Gelatin/heparin coated bio-inspired polyurethane composite fibers to construct small-caliber artificial blood vessel grafts.

Long-term patency and ability for revascularization remain challenges for small-caliber blood vessel grafts to treat cardiovascular diseases clinically. Here, a gelatin/heparin coated bio-inspired polyurethane composite fibers-based artificial blood vessel with continuous release of NO and biopeptides to regulate vascular tissue repair and maintain long-term patency is fabricated. A biodegradable polyurethane elastomer that can catalyze S-nitrosothiols in the blood to release NO is synthesized (NPU). Then, the NPU core-shell structured nanofiber grafts with requisite mechanical properties and biopeptide release for inflammation manipulation are fabricated by electrospinning and lyophilization. Finally, the surface of tubular NPU nanofiber grafts is coated with heparin/gelatin and crosslinked with glutaraldehyde to obtain small-caliber artificial blood vessels (ABVs) with the ability of vascular revascularization. We demonstrate that artificial blood vessel grafts promote the growth of endothelial cells but inhibit the growth of smooth muscle cells by the continuous release of NO; vascular grafts can regulate inflammatory balance for vascular tissue remodel without excessive collagen deposition through the release of biological peptides. Vascular grafts prevent thrombus and vascular stenosis to obtain long-term patency. Hence, our work paves a new way to develop small-caliber artificial blood vessel grafts that can maintain long-term patency in vivo and remodel vascular tissue successfully.

Myricetin Oligomer Triggers Multi-Receptor Mediated Penetration and Autophagic Restoration of Blood-Brain Barrier for Ischemic Stroke Treatment.

Restoration of blood-brain barrier (BBB) dysfunction, which drives worse outcomes of ischemic stroke, is a potential target for therapeutic opportunities, whereas a sealed BBB blocks the therapeutics entrance into the brain, making the BBB protection strategy paradoxical. Post ischemic stroke, hypoxia/hypoglycemia provokes the up-regulation of transmembrane glucose transporters and iron transporters due to multiple metabolic disorders, especially in brain endothelial cells. Herein, we develop a myricetin oligomer-derived nanostructure doped with Ce to bypass the BBB which is cointermediated by glucose transporters and iron transporters such as glucose transporters 1 (GLUT1), sodium/glucose cotransporters 1 (SGLT1), and transferrin(Tf) reporter (TfR). Moreover, it exhibits BBB restoration capacity by regulating the expression of tight junctions (TJs) through the activation of protective autophagy. The myricetin oligomers scaffold not only acts as targeting moiety but is the prominent active entity that inherits all diverse pharmacological activities of myricetin. The suppression of oxidative damage, M1 microglia activation, and inflammatory factors makes it a multitasking nanoagent with a single component as the scaffold, targeting domain and curative components.

Hitchhiking of Cas9 with nucleus-localized proteins impairs its controllability and leads to efficient genome editing of NLS-free Cas9.

CRISPR-Cas9 is the most commonly used genome-editing tool in eukaryotic cells. To modulate Cas9 entry into the nucleus to enable control of genome editing, we constructed a light-controlled CRISPR-Cas9 system to control exposure of the Cas9 protein nuclear localization signal (NLS). Although blue-light irradiation was found to effectively control the entry of Cas9 protein into the nucleus with confocal microscopy observation, effective gene editing occurred in controls with next-generation sequencing analysis. To further clarify this phenomenon, a CRISPR-Cas9 editing system without the NLS and a CRISPR-Cas9 editing system containing a nuclear export signal were also constructed. Interestingly, both Cas9 proteins could achieve effective editing of target sites with significantly reduced off-target effects. Thus, we speculated that other factors might mediate Cas9 entry into the nucleus. However, NLS-free Cas9 was found to produce effective target gene editing even following inhibition of cell mitosis to prevent nuclear import caused by nuclear membrane disassembly. Furthermore, multiple nucleus-localized proteins were found to interact with Cas9, which could mediate the "hitchhiking" of NLS-free Cas9 into the nucleus. These findings will inform future attempts to construct controllable gene-editing systems and provide new insights into the evolution of the nucleus and compatible protein functions.

