Sustained release of 5-aminosalicylic acid from azoreductase-responsive polymeric prodrugs for prolonged colon-targeted colitis therapy.

Ulcerative colitis (UC) is a challenging inflammatory gastrointestinal disorder, whose therapies encounter limitations in overcoming insufficient colonic retention and rapid systemic clearance. In this study, we report an innovative polymeric prodrug nanoformulation for targeted UC treatment through sustained 5-aminosalicylic acid (5-ASA) delivery. Amphiphilic polymer-based 13.5 nm micelles were engineered to incorporate azo-linked 5-ASA prodrug motifs, enabling cleavage via colonic azoreductases. In vitro, micelles exhibited excellent stability under gastric/intestinal conditions while demonstrating controlled 5-ASA release over 24 h in colonic fluids. Orally administered micelles revealed prolonged 24-h retention and a high accumulation within inflamed murine colonic tissue. At an approximately 60% dose reduction from those most advanced recent studies, the platform halted DSS colitis progression and outperformed standard 5-ASA therapy through a 77-97% suppression of inflammatory markers. Histological analysis confirmed intact colon morphology and restored barrier protein expression. This integrated prodrug nanoformulation addresses limitations in colon-targeted UC therapy through localized bioactivation and tailored pharmacokinetics, suggesting the potential of nanotechnology-guided precision delivery to transform disease management.

Material priority engineered metal-polyphenol networks: mechanism and platform for multifunctionalities.

Engineering the surface of materials with desired multifunctionalities is an effective way to fight against multiple adverse factors during tissue repair process. Recently, metal-polyphenol networks (MPNs) have gained increasing attention because of their rapid and simple deposition process onto various substrates (silicon, quartz, gold and polypropylene sheets, etc.). However, the coating mechanism has not been clarified, and multifunctionalized MPNs remain unexplored. Herein, the flavonoid polyphenol procyanidin (PC) was selected to form PC-MPN coatings with Fe3+, and the effects of different assembly parameters, including pH, molar ratio between PC and Fe3+, and material priority during coating formation, were thoroughly evaluated. We found that the material priority (addition sequence of PC and Fe3+) had a great influence on the thickness of the formed PC-MPNs. Various surface techniques (e.g., ultraviolet-visible spectrophotometry, quartz crystal microbalance, X-ray photoelectron spectroscopy, atomic force microscopy, and scanning electron microscopy) were used to investigate the formation mechanism of PC-MPNs. Then PC-MPNs were further engineered with multifunctionalities (fastening cellular attachment in the early stage, promoting long-term cellular proliferation, antioxidation and antibacterial activity). We believe that these findings could further reveal the coating formation mechanism of MPNs and guide the future design of MPN coatings with multifunctionalities, thereby greatly broadening their application prospects, such as in sensors, environments, drug delivery, and tissue engineering.

Photopotentiation of the GABAA receptor with caged diazepam.

As the inhibitory γ-aminobutyric acid-ergic (GABAergic) transmission has a pivotal role in the central nervous system (CNS) and defective forms of its synapses are associated with serious neurological disorders, numerous versions of caged GABA and, more recently, photoswitchable ligands have been developed to investigate such transmission. While the complementary nature of these probes is evident, the mechanisms by which the GABA receptors can be photocontrolled have not been fully exploited. In fact, the ultimate need for specificity is critical for the proper synaptic exploration. No caged allosteric modulators of the GABAA receptor have been reported so far; to introduce such an investigational approach, we exploited the structural motifs of the benzodiazepinic scaffold to develop a photocaged version of diazepam (CD) that was tested on basolateral amygdala (BLa) pyramidal cells in mouse brain slices. CD is devoid of any intrinsic activity toward the GABAA receptor before irradiation. Importantly, CD is a photoreleasable GABAA receptor-positive allosteric modulator that offers a different probing mechanism compared to caged GABA and photoswitchable ligands. CD potentiates the inhibitory signaling by prolonging the decay time of postsynaptic GABAergic currents upon photoactivation. Additionally, no effect on presynaptic GABA release was recorded. We developed a photochemical technology to individually study the GABAA receptor, which specifically expands the toolbox available to study GABAergic synapses.

