Engineering APOBEC3A deaminase for highly accurate and efficient base editing.

Cytosine base editors (CBEs) are effective tools for introducing C-to-T base conversions, but their clinical applications are limited by off-target and bystander effects. Through structure-guided engineering of human APOBEC3A (A3A) deaminase, we developed highly accurate A3A-CBE (haA3A-CBE) variants that efficiently generate C-to-T conversion with a narrow editing window and near-background level of DNA and RNA off-target activity, irrespective of methylation status and sequence context. The engineered deaminase domains are compatible with PAM-relaxed SpCas9-NG variant, enabling accurate correction of pathogenic mutations in homopolymeric cytosine sites through flexible positioning of the single-guide RNAs. Dual adeno-associated virus delivery of one haA3A-CBE variant to a mouse model of tyrosinemia induced up to 58.1% editing in liver tissues with minimal bystander editing, which was further reduced through single dose of lipid nanoparticle-based messenger RNA delivery of haA3A-CBEs. These results highlight the tremendous promise of haA3A-CBEs for precise genome editing to treat human diseases.

Engineering a precise adenine base editor with minimal bystander editing.

Adenine base editors (ABEs) catalyze A-to-G transitions showing broad applications, but their bystander mutations and off-target editing effects raise safety concerns. Through structure-guided engineering, we found ABE8e with an N108Q mutation reduced both adenine and cytosine bystander editing, and introduction of an additional L145T mutation (ABE9), further refined the editing window to 1-2 nucleotides with eliminated cytosine editing. Importantly, ABE9 induced very minimal RNA and undetectable Cas9-independent DNA off-target effects, which mainly installed desired single A-to-G conversion in mouse and rat embryos to efficiently generate disease models. Moreover, ABE9 accurately edited the A5 position of the protospacer sequence in pathogenic homopolymeric adenosine sites (up to 342.5-fold precision over ABE8e) and was further confirmed through a library of guide RNA-target sequence pairs. Owing to the minimized editing window, ABE9 could further broaden the targeting scope for precise correction of pathogenic single-nucleotide variants when fused to Cas9 variants with expanded protospacer adjacent motif compatibility. bpNLS, bipartite nuclear localization signals.

Reversible Assembly of Proteins and Phenolic Polymers for Intracellular Protein Delivery with Serum Stability.

The design of intracellular delivery systems for protein drugs remains a challenge due to limited delivery efficacy and serum stability. Herein, we propose a reversible assembly strategy to assemble cargo proteins and phenolic polymers into stable nanoparticles for this purpose using a heterobifunctional adaptor (2-formylbenzeneboronic acid). The adaptor is easily decorated on cargo proteins via iminoboronate chemistry and further conjugates with catechol-bearing polymers to form nanoparticles via boronate diester linkages. The nanoparticles exhibit excellent serum stability in culture media but rapidly release the cargo proteins triggered by lysosomal acidity and GSH after endocytosis. In a proof-of-concept animal model, the strategy successfully transports superoxide dismutase to retina via intravitreal injection and efficiently ameliorates the oxidative stress and cellular damage in the retina induced by ischemia-reperfusion (I/R) with minimal adverse effects. The reversible assembly strategy represents a robust and efficient method to develop serum-stable systems for the intracellular delivery of biomacromolecules.

A Fluorous Peptide Amphiphile with Potent Antimicrobial Activity for the Treatment of MRSA-induced Sepsis and Chronic Wound Infection.

The rising prevalence of global antibiotic resistance evokes the urgent need for novel antimicrobial candidates. Cationic lipopeptides have attracted much attention due to their strong antimicrobial activity, broad-spectrum and low resistance tendency. Herein, a library of fluoro-lipopeptide amphiphiles was synthesized by tagging a series of cationic oligopeptides with a fluoroalkyl tail via a disulfide spacer. Among the lipopeptide candidates, R6F bearing six arginine moieties and a fluorous tag shows the highest antibacterial activity, and it exhibits an interesting fluorine effect as compared to the non-fluorinated lipopeptides. The high antibacterial activity of R6F is attributed to its excellent bacterial membrane permeability, which further disrupts the respiratory chain redox stress and cell wall biosynthesis of the bacteria. By co-assembling with lipid nanoparticles, R6F showed high therapeutic efficacy and minimal adverse effects in the treatment of MRSA-induced sepsis and chronic wound infection. This work provides a novel strategy to design highly potent antibacterial peptide amphiphiles for the treatment of drug-resistant bacterial infections.

