In this paper we study two lysine-based peptide dendrimers with Lys-His-Arg and Lys-Arg-His repeating units and terminal lysine groups. Combination of His and Arg properties in a dendrimer could be important for biomedical applications, especially for prevention of dendrimer aggregation and for penetration of dendrimers through various cell membranes. We describe the synthesis of these dendrimers and the confirmation of their structure using 1D and 2D Nuclear Magnetic Resonance (NMR) spectroscopy. NMR spectroscopy and relaxation are used to study the structural and dynamic properties of these macromolecules and to compare them with properties of previously studied dendrimers with Lys-2Arg and Lys-2His repeating units. Our results demonstrate that both Lys-His-Arg and Lys-Arg-His dendrimers have pH sensitive conformation and dynamics. However, properties of Lys-His-Arg at normal pH are more similar to those of the more hydrophobic Lys-2His dendrimer, which has tendency towards aggregation, while the Lys-Arg-His dendrimer is more hydrophilic. Thus, the conformation with the same amino acid composition of Lys-His-Arg is more pH sensitive than Lys-Arg-His, while the presence of Arg groups undoubtedly increases its hydrophilicity compared to Lys-2His. Hence, the Lys-His-Arg dendrimer could be a more suitable (in comparison with Lys-2His and Lys-Arg-His) candidate as a pH sensitive nanocontainer for drug delivery.

Mechanical relaxation of functionalized carbosilane dendrimer melts.

Functionalizing the internal structure of classical dendrimers is a new way of tailoring their properties. Using atomistic molecular dynamics simulations, we investigate the rheological behavior of functionalized dendrimer (FD) melts obtained by modifying the branching of carbosilane dendrimers (CSD). The time (relaxation modulus G(t)) and frequency (storage G' and loss G'' moduli) dependencies of the dynamic modulus are obtained. Fourth generation FD melts present a region where G' > G''. In contrast, their non-functionalized counterparts (i.e., classical dendrimers with regular branching) do not show such a region. The comparative analysis of FD and CSD suggests that the internal densification due to functionalization prevents the penetration of branches and causes FD to behave like colloidal particles in a crowded environment. Since CSD have no special interactions, we expect that this effect will be common for other dendrimer macromolecules.

Size and Structure of Empty and Filled Nanocontainer Based on Peptide Dendrimer with Histidine Spacers at Different pH.

Novel peptide dendrimer with Lys-2His repeating units was recently synthesized, studied by NMR (Molecules, 2019, 24, 2481) and tested as a nanocontainer for siRNA delivery (Int. J. Mol. Sci., 2020, 21, 3138). Histidine amino acid residues were inserted in the spacers of this dendrimer. Increase of their charge with a pH decrease turns a surface-charged dendrimer into a volume-charged one and should change all properties. In this paper, the molecular dynamics simulation method was applied to compare the properties of the dendrimer in water with explicit counterions at two different pHs (at normal pH with neutral histidines and at low pH with fully protonated histidines) in a wide interval of temperatures. We obtained that the dendrimer at low pH has essentially larger size and size fluctuations. The electrostatic properties of the dendrimers are different but they are in good agreement with the theoretical soft sphere model and practically do not depend on temperature. We have shown that the effect of pairing of side imidazole groups is much stronger in the dendrimer with neutral histidines than in the dendrimer with protonated histidines. We also demonstrated that the capacity of a nanocontainer based on this dendrimer with protonated histidines is significantly larger than that of a nanocontainer with neutral histidines.

Why the Orientational Mobility in Arginine and Lysine Spacers of Peptide Dendrimers Designed for Gene Delivery Is Different?

New peptide dendrimer with Lys-2Arg repeating units was recently studied experimentally by NMR (RSC Advances, 2019, 9, 18018) and tested as gene carrier successfully (Int. J. Mol. Sci., 2020, 21, 3138). The unusual slowing down of the orientational mobility of 2Arg spacers in this dendrimer was revealed. It has been suggested that this unexpected behavior is caused by the Arg-Arg pairing effect in water, which leads to entanglements between dendrimer branches. In this paper, we determine the reason for this slowing down using atomistic molecular dynamics simulation of this dendrimer. We present that the structural properties of Lys-2Arg dendrimer are close to those of the Lys-2Lys dendrimer at all temperatures (Polymers, 2020, 12, 1657). However, the orientational mobility of the H-H vector in CH2-N groups of 2Arg spacers in Lys-2Arg dendrimer is significantly slower than the mobility of the same vector in the Lys-2Lys dendrimer. This result is in agreement with the recent NMR experiments for the same systems. We revealed that this difference is not due to the arginine-arginine pairing, but is due to the semiflexibility effect associated with the different contour length from CH2-N group to the end of the side arginine or lysine segment in spacers.

Comparison of Structure and Local Dynamics of Two Peptide Dendrimers with the Same Backbone but with Different Side Groups in Their Spacers.

