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Gene therapies represent promising new therapeutic options for a variety of indications. However, despite several approved drugs, its potential remains untapped. For polymeric gene delivery, endosomal escape represents a bottleneck. SO1861, a naturally occurring triterpene saponin with endosomal escape properties isolated from Saponaria officinalis L., has been described as additive agent to enhance transfection efficiency (sapofection). However, the challenge to synchronize the saponin and gene delivery system in vivo imposes limitations. Herein, we address this issue by conjugating SO1861 to a peptide-based gene vector using a pH-sensitive hydrazone linker programmed to release SO1861 at the acidic pH of the endosome. Nanoplexes formulated with SO1861-equipped peptides were investigated for transfection efficiency and tolerability in vitro and in vivo . In all investigated cell lines, SO1861-conjugated nanoplexes have shown superior transfection efficiency and cell viability over supplementation of transfection medium with free SO1861. Targeted SO1861-equipped nanoplexes incorporating a targeting peptide were tested in vitro and in vivo in an aggressively growing neuroblastoma allograft model in mice. Using a suicide gene vector encoding the cytotoxic protein saporin, a slowed tumor growth and improved survival rate were observed for targeted SO1861-equipped nanoplexes compared to vehicle control.

Gene therapy provides exciting new therapeutic opportunities beyond the reach of traditional treatments. Despite the tremendous progress of viral vectors, their high cost, complex manufacturing, and side effects have encouraged the development of nonviral alternatives, including cationic polymers. However, these are less efficient in overcoming cellular barriers, resulting in lower transfection efficiencies. Although the exquisite structural tunability of polymers might be envisaged as a versatile tool for improving transfection, the need to fine-tune several structural parameters represents a bottleneck in current screening technologies. By taking advantage of the fast-forming and strong boronate ester bond, an archetypal example of dynamic covalent chemistry, a highly adaptable gene delivery platform is presented, in which the polycation synthesis and pDNA complexation occur in situ . The robustness of the strategy entitles the simultaneous evaluation of several structural parameters at will, enabling the accelerated screening and adaptive optimization of lead polymeric vectors using dynamic covalent libraries.

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Anfray, Varela, Ummarino, Maeda, Sironi, Gandoy, Brea, Loza, León, Calvo, Correa, Fernandez-Megia, Alonso, Allavena, Crecente-Campo and Andón.)

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., (© 2022. The Author(s).)

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 C 8, 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 C 8 . Additionally, we demonstrated that the iodine atom of the C 8 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 C 8 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 C 8 ligand. The rapid internalization of the aptadendrimers and the fluorescence properties make them attractive for the development of potential nanocarriers.

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 Gd 3+ as the sole PSR agent, restricting the paramagnetic filtering repertoire. Herein, we present Cu 2+ as a robust PSR agent with characteristic filtering properties. While Gd 3+ depends on unspecific ion-pair interactions with anionic components, Cu 2+ stands out for filtering species via ordered coordination complexes. An evaluation of the paramagnetic effect of Cu 2+ over more than 50 small molecules and polymers has unveiled different sensitivities to Cu 2+ (especially high for pyridines, diamines, polyamines, and amino alcohols) and precise filtering conditions for mixtures ( 1 H, COSY, and HMQC) that were challenged with a test bed of commercial drugs. The advantage of integrating Cu 2+ and Gd 3+ for the stepwise PSR filtering of complex mixtures is also shown.

The self-assembly of amyloid-β (Aβ) generates cytotoxic oligomers linked to the onset and progression of Alzheimer's disease (AD). As many fundamental molecular pathways that control Aβ aggregation are yet to be unraveled, an important strategy to control Aβ cytotoxicity is the development of bioactive synthetic nanotools capable of interacting with the heterogeneous ensemble of Aβ 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 Aβ 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 Aβ, inhibiting the primary and secondary nucleation of Aβ fibrillization and disrupting the Aβ preformed fibrils.