Single-injection subunit vaccine for rabies prevention using lentinan as adjuvant.

Current rabies vaccines require 5 doses to provide full protection from the deadly virus, which significantly reduce the compliance of recipients. To minimize the number of immunizations herein single injection vaccines were developed. First a single injection vaccine was designed using rabies virus glycoprotein (G protein) as antigen. A time-controlled release system which uses dynamic layer-by-layer films as erodible coating was employed to accomplish multiply pulsatile releases of G protein. The single-injection vaccine elicits potent humoral and cellular immune responses comparable to the corresponding multi-dose ordinary vaccines because of their similar release pattern of G protein. To further improve its performance, a second single injection vaccine, in which lentinan was added as adjuvant, was designed. This single-injection vaccine again elicits humoral and cellular immune responses comparable to the corresponding multi-dose ordinary vaccines because of their similar release pattern of antigen and adjuvant. In addition, the second single-injection vaccine elicits higher level immune response and provides higher efficiency on virus inhibition than the first one because lentinan can booster immune response.

Advances in Off-Target Detection for CRISPR-Based Genome Editing.

The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-based genome editing system exhibits marked potential for both gene editing and gene therapy, and its continuous improvement contributes to its great clinical potential. However, the largest hindrance to its application in clinical practice is the presence of off-target effects (OTEs). Thus, in addition to continuous optimization of the CRISPR system to reduce and eventually eliminate OTEs, further development of unbiased genome-wide detection of OTEs is key for its successful clinical application. This article summarizes detection strategies for OTEs of different CRISPR systems, to provide detailed guidance for the detection of OTEs in CRISPR-based genome editing.

Construction of single-injection vaccine using new time-controlled release system.

Single-injection vaccines may overcome issues, such as high cost and poor patient compliance, of the multi-bolus regimes dominantly used in vaccination. However no such vaccine has been commercialized because time-controlled release, an unconventional release kinetics, is difficult to achieve. Here a new time-controlled release system using dynamic layer-by-layer (LBL) film as erodible coating was used to design single-injection vaccine. Unlike commonly used degradable polymers, dynamic LBL film disintegrates at a constant rate, thus allowing distinct pulsatile release of antigen at predetermined intervals. The release pattern of the single-injection vaccine mimics closely to that of ordinary multi-dose regimes. It elicits both humoral and cellular immune responses which are comparable to or even stronger than the corresponding multi-dose regime. In addition, it inhibits tumor growth more effectively. The new vaccine will not only improve patient compliance but also therapeutic outcome.

Single-injection COVID-19 subunit vaccine elicits potent immune responses.

Current vaccination schedules, including COVID-19 vaccines, require multiple doses to be administered. Single injection vaccines eliciting equivalent immune response are highly desirable. Unfortunately because unconventional release kinetics are difficult to achieve it still remains a huge challenge. Herein a single-injection COVID-19 vaccine was designed using a highly programmable release system based on dynamic layer-by-layer (LBL) films. The antigen, S1 subunit of SARS-CoV-2 spike protein, was loaded in CaCO3 microspheres, which were further coated with tannic acid (TA)/polyethylene glycol (PEG) LBL films. The single-injection vaccine was obtained by mixing the microspheres coated with different thickness of TA/PEG films. Because of the unique constant-rate erosion behavior of the TA/PEG coatings, this system allows for distinct multiple pulsatile release of antigen, closely mimicking the release profile of antigen in conventional multiple dose vaccines. Immunization with the single injection vaccine induces potent and persistent S1-specific humoral and cellular immune responses in mice. The sera from the vaccinated animal exhibit robust in vitro viral neutralization ability. More importantly, the immune response and viral inhibition induced by the single injection vaccine are as strong as that induced by the corresponding multiple dose vaccine, because they share the same antigen release profile. STATEMENT OF SIGNIFICANCE: Vaccines are the most powerful and cost-effective weapons against infectious diseases such as COVID-19. However, current vaccination schedules, including the COVID-19 vaccines, require multiple doses to be administered. Herein a single-injection COVID-19 vaccine is designed using a highly programmable release system. This vaccine releases antigens in a pulsatile manner, closely mimicking the release pattern of antigens in conventional multiple dose vaccines. As a result, one single injection of the new vaccine induces an immune response and viral inhibition similar to that induced by the corresponding multiple-dose vaccine approach.