Far-Red Photoactivatable BODIPYs for the Super-Resolution Imaging of Live Cells.

The photoinduced disconnection of an oxazine heterocycle from a borondipyrromethene (BODIPY) chromophore activates bright far-red fluorescence. The high brightness of the product and the lack of autofluorescence in this spectral region allow its detection at the single-molecule level within the organelles of live cells. Indeed, these photoactivatable fluorophores localize in lysosomal compartments and remain covalently immobilized within these organelles. The suppression of diffusion allows the reiterative reconstruction of subdiffraction images and the visualization of the labeled organelles with excellent localization precision. Thus, the combination of photochemical, photophysical and structural properties designed into our fluorophores enable the visualization of live cells with a spatial resolution that is inaccessible to conventional fluorescence imaging.

Photochemical Barcodes.

A photochemical strategy to encode fluorescence signals in vivo with spatial control was designed around the unique properties of a photoactivatable borondipyrromethene (BODIPY). The photoinduced disconnection of two oxazines, flanking a single BODIPY, in two consecutive steps produces a mixture of three emissive molecules with resolved fluorescence inside polymer beads. The relative amounts and emission intensities of the three fluorophores can be regulated precisely in each bead by adjusting the dose of activating photons to mark individual particles with distinct codes of fluorescence signals. The visible wavelengths and mild illumination sufficient to induce these transformations permit the photochemical barcoding of beads also in living nematodes. Different regions of the same animal can be labeled with distinct barcodes to allow the monitoring of their dynamics for long times with no toxic effects. Thus, our photochemical strategy for the generation of fluorescence barcodes can produce multiple and distinguishable labels in the same biological sample to enable the spatiotemporal tracking of, otherwise indistinguishable, targets.

Self-Assembling Nanoparticles of Amphiphilic Polymers for In Vitro and In Vivo FRET Imaging.

Self-assembling nanoparticles of amphiphilic polymers are viable delivery vehicles for transporting hydrophobic molecules across hydrophilic media. Noncovalent contacts between the hydrophobic domains of their macromolecular components are responsible for their formation and for providing a nonpolar environment for the encapsulated guests. However, such interactions are reversible and, as a result, these supramolecular hosts can dissociate into their constituents amphiphiles to release the encapsulated cargo. Operating principles to probe the integrity of the nanocarriers and the dynamic exchange of their components are, therefore, essential to monitor the fate of these supramolecular assemblies in biological media. The co-encapsulation of complementary chromophores within their nonpolar interior offers the opportunity to assess their stability on the basis of energy transfer and fluorescence measurements. Indeed, the exchange of excitation energy between the entrapped chromophores can only occur if the nanoparticles retain their integrity to maintain donors and acceptors in close proximity. In fact, energy-transfer schemes are becoming invaluable protocols to elucidate the transport properties of these fascinating supramolecular constructs in a diversity of biological preparations and can facilitate the identification of strategies to deliver contrast agents and/or drugs to target locations in living organisms for potential diagnostic and/or therapeutic applications.

A Photoswitchable Fluorophore for the Real-Time Monitoring of Dynamic Events in Living Organisms.

This study reports the synthesis of a photoactivatable fluorophore with optimal photochemical and photophysical properties for the real-time tracking of motion in vivo. The photoactivation mechanism designed into this particular compound permits the conversion of an emissive reactant into an emissive product with resolved fluorescence, under mild illumination conditions that are impossible to replicate with conventional switching schemes based on bleaching. Indeed, the supramolecular delivery of these photoswitchable probes into the cellular blastoderm of Drosophila melanogaster embryos allows the real-time visualization of translocating molecules with no detrimental effects on the developing organisms. Thus, this innovative mechanism for fluorescence photoactivation can evolve into a general chemical tool to monitor dynamic processes in living biological specimens.

Structural Implications on the Properties of Self-Assembling Supramolecular Hosts for Fluorescent Guests.