Fluorination Promotes the Cytosolic Delivery of Genes, Proteins, and Peptides.

The cytosolic delivery of biomolecules such as genes, proteins, and peptides is of great importance for biotherapy but usually limited by multiple barriers during the process. Cell membrane with high hydrophobic character is one of the representative biological barriers for cytosolic delivery. The introduction of hydrophobic ligands such as aliphatic lipids onto materials or biomolecules could improve their membrane permeability. However, these ligands are lipophilic and tend to interact with the phospholipids in the membrane as well as serum proteins, which may hinder efficient intracellular delivery. To solve this issue, our research group proposed the use of fluorous ligands with both hydrophobicity and lipophobicity as ideal alternatives to aliphatic lipids to promote cytosolic delivery.In our first attempt, fluorous ligands were conjugated onto cationic polymers to increase their gene delivery efficacy. The fluorination dramatically increased the gene delivery performance at low polymer doses. In addition, the strategy greatly improved the serum tolerance of cationic polymers, which is critical for efficient gene delivery in vivo. Besides serum tolerance, mechanism studies revealed that fluorination increases multiple steps such as cellular uptake and endosomal escape. Fluorination also allowed the assembly of low-molecular-weight polymers and achieved highly efficient gene delivery with minimal material toxicity. The method showed robust efficiency for polymers, including linear polymers, branched polymers, dendrimers, bola amphiphilies, and dendronized polymers.Besides gene delivery, fluorinated polymers were also used for intracellular protein delivery via a coassembly strategy. For this purpose, two lead fluoropolymers were screened from a library of amphiphilic materials. The fluoropolymers are greatly superior to their nonfluorinated analogues conjugated with aliphatic lipids. The fluorous lipids are beneficial for polymer assembly and protein encapsulation, reduced protein denaturation, facilitated endocytosis, and decreased polymer toxicity compared to nonfluorinated lipids. The materials exhibited potent efficacy in therapeutic protein and peptide delivery to achieve cancer therapy and were able to fabricate a personalized nanovaccine for cancer immunotherapy. Finally, the fluorous lipids were directly conjugated to peptides via a disulfide bond for cytosolic peptide delivery. Fluorous lipids drive the assembly of cargo peptides into uniform nanoparticles with much improved proteolytic stability and promote their delivery into various types of cells. The delivery efficacy of this strategy is greatly superior to traditional techniques such as cell-penetrating peptides both in vitro and in vivo. Overall, the fluorination techniques provide efficient and promising strategies for the cytosolic delivery of biomolecules.

ATG4B and pS383/392-ATG4B serve as potential biomarkers and therapeutic targets of colorectal cancer.

BACKGROUND: Autophagy related protease 4B (ATG4B) is a protease required for autophagy processing, which is strongly implicated in cancer progression.  Phosphorylation of ATG4B is crucial for activation of its protease activity.  However, little is known about the relationship of ATG4B and its phosphorylated form at Ser 383 and 392 sites (pS383/392-ATG4B), with clinical outcomes, particularly in colorectal cancer (CRC). METHODS: The ATG4B gene expression in CRC patients was obtained from The Cancer Genome Atlas (TCGA) database to analyze its clinical relevance. Tissue microarrays composed of 118 CRC patient specimens were used to determine the associations of ATG4B and pS383/392-ATG4B protein levels with prognosis. The biological functions of ATG4B in CRC cells were inspected with cell proliferation, mobility and spheroid culture assays. RESULTS: ATG4B gene expression was elevated in tumor tissues of CRC patients compared to that in adjacent normal tissues and high level of ATG4B expression was associated with poor survival. Similarly, protein levels of ATG4B and pS383/392-ATG4B were highly correlated with worse overall survival and disease-free survival. Stratification analysis results showed that high level of ATG4B had significantly higher risk of mortality in males and elderly patients compared to those female patients and patients 60 years or younger. In contrast, multivariate Cox's regression analysis indicated that high level of pS383/392-ATG4B was significantly linked to unfavorable overall survival and disease-free survival of males and elderly patients, whereas, it had no correlation with female patients and patients 60 years or younger. Moreover, high level of ATG4B was positively associated with increased mortality risk in patients with advanced AJCC stages (III and IV) and lymph node invasion (N1 and N2) for both overall survival and disease-free survival. Nevertheless, high level of pS383/392-ATG4B was positively correlated with increased mortality risk in patients with early AJCC stages (I and II) and without lymph node invasion (N0). In addition, silencing ATG4B attenuated migration, invasion, and further enhanced the cytotoxic effects of chemotherapeutic drugs in two and three-dimensional cultures of CRC cells. CONCLUSIONS: Our results suggest that ATG4B and pS383/392-ATG4B might be suitable biomarkers and therapeutic targets for CRC.