The observation of signals in solution NMR requires nuclei with sufficiently large transverse relaxation times ( T 2 ). 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 1 H NMR spectra of polymers. The saturation of a polymer in a region of the NMR spectrum with IF (very short 1 H T 2 ) results in an efficient propagation of the magnetization by spin diffusion through the network of protons to a visible-invisible interphase with larger 1 H T 2 (STD on ). Subtracting this spectrum from one recorded without saturation (STD off ) produces a difference spectrum (STD off-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.

The progressive accumulation of amyloid-beta (Aβ) 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 Aβ levels and ameliorates contextual memory deficit, concomitant hypercholesterolemia/hypertriglyceridemia limits their clinical application. DMHCA ( N , N -dimethyl-3β-hydroxycholenamide) is an LXR partial agonist that, despite inducing the expression of apolipoprotein E (main responsible of Aβ 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 Aβ oligomers without affecting plasma lipid levels. These results represent a proof of principle for further clinical developments of DMHCA delivery systems.

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 1 H 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 1 H signals and lower T 2 and diffusion coefficient). The interaction parameters of the encapsulation process ( K ∼ 4.51 × 10 4 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., Competing Interests: The authors declare no competing financial interest., (© 2021 American Chemical Society.)

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 pK h 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., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 × 10 3 M -1 , and those of the [Gn]-PhCO 2 Na dendrimers were similar to those of NUPR1 for its natural partners (0.1-1 × 10 6 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.

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.

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 Gd 3+ and a polymer with a high transverse relaxation enhancement (R 2p , which gives a measure of the Gd 3+ -complexing ability) results in the suppression of signals from any polymer-interacting component in mixtures, irrespective of their R 2p . By using poly(acrylic acid) (PAA) as a model system, we demonstrate selective filtering of the signals of typical low-R 2p species (insensitive to Gd 3+ ), such as molecular/polymeric cations and non-ionic polymers, which, through PAA recognition (electrostatic/hydrogen-bonding interactions), become exposed to the paramagnetic effect of Gd 3+ , 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 Gd 3+ and T 2 -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., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 Gd 3+ 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., Competing Interests: The authors declare no competing financial interest.

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.

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.

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.

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.

An in situ template fabrication of inorganic nanoparticles using carboxylated PEG-dendritic block copolymers of the GATG family is described as a function of the dendritic block generation, the metal (Au, CdSe) and metal molar ratio. The biocompatibility of the generated nanoparticles analysed in terms of their aggregation in physiological media, cytotoxicity and uptake by macrophages relates to the PEG density of the surface of the hybrids.

Although growth factors and anti-apoptotic peptides have been shown to be neuroprotective in stroke models, translation of these experimental findings to clinic is hampered by limited penetration of peptides to the brain. Here, we show that a large peptide like the basic fibroblast growth factor (bFGF) and a small peptide inhibitor of caspase-3 (z-DEVD-FMK) can effectively be transported to the brain after systemic administration by incorporating these peptides to brain-targeted nanoparticles (NPs). Chitosan NPs were loaded with peptides and then functionalized by conjugating with antibodies directed against the transferrin receptor-1 on brain endothelia to induce receptor-mediated transcytosis across the blood-brain barrier (BBB). Pre-ischemic systemic administration of bFGF- or z-DEVD-FMK-loaded NPs significantly decreased the infarct volume after 2-hour middle cerebral artery occlusion and 22-hour reperfusion in mice. Co-administration of bFGF- or z-DEVD-FMK-loaded NPs reduced the infarct volume further and provided a 3-hour therapeutic window. bFGF-loaded NPs were histologically detected in the brain parenchyma and also restored ischemia-induced Akt dephosphorylation. The neuroprotection was not observed when receptor-mediated transcytosis was inhibited with imatinib or when bFGF-loaded NPs were not conjugated with the targeting antibody, which enables them to cross the BBB. Nanoparticles targeted to brain are promising drug carriers to transport large as well as small BBB-impermeable therapeutics for neuroprotection against stroke.

