Self-assembly of enzymes and prodrugs with clickable amino acids for nucleus-targeted cancer therapy.

A nucleus-targeted nanocomposite was prepared by clickable amino acid-tuned one-step co-assembly of proteins and chemotherapeutics. The nanocomposite with favorable pharmacokinetic behavior can effectively accumulate in the nucleus, thereby significantly enhancing the anticancer therapeutic effect both in vitro and in vivo.

Research progress in nucleus-targeted tumor therapy.

The nucleus is considered the most important organelle in the cell as it plays a central role in controlling cell reproduction, metabolism, and the cell cycle. The successful delivery of drugs into the nucleus can achieve excellent therapeutic effects, which reveals the potential of nucleus-targeted therapy in precision medicine. However, the transportation of therapeutics into the nucleus remains a significant challenge due to various biological barriers. Herein, we summarize the recent progress in the nucleus-targeted drug delivery system (NDDS). The structures of the nucleus and nuclear envelope are first described in order to understand the mechanisms by which drugs cross the nuclear envelope. Then, various drug delivery strategies based on the mechanisms and their applications are discussed. Finally, the challenges and solutions in the field of nucleus-targeted drug delivery are raised for developing a more efficient NDDS and promoting its clinical transformation.

Nucleus-selective self-augmenting cascade nanoassemblies for targeted synergistic photo-chemo therapy of tumors.

A nucleus-targeted enzyme prodrug nanocomposite, assembled from ß-cyclodextrin-lysine (CL), catalase (CAT), Pt(IV) and chlorin e6 (Ce6), was developed for self-augmenting cascade photo-chemo therapy of tumors. It can effectively transport through the cytoplasm and accumulate in the nucleus, thereby significantly inhibiting tumor growth and lung metastasis.

Biomimetic Antithrombotic Tissue-Engineered Vascular Grafts for Converting Cholesterol and Free Radicals into Nitric Oxide.

Small-diameter tissue-engineered vascular grafts (sdTEVGs) are essential materials used in bypass or replacement surgery for cardiovascular diseases; however, their application efficacy is limited because of patency rates, especially under hyperlipidemia, which is also clinically observed in patients with cardiovascular diseases. In such cases, improving sdTEVG patency is challenging because cholesterol crystals easily cause thrombosis and impede endothelialization. Herein, the development of a biomimetic antithrombotic sdTEVG incorporating cholesterol oxidase and arginine into biomineralized collagen-gold hydrogels on a sdTEVG surface is described. Biomimetic antithrombotic sdTEVGs represent a multifunctional substrate for the green utilization of hazardous substances and can convert cholesterol into hydrogen peroxide, which can react with arginine to generate nitric oxide (NO). NO is a vasodilator that can simulate the antithrombotic action of endothelial cells under hyperlipidemic conditions. In vivo studies show that sdTEVGs can rapidly produce large amounts of NO via a cholesterol catalytic cascade to inhibit platelet aggregation, thereby improving the blood flow velocity and patency rates 60 days after sdTEVG transplantation. A practical and reliable strategy for transforming "harmful" substances into "beneficial" factors at early transplantation stages is presented, which can also promote vascular transplantation in patients with hyperlipidemia.

Delivery of Transcriptional Factors for Activating Antioxidant Defenses against Inflammatory Bowel Disease.

Oxidative stress caused by the overproduction of reactive oxygen species (ROS) plays an important role in inflammatory bowel disease (IBD). It is well-known that the Nrf2-ARE (antioxidative response element) pathway is important in the regulation mechanism of antioxidant defense. Therefore, Nrf2 activation may be an effective therapeutic strategy for IBD. Here, we reported the development of a nucleus-targeted Nrf2 delivery nanoplatform, termed N/LC, that could accumulate in inflamed colonic epithelium, reduce inflammatory responses, and restore epithelium barriers in a murine model of acute colitis. N/LC nanocomposites could quickly escape from lysosomes, so Nrf2 largely accumulated in the nucleus of colonic cells, activated the Nrf2-ARE signaling pathway, further elevated the expression levels of downstream detoxification and antioxidant genes, and protected cells from oxidative damage. These results suggested that N/LC might be a potential nanoplatform for IBD therapy. The study provided the basis for the biomedical applications of Nrf2-based therapeutics in various diseases.

