Electrochemiluminescence aptasensing method for ultrasensitive determination of lipopolysaccharide based on CRISPR-Cas12a accessory cleavage activity.

In this study, an ultrasensitive electrochemiluminescence (ECL) aptasensing method was developed for lipopolysaccharide (LPS) determination based on CRISPR-Cas12a accessory cleavage activity. Tris (2,2'-bipyridine) dichlororuthenium (II) (Ru(bpy)32+) was adsorbed on the surface of a glassy carbon electrode (GCE) coated with a mixture of gold nanoparticles (AuNPs) and Nafion film via electrostatic interaction. The obtained ECL platform (Ru(bpy)32+/AuNP/Nafion/GCE) exhibited strong ECL emission. Thiol-functionalized single-stranded DNA (ssDNA) was modified with a ferrocenyl (Fc) group and autonomously assembled on the ECL platform of Ru(bpy)32+/AuNP/Nafion/GCE via thiol-gold bonding, resulting in the quenching of ECL emission. After hybridization of the LPS aptamer strand (AS) with its partial complementary strand (CS), the formed double-stranded DNA (dsDNA) could activate CRISPR-Cas12a to indiscriminately cleave ssDNA-Fc on the surface of Ru(bpy)32+/AuNP/Nafion/GCE, resulting in recovery of the ECL intensity of Ru(bpy)32+ due to the increasing distance between Fc and the electrode surface. The combination of LPS and AS suppressed the formation of dsDNA, inhibited the activation of CRISPR-Cas12a, and prevented further cleavage of ssDNA-Fc. This mechanism aided in upholding the integrity of ssDNA-Fc on the surface of the electrode and was combined with ECL quenching induced by the target. The ECL intensity decreased linearly as the concentration of LPS increased from 1 to 50,000 pg/mL and followed a logarithmic relationship. This method exhibited a remarkably low detection limit of 0.24 pg/mL, which meets the requirement for low-concentration detection of LPS in the human body. The proposed method demonstrates the capacity of CRISPR-Cas12a to perform non-specific cutting of single-stranded DNA and transform the resultant cutting substances into changes in the ECL signal. By amalgamating this approach with the distinct identification abilities of LPS and its aptamers, a simple, responsive, and discriminatory LPS assay was established that holds immense significance for clinical diagnosis.

An injectable hemostatic PEG-based hydrogel with on-demand dissolution features for emergency care.

Uncontrolled bleeding from internal noncompressible wounds is a major cause of prehospital death in military personnel and civilian populations. An ideal hemostatic sealant for emergency care should quickly control blood loss and be removed without debridement for the follow-up treatment in the operating room, yet the lack of suitable materials to meet both requirements is the bottleneck. Herein, we suggest an injectable and dissolvable hydrogel sealant for hemorrhage management of noncompressible wounds. To this end, a 4-arm poly(ethylene glycol) (PEG) crosslinker modified with thioester linkages and terminated with aldehyde groups is designed and synthesized, and to modulate the gel properties and make it suitable as a hemostatic sealant, a mixed amino component composed of poly(ethylene imine) and adipic dihydrazide is employed to react with the PEG crosslinker to form the adhesive and elastic sealant for the first time. The aldehyde groups provide the adhesion to the tissues, and the amino component affords the procoagulant ability. More importantly, the thioester moieties allow the on-demand dissolution of sealant via a thiol-thioester exchange reaction upon exposure to an exogenous thiolate solution. In the rat femoral artery puncture and liver injury models, the administration of the hydrogel sealant dramatically reduces blood loss, and its subsequent removal does not induce rebleeding. Consequently, this hydrogel sealant with the unique feature of on-demand dissolution can not only efficiently control bleeding in emergent scenarios, but also allow non-traumatic re-exposure of wounds during subsequent surgical care. STATEMENT OF SIGNIFICANCE: Sealants, adhesives or hemostatic dressings currently used in emergency situations not only require manual pressure to control bleeding, but also face removal by cutting and mechanical debridement to enable eventual surgical treatment. In this study, we design and develop an injectable and adhesive hydrogel sealant with good procoagulant capacity and on-demand dissolution feature. The application of the hydrogel sealant substantially reduces bleeding from internal noncompressible wounds without the need for direct pressure, and demonstrates for the first time that its controlled removal without debridement does not cause rebleeding. Considering that there are currently no commercial wound sealant systems with the feature of on-demand dissolution, the hydrogel sealant developed by us is expected to address an unmet clinical need.

PEG-based thermosensitive and biodegradable hydrogels.