The paramagnetic spin relaxation (PSR) filter allows the suppression of the NMR resonances of individual components in mixtures according to their Gd(3+)-complexing ability. The difficulty in predicting this property hampers, however, the widespread application of this filter. Herein we describe that the PSR filter is dominated by the transverse relaxation enhancement (R(2p)) experienced by nuclei in the presence of Gd(3+), so that R(2p) represents a reliable predictive tool of suppression in the 1D and 2D PSR filter of complex mixtures. The robustness of R(2p) as a predictive tool in PSR filters has been demonstrated at different magnetic fields and for the (1)H, (13)C, COSY, and HMQC filtering of commercial multicomponent compositions, including beverages and drugs.

Dendrimers are synthetic macromolecules composed of repetitive layers of branching units that emerge from a central core. They are characterized by a tunable size and precise number of peripheral groups which determine their physicochemical properties and function. Their high multivalency, functional surface, and globular architecture with diameters in the nanometer scale makes them ideal candidates for a wide range of applications. Gallic acid-triethylene glycol (GATG) dendrimers have attracted our attention as a promising platform in the biomedical field because of their high tunability and versatility. The presence of terminal azides in GATG dendrimers and poly(ethylene glycol) (PEG)-dendritic block copolymers allows their efficient functionalization with a variety of ligands of biomedical relevance including anionic and cationic groups, carbohydrates, peptides, or imaging agents. The resulting functionalized dendrimers have found application in drug and gene delivery, as antiviral agents and for the treatment of neurodegenerative diseases, in diagnosis and as tools to study multivalent carbohydrate recognition and dendrimer dynamics. Herein, we present an account on the preparation and recent applications of GATG dendrimers in these fields.

The characteristic distribution of transverse relaxation times (T2) within dendrimers (shorter values at the core than the periphery) can be exploited in T2-edited 1D and 2D NMR experiments for the stepwise filtering of internal nuclei according to their topology within the dendritic structure. The resulting filtered spectra, which can be conceived as corresponding to virtual hollow dendrimers, benefit from reduced signal overlap, thus facilitating signal assignment and characterization. The generality of the method as a powerful tool in structural and end-group analysis has been confirmed with various dendritic families and nuclei ((1)H, (13)C, (31)P).

An unexpected (1)H NMR invisible fraction (IF) for chitosan (CS) and CS-g-PEG is reported. The presence of this IF is remarkable considering that solution NMR is recognized as the method of choice for studying structural modifications in CS, including the degrees of acetylation (DA) and substitution (DS). In spite of IF figures as high as 50%, this IF does not interfere in the correct determination of the DA by (1)H NMR, pointing to a homogeneous distribution of acetyl groups along the visible and invisible fractions. Quite in contrast, the IF negatively biases the determination of the DS in CS-g-PEG, with relative errors as high as 150% in a broad range of temperatures, pH values, and concentrations. This fact raises concerns about the accuracy of previously reported DS data for CS-g-PEG and many other CS copolymers. Efficient user-friendly conditions have been developed for the correct determination of the DS of CS-g-PEG by depolymerization by nitrous acid.

A critical objective in cancer therapy is to reduce the systemic toxicity through the modification of the biodistribution of anticancer drugs. Herein, we disclose a new biodegradable nanocarrier, polyglutamic acid (PGA) nanocapsules, and present the in vivo pharmacokinetics/toxicity proof-of-concept for the anticancer drug plitidepsin. These novel nanocapsules were prepared using a modified solvent displacement technique where the polyamino acid was electrostatically deposited onto the lipid core. The nanocapsules exhibited an average size of 200 nm, a negative zeta potential and a great capacity for the encapsulation of plitidepsin (encapsulation efficiency above 90%). In addition, the nanocapsules could be freeze-dried and showed an adequate stability profile upon storage. Finally, the in vivo proof-of-concept studies performed in mice indicated that the encapsulation provided the drug with a prolonged blood circulation and a significantly reduced toxicity. In fact, the maximum tolerated dose of the nanoencapsulated drug was more than 3 times that of the reference formulation (Cremophor® EL plitidepsin solution). Overall, beyond the value of this specific formulation, the work reported here represents the evidence of the potential of polyamino acid nanocapsules in nano-oncological therapy.

