Modular Synthesis of PEG-Dendritic Block Copolymers by Thermal Azide-Alkyne Cycloaddition with Internal Alkynes and Evaluation of their Self-Assembly for Drug Delivery Applications.

Linear-dendritic block copolymers assemble in solution due to differences in the solubility or charge properties of the blocks. The monodispersity and multivalency of the dendritic block provide unparalleled control for the design of drug delivery systems when incorporating poly(ethylene glycol) (PEG) as a linear block. An accelerated synthesis of PEG-dendritic block copolymers based on the click and green chemistry pillars is described. The tandem composed of the thermal azide-alkyne cycloaddition with internal alkynes and azide substitution is revealed as a flexible, reliable, atom-economical, and user-friendly strategy for the synthesis and functionalization of biodegradable (polyester) PEG-dendritic block copolymers. The high orthogonality of the sequence has been exploited for the preparation of heterolayered copolymers with terminal alkenes and alkynes, which are amenable for subsequent functionalization by thiol-ene and thiol-yne click reactions. Copolymers with tunable solubility and charge were so obtained for the preparation of various types of nanoassemblies with promising applications in drug delivery.

Mannose-modified hyaluronic acid nanocapsules for the targeting of tumor-associated macrophages.

Tumor-associated macrophages (TAMs), a class of immune cells that play a key role in tumor immunosuppression, are recognized as important targets to improve cancer prognosis and treatment. Consequently, the engineering of drug delivery nanocarriers that can reach TAMs has acquired special relevance. This work describes the development and biological evaluation of a panel of hyaluronic acid (HA) nanocapsules (NCs), with different compositions and prepared by different techniques, designed to target macrophages. The results showed that plain HA NCs did not significantly influence the polarization of M0 and M2-like macrophages towards an M1-like pro-inflammatory phenotype; however, the chemical functionalization of HA with mannose (HA-Man) led to a significant increase of NCs uptake by M2 macrophages in vitro and to an improved biodistribution in a MN/MNCA1 fibrosarcoma mouse model with high infiltration of TAMs. These functionalized HA-Man NCs showed a higher accumulation in the tumor compared to non-modified HA NCs. Finally, the pre-administration of the liposomal liver occupying agent Nanoprimer™ further increased the accumulation of the HA-Man NCs in the tumor. This work highlights the promise shown by the HA-Man NCs to target TAMs and thus provides new options for the development of nanomedicine and immunotherapy-based cancer treatments.

Polymeric nanocapsules loaded with poly(I:C) and resiquimod to reprogram tumor-associated macrophages for the treatment of solid tumors.

Background: In the tumor microenvironment (TME), tumor-associated macrophages (TAMs) play a key immunosuppressive role that limits the ability of the immune system to fight cancer. Toll-like receptors (TLRs) ligands, such as poly(I:C) or resiquimod (R848) are able to reprogram TAMs towards M1-like antitumor effector cells. The objective of our work has been to develop and evaluate polymeric nanocapsules (NCs) loaded with poly(I:C)+R848, to improve drug stability and systemic toxicity, and evaluate their targeting and therapeutic activity towards TAMs in the TME of solid tumors. Methods: NCs were developed by the solvent displacement and layer-by-layer methodologies and characterized by dynamic light scattering and nanoparticle tracking analysis. Hyaluronic acid (HA) was chemically functionalized with mannose for the coating of the NCs to target TAMs. NCs loaded with TLR ligands were evaluated in vitro for toxicity and immunostimulatory activity by Alamar Blue, ELISA and flow cytometry, using primary human monocyte-derived macrophages. For in vivo experiments, the CMT167 lung cancer model and the MN/MCA1 fibrosarcoma model metastasizing to lungs were used; tumor-infiltrating leukocytes were evaluated by flow cytometry and multispectral immunophenotyping. Results: We have developed polymeric NCs loaded with poly(I:C)+R848. Among a series of 5 lead prototypes, protamine-NCs were selected based on their physicochemical properties (size, charge, stability) and in vitro characterization, showing good biocompatibility on primary macrophages and ability to stimulate their production of T-cell attracting chemokines (CXCL10, CCL5) and to induce M1-like macrophages cytotoxicity towards tumor cells. In mouse tumor models, the intratumoral injection of poly(I:C)+R848-protamine-NCs significantly prevented tumor growth and lung metastasis. In an orthotopic murine lung cancer model, the intravenous administration of poly(I:C)+R848-prot-NCs, coated with an additional layer of HA-mannose to improve TAM-targeting, resulted in good antitumoral efficacy with no apparent systemic toxicity. While no significant alterations were observed in T cell numbers (CD8, CD4 or Treg), TAM-reprogramming in treated mice was confirmed by the relative decrease of interstitial versus alveolar macrophages, having higher CD86 expression but lower CD206 and Arg1 expression in the same cells, in treated mice. Conclusion: Mannose-HA-protamine-NCs loaded with poly(I:C)+R848 successfully reprogram TAMs in vivo, and reduce tumor progression and metastasis spread in mouse tumors.

