Tet1 peptide and zinc (II)-adenine multifunctional module functionalized polycations as efficient siRNA carriers for Parkinson's disease.

A bioreducible Zn (II)-adenine multifunctional module (BS) and Tet1 peptide were used to modify low-molecular-weight PEI3.5k (polyethyleneimine with molecular weight of 3.5 kDa)into a siRNA vector Zn-PB-T with high transfection efficiency in neurons. A GSH-responsive breakable disulfide spacer was introduced into BS to realize the controlled release of siRNA from the polyplexes in cytoplasm. Zn-PB showed >90% transfection rates in multiple cell lines (3 T3, HK-2, HepG2, 293 T, HeLa, PANC-1)，and 1.8-folds higher EGFP knockdown rates than commercial Lipo2k in normal cell line 293 T and cancer cell line HepG2. And Zn-PB-T1 showed 4.7-4.9- and 8.0-8.1-folds higher transfection efficiency comparing to commercial Lipo2k and PEI25k (polyethyleneimine with molecular weight of 25 kDa) in PC12 cells respectively, 2.1-fold EGFP gene silencing efficiency (96.6% EGFP knockdown rates) superior to commercial Lipo2k in neurons. In Parkinson's model, Zn-PB-T1/SNCA-siRNA can effectively protect neurons against MPP+-induced cell death and apoptosis, increasing the cell survival rate to 84.6% and reducing the cell apoptosis rate to 10.8%. This work demonstrated the promising application prospects of the resulting efficient siRNA carriers in siRNA-mediated gene therapy of Parkinson's disease.

Zinc(II)-Quinoline module and ROS cleavable crosslinker functionalized self-fluorescent siRNA vectors for selective gene silencing of cancer cells.

A highly efficient siRNA vector (Zn-PQD) capable of selectively silencing genes in cancer cells was obtained by using ROS-cleavable DED to crosslink low molecular weight (LMW) polyethylene imine (PEI) modified by self-fluorescent metal coordinatied multifunctional module Zn-QS. Under the combined action of DED cross-linking and Zn-QS modification, Zn-PQD performs well in the siRNA delivery process in cancer cells, including siRNA condensation, cell uptake, endosome escape, and siRNA release. Zn-PQD exhibited higher transfection efficiency than commercial PEI25k and Lipo2k in multiple cancer cell lines including HepG2, HeLa, 4 T1, H520 and PANC-1, as well as cancer treatment-related stem cell rADSC. Ultimately, Zn-PQD can achieve extremely high and selective gene silencing effects in cancer cells (with a gene silencing rate of 98.3% in HepG2). This work is expected to provide an efficient and safe siRNA carrier for the future tumor siRNA therapy and its study of fluorescence mediated mechanism.

Molecularly imprinted self-buffering double network hydrogel containing bi-amidoxime functional groups for the rapid hydrolysis of organophosphates.

The development of high-performance catalyst materials with high catalytic activity for the hydrolysis of organophosphorus toxicants without additional pH buffer conditions has become an urgent need for practical application. Here, a multifunctional molecularly imprinted polymer double network hydrogel (MIP-DN) material has been prepared by integrating the first polymer network containing the functional group of bi-amidoxime as the catalytic active center and the cationic polymer polyethyleneimine (PEI) with pH buffer function as the main component of the second network. Advantageously, the resultant MIP-DN hydrogel showed excellent catalytic performance without additional pH buffer conditions, exhibiting a half-life of 25 min for the hydrolysis of paraoxon in pure water. Together with multi-functions of high catalytic activity, self-buffering function and excellent processability, the MIP-DN hydrogel prepared in this work provides a new strategy for the preparation of catalytic materials with practical application value toward toxic organophosphates.

Zinc(II)-Cyclen Multifunctional Complex Module-Mediated Polycation-Based High-Performance pDNA Vectors.

A kind of novel multifunctional modules based on zinc(II)-coordinative cyclen has been developed, which is utilized to modify low molecular weight polyethylenimine (LMWPEI) obtaining high-performance DNA vectors. A series of in vitro experiments were carried out to explore the performance of the module in improving the key process of gene transfection, such as DNA condensation, serum resistance, cellular uptake, and endosomal escape. The results demonstrate that there is a significant synergistic effect between the functional module and PEI2.5k in the process of breaking through the key barriers of gene transfection. The optimal Zn-PCD mediates 160-fold higher gluciferase activity than commercial transfection reagents PEI25k in ADSC stem cells with more than 90% cell viability and achieves excellent transfection efficiency in diverse cell types, for instance, HepG2 cells, 293T cells, and 293F suspension cells.