Multivalency is a key, ubiquitous phenomenon in nature characterized by a complex combination of binding mechanisms, with special relevance in carbohydrate-lectin recognition. Herein we introduce an original surface plasmon resonance kinetic approach to analyze multivalent interactions that has been validated with dendrimers as monodisperse multivalent analytes binding to lectin clusters. The method, based on the analysis of early association and late dissociation phases of the sensorgrams provides robust information of the glycoconjugate binding efficiency and real-time structural data of the binding events under the complex scenario of the glyco-cluster effect. Notably, it reveals the dynamic nature of the interaction and offers experimental evidence on the contribution of binding mechanisms.

The development and evaluation of PEGylated chitosan (CS) nanocapsules (NCs) conjugated to a monoclonal antibody anti-TMEFF-2 (CS-PEG-anti-TMEFF-2 mAb NCs) for targeted delivery of docetaxel (DCX) is presented. CS-PEG-Biotin NCs, displaying biotin tags at their surface, were obtained and efficiently functionalized with an anti-TMEFF-2 mAb through a convenient avidin-biotin approach. Cell cycle analysis after treatment with different DCX-loaded CS-PEG NC formulations indicated that the encapsulated drug remained fully active, showing a similar functional behavior to free DCX. In vivo efficacy studies using a non-small cell lung carcinoma xenograft revealed that CS-PEG-anti-TMEFF-2 NCs resulted as effective as free DCX (Taxotere®). Interestingly, differences on the pharmacodynamic behavior among the different DCX formulations were observed. Thus, while free DCX exhibited a fast and short effect on tumor volume reduction, CS-PEG-anti-TMEFF-2 mAb NCs showed a delayed and prolonged action, with no significant side effects of treatments., (Copyright © 2012 Elsevier B.V. All rights reserved.)

NMR is a powerful tool to study the dynamics of dendrimers. By analogy to linear polymers, shorter T(1) relaxation times have been traditionally associated to less mobile nuclei and hence, dendrimers described with reduced local motions at either the core or the periphery. Herein we report a NMR relaxation study [(1)H and (13)C T(1), T(2); (13)C{(1)H}NOE; various fields and temperatures] which reveals profound differences between the relaxation behavior of dendrimers and linear polymers. Dendrimers show slower dynamics at internal layers and on increasing generation and may display internal nuclei in the slow motional regime with larger T(1) values than the periphery. In contrast to the relaxation properties of linear polymers, these T(1) increments should not be interpreted as resulting from faster dynamics. Only the recording of T(1) data at various temperatures (alternatively, T(2) or NOE at one temperature) ensures the correct interpretation of dendrimer dynamics.

The synthesis of a new family of amino-functionalized gallic acid-triethylene glycol (GATG) dendrimers and their block copolymers with polyethylene glycol (PEG) has recently being disclosed. In addition, these dendrimers have shown potential for gene delivery applications, as they efficiently complex nucleic acids and form small and homogeneous dendriplexes. On this basis, the present study aimed to explore the interaction of the engineered dendriplexes with blood components, as well as their stability, cytotoxicity and ability to enter and transfect mammalian cells. Results show that GATG dendrimers can form stable dendriplexes, protect the associated pDNA from degradation, and are biocompatible with HEK-293T cells and erythrocytes. More importantly, dendriplexes are effectively internalized by HEK-293T cells, which are successfully transfected. Besides, PEGylation has a marked influence on the properties of the resulting dendriplexes. While PEGylated GATG dendrimers have improved biocompatibility, the long PEG chains limit their uptake by HEK-293T cells, and thus, their ability to transfect them. As a consequence, the degree of PEGylation in dendriplexes containing dendrimer/block copolymer mixtures emerges as an important parameter to be modulated in order to obtain an optimized stealth formulation able to effectively induce the expression of the encoded protein.