Gallic Acid-Triethylene Glycol Aptadendrimers Synthesis, Biophysical Characterization and Cellular Evaluation.

Herein, we describe the synthesis of an aptadendrimer by covalent bioconjugation of a gallic acid-triethylene glycol (GATG) dendrimer with the G-quadruplex (G4) AT11 aptamer (a modified version of AS1411) at the surface. We evaluated the loading and interaction of an acridine orange ligand, termed C8, that acts as an anticancer drug and binder/stabilizer of the G4 structure of AT11. Dynamic light scattering experiments demonstrated that the aptadendrimer was approximately 3.1 nm in diameter. Both steady-state and time-resolved fluorescence anisotropy evidenced the interaction between the aptadendrimer and C8. Additionally, we demonstrated that the iodine atom of the C8 ligand acts as an effective intramolecular quencher in solution, while upon complexation with the aptadendrimer, it adopts a more extended conformation. Docking studies support this conclusion. Release experiments show a delivery of C8 after 4 h. The aptadendrimers tend to localize in the cytoplasm of various cell lines studied as demonstrated by confocal microscopy. The internalization of the aptadendrimers is not nucleolin-mediated or by passive diffusion, but via endocytosis. MTT studies with prostate cancer cells and non-malignant cells evidenced high cytotoxicity mainly due to the C8 ligand. The rapid internalization of the aptadendrimers and the fluorescence properties make them attractive for the development of potential nanocarriers.

Filtering the NMR Spectra of Mixtures by Coordination to Paramagnetic Cu2.

The paramagnetic spin relaxation (PSR) filter allows the selective NMR signal suppression of components in mixtures according to their complexation ability to a paramagnetic ion. It relies on the faster relaxation of nuclei in paramagnetic environments and thus is complementary to classical diffusion and relaxation filters. So far, the PSR filter has established Gd3+ as the sole PSR agent, restricting the paramagnetic filtering repertoire. Herein, we present Cu2+ as a robust PSR agent with characteristic filtering properties. While Gd3+ depends on unspecific ion-pair interactions with anionic components, Cu2+ stands out for filtering species via ordered coordination complexes. An evaluation of the paramagnetic effect of Cu2+ over more than 50 small molecules and polymers has unveiled different sensitivities to Cu2+ (especially high for pyridines, diamines, polyamines, and amino alcohols) and precise filtering conditions for mixtures (1H, COSY, and HMQC) that were challenged with a test bed of commercial drugs. The advantage of integrating Cu2+ and Gd3+ for the stepwise PSR filtering of complex mixtures is also shown.

Functional Gallic Acid-Based Dendrimers as Synthetic Nanotools to Remodel Amyloid-ß-42 into Noncytotoxic Forms.