Strategies for Optimization of the Clustered Regularly Interspaced Short Palindromic Repeat-Based Genome Editing System for Enhanced Editing Specificity.

The clustered regularly interspaced short palindromic repeats (CRISPR) system is inarguably the most valuable gene editing tool ever discovered. Currently, three classes of CRISPR-based genome editing systems have been developed for gene editing, including CRISPR/CRISPR associate system (Cas) nucleases, base editors, and prime editors. Ever-evolving CRISPR technology plays an important role in medicine; however, the biggest obstacle to its use in clinical practice is the induction of off-target effects (OTEs) during targeted editing. Therefore, continuous improvement and optimization of the CRISPR system for reduction of OTEs is a major focus in the field of CRISPR research. This review aims to provide a comprehensive guide for optimization of the CRISPR-based genome editing system.

Tough, Resilient, Adhesive, and Anti-Freezing Hydrogels Cross-Linked with a Macromolecular Cross-Linker for Wearable Strain Sensors.

Ideal conductive hydrogels for flexible, wearable strain sensors should be tough, highly resilient, adhesive, and anti-freezing. However, such hydrogels are difficult to design. Herein, a multifunctional macromolecular cross-linker (MC) based on poly(hydroxyethyl-l-glutamine) was designed and used to synthesize the hydrogels. Cross-linking with the MC leads to a reduced inhomogeneity of the gel network. Therefore, the mechanical properties of the gels are significantly improved compared with the ordinary hydrogels cross-linked with the conventional cross-linker N,N-methylenebisacrylamide (BIS). The MC-cross-linked gels also exhibit high resilience. At the same time, replacing BIS with MC significantly improves the adhesive properties of the gel, which is attributed to the introduction of a large amount of adhesive groups with the MC. The gels can stick to various substrates including skin. The good tissue adhesiveness of the gel allows it to stick to skin by itself without using any straps or adhesive tapes when used as a flexible wearable strain sensor. Both large and subtle human movements were successfully monitored using the sensor. The signals are highly stable and reliable, thanks to the high resilience of the gel. The introduction of the polar groups also improved dramatically the anti-freezing properties of the gels. Even at -20 °C, the gels still remained highly flexible and stretchable, therefore allowing the gel-based sensor to work at sub-zero temperatures. The excellent toughness, resilience, tissue-adhesiveness, and anti-freezing properties of the gel make it a good choice for a flexible wearable sensor.

A highly programmable platform for sequential release of protein therapeutics.

Drug carriers capable of releasing multiple protein therapeutics in an appropriate sequence are highly desirable for the treatment of many diseases. However current systems only allow the sequential release of two or three proteins, and it is difficult to adjust the time intervals between them. Here to solve these problems a new system is designed. The proteins are first encapsulated in CaCO3 microspheres. Then the microspheres are coated with hydrogen-bonded tannic acid (TA)/polyethylene glycol (PEG) layer-by-layer films. The encapsulated protein does not release from the microsphere until the TA/PEG coating is fully disintegrated. As the TA/PEG coating is eroded at a constant rate, the lag time for protein release is proportional to the coating thickness. To achieve sequential release, one can simply coat the protein-encapsulated microspheres with different thickness TA/PEG films and then mix them. Both in vitro and in vivo tests demonstrate that the proteins can be released from the mixed samples in a sequence according to the thickness of the TA/PEG coatings. The time intervals between the protein releases can be facilely adjusted by adjusting the thickness of the TA/PEG coatings. In addition, sequential release of more than 3 proteins can be facilely achieved.