Dibenzocyclooctyne linked lysine-cyclodextrin for efficient intranucleus delivery of proteins.

Intranuclear protein delivery shows great prospects in broadening the application scope of protein therapy and revolutionizing medicine, however, effective delivery of native proteins into the nucleus of cells remains a great challenge. Herein, we report the supramolecular nanoparticles based on the self-assembly of dibenzocyclooctyne (DIBO) linked lysine-cyclodextrin (DLC) for efficient intranucleus delivery of proteins. Coordination-driven self-assembly of DLCs in aqueous solution enables efficient encapsulation of proteins just by simple mixing, so as to maintain their biological activity in a reliable way. DLC nanoparticles ensure effective intranuclear protein delivery for therapeutic applications and gene regulation. This rationally designed DIBO containing amino acid-cyclodextrin derivative allows the development of a convenient and universal nanoplatform for intranuclear delivery of native proteins.

Nucleus-selective codelivery of proteins and drugs for synergistic antitumor therapy.

Subcellular organelle targeted transport is of great significance for accurately delivering drugs to active sites for better pharmacological effects, but there are still a lot of challenges due to transport problems. In addition, the killing effect of one kind of drug on cells is limited. Therefore, it is necessary to develop a multifunctional nanoplatform that can co-deliver synergistic therapeutic agents. Here, we prepare a simple amphiphilic nanocarrier (LC) with rapid endosomal escape ability for nucleus-selective delivery of hydrophilic active protein deoxyribonuclease I (DNase I) and hydrophobic anticancer drug doxorubicin (DOX). LC has been applied to effectively encapsulate DNase I just by simply mixing their aqueous solutions together. In addition, DOX modified with adamantane groups via a redox-responsive linker is incorporated into the architecture of DNase I nanoformulations through host-guest interaction. This multi-component nanoplatform can quickly escape from the endolysosomes into the cytoplasm and make DNase I and DOX highly accumulate in the nucleus and consequently induce strong synergistic anticancer efficacy both in vitro and in vivo. This work illustrates a new platform for codelivery of proteins and drugs that target subcellular compartments for functions.

Self-Assembly of Intelligent Nanoplatform for Endogenous H2S-Triggered Multimodal Cascade Therapy of Colon Cancer.

The specific in situ generation and activation of therapeutic agents with high spatiotemporal precision is expected to revolutionize cancer treatment. Here, we develop an intelligent nanoplatform (termed as NP-Cu), which is constructed by assembling photosensitizer chlorin e6 (Ce6), hypoxia-responsive prodrug banoxantrone (AQ4N) with clickable dibenzocyclooctyne (DIBO) functionalized lysine (D-K), and cyclen-Cu2+ complex, for improving combination anticancer therapy. Cyclen-Cu2+ complex-induced photodynamic therapy (PDT) quenching in NP-Cu can be effectively and selectively activated by tumor-overproduced hydrogen sulfide (H2S). More importantly, the reaction of endogenous H2S with Cu2+ can generate photothermal agent copper sulfide (CuS) for photothermal therapy (PTT). Furthermore, with the activation of PTT and PDT, intracellular hypoxic stress is amplified to trigger AQ4N-associated chemodynamic therapy (CDT), leading to light-enhanced cascade therapy of PDT, PTT and CDT. Therefore, we present a simple and practical strategy for developing pathological stimuli responsive combination therapy, which has the potential of advancing precision cancer medicine.

The First FRET-Based RNA Aptamer NanoKit for Sensitively and Specifically Detecting c-di-GMP.