The self-assembly of amyloid-ß (Aß) generates cytotoxic oligomers linked to the onset and progression of Alzheimer's disease (AD). As many fundamental molecular pathways that control Aß aggregation are yet to be unraveled, an important strategy to control Aß cytotoxicity is the development of bioactive synthetic nanotools capable of interacting with the heterogeneous ensemble of Aß species and remodel them into noncytotoxic forms. Herein, the synthesis of nanosized, functional gallic acid (Ga)-based dendrimers with a precise number of Ga at their surface is described. It is shown that these Ga-terminated dendrimers interact by H-bonding with monomeric/oligomeric Aß species at their Glu, Ala, and Asp residues, promoting their remodeling into noncytotoxic aggregates in a process controlled by the Ga units. The multivalent presentation of Ga on the dendrimer surface enhances their ability to interact with Aß, inhibiting the primary and secondary nucleation of Aß fibrillization and disrupting the Aß preformed fibrils.

Dendrimers as Color-Stabilizers of Pyranoanthocyanins: The Dye Concentration Governs the Host-Guest Interaction Mechanisms.

Anionic dendrimers have recently emerged as hosts (H) for the color stabilization of the flavylium cation of anthocyanin guests (G). The interaction with a promising, more hydrophobic pyranoanthocyanin illustrates how the structure and concentration of the dye modulate the host-guest interaction mechanisms. NMR and UV-vis titrations (host over guest, from G/H ratio 2089 to 45) showed that at relatively low dendrimer-to-dye concentrations, ion pairs at the dendrimer periphery prevail over dye encapsulation. This promotes the deaggregation of the dye, not previously observed with anthocyanins, and related to the more hydrophobic nature of this dye (deshielding of the dye 1H signals, higher T 2 relaxation times, constant diffusion coefficient). As the dendrimer concentration increases, the dye encapsulation, earlier unseen with structurally simpler flavylium dyes, becomes dominant (shielding and broadening of the dye 1H signals and lower T 2 and diffusion coefficient). The interaction parameters of the encapsulation process (K ∼ 4.51 × 104 M-1, n ∼ 150) indicate the binding of ca. one pyranoanthocyanin molecule by each sulfate terminal group. Our results provide insights into the ability of dendrimers to host structurally diverse pyranoflavylium-based dyes and how the structure of the latter modulates the range of interactions involved. The encapsulation ability of this dendrimer to such pH-sensitive dyes is envisioned for the host-guest sensing applications such as pH-responsive systems used for example in food smart packaging.

Liver X Receptor Activation with an Intranasal Polymer Therapeutic Prevents Cognitive Decline without Altering Lipid Levels.

The progressive accumulation of amyloid-beta (Aß) in specific areas of the brain is a common prelude to late-onset of Alzheimer's disease (AD). Although activation of liver X receptors (LXR) with agonists decreases Aß levels and ameliorates contextual memory deficit, concomitant hypercholesterolemia/hypertriglyceridemia limits their clinical application. DMHCA (N,N-dimethyl-3ß-hydroxycholenamide) is an LXR partial agonist that, despite inducing the expression of apolipoprotein E (main responsible of Aß drainage from the brain) without increasing cholesterol/triglyceride levels, shows nil activity in vivo because of a low solubility and inability to cross the blood brain barrier. Herein, we describe a polymer therapeutic for the delivery of DMHCA. The covalent incorporation of DMHCA into a PEG-dendritic scaffold via carboxylate esters produces an amphiphilic copolymer that efficiently self-assembles into nanometric micelles that exert a biological effect in primary cultures of the central nervous system (CNS) and experimental animals using the intranasal route. After CNS biodistribution and effective doses of DMHCA micelles were determined in nontransgenic mice, a transgenic AD-like mouse model of cerebral amyloidosis was treated with the micelles for 21 days. The benefits of the treatment included prevention of memory deterioration and a significant reduction of hippocampal Aß oligomers without affecting plasma lipid levels. These results represent a proof of principle for further clinical developments of DMHCA delivery systems.

Unveiling an NMR-Invisible Fraction of Polymers in Solution by Saturation Transfer Difference.