Injectable thermosensitive hydrogels are free-flowing polymer solutions at low or room temperature, making them easy to encapsulate the therapeutic payload or cells via simply mixing. Upon injection into the body, in situ forming hydrogels triggered by body temperature can act as drug-releasing reservoirs or cell-growing scaffolds. Finally, the hydrogels are eliminated from the administration sites after they accomplish their missions as depots or scaffolds. This review outlines the recent progress of poly(ethylene glycol) (PEG)-based biodegradable thermosensitive hydrogels, especially those composed of PEG-polyester copolymers, PEG-polypeptide copolymers and poly(organophosphazene)s. The material design, performance regulation, thermogelation and degradation mechanisms, and corresponding applications in the biomedical field are summarized and discussed. A perspective on the future thermosensitive hydrogels is also highlighted. STATEMENT OF SIGNIFICANCE: Thermosensitive hydrogels undergoing reversible sol-to-gel phase transitions in response to temperature variations are a class of promising biomaterials that can serve as minimally invasive injectable systems for various biomedical applications. Hydrophilic PEG is a main component in the design and fabrication of thermoresponsive hydrogels due to its excellent biocompatibility. By incorporating hydrophobic segments, such as polyesters and polypeptides, into PEG-based systems, biodegradable and thermosensitive hydrogels with adjustable properties in vitro and in vivo have been developed and have recently become a research hotspot of biomaterials. The summary and discussion on molecular design, performance regulation, thermogelation and degradation mechanisms, and biomedical applications of PEG-based thermosensitive hydrogels may offer a demonstration of blueprint for designing new thermogelling systems and expanding their application scope.

Biological sealing and integration of a fibrinogen-modified titanium alloy with soft and hard tissues in a rat model.

Percutaneous or transcutaneous devices are important and unique, and the corresponding biological sealing at the skin-implant interface is the key to their long-term success. Herein, we investigated the surface modification to enhance biological sealing, using a metal sheet and screw bonded by biomacromolecule fibrinogen mediated via pre-deposited synthetic macromolecule polydopamine (PDA) as a demonstration. We examined the effects of a Ti-6Al-4V titanium alloy modified with fibrinogen (Ti-Fg), PDA (Ti-PDA) or their combination (Ti-PDA-Fg) on the biological sealing and integration with skin and bone tissues. Human epidermal keratinocytes (HaCaT), human foreskin fibroblasts (HFF) and preosteoblasts (MC3T3-E1), which are closely related to percutaneous implants, exhibited better adhesion and spreading on all the three modified sheets compared with the unmodified alloy. After three-week subcutaneous implantation in Sprague-Dawley (SD) rats, the Ti-PDA-Fg sheets could significantly attenuate the soft tissue response and promote angiogenesis compared with other groups. Furthermore, in the model of percutaneous tibial implantation in SD rats, the Ti-PDA-Fg screws dramatically inhibited epithelial downgrowth and promoted new bone formation. Hence, the covalent immobilization of fibrinogen through the precoating of PDA is promising for enhanced biological sealing and osseointegration of metal implants with soft and hard tissues, which is critical for an orthopedic percutaneous medical device.

An injectable thermosensitive hydrogel loaded with an ancient natural drug colchicine for myocardial repair after infarction.

Localized administration of anti-inflammatory agents benefits patients after myocardial infarction (MI) by repressing/modulating inflammatory response of the MI region and thus accelerating repair of the impaired tissues. Colchicine (Col), an ancient natural drug, has excellent anti-inflammatory effects; however, its utilization is strictly limited due to its severe systemic toxicity and narrow therapeutic window. In this study, we developed an intramyocardial delivery system of Col using an injectable, thermosensitive poly(lactide-co-glycolide)-poly(ethylene glycol)-poly(lactide-co-glycolide) (PLGA-PEG-PLGA) polymer hydrogel as the vehicle for the treatment of MI while minimizing its systemic toxicity. The aqueous PLGA-PEG-PLGA solution loaded with Col (Col@Gel) underwent a sol-gel transition at 35 °C and maintained a gel state at body temperature. Col was released from the Col@Gel in an initial burst followed by a sustained release manner for over 8 days. The in vitro cell tests showed that the Col@Gel system significantly inhibited macrophage proliferation and migration. In a mouse model of MI, a single intramyocardial administration of the Col@Gel effectively alleviated cardiac inflammation, inhibited myocardial apoptosis and fibrosis, improved cardiac function and structure, and increased mouse survival without inducing severe systemic toxicity, which was observed following intraperitoneal administration of Col solution. These results suggested that the Col@Gel system is a reliable drug delivery system for the sustained local release of Col and has great potential as an anti-inflammatory therapy for the treat of MI.