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.

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.

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.

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.

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.

TP53 mutations in functional corticotroph tumors are linked to invasion and worse clinical outcome.

Corticotroph macroadenomas are rare but difficult to manage intracranial neoplasms. Mutations in the two Cushing's disease mutational hotspots USP8 and USP48 are less frequent in corticotroph macroadenomas and invasive tumors. There is evidence that TP53 mutations are not as rare as previously thought in these tumors. The aim of this study was to determine the prevalence of TP53 mutations in corticotroph tumors, with emphasis on macroadenomas, and their possible association with clinical and tumor characteristics. To this end, the entire TP53 coding region was sequenced in 86 functional corticotroph tumors (61 USP8 wild type; 66 macroadenomas) and the clinical characteristics of patients with TP53 mutant tumors were compared with TP53/USP8 wild type and USP8 mutant tumors. We found pathogenic TP53 variants in 9 corticotroph tumors (all macroadenomas and USP8 wild type). TP53 mutant tumors represented 14% of all functional corticotroph macroadenomas and 24% of all invasive tumors, were significantly larger and invasive, and had higher Ki67 indices and Knosp grades compared to wild type tumors. Patients with TP53 mutant tumors had undergone more therapeutic interventions, including radiation and bilateral adrenalectomy. In conclusion, pathogenic TP53 variants are more frequent than expected, representing a relevant amount of functional corticotroph macroadenomas and invasive tumors. TP53 mutations associated with more aggressive tumor features and difficult to manage disease.

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.

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.

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.

When aggregation-induced emission meets protein aggregates.

There is an unmet demand for research tools to monitor the multistep protein aggregation process in live cells, a process that has been associated with a growing number of human diseases. Recently, AIEgens have been developed to directly monitor the entire protein aggregation process in test tubes and live cells. Future application of AIEgens is expected to shed light on both diagnosis and treatment of disease rooted in protein aggregation.

Direct visualization and profiling of protein misfolding and aggregation in live cells.

Over the past few years, research tools have been developed to monitor the multistep protein aggregation process in live cells, a process that has been associated with a growing number of human diseases. Herein, we describe recent advances in methods that can either survey the distribution of aggregation at the level of the cellular proteome using mass spectroscopy or discern the multistep aggregation process of specific proteins of interest via fluorescence signals. Future development and application of such technologies are expected to provide insights on mechanisms, diagnosis, and treatment of diseases rooted in protein aggregation.

Preconditioning increases brain resistance against acute brain injury via neuroinflammation modulation.

Acute brain injury (ABI) is a broad concept mainly comprised of sudden parenchymal brain injury. Acute brain injury outcomes are dependent not only on the severity of the primary injury, but the delayed secondary injury that subsequently follows as well. These are both taken into consideration when determining the patient's prognosis. Growing clinical and experimental evidence demonstrates that "preconditioning," a prophylactic approach in which the brain is exposed to various pre-injury stressors, can induce varying degrees of "tolerance" against the impact of the ABI by modulating neuroinflammation. In this review, we will summarize the pathophysiology of ABI, and discuss the involved mechanisms of neuroinflammation in ABI, as well as existing experimental and clinical studies demonstrating the efficacy of preconditioning methods in various types of ABI by modulating neuroinflammation.

The Role of Iron, Its Metabolism and Ferroptosis in Traumatic Brain Injury.

Traumatic brain injury (TBI) is a structural and physiological disruption of brain function caused by external forces. It is a major cause of death and disability for patients worldwide. TBI includes both primary and secondary impairments. Iron overload and ferroptosis highly involved in the pathophysiological process of secondary brain injury. Ferroptosis is a form of regulatory cell death, as increased iron accumulation in the brain leads to lipid peroxidation, reactive oxygen species (ROS) production, mitochondrial dysfunction and neuroinflammatory responses, resulting in cellular and neuronal damage. For this reason, eliminating factors like iron deposition and inhibiting lipid peroxidation may be a promising therapy. Iron chelators can be used to eliminate excess iron and to alleviate some of the clinical manifestations of TBI. In this review we will focus on the mechanisms of iron and ferroptosis involving the manifestations of TBI, broaden our understanding of the use of iron chelators for TBI. Through this review, we were able to better find novel clinical therapeutic directions for further TBI study.

Far-red photoactivatable BODIPYs for the super-resolution imaging of live cells.

The identification of viable designs to construct switchable fluorescent probes and operate them in the interior of live cells is essential to allow the acquisition of SMLM images and permit the visualization of cellular components with sub-diffraction resolution. Our laboratories developed a mechanism to switch the fluorescence of BODIPY chromophores with the photoinduced cleavage of oxazine heterocycles under mild 405-nm illumination. With appropriate structural modifications, these switchable molecules can be engineered to immobilize covalently on large biomolecules within lysosomal compartments of live COS-7 cells and produce bright far-red fluorescence with optimal contrast upon activation. Such a combination of properties permits the acquisition of PALM images of the labeled organelles with localization precision of ca. 15nm. This article reports the experimental protocols for the synthesis of and live-cell labeling with these compounds as well as for the reconstruction of super-resolution images of the resulting biological preparations.

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.

An all-photonic full color RGB system based on molecular photoswitches.

On-command changes in the emission color of functional materials is a sought-after property in many contexts. Of particular interest are systems using light as the external trigger to induce the color changes. Here we report on a tri-component cocktail consisting of a fluorescent donor molecule and two photochromic acceptor molecules encapsulated in polymer micelles and we show that the color of the emitted fluorescence can be continuously changed from blue-to-green and from blue-to-red upon selective light-induced isomerization of the photochromic acceptors to the fluorescent forms. Interestingly, isomerization of both acceptors to different degrees allows for the generation of all emission colors within the red-green-blue (RGB) color system. The function relies on orthogonally controlled FRET reactions between the blue emitting donor and the green and red emitting acceptors, respectively.

Long-term outcomes of surgical resection with or without adjuvant therapy for treatment of primary spinal peripheral primitive neuroectodermal tumors.

OBJECTIVE: We sought to assess the use of surgical treatment, the effect of postoperative adjuvant therapy, and the prognostic factors for survival of patients with primary spinal peripheral primitive neuroectodermal tumors (pPNETs). PATIENTS AND METHODS: The clinical data of 24 patients, who had been surgically treated from April 2003 to February 2018 and in whom immunohistochemical staining results had confirmed the diagnosis of primary spinal pPNETs, were retrospectively analyzed. To analyze the factors related to prognosis, the Kaplan-Meier method was used for univariate analysis, the log-rank method was used to test the significance of difference, and multivariate analysis was performed using Cox regression. RESULTS: The overall 1-year, 2-year, and 5-year survival rates were 73.2%, 48.1%, and 12.0%, respectively. The median survival time (MST) of all patients was 21 months. Univariate analysis showed that the extent of tumor resection, adjuvant radiotherapy, and chemotherapy were the factors influencing patient prognosis after surgery (all P < 0.05); sex, age, tumor location, and preoperative Karnofsky performance scale (KPS) scores were not the influential factors for prognosis of patients after surgery (all P > 0.05). Multivariate analysis showed that gross total resection (GTR) of tumors and adjuvant radiotherapy were independent factors influencing the prognosis of patients with pPNETs (all P < 0.05). CONCLUSIONS: Primary spinal pPNETs are extremely rare, and they have a poor prognosis. Microsurgical GTR of the tumor is the preferred method of treatment. Radiotherapy plays an important role in improving the prognosis of patients with pPNETs. GTR combined with radiotherapy and chemotherapy may be the best treatment modality.

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.