Synthesis and Nuclear Magnetic Resonance Structural Evaluation of Oxime-Linked Oligosialic Acid-Based Glycodendrimers.

A series of four oxime-linked octavalent sialic acid and oligosialic acid poly(ether amidoamine) glycodendrimers were synthesized. In the attachment of the sialic acids to the dendrimer core, chemoselective oxime bonds were formed between the unprotected sugars (sialic acid or α-2,8-linked di- through tetra-sialic acids) and the aminooxy-terminated dendrimer core in a microwave-mediated reaction, resulting in good to excellent yields (58-100%) of the fully functionalized octavalent glycodendrimers. Next, using a combination of 1D and 2D nuclear magnetic resonance and working from the inside outward, we employed a systematic method to assign the proton and carbon signals starting with the smallest linkers and dendrimer cores and moving gradually up to the completed octavalent glycodendrimers. Through this approach, the assignment of the protons and carbons was possible, including the E- and Z-isomers related to the oxime dendrimer to sugar connections and relative quantities of each. These glycodendrimers were designed as broad-spectrum inhibitors of viral pathogens.

Unveiling the G4-PAMAM capacity to bind and protect Ang-(1-7) bioactive peptide by molecular dynamics simulations.

Angiotensin-(1-7) re-balance the Renin-Angiotensin system affected during several pathologies, including the new COVID-19; cardiovascular diseases; and cancer. However, one of the limiting factors for its therapeutic use is its short half-life, which might be overcome with the use of dendrimers as nanoprotectors. In this work, we addressed the following issues: (1) the capacity of our computational protocol to reproduce the experimental structural features of the (hydroxyl/amino)-terminated PAMAM dendrimers as well as the Angiotensin-(1-7) peptide; (2) the coupling of Angiotensin-(1-7) to (hydroxyl/amino)-terminated PAMAM dendrimers in order to gain insight into the structural basis of its molecular binding; (3) the capacity of the dendrimers to protect Angiotensin-(1-7); and (4) the effect of pH changes on the peptide binding and covering. Our Molecular-Dynamics/Metadynamics-based computational protocol well modeled the structural experimental features reported in the literature and our double-docking approach was able to provide reasonable initial structures for stable complexes. At neutral pH, PAMAM dendrimers with both terminal types were able to interact stably with 3 Angiotensin-(1-7) peptides through ASP1, TYR4 and PRO7 key amino acids. In general, they bind on the surface in the case of the hydroxyl-terminated compact dendrimer and in the internal zone in the case of the amino-terminated open dendrimer. At acidic pH, PAMAM dendrimers with both terminal groups are still able to interact with peptides either internalized or in its periphery, however, the number of contacts, the percentage of coverage and the number of hydrogen bonds are lesser than at neutral pH, suggesting a state for peptide release. In summary, amino-terminated PAMAM dendrimer showed slightly better features to bind, load and protect Angiotensin-(1-7) peptides.

The Unexpected Importance of the Primary Structure of the Hydrophobic Part of One-Component Ionizable Amphiphilic Janus Dendrimers in Targeted mRNA Delivery Activity.

Viral and synthetic vectors for delivery of nucleic acids impacted genetic nanomedicine by aiding the rapid development of the extraordinarily efficient Covid-19 vaccines. Access to targeted delivery of nucleic acids is expected to expand the field of nanomedicine beyond most expectations. Both viral and synthetic vectors have advantages and disadvantages. The major advantage of the synthetic vectors is their unlimited synthetic capability. The four-component lipid nanoparticles (LNPs) are the leading nonviral vector for mRNA used by Pfizer and Moderna in Covid-19 vaccines. Their synthetic capacity inspired us to develop a one-component multifunctional sequence-defined ionizable amphiphilic Janus dendrimer (IAJD) delivery system for mRNA. The first experiments on IAJDs provided, through a rational-library design combined with orthogonal-modular accelerated synthesis and sequence control in their hydrophilic part, some of the most active synthetic vectors for the delivery of mRNA to lung. The second experiments employed a similar strategy, generating, by a less complex hydrophilic structure, a library of IAJDs targeting spleen, liver, and lung. Here, we report preliminary studies designing the hydrophobic region of IAJDs by using dissimilar alkyl lengths and demonstrate the unexpectedly important role of the primary structure of the hydrophobic part of IAJDs by increasing up to 90.2-fold the activity of targeted delivery of mRNA to spleen, lymph nodes, liver, and lung. The principles of the design strategy reported here and in previous publications indicate that IAJDs could have a profound impact on the future of genetic nanomedicine.

PAMAM-calix-dendrimers: Synthesis and Thiacalixarene Conformation Effect on DNA Binding.

A convenient method for the synthesis of the first generation PAMAM dendrimers based on the thiacalix[4]arene has been developed for the first time. Three new PAMAM-calix-dendrimers with the macrocyclic core in cone, partial cone, and 1,3-alternate conformations were obtained with high yields. The interaction of the obtained compounds with salmon sperm DNA resulted in the formation of the associates of the size up to 200 nm, as shown by the UV-Vis spectroscopy, DLS, and TEM. It was demonstrated by the CD method that the structure of the DNA did not undergo significant changes upon binding. The PAMAM-calix-dendrimer based on the macrocycle in cone conformation stabilized DNA and prevented its degradation.