Ag(I) Pyridine-Amidoxime Complex as the Catalysis Activity Domain for the Rapid Hydrolysis of Organothiophosphate-Based Nerve Agents: Mechanistic Evaluation and Application.

Two novel Ag(I) complexes containing synergistic pyridine and amidoxime ligands (Ag-DPAAO and Ag-PAAO) were first designed as complex monomers. Taking advantage of the molecular imprinting technique and solvothermal method, molecular imprinted porous cross-linked polymers (MIPCPs) were developed as a robust platform for the first time to incorporate Ag-PAAO into a polymer material as a recyclable catalyst. Advantageously, the observed pseudo first-order rate constant (kobs) of MIPCP-Ag-PAAO-20% for ethyl-parathion (EP) hydrolysis is about 1.2 × 104-fold higher than that of self-hydrolysis (30 °C, pH = 9). Furthermore, the reaction mechanism of the MIPCP-containing Ag-PAAO-catalyzed organothiophosphate was analyzed in detail using density functional theory and experimental spectra, indicating that the amidoxime can display dual roles for both the key coordination with the silver ion and nucleophilic attack to weaken the P-OAr bond in the catalytic active site.

Zinc(II)-Dipicolylamine Analogs Mediated PEI1.8k/pDNA Vector: Effect of Ligand Structure on the Gene Transport Process.

Zinc ion complexes of dipicolylamine analogs, due to the strong synergistic effect between the Zn2+ complex of containing polypyridine derivatives and polycations in each key step of pDNA transport, have been used as the third component to mediate polyethyleneimine with molecular weight 1.8 kDa (PEI1.8k)/DNA gene delivery system. And the effects of different structural characteristics, such as the number of pyridinamine ligands, the hydrophilic-hydrophobicity of the adjacent groups, on the in vitro transfection performance of the ternary complex are systematically investigated. This ternary hybrid system provides an effective strategy to improve the gene delivery of cationic polymers.

Zn(ii)-Dipicolylamine analogues with amphiphilic side chains endow low molecular weight PEI with high transfection performance.

To investigate the effect of amphiphilic balance of Zn(ii)-dipicolylamine analogues on the transfection process, we fabricated a series of Zn(ii)-dipicolylamine functional modules (DDAC-Rs) with different hydrophilic-phobic side chains to modify low molecular weight PEI (Zn-DP-Rs) by the Michael addition reaction. Zn-DP-Rs with hydrophilic terminal hydroxy group side chains demonstrate superior overall performance compared to those of hydrophobic alkyl side chains. In terms of the influence of the chain lengths in DDAC-Rs, from Zn-DP-A/OH-3 to Zn-DP-A/OH-5, the corresponding transfection efficiency shows an upward trend as the lengths increase. However, decreasing efficacy is observed with further increase in the length of side chains. In addition, the Zn-DP-Rs with amphiphilic side chains show prominent performance in every respect, highlighting the significance of balance in the amphipathy of side chains in DDAC-Rs. This work is of great significance for the development of polycationic gene carrier materials with excellent performance.

Highly branched poly(ß-amino ester) delivery of minicircle DNA for transfection of neurodegenerative disease related cells.

Current therapies for most neurodegenerative disorders are only symptomatic in nature and do not change the course of the disease. Gene therapy plays an important role in disease modifying therapeutic strategies. Herein, we have designed and optimized a series of highly branched poly(ß-amino ester)s (HPAEs) containing biodegradable disulfide units in the HPAE backbone (HPAESS) and guanidine moieties (HPAESG) at the extremities. The optimized polymers are used to deliver minicircle DNA to multipotent adipose derived stem cells (ADSCs) and astrocytes, and high transfection efficiency is achieved (77% in human ADSCs and 52% in primary astrocytes) whilst preserving over 90% cell viability. Furthermore, the top-performing candidate mediates high levels of nerve growth factor (NGF) secretion from astrocytes, causing neurite outgrowth from a model neuron cell line. This synergistic gene delivery system provides a viable method for highly efficient non-viral transfection of ADSCs and astrocytes.