Smart microneedle patches for rapid, and painless transdermal insulin delivery.

Insulin administration at mealtimes for the control of postprandial glucose is a major part of basal-bolus insulin therapy; however, painful subcutaneous (SC) injections lead to poor patient compliance. The microneedle (MN) patch, which allows painless transdermal drug delivery, is a promising substitute; however, it remains a big challenge to deliver insulin as rapidly as by SC injection. Here a novel MN patch is designed in which the MNs are coated with insulin/poly-l-glutamic acid (PGA) layer-by-layer (LBL) films at pH 3.0. This coating is pH-sensitive because the net charge of insulin turns from positive to negative when the pH increases from 3.0 to 7.4. As a result, when transferred to pH 7.4 media, e.g., when inserted into skin, the coating dissociates instantly and releases insulin rapidly. A brief epidermal application (<1 min) of the coated MNs is enough for complete film dissociation. More importantly, the coated MN patch exhibits a pharmacokinetic and a pharmacodynamic profile comparable to that of insulin administrated by SC injection, suggesting the coated MN patch can deliver insulin as rapidly as the SC injection. In addition, the patch exhibits excellent biocompatibility and storage stability. The new MN patch is expected to become a painless, convenient method for the control of postprandial glucose.

Precise and tunable time-controlled drug release system using layer-by-layer films as erodible coatings.

Unlike conventional drug carriers, time-controlled release systems do not release drug immediately, but start to release drug after a predetermined lag time. Coating a drug-loaded core with an erodible barrier is a valid way to defer drug release, however, the complicated erosion behavior of the erodible coatings makes it difficult to predict and tune the lag time. Herein we proposed that dynamic layer-by-layer films, using hydrogen-bonded poly(ethylene glycol)/tannic acid (PEG/TA) film as an example, are ideal erodible coatings, because their erosion mechanism is clear and simple, and they disintegrate at constant rate. As a proof, we demonstrated that the release of bovine serum albumin (BSA) from BMS spheres can be deferred by PEG/TA coating. More importantly, the lag time can be simply tuned by the thickness of the coating. By mixing bimodal mesoporous silica (BMS) spheres coated with different thickness PEG/TA films, multiple pulse release was achieved. Similar release patterns were also successfully achieved in vivo.

Development of a Self-Restricting CRISPR-Cas9 System to Reduce Off-Target Effects.

Development of the CRISPR-Cas9 gene-editing system has given rise to a new era of gene editing with wide applications in biology, medicine, agriculture, and other fields. However, the overexpression of Cas9 nuclease causes off-target effects and may trigger an immune response in vivo. Therefore, we constructed a self-restricting CRISPR-Cas9 system, where the target gene sequence corresponding to the guide RNA (gRNA) is inserted on either end of the Cas9 promoter. When double-strand breaks (DSBs) are induced in the target gene sequence, the Cas9 promoter is cut off and transcription ceases. With this system, expression of Cas9 protein at 60 h after transfection is only 10% that of the wild-type system, with about 70% promoter deletion efficiency. The target site editing efficiency and homologous recombination efficiency of the self-restricting system remain at about 50% and 30%, respectively, while the frequency of off-target indel formation decreased by 76.7%. Further, the number of indel types was also reduced from 13 to 2. Because this system does not include additional gRNA sequences, the possibility of introducing new off-target mutations is decreased. Importantly, this system is composed of a single plasmid, which could potentially be easily introduced in vivo using a viral vector or nanoparticles.

Biomimetic nano-NOS mediated local NO release for inhibiting cancer-associated platelet activation and disrupting tumor vascular barriers.