The observation of signals in solution NMR requires nuclei with sufficiently large transverse relaxation times (T2). Otherwise, broad signals embedded in the baseline afford an invisible fraction of nuclei (IF). Based on the STD (saturation transfer difference) sequence, IF-STD is presented as a quick tool to unveil IF in the 1H NMR spectra of polymers. The saturation of a polymer in a region of the NMR spectrum with IF (very short 1H T2) results in an efficient propagation of the magnetization by spin diffusion through the network of protons to a visible-invisible interphase with larger 1H T2 (STDon). Subtracting this spectrum from one recorded without saturation (STDoff) produces a difference spectrum (STDoff-on), with the nuclei at the visible-invisible interphase, that confirms the presence of an IF. Analysis of a wide collection of polymers by IF-STD reveals IF more common than previously thought, with relevant IF figures when STD > 0.4% at 750 MHz. A fundamental property of the IF-STD experiment is that the signal is generated within a single state comprising polymer domains with different dynamics, as opposed to several states in exchange with different degrees of aggregation. Contrary to a reductionist visible-invisible dichotomy, our results confirm a continuous distribution of nuclei with diverse dynamics. Since nuclei observed (edited) by IF-STD at the visible-invisible interphase are in close spatial proximity to the IF (tunable with the saturation time), they emerge as a privileged platform from which gaining an insight into the IF itself.

Impact of a Water-Soluble Gallic Acid-Based Dendrimer on the Color-Stabilizing Mechanisms of Anthocyanins.

The interaction of two anthocyanins with a water-soluble polyanionic dendrimer was studied through UV/Vis, stopped-flow, and NMR spectroscopy. Cyanidin-3-glucoside (cy3glc) revealed a stronger interaction than malvidin-3-glucoside (mv3glc) at pH 1 according to their apparent association constants. A higher color increased was also obtained for cy3glc at pH 3.5 as a result of this stronger interaction. A high-frequency chemical shift of the cy3glc aromatic protons suggest the formation of ionic pairs. The interaction parameters (K≈700 m-1 , n≈295) indicated the binding of approximately two anthocyanin molecules by each sulfate group. The equilibrium and rate constants of cy3glc in the presence of dendrimer showed an increased stability of the flavylium cation and a higher protection of this species from hydration (pK'a and pKh increased almost one pH unit). The tuning and color stabilization of anthocyanins by using this dendrimer allow novel applications as colorimetric sensors for food packaging.

Dendrimers as Competitors of Protein-Protein Interactions of the Intrinsically Disordered Nuclear Chromatin Protein NUPR1.

NUPR1 is a protumoral multifunctional intrinsically disordered protein, which is activated during the acute phases of pancreatitis, interacting with several biomolecules through residues around Ala33 and Thr68. Because of the large size of this hot-spot, designed small molecules could be insufficient to modulate all NUPR1 functions. In this work, we studied NUPR1 interactions with dendrimers by using biophysical techniques and in silico methods. Our results, obtained with different functionalized dendrimers (anionic, cationic and neutral) and several of their generations, indicate that NUPR1 was bound to the dendrimers. Functionalities at the dendrimer periphery modulated the affinity for NUPR1, and for any dendrimer, the affinity increased with generation. The affinities of most of the dendrimers were in the range 4-40 × 103 M-1, and those of the [Gn]-PhCO2Na dendrimers were similar to those of NUPR1 for its natural partners (0.1-1 × 106 M-1). In all dendrimers, the residues of NUPR1 first affected upon binding were located around Ala33, indicating that NUPR1 employs the same hot-spot to recognize any natural or synthetic molecule.

Multivalent Affidendrons with High Affinity and Specificity toward Staphylococcus aureus as Versatile Tools for Modulating Multicellular Behaviors.

Multivalency is a widely occurring natural phenomenon often exploited in nanotechnology to enhance biorecognition. We report the preparation and characterization of versatile, multivalent Affitin-dendrimer conjugates (Affidendrons) showcased by a set targeting Staphylococcus aureus ( S. aureus), an opportunistic pathogen causing numerous hospital- and community-acquired infections. Affitins are small affinity proteins characterized by higher stability and lower cost-effective production than antibodies. The strategy presented provides a platform for the rational design of multivalent nanodevices that, retaining the ability of Affitins to recognize their target with high specificity, achieve a largely enhanced affinity. Affidendrons with precisely designed size and valency have been exploited to modulate complex multicellular behaviors of S. aureus, such as agglutination and biofilm formation. Agglutination assays showed that Affidendrons rapidly cross-link S. aureus strains with high bacterial cell selectivity. Moreover, remarkably low concentrations of Affidendrons were able to effectively prevent biofilm formation. Overall, Affidendrons represent a promising platform for the rapid and selective pathogen identification, infection imaging, and theranostic applications.