A Cu+ /Thiourea Dendrimer Achieves Excellent Cytosolic Protein Delivery via Enhanced Cell Uptake and Endosome Escape.

Intracellular protein delivery has attracted considerable attention in the development of protein-based therapeutics, however, the design of highly efficient materials for robust delivery of native proteins remains challenging. This study proposes a Cu+ -based coordination polymer for cytosolic protein delivery with high efficacy and robustness. The phenylthiourea grafted dendrimer is coordinated with cuprous ions to prepare the polymeric carrier, which efficiently bind cargo proteins via a combination of coordination, ionic and hydrophobic interactions. The incorporation of Cu+ ions in the polymer greatly improves its cellular uptake and endosomal escape. The cuprous-based coordination polymer successfully delivered a variety of structurally diverse proteins into various cell lines with reserved bioactivities. This study provides a new type of coordination polymers for cytosolic delivery of biomacromolecules.

Polycatechols with Robust Efficiency in Cytosolic Peptide Delivery via Catechol-Boronate Chemistry.

Cytosolic delivery of peptides remains a challenging task because of the limited binding sites on peptides and the existence of multiple intracellular barriers. Here, we proposed the use of polycatechols with a high cell permeability to deliver peptides of different physicochemical properties using the catechol-boronate chemistry. Peptides were decorated with boronate moieties via three strategies, and the introduced boronate groups greatly increased the binding affinity of cargo peptides with polycatechols. The loading peptides could be released under the endolysosomal acidity. When the cargo peptide was modified with boronate moiety via a p-hydroxybenzylcarbamate self-immolative spacer, it could be loaded by polycatechols and released in a traceless manner triggered by reactive oxygen species. The proposed strategies greatly promote the cytosolic delivery efficiency of different peptides into various cell lines and restored their biofunctions after intracellular delivery and release. This study provides a general and robust platform for the intracellular delivery of membrane-impermeable peptides.

Robust Reversible Cross-Linking Strategy for Intracellular Protein Delivery with Excellent Serum Tolerance.

Intracellular protein delivery has attracted increasing attentions in biomedical applications. However, current delivery systems usually have poor serum stability due to the competitive binding of serum proteins to the polymers during delivery. Here, we report a reversible cross-linking strategy to improve the serum stability of polymers for robust intracellular protein delivery. In the proposed delivery system, nanoparticles are assembled by cargo proteins and cationic polymers and further stabilized by a glutathione-cleavable and traceless cross-linker. The cross-linked nanoparticles show high stability and efficient cell internalization in serum containing medium and can release the cargo proteins in response to intracellular glutathione and acidic pH in a traceless manner. The generality and versatility of the proposed strategy were demonstrated on different types of cationic polymers, cargo proteins, as well as cell lines. The study provides a facile and efficient method for improving the serum tolerance of cationic polymers in intracellular protein delivery.

Fluorous-Tagged Peptide Nanoparticles Ameliorate Acute Lung Injury via Lysosomal Stabilization and Inflammation Inhibition in Pulmonary Macrophages.

Acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) is a common respiratory critical syndrome that currently has no effective therapeutic interventions. Pulmonary macrophages play a principal role in the initiation and progression of the overwhelming inflammation in ALI/ARDS. Here, a type of fluorous-tagged bioactive peptide nanoparticle termed CFF13F is developed, which can be efficiently internalized by macrophages and suppress the excessive expression of cytokines and the overproduction of reactive oxygen species (ROS) triggered by lipopolysaccharide (LPS). The cytoprotective effect of CFF13F may be attributed to the lysosomal-stabilization property and regulation of the antioxidative system. Moreover, intratracheal pretreatment with CFF13F can effectively reduce local and systematic inflammation, and ameliorate pulmonary damage in an LPS-induced ALI murine model. The therapeutic efficacy of CFF13F is affected by the administration routes, and the local intratracheal injection is found to be the optimal choice for ALI treatment, with preferred biodistribution profiles. The present study provides solid evidence of the potent immunomodulatory bioactivity of the fluorous-tagged peptide nanoparticles CFF13F in vitro and in vivo, and sheds light on the development of novel efficient nanodrugs for ALI/ARDS.