The influence of a GATG (gallic acid-triethylene glycol) dendrimer decorated with 27 terminal morpholine groups ([G3]-Mor) on the aggregation process of Alzheimer's peptide has been investigated. Amyloid fibrils were formed from the Aβ 1-28 peptide and the process was monitored by a ThT assay, changes in CD spectra, and transmission electron microscopy. In the presence of [G3]-Mor, more fibrils were built and the process significantly accelerated compared with a control. The cytotoxicity of (1) Aβ and (2) the system [G3]-Mor/Aβ was monitored at different stages of the aggregation process. Prefibrillar species were more toxic than mature fibrils. [G3]-Mor significantly reduced the toxicity of Aβ, probably because of lowering the amount of prefibrillar forms in the system by speeding up the process of fibril formation., From the Clinical Editor: In this study, GATG dendrimer decorated with 27 terminal morpholine groups was able to reduce beta-amyloid fibril formation, which might represent a new method to address the key pathology in Alzheimer's disease., (Copyright © 2012 Elsevier Inc. All rights reserved.)

GATG (gallic acid-triethylene glycol) dendrimers represent appealing nanostructures for biomedical applications. The incorporation of specific ligands and targeting and imaging agents on their surface has resulted in promising tools in diagnosis and drug delivery. With the aim to further explore the versatility of GATG dendrimers in the biomedical field, in this work we study the effect of peripheral substitution on their uptake and intracellular trafficking in living cells. To this end, peripheral groups with different physicochemical properties and biological relevance have been installed on the surface of GATG dendrimers, and their interactions, uptake efficacy, and specificity for certain cell populations studied by confocal microscopy. Finally, this information was used to design a pH-sensitive drug delivery system for the selective release of cargo molecules inside cells after lysosomal localization. These results along with the easy functionalization and modular architecture of GATG dendrimers reveal these systems as promising nanotools in biomedicine.

During the last decades, great efforts have been devoted to design polymers for reducing the toxicity, increasing the absorption, and improving the release profile of drugs. Advantage has been also taken from the inherent multivalency of polymers and dendrimers for the incorporation of diverse functional molecules of interest in targeting and diagnosis. In addition, polymeric hydrogels with the ability to encapsulate drugs and cells have been developed for drug delivery and tissue engineering applications. In the long road to this successful story, pharmaceutical sciences have been accompanied by parallel advances in synthetic methodologies allowing the preparation of precise polymeric materials with enhanced properties. In this context, the introduction of the click concept by Sharpless and coworkers in 2001 focusing the attention on modularity and orthogonality has greatly benefited polymer synthesis, an area where reaction efficiency and product purity are significantly challenged. The purpose of this Expert Review is to discuss the impact of click chemistry in the preparation and functionalization of polymers, dendrimers, and hydrogels of interest in drug delivery.

Introduction: Advances achieved over the last few years in drug delivery have provided novel and versatile possibilities for the treatment of various diseases. Among the biomaterials applied in this field, it is worth highlighting the increasing importance of polyaminoacids and polypeptides. The appealing properties of these polymers are very promising for the design of novel compositions in a variety of drug delivery applications., Areas Covered: This review provides an overview on the general characteristics of polyaminoacids and polypeptides and briefly discusses different synthetic pathways for their production. This is followed by a detailed description of different drug delivery applications of these polymers, emphasizing those examples that already reached advanced preclinical development or have entered clinical trials., Expert Opinion: Polyaminoacids and polypeptides are gaining much attention in drug delivery due to their exceptional properties. Their application as polymers for drug delivery purposes has been sped up by the significant achievements related to their synthesis. Certainly, cancer therapy has benefited the most from these advances, although other fields such as vaccine delivery and alternative administration routes are also being successfully explored. The design of new entities based on polyaminoacids and polypeptides and the improved insight gained in drug delivery guarantee exciting findings in the near future.

The purpose of this Expert Review is to discuss the impact of click chemistry in nanosized drug delivery systems. Since the introduction of the click concept by Sharpless and coworkers in 2001, numerous examples of click reactions have been reported for the preparation and functionalization of polymeric micelles and nanoparticles, liposomes and polymersomes, capsules, microspheres, metal and silica nanoparticles, carbon nanotubes and fullerenes, or bionanoparticles. Among these click processes, Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) has attracted most attention based on its high orthogonality, reliability, and experimental simplicity for non-specialists. A renewed interest in the use of efficient classical transformations has been also observed (e.g., thiol-ene coupling, Michael addition, Diels-Alder). Special emphasis is also devoted to critically discuss the click concept, as well as practical aspects of application of CuAAC to ensure efficient and harmless bioconjugation.