Nine amphiphilic macromolecules with decyl and oligo(ethylene glycol) side chains, randomly distributed along a common poly(methacrylate) backbone, were synthesized from the radical copolymerization of appropriate methacrylate monomers. The resulting amphiphilic constructs differ in (1) the ratio between their hydrophobic and hydrophilic components, (2) the length of their oligo(ethylene glycol) chains, and/or (3) the molecular weight. When the ratio between hydrophobic and hydrophilic segments is comprised between 6:1 and 1:2, the macromolecules assemble spontaneously into particles with nanoscaled dimensions in neutral buffer and capture hydrophobic borondipyrromethene chromophores in their interior. However, the critical concentration required for the assembly of these supramolecular hosts as well as their hydrodynamic diameter, supramolecular weight, and number of constituent macromolecular building blocks all vary monotonically with the ratio between hydrophobic and hydrophilic components. Specifically, the critical concentration decreases and the other three parameters increase as the relative hydrophobic content raises. Furthermore, an increase in the relative hydrophobic content also discourages interchromophoric interactions between entrapped guests in both ground and excited states as well as delays access of potential quenchers. In fact, these observations demonstrate that the hydrophobic components must be in excess over their hydrophilic counterparts for optimal supramolecular hosts to assemble. Indeed, a ratio of 6:1 between the numbers of decyl and oligo(ethylene glycol) side chains appears to be ideal for this particular structural design. Under these conditions, supramolecular hosts assemble spontaneously even at relatively low polymer concentrations and their fluorescent guests do not escape into the bulk aqueous solution, despite the reversibility of the noncovalent interactions holding the supramolecular container together. Thus, these systematic investigations provide invaluable structural guidelines to design self-assembling supramolecular hosts with optimal composition for the effective encapsulation of fluorescent guests and can lead to ideal delivery vehicles for the transport of imaging probes to target locations in biological samples.

Photoactivatable BODIPYs designed to monitor the dynamics of supramolecular nanocarriers.

Self-assembling nanoparticles of amphiphilic polymers can transport hydrophobic molecules across hydrophilic media and, as a result, can be valuable delivery vehicles for a diversity of biomedical applications. Strategies to monitor their dynamics noninvasively and in real time are, therefore, essential to investigate their translocation within soft matrices and, possibly, rationalize the mechanisms responsible for their diffusion in biological media. In this context, we designed molecular guests with photoactivatable fluorescence for these supramolecular hosts and demonstrated that the activation of the fluorescent cargo, under optical control, permits the tracking of the nanocarrier translocation across hydrogel matrices with the sequential acquisition of fluorescence images. In addition, the mild illumination conditions sufficient to implement these operating principles permit fluorescence activation within developing Drosophila melanogaster embryos and enable the monitoring of the loaded nanocarriers for long periods of time with no cytotoxic effects and no noticeable influence on embryogenesis. These photoresponsive compounds combine a borondipyrromethene (BODIPY) chromophore and a photocleavable oxazine within their covalent skeleton. Under illumination at an appropriate activation wavelength, the oxazine ring cleaves irreversibly to bring the adjacent BODIPY fragment in conjugation with an indole heterocycle. This structural transformation shifts bathochromically the BODIPY absorption and permits the selective excitation of the photochemical product with concomitant fluorescence. In fact, these operating principles allow the photoactivation of BODIPY fluorescence with large brightness and infinite contrast. Thus, our innovative structural design translates into activatable fluorophores with excellent photochemical and photophysical properties as well as provides access to a general mechanism for the real-time tracking of supramolecular nanocarriers in hydrophilic matrices.

Energy-Transfer Schemes To Probe Fluorescent Nanocarriers and Their Emissive Cargo.

A strategy to probe supramolecular nanocarriers and their cargo in the intracellular space was developed on the basis of fluorescence measurements and energy transfer. It relies on the covalent attachment of an energy donor, or acceptor, to the macromolecular backbone of amphiphilic polymers and the noncovalent encapsulation of a complementary acceptor, or donor, in the resulting micelles. In aqueous environments, these macromolecules self-assemble into nanostructured constructs and bring the complementary chromophores in close proximity to enable efficient energy transfer. These supramolecular assemblies travel from the extracellular to the intracellular space and retain their integrity in the process. Indeed, donors and acceptors remain close to each other after internalization, and excitation of the former chromophores translates into significant intracellular emission from the latter. Furthermore, these supramolecular assemblies exchange their components with fast kinetics in aqueous dispersions because of the reversible character of the noncovalent contacts holding them together. As a result, micelles incorporating exclusively the donors and nanocarriers containing only the acceptors scramble their chromophoric building blocks, upon mixing, to allow the transfer of energy. These dynamic processes can be reproduced in the intracellular environment with the sequential incubation of cells with the two sets of complementary nanostructured assemblies. Thus, these operating principles and choice of supramolecular synthons are particularly valuable to monitor self-assembling nanocarriers and their cargo inside living cells and can facilitate the elucidation of the behavior of these promising delivery vehicles in a diversity of biological specimens.