Fluoropolymers in biomedical applications: state-of-the-art and future perspectives.

Fluoropolymers have unique physicochemical properties such as hydrophobicity and lipophobicity, good chemical stability and bio-inertness, low surface energy and phase segregation. Owing to these properties, fluoropolymers have been widely used to prepare high performance materials. Especially, the use of fluoropolymers in biomedical applications has grown rapidly during the past decade. This critical review focuses on the recent advances of fluoropolymers in gene delivery, cytosolic protein delivery, drug delivery, magnetic resonance imaging, photodynamic therapy, anti-fouling and anti-bacterial applications, and tissue engineering. The mechanisms and features of fluoropolymers in these specific applications are discussed. Besides, we have reviewed the methods to synthesize water-soluble fluoropolymers for the applications and explained their supramolecular assembly behaviors in solutions. Finally, the opportunities and challenges of fluoropolymers in biomedical applications are discussed.

Stimuli-responsive polydopamine-based smart materials.

Stimuli responsiveness has long been a fascinating feature of smart material design. Polydopamine (PDA), a nature inspired polymeric pigment, exhibits excellent photo-responsive properties and has active surface functionality for loading various responsive motifs, making it a promising candidate for the construction of stimuli-responsive smart functional materials. PDA has long been considered as a robust coating material, but its responsive feature has rarely been emphasized in the past reviews. Herein, we present the first effort to summarize recent advances in the design strategies, responsive mechanisms, and diverse applications of stimuli-responsive PDA-based smart materials; the stimuli include light, pH, chemicals, temperature, humidity, electric fields, mechanical force, magnetic fields, and ultrasound. Moreover, the current trends, challenges, and future directions of stimuli-responsive PDA-based materials are also elaborated.

A pH-Responsive Phase-Transition Polymer with High Serum Stability in Cytosolic Protein Delivery.

Polymer-mediated intracellular protein delivery systems are important for the development of protein-based biotechnologies and therapeutics. However, intracellular delivery of cargo proteins in the presence of serum remains challenging due to competitive binding of serum proteins with the polymers. Here, we reported a dendrimer engineered with a high density of 4-diethylaminophenyl groups on the surface to address this issue. The dendrimer showed a pH-responsive phase-transition behavior and could assemble with cargo proteins into stable nanoparticles in serum solutions. It efficiently delivered cargo proteins into living cells, and exhibited a pH-responsive disassembly behavior after cell internalization. As a result, various cargo proteins were delivered into the cytosol of living cells with maintained bioactivity. This study provided a convenient and efficient strategy to design polymers with high serum-tolerance in cytosolic protein delivery.

A Duplex CRISPR-Cas9 Ribonucleoprotein Nanomedicine for Colorectal Cancer Gene Therapy.

Based on the high frequency of concurrent adenomatous polyposis coli (APC) and KRAS mutations and their strong cooperative interaction in human colorectal cancer (CRC) promotion, we herein develop a CRISPR-Cas9-based genome-editing nanomedicine to target both APC and KRAS mutations for the treatment of CRC. To this end, a hyaluronic acid (HA)-decorated phenylboronic dendrimer (HAPD) was designed for the targeted delivery of Cas9 ribonucleoprotein (RNP), by which both APC and KRAS genetic mutations harboring in CRC cells can be synergistically disrupted. Systemic administration of Cas9 RNP targeting APC and KRAS enabled by HAPD significantly inhibits tumor growth on xenografted and orthotopic CRC mouse models and also greatly prevents CRC-induced liver metastasis and lung metastasis. Thus, this duplex genome-editing system provides a promising gene therapy strategy for the treatment of human CRC and can be extended to other types of cancers with activated Wnt/ß-catenin and RAS/extracellular signal-regulated kinase (ERK) pathways.

Therapeutic Nanoparticles from Grape Seed for Modulating Oxidative Stress.

The therapeutic potential of nanomaterials toward oxidative damage relevant diseases has attracted great attentions by offering promising advantages compared with conventional antioxidants. Although different kinds of nanoantioxidants have been well developed, the facile fabrication of robust and efficient nanoscavengers is still met with challenges like the use of toxic and high-cost subunits, the involvement of multistep synthetic process, and redundant purification work. Herein, a direct fabrication strategy toward polyphenol nanoparticles with tunable size, excellent biocompatibility, and reactive oxygen species (ROS) scavenging capacities from grape seed via an enzymatic polymerization method is reported. The resulting nanoparticles can efficiently prevent cell damage from ROS and exert promising in vivo antioxidant therapeutic effects on several oxidative stress-related diseases, including accelerating wound healing, inhibiting ulcerative colitis, and regulating the oxidative stress in dry eye disease. This study can stimulate the development of more kinds of low-cost, safe, and efficient biomass-based antioxidative nanomaterials via similar fabrication methodologies.