Filtering the NMR Spectra of Complex Mixtures through Polymer-Mediated Paramagnetic Spin Relaxation.

A polymer-mediated paramagnetic spin relaxation (PSR) filter is presented for the selective suppression of signals from polymer-interacting species in the 1D and 2D NMR spectra of mixtures. The combined use of Gd3+ and a polymer with a high transverse relaxation enhancement (R2p , which gives a measure of the Gd3+ -complexing ability) results in the suppression of signals from any polymer-interacting component in mixtures, irrespective of their R2p . By using poly(acrylic acid) (PAA) as a model system, we demonstrate selective filtering of the signals of typical low-R2p species (insensitive to Gd3+ ), such as molecular/polymeric cations and non-ionic polymers, which, through PAA recognition (electrostatic/hydrogen-bonding interactions), become exposed to the paramagnetic effect of Gd3+ , while leaving non-PAA-interacting species unaffected. Typical suppression conditions involve PAA (approximately equimolar amount with respect to the species to be filtered) accompanied by sub-mm concentrations of Gd3+ and T2 -filters ≤100 ms. Overall, by exploiting the PSR principles and the recognition properties of polymers, selective NMR filtrations that are not attainable by diffusion, relaxation, or direct PSR filters, can be achieved.

Tuning the Size of Nanoassembles: A Hierarchical Transfer of Information from Dendrimers to Polyion Complexes.

The generation of dendrimers is a powerful tool in the control of the size and biodistribution of polyion complexes (PIC). Using a combinatorial screening of six dendrimers (18-243 terminal groups) and five oppositely charged PEGylated copolymers, a dendrimer-to-PIC hierarchical transfer of structural information was revealed with PIC diameters that increased from 80 to 500 nm on decreasing the dendrimer generation. This rise in size, which was also accompanied by a micelle-to-vesicle transition, is interpreted according to a cone- to rod-shaped progression in the architecture of the unit PIC (uPIC). This precise size tuning enabled dendritic PICs to act as nanorulers for controlled biodistribution. Overall, a domino-like control of the size and biological properties of PIC that is not attainable with linear polymers is feasible through dendrimer generation.

Fast NMR Screening of Macromolecular Complexes by a Paramagnetic Spin Relaxation Filter.

The paramagnetic spin relaxation filter is described for the rapid NMR screening of intermolecular interactions between ligands and macromolecular anionic receptors with large transverse relaxation enhancements (R 2p). The addition of micromolar concentrations of Gd3+ to the mixture produces the immediate broadening/suppression of the NMR signals of interacting species while leaving unaffected those of noncompetitive binders (one-dimensional and two-dimensional experiments). The method is highly sensitive, unveiling interactions that are too weak to generate changes in chemical shifts or relaxation times. It is operationally very simple and hence, it is amenable to ready implementation by nonspecialists. Examples of application such as detecting the formation of interpolymer complexes, cyclodextrin host-guest interactions, and the screening of DNA ligands are included that demonstrate the reliability and broad applicability of the method.

Preparation and Characterization of Biocompatible Chitosan Nanoparticles for Targeted Brain Delivery of Peptides.

Here, we describe a nanocarrier system that can transfer chitosan nanoparticles loaded with either small peptides such as the caspase inhibitor Z-DEVD-FMK or a large peptide like basic fibroblast growth factor across the blood-brain barrier. The nanoparticles are selectively directed to the brain and are not measurably taken up by the liver and spleen. Intravital fluorescent microscopy provides an opportunity to study the penetration kinetics of nanoparticles loaded with fluorescent agents such as Nile red. Nanoparticles functionalized with anti-transferrin antibody and loaded with peptides efficiently provided neuroprotection when systemically administered either as a formulation bearing a single peptide or a mixture of them. Failure of brain permeation of the nanoparticles after inhibition of vesicular transcytosis by imatinib as well as when nanoparticles were not functionalized with anti-transferrin antibody indicates that this nanomedicine formulation is rapidly transported across the blood-brain barrier by receptor-mediated transcytosis.