Optimization of boronic ester-based amphiphilic copolymers for ROS-responsive drug delivery.

This study introduces boronic ester-based ROS-responsive amphiphilic copolymers for antioxidant drug delivery. Tuning the hydrophobic/hydrophilic balance optimized the size, curcumin encapsulation, ROS-triggered release, cellular uptake, and intracellular ROS scavenging. The lead P1b formulation self-assembled into stable 10 nm micelles enabling rapid ROS-triggered curcumin release and preferential cellular internalization. P1b eliminated over 90% of pathogenic intracellular ROS within 10 minutes, demonstrating a rapid antioxidant therapy.

Enhanced Ocular Delivery of Beva via Ultra-Small Polymeric Micelles for Noninvasive Anti-VEGF Therapy.

Pathological ocular neovascularization resulting from retinal ischemia constitutes a major cause of vision loss. Current anti-VEGF therapies rely on burdensome intravitreal injections of Bevacizumab (Beva). Herein ultrasmall polymeric micelles encapsulating Beva (P@Beva) are developed for noninvasive topical delivery to posterior eye tissues. Beva is efficiently loaded into 11 nm micelles fabricated via self-assembly of hyperbranched amphiphilic copolymers. The neutral, brush-like micelles demonstrate excellent drug encapsulation and colloidal stability. In vitro, P@Beva enhances intracellular delivery of Beva in ocular cells versus free drug. Ex vivo corneal and conjunctival-sclera-choroidal tissues transport after eye drops are improved 23-fold and 7.9-fold, respectively. Anti-angiogenic bioactivity is retained with P@Beva eliciting greater inhibition of endothelial tube formation and choroid sprouting over Beva alone. Remarkably, in an oxygen-induced retinopathy (OIR) model, topical P@Beva matching efficacy of intravitreal Beva injection, is the clinical standard. Comprehensive biocompatibility verifies safety. Overall, this pioneering protein delivery platform holds promise to shift paradigms from invasive intravitreal injections toward simplified, noninvasive administration of biotherapeutics targeting posterior eye diseases.

Gelatin-based dynamic response antioxidant, anti-inflammatory multifunctional hydrogel for enhanced diabetic wound repair.

Diabetic wound therapy presents significant challenges in the clinical environment, where persistent bleeding, disturbed inflammatory regulation, impaired cellular proliferation, and impaired tissue remodeling are major features of diabetic wound healing. However, current treatment strategies need to be considered in the context of the dynamic and complex needs of chronic wound healing. Here, multifunctional dynamic boronic acid cross-linked hydrogels were prepared by the reaction of gelatin (Gel) inoculated with 5-carboxy 3-nitrophenylboronic acid (NPBA) and Epigallocatechin gallate (EGCG) to achieve rapid gelation at pH = 7.4, EGCG could interact electrostatically with cationic antimicrobial peptides (AMP) to achieve the effective loading of AMP in the hydrogels. This hydrogel can be injected and adhered to skin defects in diabetic patients to provide a barrier and rapid hemostasis. In a high glucose microenvironment, the rapid release of AMP effectively kills bacteria, while the responsive release of EGCG eliminates reactive oxygen species (ROS) and promotes macrophage M2 polarization. In addition, the hydrogel had excellent biocompatibility and degradability properties, degraded completely after 3 days of subcutaneous injection, and was non-toxic in H&E staining of major organs and serum liver function indices in mice. This multifunctional injectable hydrogel accelerates diabetic skin wound repair and is a promising dressing for the precise treatment of diabetic wounds.

Self-Report Amphiphilic Polymer-Based Drug Delivery System with ROS-Triggered Drug Release for Osteoarthritis Therapy.