Multifunctional oligomer immobilized on quartz crystal microbalance: a facile and stabilized molecular imprinting strategy for glycoprotein detection.

Glycoprotein detection holds great potential for early diagnosis of diverse diseases. For this purpose, the combination of quartz crystal microbalance (QCM) sensor and molecular imprinting has attracted increasing attention. Nonetheless, the recently common imprinted films fabricated on QCM electrode are thick and rigid, lacking flexibility in aqueous phase. Alternatively, small molecules immobilized on the electrode to construct molecular scale film could address this problem, while stabilization of the imprinted sites remains challenging. Herein, a co-assembly complex was obtained by the mixture of template and multifunctional oligomer, which was then immobilized on the amino-modified transducer surface through epoxy-amino reaction to form a protein-imprinted film. Afterward, the remaining epoxy groups in oligomer chains were cross-linked to conserve and stabilize the orientation of imprinted sites after template elution. Template rebinding tests show that cross-linked film has much higher imprinting factors than that of the non-cross-linked counterpart. Furthermore, control proteins that are distinct in properties and structures were employed to demonstrate the selectivity of this approach, and the imprinted assay reveals high affinity and specificity towards template protein. Graphical Abstract.

Zinc Coordinated Cationic Polymers Break Up the Paradox between Low Molecular Weight and High Transfection Efficacy.

Cationic polymers have emerged as appealing nonviral gene vectors for decades, which, however, suffer from the paradox between low molecular weight and high transfection efficacy. Low molecular weight cationic polymers (LCPs) are well cell tolerated but are perplexed by orders-of-magnitude less efficacy compared to their macromolecular counterparts. The deficiency mainly lies in weak DNA binding of polymers and difficulty in endosomal escape of formulated polyplexes. Herein, we demonstrate that, through zinc (Zn) coordinated modification of LCPs, the high transfection efficiency and low molecular weight (thus low cytotoxicity) can be achieved simultaneously. The Zn coordinated ligand shows a high affinity to phosphate components and therefore will largely benefit the DNA packaging and endosomal membrane destabilization, addressing the defects of LCPs in gene delivery. Zn coordinative functionalization of LCPs breaks up the "efficacy-toxicity" paradox and provides great promise for the development of clinically efficient and safe nonviral gene vectors.

Virus Spike and Membrane-Lytic Mimicking Nanoparticles for High Cell Binding and Superior Endosomal Escape.

Virus-inspired mimics for gene therapy have attracted increasing attention because viral vectors show robust efficacy owing to the highly infectious nature and efficient endosomal escape. Nonetheless, until now, synthetic materials have failed to achieve high "infectivity," and especially, the mimicking of virus spikes for "infection" is underappreciated. Herein, a virus spike mimic by a zinc (Zn) coordinative ligand that shows high affinity toward phosphate-rich cell membranes is reported. Surprisingly, this ligand also demonstrates superior functionality of destabilizing endosomes. Therefore, the Zn coordination is more likely to imitate the virus nature with high cell binding and endosomal membrane disruption. Following this, the Zn coordinative ligand is functionalized on a bioreducible cross-linked peptide with alkylation that imitates the viral lipoprotein shell. The ultimate virus-mimicking nanoparticle closely imitates the structures and functions of viruses, leading to robust transfection efficiency both in vitro and in vivo. More importantly, apart from targeting ligand- and cell-penetrating peptide, the metal coordinative ligand may provide another option to functionalize diverse biomaterials for enhanced efficacy, demonstrating its broad referential significance to pursue nonviral vectors with high performance.

Zinc Coordination Substitute Amine: A Noncationic Platform for Efficient and Safe Gene Delivery.