Targeted Delivery of mRNA with One-Component Ionizable Amphiphilic Janus Dendrimers.

Targeted and efficient delivery of nucleic acids with viral and synthetic vectors is the key step of genetic nanomedicine. The four-component lipid nanoparticle synthetic delivery systems consisting of ionizable lipids, phospholipids, cholesterol, and a PEG-conjugated lipid, assembled by microfluidic or T-tube technology, have been extraordinarily successful for delivery of mRNA to provide Covid-19 vaccines. Recently, we reported a one-component multifunctional sequence-defined ionizable amphiphilic Janus dendrimer (IAJD) synthetic delivery system for mRNA relying on amphiphilic Janus dendrimers and glycodendrimers developed in our laboratory. Amphiphilic Janus dendrimers consist of functional hydrophilic dendrons conjugated to hydrophobic dendrons. Co-assembly of IAJDs with mRNA into dendrimersome nanoparticles (DNPs) occurs by simple injection in acetate buffer, rather than by microfluidic devices, and provides a very efficient system for delivery of mRNA to lung. Here we report the replacement of most of the hydrophilic fragment of the dendron from IAJDs, maintaining only its ionizable amine, while changing its interconnecting group to the hydrophobic dendron from amide to ester. The resulting IAJDs demonstrated that protonated ionizable amines play dual roles of hydrophilic fragment and binding ligand for mRNA, changing delivery from lung to spleen and/or liver. Replacing the interconnecting ester with the amide switched the delivery back to lung. Delivery predominantly to liver is favored by pairs of odd and even alkyl groups in the hydrophobic dendron. This simple structural change transformed the targeted delivery of mRNA mediated with IAJDs, from lung to liver and spleen, and expands the utility of DNPs from therapeutics to vaccines.

One-Component Multifunctional Sequence-Defined Ionizable Amphiphilic Janus Dendrimer Delivery Systems for mRNA.

Efficient viral or nonviral delivery of nucleic acids is the key step of genetic nanomedicine. Both viral and synthetic vectors have been successfully employed for genetic delivery with recent examples being DNA, adenoviral, and mRNA-based Covid-19 vaccines. Viral vectors can be target specific and very efficient but can also mediate severe immune response, cell toxicity, and mutations. Four-component lipid nanoparticles (LNPs) containing ionizable lipids, phospholipids, cholesterol for mechanical properties, and PEG-conjugated lipid for stability represent the current leading nonviral vectors for mRNA. However, the segregation of the neutral ionizable lipid as droplets in the core of the LNP, the "PEG dilemma", and the stability at only very low temperatures limit their efficiency. Here, we report the development of a one-component multifunctional ionizable amphiphilic Janus dendrimer (IAJD) delivery system for mRNA that exhibits high activity at a low concentration of ionizable amines organized in a sequence-defined arrangement. Six libraries containing 54 sequence-defined IAJDs were synthesized by an accelerated modular-orthogonal methodology and coassembled with mRNA into dendrimersome nanoparticles (DNPs) by a simple injection method rather than by the complex microfluidic technology often used for LNPs. Forty four (81%) showed activity in vitro and 31 (57%) in vivo. Some, exhibiting organ specificity, are stable at 5 °C and demonstrated higher transfection efficiency than positive control experiments in vitro and in vivo. Aside from practical applications, this proof of concept will help elucidate the mechanisms of packaging and release of mRNA from DNPs as a function of ionizable amine concentration, their sequence, and constitutional isomerism of IAJDs.

Developments in Treatment Methodologies Using Dendrimers for Infectious Diseases.

Dendrimers comprise a specific group of macromolecules, which combine structural properties of both single molecules and long expanded polymers. The three-dimensional form of dendrimers and the extensive possibilities for use of additional substrates for their construction creates a multivalent potential and a wide possibility for medical, diagnostic and environmental purposes. Depending on their composition and structure, dendrimers have been of interest in many fields of science, ranging from chemistry, biotechnology to biochemical applications. These compounds have found wide application from the production of catalysts for their use as antibacterial, antifungal and antiviral agents. Of particular interest are peptide dendrimers as a medium for transport of therapeutic substances: synthetic vaccines against parasites, bacteria and viruses, contrast agents used in MRI, antibodies and genetic material. This review focuses on the description of the current classes of dendrimers, the methodology for their synthesis and briefly drawbacks of their properties and their use as potential therapies against infectious diseases.

Engineering of Janus-Like Dendrimers with Peptides Derived from Glycoproteins of Herpes Simplex Virus Type 1: Toward a Versatile and Novel Antiviral Platform.

Novel antiviral nanotherapeutics, which may inactivate the virus and block it from entering host cells, represent an important challenge to face viral global health emergencies around the world. Using a combination of bioorthogonal copper-catalyzed 1,3-dipolar alkyne/azide cycloaddition (CuAAC) and photoinitiated thiol-ene coupling, monofunctional and bifunctional peptidodendrimer conjugates were obtained. The conjugates are biocompatible and demonstrate no toxicity to cells at biologically relevant concentrations. Furthermore, the orthogonal addition of multiple copies of two different antiviral peptides on the surface of a single dendrimer allowed the resulting bioconjugates to inhibit Herpes simplex virus type 1 at both the early and the late stages of the infection process. The presented work builds on further improving this attractive design to obtain a new class of therapeutics.