Platelets attribute to the hypercoagulation of blood and maintenance of the tumor vascular integrity, resulting in limited intratumoral perfusion of nanoparticle into solid tumors. To overcome these adversities, we herein present an antiplatelet strategy based on erythrocyte membrane-enveloped proteinic nanoparticles that biomimic nitric oxide synthase (NOS)with co-loading of l-Arginine (LA) and photosensitizer IR783 for local NO release and inhibition of the activation of tumor-associated platelets specifically, thereby enhancing vascular permeability and accumulation of the nanoparticles in tumors. A cRGD-immobolized membrane structure is constructed to actively target platelets and cancer cells respectively, through overexpressed integrin receptors such as integrin αIIbß3 and αvß3, accelerating the inhibition of platelet activation and endocytosis of nanoparticles by tumor cells. Bio-mimicking the arginine/NO pathway in vivo, synergistical delivery of LA and IR783 enables LA molecules readily oxidize to NO with O2 that is mediated by activated IR783, the resulted NO not only retards the activity of platelets to disrupt the vascular integrity of tumor but also enhances toxicity to cancer cells. In addition, NIR-controlled release localizes the NO spatiotemporally to tumor-associated platelets and prevents undesirable systemic bleeding substantially. The reduction of the hypercoagulable state is further demonstrated by the down-regulation of tissues factor (TF) expression in tumor cells. Our study describes a promising approach to combat cancer, which advances the biomimetic NOS system as the potent therapeutic forces toward clinic applications.

Reduction of thrombotic and inflammatory complications of polystyrene-block-polyisoprene-block-polystyrene (SIS) with one-step electrospinning.

Polystyrene-block-polyisoprene-block-polystyrene (SIS) has been used as biomaterials due to its soft and stable properties under physiological conditions. However, the thrombotic and inflammatory complications caused by SIS restrain its application as blood-contacting implant. To overcome this problem, the hydrophilic core-shell structured SIS-based microfiber with antioxidant encapsulation is fabricated with one-step reactive electrospinning. We demonstrate that the phase separation of SIS and acylated Pluronic F127 (F127-DA) components and crosslinking during electrospinning renders the microfiber blood compatible and stable under physiological condition; the encapsulation of 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G) in microfiber and subsequent release of AA-2G detoxifies the excess reactive oxygen species (ROS). The microfibers are nontoxic to cells and promote the fast growth and proliferation of human umbilical vein endothelial cells (HUVECs) in the presence of ROS; the thrombotic and inflammatory complications are effectively reduced with implant evaluation in vivo. Therefore, our work paves a new way to improve the biocompatibility of SIS, making it a promising candidate for blood contact materials.

Biomimicking Dual-Responsive Extracellular Matrix Restoring Extracellular Balance through the Na/K-ATPase Pathway.

Biomedical implant mimicking the physiological extracellular matrix (ECM) is a new strategy to modulate the cell microenvironment to improve implant integrity and longevity. However, the biomimicking ECM suffers from low sensitivity to pathological change and low efficiency to restore the physiological state in vivo. To overcome these problems, reactive oxygen species (ROS) and K+ dual-responsive micro-/nanofibers that encapsulate ascorbic acid-2-glucoside (AA-2G) are fabricated on an elastomer substrate with electrospinning to mimic the ECM. The strategy is based on the fact that ROS and K+ dual responsiveness enhance the sensitivity of the ECM to pathological changes and delivery of AA-2G from the ECM to cell membrane promotes reactivating Na/K-ATPase and shifting cellular diseased conditions to the normal state. We demonstrate that the ROS and K+-responsive tripolymer of poly(ethylene glycol)diacrylate, 1,2-ethanedithiol, and 4-nitrobenzo-18-crown-6-ether (PEGDA-EDT-BCAm) are synthesized successfully; the ECM composed of acylated poly(caprolactone)/PEGDA-EDT-BCAm/AA-2G micro-/nanofibers is prepared through reactive electrospinning; the ECM is sensitive to ROS and K+ concentration in the microenvironment to release AA-2G, which targets the membrane to remove the excessive ROS and reactivate Na/K-ATPase; as a result, the ECM reduces oxidative stress and restores the extracellular physiological state both in vitro and in vivo. This work provides basic principles to design an implant that can adjust the extracellular microenvironment while avoiding pathogenicity to improve implant integrity and longevity in vivo.