An effective method to identify c-di-GMP may significantly facilitate the exploration of its signaling pathways and bacterial pathogenesis. Herein, we have developed the first conjugated polymer-amplified RNA aptamer NanoKit with a unique core-shell-shell architecture, which combines the advantages of high selectivity of RNA aptamers and high sensitivity of strong fluorescence resonance energy transfer (FRET) effect, for precisely detecting c-di-GMP. We identified that NanoKit could selectively detect c-di-GMP with a low detection limit of 50 pM. Importantly, NanoKit could identify bacterial species and physiological states, such as planktonic, biofilm, and even antibiotic-resistance, on the basis of their different c-di-GMP expression patterns. Particularly, NanoKit could distinguish bacterial infection and inflammation and identify Pseudomonas aeruginosa associated pneumonia and sepsis, thereby guiding treatment choice and monitoring antibiotic effects. Therefore, NanoKit provides a promising strategy to rapidly identify c-di-GMP and its associated diseases and may benefit for pathophoresis management.

Programmable dual responsive system reconstructing nerve interaction with small-diameter tissue-engineered vascular grafts and inhibiting intimal hyperplasia in diabetes.

Small-diameter tissue-engineered vascular grafts (sdTEVGs) with hyperglycemia resistance have not been constructed. The intimal hyperplasia caused by hyperglycemia remains problem to hinder the patency of sdTEVGs. Here, inspired by bionic regulation of nerve on vascular, we found the released neural exosomes could inhibit the abnormal phenotype transformation of vascular smooth muscle cells (VSMCs). The transformation was a prime culprit causing the intimal hyperplasia of sdTEVGs. To address this concern, sdTEVGs were modified with an on-demand programmable dual-responsive system of ultrathin hydrogels. An external primary Reactive Oxygen Species (ROS)-responsive Netrin-1 system was initially triggered by local inflammation to induce nerve remolding of the sdTEVGs overcoming the difficulty of nerve regeneration under hyperglycemia. Then, the internal secondary ATP-responsive DENND1A (guanine nucleotide exchange factor) system was turned on by the neurotransmitter ATP from the immigrated nerve fibers to stimulate effective release of neural exosomes. The results showed nerve fibers grow into the sdTEVGs in diabetic rats 30 days after transplantation. At day 90, the abnormal VSMCs phenotype was not detected in the sdTEVGs, which maintained long-time patency without intima hyperplasia. Our study provides new insights to construct vascular grafts resisting hyperglycemia damage.

Clickable amino acid tuned self-assembly of a nucleus-selective multi-component nanoplatform for synergistic cancer therapy.

Nucleus-targeted therapy holds great promise in cancer treatment; however, a lack of effective nucleus-specific delivery significantly limits its application potential. Here, we report a nucleus-targeted synergistic chemo-photodynamic therapy based on the self-assembly of chlorin e6 (Ce6) and doxorubicin (DOX) tuned by clickable dibenzocyclooctyne (DIBO) functionalized lysine (D-K) and subsequent reaction with crosslinkers. The assembled nanodrugs with high loading efficiency and long-term stability show enhanced cellular uptake and accumulation in the nucleus, resulting in greatly improved in vitro and in vivo chemo-photodynamic efficacy. Notably, D-K can promote the rapid self-assembly of Ce6 and DOX in aqueous solution, avoiding the introduction of organic solvents or tedious preparations. In addition, the introduction of the DIBO group can effectively expand the types of self-assembly material and enhance the self-assembly behaviour through a copper-free click reaction. Therefore, we present an effective nucleus-targeted combination drug delivery strategy, which has great potential in the treatment of many diseases.

Clickable amino acid derivative tuned self-assembly of antigen and adjuvant for cancer immunotherapy.

Amino acid-tuned self-assembly has become an attractive strategy for constructing various functional materials. Here, a series of dibenzocyclooctyne (DIBO) functionalized amphiphilic amino acid derivatives are designed and screened as building blocks of functional supramolecular self-assembly nanoparticles for cancer immunotherapy. One top-performing supramolecular self-assembly material (named DA6C1) is identified through combinatorial screening, and spherical nanoparticles can be easily prepared by this material tuned multicomponent synergistic self-assembly of ovalbumin (OVA) and CpG oligonucleotide. DA6C1 based nanovaccine can significantly enhance the cellular uptake of OVA and CpG into the same bone marrow derived dendritic cells (BMDCs) and greatly improve the activation of DCs. Moreover, after subcutaneous injection, this nanovaccine flows rapidly to the lymph nodes and elicits strong immune responses to achieve effective prophylactic and therapeutic effect. Therefore, our work highlights the great potential of clickable amino acid derivatives as a convenient and powerful tool to construct nanovaccine for effective immunotherapy.