Activation of FAK/Rac1/Cdc42-GTPase signaling ameliorates impaired microglial migration response to Aß42 in triggering receptor expressed on myeloid cells 2 loss-of-function murine models.

Mutation of Triggering receptor expressed on myeloid cells 2 (TREM2) impairs the response of microglia to amyloid-ß (Aß) pathology in Alzheimer's disease (AD), although the mechanism governing TREM2-regulated microglia recruitment to Aß plaques remains unresolved. Here, we confirm that TREM2 mutation attenuates microglial migration. Then, using Trem2-/- mice and an R47H variant mouse model for AD generated for this study, we show that TREM2 deficiency or the AD-associated R47H mutation results in inhibition of FAK and Rac1/Cdc42-GTPase signaling critical for cell migration. Intriguingly, treatment with CN04, a Rac1/Cdc42-GTPase activator, partially enhances microglial migration in response to oligomeric Aß42 in Trem2-/- or R47H microglia both in vitro and in vivo. Our study shows that the dysfunction of microglial migration in the AD-associated TREM2 R47H variant is caused by FAK/Rac1/Cdc42 signaling disruption, and that activation of this signaling ameliorates impaired microglial migration response to Aß42 , suggesting a therapeutic target for R47H-bearing patients with high risk of AD.

Bifunctional and Bioreducible Dendrimer Bearing a Fluoroalkyl Tail for Efficient Protein Delivery Both In Vitro and In Vivo.

Rational design of stimuli-responsive polymers for cytosolic protein delivery with robust efficiency is of great importance but remains a challenging task. Here, we reported a bioreducible and amphiphilic dendrimer bearing a fluoroalkyl tail for this purpose. The fluorolipid was conjugated to the focal point of a cysteamine-cored polyamidoamine dendrimer via disulfide bond, while phenylboronic acid moieties were decorated on dendrimer surface for efficient protein binding. The yielding polymer showed high protein binding capability and complex stability and could efficiently release the cargo proteins in a glutathione-responsive manner. The designed polymer was effective in the delivery of various membrane-impermeable proteins into living cells with reserved bioactivities. In addition, the polymer efficiently delivered a toxin protein saporin into 4T1 breast cancer cells and inhibited the tumor growth in vivo after intravenous injection. The developed polymer in this study is a promising vector for the delivery of membrane-impermeable proteins to treat various diseases.

Natural polyphenol assisted delivery of single-strand oligonucleotides by cationic polymers.

Single-strand oligonucleotides provide promising potential as new therapeutics towards various diseases. However, the efficient delivery of oligonucleotide therapeutics is still challenging due to their susceptibility to nuclease degradation and the lack of effective carriers for condensation. In this study, we reported the use of natural polyphenol to facilitate the condensation of single-strand oligonucleotides by cationic polymers. Green tea catechin complexed with single-strand oligonucleotides to form anionic nanoparticles, which were further coated by low molecular weight cationic polymers to increase their cell internalization. The resulting core-shell structured nanoparticles, so-called green nanoparticles (GNPs), showed improved cargo stability, and achieved high efficiency in the delivery of several types of single-strand oligonucleotides including antisense oligonucleotides, anti-miRNA, and DNAzyme. This study provides a facile strategy for the efficient delivery of single-strand oligonucleotides.

Metal-Containing Polydopamine Nanomaterials: Catalysis, Energy, and Theranostics.

Polydopamine (PDA) is a major type of artificial melanin material with many interesting properties such as antioxidant activity, free-radical scavenging, high photothermal conversion efficiency, and strong metal-ion chelation. The high affinity of PDA to a wide range of metals/metal ions has offered a new class of functional metal-containing polydopamine (MPDA) nanomaterials with promising functions and extensive applications. Understanding and controlling the metal coordination environment is vital to achieve desirable functions for which such materials can be exploited. MPDA nanomaterials with metal/metal ions as the active functions are reviewed, including their synthesis and metal coordination environment and their applications in catalysis, batteries, solar cells, capacitors, medical imaging, cancer therapy, antifouling, and antibacterial coating. The current trends, limitations, and future directions of this area are also explored.