Construction of K+ responsive surface on SEBS to reduce the hemolysis of preserved erythrocytes.

Hemolysis of stored erythrocytes is a big obstacle for the development of new plasticizer-free polymer containers. Hemolysis is mainly caused by cell membrane oxidation and cation leaks from the intracellular fluid during storage. To construct an anti-hemolytic surface for a plasticizer-free polymer, we fabricated 2-O-α-d-glucopyranosyl-l-ascorbic acid (AA-2G)-loaded polycaprolactone (PCL)-crown ether micro/nanofibers on the surface of styrene-b-(ethylene-co-butylene)-b-styrene (SEBS). Our strategy is based on the sensitive response of the crown ether to leaked potassium, causing the release of AA-2G, the AA-2G can then remove the excess ROS, maintaining the Na/K-pump activity and the cell integrity. We demonstrated that the PCL-crown ether micro/nanofibers have been well prepared on the surface of SEBS; the micro/nanofibers provide a sensitive response to excess K+ and trigger the rapid release of AA-2G. AA-2G then acts as an antioxidant to reduce the excess ROS and maintain the Na/K-pump activity to mitigate cation leaks, resulting in the reduced hemolysis of the preserved erythrocytes. Our work thus provides a novel method for the development of plasticizer-free polymers for the storage of erythrocytes, and has the potential to be used to fabricate long-term anti-hemolytic biomaterials for in vivo use.

Multiple microarrays of non-adherent cells on a single 3D stimuli-responsive binary polymer-brush pattern.

We have developed a 3D smart binary polymer-brush pattern on the polymer substrate for inducing multiple cell microarrays aided by a lectin and temperature. The binary polymer-brush pattern composed of poly(N-isopropylacrylamide) (PNIPAM) and poly(d-gluconamidoethyl methacrylate) (PGAMA) brushes is fabricated by combining the photolithography technique with a surface-initiated photo-polymerization (SIPP) method. We demonstrate that well-defined binary polymer-brush patterns with high resolution are fabricated using this facile method. The patterned hierarchical PNIPAM brushes exhibit reversible switching to the adhesion of red blood cells (RBCs) induced by thermo-responsiveness. The PGAMA brush domains resist adhesion of RBCs but bind specifically with concanavalin A (Con A), forming a lectin pattern to capture RBCs in a site-specific manner. Therefore, multiple cell microarrays on a single platform are generated with the aid of Con A and temperature. This novel platform composed of a smart binary polymer-brush pattern is versatile and specific, which opens up pathways to potential applications such as microsensors, biochips and bioassays.

Bioinspired Antioxidant Defense System Constructed by Antioxidants-Eluting Electrospun F127-Based Fibers.

Cells were continuously exposed to oxidative damage by overproduction of reactive oxygen species (ROS) when they contacted implanted biomaterials. The strategy to prevent cells from oxidative injures remains a challenge. Inspired by the antioxidant defense system of cells, we constructed a biocompatible and ROS-responsive architecture on the substrate of styrene-b-(ethylene-co-butylene)-b-styrene elastomer (SEBS). The strategy was based on fabrication of architectures through reactive electrospinning of mixture including SEBS, acylated Pluronic F127, copolymer of poly(ethylene glycol) diacrylate and 1,2-ethanedithiol (PEGDA-EDT), and antioxidants (AA-2G) and ROS-triggered release of AA-2G from microfibers to detoxify the excess ROS. We demonstrated that the stable and hydrophilic architecture was constructed by phase separation of SEBS/F127 components and cross-linking between polymer chains during electrospinning; the ROS-responsive fibers controlled the release of AA-2G and the interaction of AA-2G with ROS reduced the oxidative damage to cells. The bioinspired architecture not only reduced mechanical and oxidative damage to cells but also maintained normal ROS level for physiological hemostasis. This work provides basic principles to design and develop antioxidative biomaterials for implantation in vivo.