The development of drug delivery systems with real-time cargo release monitoring capabilities is imperative for optimizing nanomedicine performance. Herein, we report an innovative self-reporting drug delivery platform based on a ROS-responsive random copolymer (P1) capable of visualizing cargo release kinetics via the activation of an integrated fluorophore. P1 was synthesized by copolymerization of pinacol boronate, PEG, and naphthalimide monomers to impart ROS-sensitivity, hydrophilicity, and fluorescence signaling, respectively. Detailed characterization verified that P1 self-assembles into 11 nm micelles with 10 µg mL-1 CMC and can encapsulate hydrophobic curcumin with 79% efficiency. Fluorescence assays demonstrated H2O2-triggered disassembly and curcumin release with concurrent polymer fluorescence turn-on. Both in vitro and in vivo studies validated the real-time visualization of drug release and ROS scavenging, as well as the therapeutic effect on osteoarthritis (OA). Overall, this nanotheranostic polymeric micelle system enables quantitative monitoring of drug release kinetics for enhanced treatment optimization across oxidative stress-related diseases.

Engineering Matrix-Free Drug Protein Nanoparticles with Promising Penetration through Biobarriers for Treating Corneal Neovascularization.

Protein drugs have been widely used in treating various clinical diseases because of their high specificity, fewer side effects, and favorable therapeutic effect, but they greatly suffer from their weak permeability through tissue barriers, high sensitivity to microenvironments, degradation by proteases, and rapid clearance by the immune system. Herein, we disrupted the standard protocol where protein drugs must be delivered as the cargo via a delivery system and innovatively developed a free entrapping matrix strategy by simply mixing bevacizumab (Beva) with zinc ions to generate Beva-NPs (Beva-Zn2+), where Beva is coordinatively cross-linked by zinc ions with a loading efficiency as high as 99.2% ± 0.41%. This strategy was universal to generating various protein NPs, with different metal ions (Cu2+, Fe3+, Mg2+, Sr2+). The synthetic conditions of Beva-NPs were optimized, and the generated mechanism was investigated in detail. The entrapment, releasing profile, and the bioactivities of released Beva were thoroughly studied. By using in situ doping of the fourth-generation polyamindoamine dendrimer (G4), the Beva-G4-NPs exhibited extended ocular retention and penetration through biobarriers in the anterior segment through transcellular and paracellular pathways, effectively inhibiting corneal neovascularization (CNV) from 91.6 ± 2.03% to 13.5 ± 1.87% in a rat model of CNV. This study contributes to engineering of protein NPs by using a facile strategy for overcoming the weaknesses of protein drugs and protein NPs, such as weak tissue barrier permeability, low encapsulation efficiency, poor loading capacity, and susceptibility to inactivation.

Hydrazone-Based Amphiphilic Brush Polymer for Fast Endocytosis and ROS-Active Drug Release.

Due to the high reactivity of reactive oxygen species (ROS), it is essential to sweep them away in time. In this study, ClO--responsible amphiphilic brush polymers were prepared by free radical polymerization using two monomers consisting of polyethylene glycol as the hydrophilic part, and an alkyl chain connected by hydrazone as the hydrophobic part. The macromolecules assemble into particles with nanoscaled dimensions in a neutral buffer, which ensures quick cellular internalization. The polymer has a low critical micellization concentration and can encapsulate hydrophobic drug molecules up to 19% wt. The micelles formed by the polymer disassemble in a ClO--rich environment and release 80% of their cargo within 2 h, which possesses a faster release rate compared to the previous systems. The relatively small size and the quick response of hydrazone toward ClO- ensure a quick uptake and elimination of ROS in vitro and in vivo.

pH-Responsive Hexa-Histidine Metal Assembly (HmA) with Enhanced Biocatalytic Cascades as the Vehicle for Glucose-Mediated Long-Acting Insulin Delivery.