Nucleus-Targeted Delivery of Multi-Protein Self-Assembly for Combined Anticancer Therapy.

Protein therapy has the potential to revolutionize medicine, but the delivery of multiple proteins is challenging because it requires the development of a strategy that enables different proteins to be combined together and transported not only into cells, but also to the desired cell compartments, such as the nucleus. Here, an efficient intranuclear protein delivery nanoplatform based on modified ribonuclease A (RNase A) tuned self-assembly is presented. RNase A bioreversibly modified with adamantane is functionalized with wind chime-like lysine modified cyclodextrin (WLC) to generate RNase A-WLC (R-WLC). R-WLC can not only enhance the cellular uptake of RNase A and accumulate it into the nucleus, but also works as nanovehicles to efficiently transport deoxyribonuclease I (DNase I) into the nucleus, resulting in greatly improved antitumor efficacy in vitro and in vivo. This protein co-assembly strategy can be applied to other functional proteins and has great prospects in the treatment of many diseases.

Oral delivery of antioxidant enzymes for effective treatment of inflammatory disease.

Oral administration of protein is very challenging for therapeutic applications due to its instability and easy degradation in the gastrointestinal tract. Herein, we reported an approach to encapsulate native anti-inflammatory proteins in wind chimes like cyclodextrin (WCC) for efficient oral protein delivery. The amphiphilic WCC can self-assemble into nanoparticles in aqueous solution and achieve superior encapsulation of two antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) by simply mixing with protein solution, avoiding any extra cumbersome steps that might inactivate protein. WCC nanovehicles can effectively protect enzyme activity and enhance their intracellular delivery. SOD and CAT co-loaded WCC nanoparticles (SC/WCC) can integrate the synergistic effect of SOD and CAT for enhancing the removal of reactive oxygen species (ROS), effectively inhibit the inflammatory response by reducing the secretion of proinflammatory factors and protect cells from ROS-induced oxidative damage. In the mouse colitis model, SC/WCC administered orally was able to efficiently accumulate in the inflamed colon, significantly inhibited the expression of proinflammatory mediators and notably alleviated the symptoms related to colitis. Therefore, we believe that the strategies we described here may provide a convenient and powerful platform for the treatment of other inflammatory diseases.

Highly uniform self-assembled microspheres from single macromolecule self-recognition for enhanced cancer immunotherapy.

Self-assembled highly uniform microspheres from star-shaped biocompatible polymers have been prepared as a local delivery system for co-loading different immunoagents together and ensuring their release in different stages, so as to realize effective cooperative immunotherapy and minimize systemic side effects.

Logic-signal-based multiplex detection of MiRNAs with high tension hybridization and multiple signal amplification.

MicroRNAs (miRNAs) are crucial regulators of gene expression. The abnormal expression of miRNA is often closely related to many diseases. However, the accurate clinical profiling of miRNA expression remains a great challenge due to the high similarity and wide variety of miRNA sequence structures. Here, we report a highly specific and sensitive multiplex miRNA detection scheme with high tension hybridization and dual signal amplification based on the recyclable autocatalytic DNAzyme and a light harvesting conjugated polymer. Multiple signals can be read out simultaneously by single excitation through the efficient multiple fluorescence resonance energy transfer (FRET) between the conjugated polymer and different small molecule dyes. In addition, different types of logic gates can also be operated by observing the emission intensities of the labeling dyes with miRNAs as inputs, thus giving rise to a new way for the specific detection of certain miRNAs according to the logic signals. Furthermore, we successfully applied the strategy for multiple miRNA detection in cell lysates and the results agree well with those of qRT-PCR. Thus, we believe that this platform holds great potential for miRNA detection in biological samples.

Stimuli-responsive self-assembled dendrimers for oral protein delivery.