Biodegradable PEG-dendritic block copolymers: synthesis and biofunctionality assessment as vectors of siRNA.

One important drawback of most of the currently used dendrimers for biomedical applications is their high stability under physiological conditions that can result in cytotoxicity or complications induced by the accumulation of non-degradable synthetic materials in the organism. Particularly in the gene therapy field, vector stability can further hinder the intracellular release of the nucleic acid from the dendriplex, consequently leading to low transfection efficiencies. Therefore, biodegradable cationic dendritic structures have been eagerly awaited. However, the development of these dendritic nanocarriers is challenging because of the undesired and/or premature degradation observed during their synthesis and/or application. Here, we report new hybrid-biodegradable, biocompatible, non-toxic, and water-soluble azide-terminated PEG-GATGE dendritic block copolymers, based on a gallic acid (GA) core and triethylene glycol (TG) butanoate arms, incorporating ester bonds (E) at the dendritic arms/shell. Their successful functionalization by "click" chemistry with unprotected alkynated amines allowed complexation and delivery of siRNA. The hydrophobic character of the GATGE building unit confers to these hydrolyzable dendritic bionanomaterials a great ability to complex, protect and mediate the cellular internalization of siRNA. Moreover, the localization of the degradation points at the dendritic periphery, close to the complexed siRNA, was found to be important for nucleic acid release from the nanoparticles, rendering a significant improvement of the transfection efficiency compared to their hydrolytically stable PEG-GATG copolymer counterparts. The present study puts forward these biodegradable PEG-dendritic block copolymers not only as suitable vectors for nucleic acids, but also as new avenues for further developments exploring their use in theranostics.

Dendrimers as Innovative Radiopharmaceuticals in Cancer Radionanotherapy.

Radiotherapy is one of the most commonly used cancer treatments, with an estimate of 40% success that could be improved further if more efficient targeting and retention of radiation at the tumor site were achieved. This review focuses on the use of dendrimers in radionanotherapy, an emerging technology aimed to improve the efficiency of radiotherapy by implementing nanovectorization, an already established praxis in drug delivery and diagnosis. The labeling of dendrimers with radionuclides also aims to reduce the dose of radiolabeled materials and, hence, their toxicity and tumor resistance. Examples of radiolabeled dendrimers with alpha, beta, and Auger electron emitters are commented, along with the use of dendrimers in boron neutron capture therapy (BNCT). The conjugation of radiolabeled dendrimers to monoclonal antibodies for a more efficient targeting and the application of dendrimers in gene delivery radiotherapy are also covered.

Dendrimer mediated clustering of bacteria: improved aggregation and evaluation of bacterial response and viability.

Here, we evaluate how cationic gallic acid-triethylene glycol (GATG) dendrimers interact with bacteria and their potential to develop new antimicrobials. We demonstrate that GATG dendrimers functionalised with primary amines in their periphery can induce the formation of clusters in Vibrio harveyi, an opportunistic marine pathogen, in a generation dependent manner. Moreover, these cationic GATG dendrimers demonstrate an improved ability to induce cluster formation when compared to poly(N-[3-(dimethylamino)propyl]methacrylamide) [p(DMAPMAm)], a cationic linear polymer previously shown to cluster bacteria. Viability of the bacteria within the formed clusters and evaluation of quorum sensing controlled phenotypes (i.e. light production in V. harveyi) suggest that GATG dendrimers may be activating microbial responses by maintaining a high concentration of quorum sensing signals inside the clusters while increasing permeability of the microbial outer membranes. Thus, the reported GATG dendrimers constitute a valuable platform for the development of novel antimicrobial materials that can target microbial viability and/or virulence.