Diabetes has been listed as one of the three major diseases that endanger human health. Accurately injecting insulin (Ins) depending on the level of blood glucose (LBG) is the standard treatment, especially controlling LBG in the long-term by a single injection. Herein, the pH-responsive hexa-histidine metal assembly (HmA) encapsulated with enzymes (GOx and CAT) and Ins (HmA@GCI) is engineered as the vehicle for glucose-mediated insulin delivery. HmA not only shows high proteins loading efficiency, but also well retained proteins activity and protect proteins from protease damage. Within HmA, the biocatalytic activities of enzymes and the efficiency of the cascade reaction between GOx and CAT are enhanced, leading to a super response to the change of LBG with insulin release and efficient clearance of harmful byproducts of GOx (H2 O2 ). In the treatment of diabetic mice, HmA@GCI reduces LBG to normal in half an hour and maintains for more than 5 days by a single subcutaneous injection, and nearly 24 days with four consecutive injections. During the test period, no symptoms of hypoglycemia and toxicity to tissues and organs are observed. These results indicate that HmA@GCI is a safe and long-acting hypoglycemic agent with prospective clinical application.

Transformation of the shape and shrinking the size of acid-resistant metal-organic frameworks (MOFs) for use as the vehicle of oral proteins.

The oral delivery of protein-based drugs is of great significance, but faces various obstacles, including the deactivation of proteins by the low pH in the stomach and the high concentration of protease, poor transport through intestinal bio-barriers, etc. Herein, we present an acid-resistant metal-organic framework (MOF), NU-1000, in which insulin (Ins, a model protein) was loaded with high capacity (Ins@NU-1000) through the pseudo second-order kinetic model and Langmuir isotherm model. Ins@NU-1000 protects Ins from deactivation in the stomach acid environment and releases it in the intestine through the transformation of the micro-sized rod particles into spherical nanoparticles. Interestingly, the rod particles exhibit long-term retention in the intestine, and Ins is efficiently transported by the shrunk nanoparticles through intestinal bio-barriers and released into the blood, resulting in significant oral hypoglycemic effects (lasting more than 16 h after a single oral administration). Our findings demonstrate that switching the physical properties of the delivery vehicle, such as the shape and size, can contribute to the success of oral protein administration.

Development of Fmoc-Protected Bis-Amino Acids toward Automated Synthesis of Highly Functionalized Spiroligomers.

We report the fluorenylmethoxycarbonyl (Fmoc) protection of functionalized bis-amino acid building blocks using a temporary Cu2+ complexation strategy, together with an efficient multikilogram-scale synthesis of bis-amino acid precursors. This allows the synthesis of stereochemically and functionally diverse spiroligomers utilizing solid-phase Fmoc/tBu chemistry to facilitate the development of applications. Four tetramers were assembled on a semiautomated microwave peptide synthesizer. We determined their secondary structures with two-dimensional nuclear magnetic resonance spectroscopy.

Improved pasireotide response in USP8 mutant corticotroph tumours in vitro.

Cushing's disease is a rare but devastating and difficult to manage condition. The somatostatin analogue pasireotide is the only pituitary-targeting pharmaceutical approved for the treatment of Cushing's disease but is accompanied by varying efficacy and potentially severe side effects. Finding means to predict which patients are more likely to benefit from this treatment may improve their management. More than half of corticotroph tumours harbour mutations in the USP8 gene, and there is evidence of higher somatostatin receptor 5 (SSTR5) expression in the USP8-mutant tumours. Pasireotide has a high affinity for SSTR5, indicating that these tumours may be more sensitive to treatment. To test this hypothesis, we examined the inhibitory action of pasireotide on adrenocorticotrophic hormone synthesis in primary cultures of human corticotroph tumour with assessed USP8 mutational status and in immortalized murine corticotroph tumour cells overexpressing human USP8 mutants frequent in Cushing's disease. Our in vitro results demonstrate that pasireotide exerts a higher antisecretory response in USP8-mutant corticotroph tumours. Overexpressing USP8 mutants in a murine corticotroph tumour cell model increased endogenous somatostatin receptor 5 (Sstr5) transcription. The murine Sstr5 promoter has two binding sites for the activating protein 1 (AP-1) and USP8 mutants possibly to mediate their action by stimulating AP-1 transcriptional activity. Our data corroborate the USP8 mutational status as a potential marker of pasireotide response and describe a potential mechanism through which USP8 mutants may regulate SSTR5 gene expression.