Oral delivery of protein is very challenging for therapeutic applications due to protein instability and degradability. Herein we synthesized benzoboroxole-containing multi-armed poly(ethylene glycol) amphiphilic dendrimers sensitive to both pH and glucose. The multi-branched structure of dendrimer facilitated its self-assembly into micelles in aqueous solution and exhibited good encapsulation of insulin just by simply mixing with insulin solution. The insulin loaded micelles were stable under strong acidic condition and showed glucose-responsive insulin release at physiological pH. The reversible binding between benzoboroxole and glucose can effectively regulate the disassembly of micelles and insulin release rate, which in turn stabilizes blood sugar fluctuations. Utilizing a mouse model of type 1 diabetes, it was demonstrated that these insulin loaded micelles could successfully control the blood glucose levels under a normoglycemic state and avoid the risk of hypoglycemia. Therefore, this smart delivery system shows great potential to significantly improve the effect of oral protein therapy.

Quantitative and high drug loading of self-assembled prodrug with defined molecular structures for effective cancer therapy.

Nanomedicines have made significant progress in the delivery of small molecular drugs, many challenges, however, still remain to be overcome, such as unsatisfactory drug loading, formulation instability, premature drug leakage, and poor blood circulation. Herein, an innovative glutathione (GSH)-sensitive amphiphilic dendritic prodrug with quantitative and high drug loading (>30 wt%) is reported. The multi-armed structure of prepared prodrug can self-assemble into nanoparticles in aqueous solution without the introduction of any organic solvents. The self-assembled prodrug nanoparticle is composed of the following key components: (i) polyethylene glycol (PEG) outer shell ensuring biocompatibility and prolonging blood circulation, (ii) prodrug inner core responding to GSH for triggered release of intact drug, (iii) multi-armed dendritic structure facilitating self-assembly and enhancing drug loading content, (iv) covalent drug conjugation avoiding drug leakage and improving stability, (v) defined chemical structures and quantitative drug loading easy for reproduction. Both in vitro and in vivo results show that these GSH-responsive prodrug nanoparticle exhibits significant inhibition of tumor cell growth, and is promising for efficient and safe chemotherapeutic delivery.

Organocatalytic Asymmetric One-Step Desymmetrizing Dearomatization Reaction of Indoles: Development and Bioactivity Evaluation.

An organocatalytic one-step desymmetrizing dearomatization reaction of indoles with in situ formed vinylidene ortho-quinone methides is reported. A set of [6-6-5] and/or [5-6-5] fused indoline heterocycles were obtained in excellent yields with excellent diastereoselectivities (>20:1 d.r.) and enantioselectivities (up to 99 % ee). Moreover, some of the obtained products were screened against a panel of cancer cell lines, and one was identified to inhibit the proliferation of all the tested cancer cells, but showed marginal effects against non-cancerous cells. The methodology provides a platform for the synthesis of new leading compounds with antitumor activity.

SiRNA Crosslinked Nanoparticles for the Treatment of Inflammation-induced Liver Injury.

RNA interference mediated by small interfering RNA (siRNA) provides a powerful tool for gene regulation, and has a broad potential as a promising therapeutic strategy. However, therapeutics based on siRNA have had limited clinical success due to their undesirable pharmacokinetic properties. This study presents pH-sensitive nanoparticles-based siRNA delivery systems (PNSDS), which are positive-charge-free nanocarriers, composed of siRNA chemically crosslinked with multi-armed poly(ethylene glycol) carriers via acid-labile acetal linkers. The unique siRNA crosslinked structure of PNSDS allows it to have minimal cytotoxicity, high siRNA loading efficiency, and a stimulus-responsive property that enables the selective intracellular release of siRNA in response to pH conditions. This study demonstrates that PNSDS can deliver tumor necrosis factor alpha (TNF-α) siRNA into macrophages and induce the efficient down regulation of the targeted gene in complete cell culture media. Moreover, PNSDS with mannose targeting moieties can selectively accumulate in mice liver, induce specific inhibition of macrophage TNF-α expression in vivo, and consequently protect mice from inflammation-induced liver damages. Therefore, this novel siRNA delivering platform would greatly improve the therapeutic potential of RNAi based therapies.