In this paper, we perform computer simulation of two lysine-based dendrimers with Lys-2Lys and Lys-2Gly repeating units. These dendrimers were recently studied experimentally by NMR (Sci. Reports, 2018, 8, 8916) and tested as carriers for gene delivery (Bioorg. Chem., 2020, 95, 103504). Simulation was performed by molecular dynamics method in a wide range of temperatures. We have shown that the Lys-2Lys dendrimer has a larger size but smaller fluctuations as well as lower internal density in comparison with the Lys-2Gly dendrimer. The Lys-2Lys dendrimer has larger charge but counterions form more ion pairs with its NH 3 + groups and reduce the bare charge and zeta potential of the first dendrimer more strongly. It was demonstrated that these differences between dendrimers are due to the lower flexibility and the larger charge (+2) of each 2Lys spacers in comparison with 2Gly ones. The terminal CH 2 groups in both dendrimers move faster than the inner CH 2 groups. The calculated temperature dependencies of the spin-lattice relaxation times of these groups for both dendrimers are in a good agreement with the experimental results obtained by NMR.

Application of new lysine-based peptide dendrimers D3K2 and D3G2 for gene delivery: Specific cytotoxicity to cancer cells and transfection in vitro.

In order to enhance intracellular uptake and accumulation of therapeutic nucleic acids for improved gene therapy methods, numerous delivery vectors have been elaborated. Based on their origin, gene carriers are generally classified as viral or non-viral vectors. Due to their significantly reduced immunogenicity and highly optimized methods of synthesis, nanoparticles (especially those imitating natural biomolecules) constitute a promising alternative for virus-based delivery devices. Thus, we set out to develop innovative peptide dendrimers for clinical application as transfection agents and gene carriers. In the present work we describe the synthesis of two novel lysine-based dendritic macromolecules (D3K2 and D3G2) and their initial characterization for cytotoxicity/genotoxicity and transfection potential in two human cell line models: cervix adenocarcinoma (HeLa) and microvascular endothelial (HMEC-1). This approach allowed us to identify more cationic D3K2 as potent delivery agent, being able to increase intracellular accumulation of large nucleic acid molecules such as plasmids. Moreover, the dendrimers exhibited specific cytotoxicity towards cancer cell line without showing significant toxic effects on normal cells. These observations are promising prognosis for future clinical application of this type of nanoparticles.

Stable Deuterium Labeling of Histidine-Rich Lysine-Based Dendrimers.

Peptide dendrimers, due to their biocompatibility and low toxicity, are highly promising candidates as nanocarriers for drugs and genes. The development of this kind of delivery system requires reliable monitoring of their metabolic and biological pathways. In this respect, hydrogen isotope labeling has tremendous importance, being a safe tool for detection of the labeled nanocarriers. In this work, we have synthesized new histidine-rich lysine-based dendrimers (Lys-2His dendrimer) with two linear histidine (His) residues in every inner segment. The presence of His residues has enabled us to perform controlled deuteration of Lys-2His dendrimers. The high deuteration degree (around 70%) does not practically change after redissolving the samples in H2O and heating them at 40 °C, which indicates the isotopic label stability.

Lysine-based dendrimer with double arginine residues.

Due to their well-defined structure, multivalency, biocompatibility, and low toxicity, lysine dendrimers can be used as safe and efficient nanocarriers for drug and gene delivery. One useful strategy for improving the gene delivery properties of dendrimers is modification with arginine amino acid (Arg) residues. Incorporation of Arg residues could be favorable for the enhancement in transfection efficiency of lysine based dendrimers. In this work, we have synthesized a new second-generation poly-l-lysine dendrimer with repeating units containing two linear Arg residues between neighboring lysine branching points (Lys-2Arg dendrimer) and studied its physicochemical properties. We confirmed the structure of Lys-2Arg dendrimer using various one- and two-dimensional 1H and 13C NMR spectroscopy methods. Comparison of T 1H relaxation data for Lys-2Arg and Lys-2Lys dendrimers showed that the replacement of double Lys residues with double Arg residues resulted in a sharp decrease in the mobility of methylene groups in side segments and in the main chain of ε-Lys inner segments. We suggest that this unexpected effect is caused by a guanidine-guanidine pairing effect in water, which leads to entanglements between dendrimer branches.

NMR studies of excluded volume interactions in peptide dendrimers.

Peptide dendrimers are good candidates for diverse biomedical applications due to their biocompatibility and low toxicity. The local orientational mobility of groups with different radial localization inside dendrimers is important characteristic for drug and gene delivery, synthesis of nanoparticles, and other specific purposes. In this paper we focus on the validation of two theoretical assumptions for dendrimers: (i) independence of NMR relaxations on excluded volume effects and (ii) similarity of mobilities of side and terminal segments of dendrimers. For this purpose we study 1H NMR spin-lattice relaxation time, T1H, of two similar peptide dendrimers of the second generation, with and without side fragments in their inner segments. Temperature dependences of 1/T1H in the temperature range from 283 to 343 K were measured for inner and terminal groups of the dendrimers dissolved in deuterated water. We have shown that the 1/T1H temperature dependences of inner groups for both dendrimers (with and without side fragments) practically coincide despite different densities of atoms inside these dendrimers. This result confirms the first theoretical assumption. The second assumption is confirmed by the 1/T1H temperature dependences of terminal groups which are similar for both dendrimers.