Amines have been extensively involved in vector design thus far, however, their clinical translation has been impeded by several obstacles: cytotoxicity, polyplex serum instability and low efficacy in vivo. In pursuit of functional groups to substitute amines in vector design to address these disadvantages is of great significance. Herein, we report well-tailored noncationic copolymers that contain hydrophilic, hydrophobic, and zinc coordinative moieties through reversible addition-fragmentation chain transfer (RAFT) polymerization for efficient and safe gene delivery. These polymers are capable of condensing DNA, enabling the formation of uncharged polyplexes. Especially, the zinc coordinative ligand can simultaneously benefit strong DNA binding, robust cellular uptake, efficacious endosomal destabilization, low cytotoxicity, and avoidance of serum protein adsorption. The coordinative module holds great promise to substitute amines and inspires the development of next-generation gene vectors. More importantly, the coordinative copolymers illuminate the possibility and potential of noncationic gene delivery systems for clinical applications.

Bioreducible Zinc(II)-Coordinative Polyethylenimine with Low Molecular Weight for Robust Gene Delivery of Primary and Stem Cells.

To transform common low-molecular-weight (LMW) cationic polymers, such as polyethylenimine (PEI), to highly efficient gene vectors would be of great significance but remains challenging. Because LMW cationic polymers perform far less efficiently than their high-molecular-weight counterparts, mainly due to weaker nucleic acid encapsulation, herein we report the design and synthesis of a dipicolylamine-based disulfide-containing zinc(II) coordinative module (Zn-DDAC), which is used to functionalize LMW PEI (Mw ≈ 1800 Da) to give a non-viral vector (Zn-PD) with high efficiency and safety in primary and stem cells. Given its high phosphate binding affinity, Zn-DDAC can significantly promote the DNA packaging functionality of PEI1.8k and improve the cellular uptake of formulated polyplexes, which is particularly critical for hard-to-transfect cell types. Furthermore, Zn-PD polymer can be cleaved by glutathione in cytoplasm to facilitate DNA release post internalization and diminish the cytotoxicity. Consequently, the optimal Zn-PD mediates 1-2 orders of magnitude higher gluciferase activity than commercial transfection reagents, Xfect and PEI25k, across diverse cell types, including primary and stem cells. Our findings provide a valuable insight into the exploitation of LMW cationic polymers for gene delivery and demonstrate great promise for the development of next-generation non-viral vectors for clinically viable gene therapy.

Alkylated branched poly(ß-amino esters) demonstrate strong DNA encapsulation, high nanoparticle stability and robust gene transfection efficacy.

A branched poly(ß-amino ester) with numerous alkyl chains (BPA) is designed and synthesized as a safe and efficient non-viral vector. The branching and hydrophobicity synergistically endow BPA with tight DNA condensation, high polyplex stability in serum, high cellular uptake and ultimately robust gene transfection efficiency, largely superior to its linear counterpart (LPA). Our results demonstrate that branching matters for gene delivery by hydrophobic gene vectors.

Biodegradable Highly Branched Poly(ß-Amino Ester)s for Targeted Cancer Cell Gene Transfection.

To enhance the gene transfection efficiency to targeted cells while reducing the side effects to untargeted cells is of great significance for clinical gene therapy. Here, biodegradable highly branched poly(ß-amino ester)s (HPAESS) are synthesized and functionalized with folate (HPAESS-FA) and lactobionic acid (HPAESS-Lac) for targeted cancer cell gene transfection. Results show that because of the triggered degradability of the vector and enhanced receptor-mediated cellular uptake of polyplexes, the HPAESS-FA and HPAESS-Lac exhibit superior gene transfection capability in specific cancer cells with negligible cytotoxicity, pointing to their promise as targeted vectors for efficient cancer gene therapy.

Multifunctional oligomer incorporation: a potent strategy to enhance the transfection activity of poly(l-lysine).

Natural polycations, such as poly(l-lysine) (PLL) and chitosan (CS), have inherent superiority as non-viral vectors due to their unparalleled biocompatibility and biodegradability. However, the application was constrained by poor transfection efficiency and safety concerns. Since previous modification strategies greatly weakened the inherent advantages of natural polycations, developing a strategy for functional group introduction with broad applicability to enhance the transfection efficiency of natural polycations without compromising their cationic properties is imperative. Herein, two uncharged functional diblock oligomers P(DMAEL-b-NIPAM) and P(DMAEL-b-Vlm) were prepared from a lactose derivative, N-iso-propyl acrylamide (NIPAM) as well as 1-vinylimidazole (Vlm) and further functionalized with four small ligands folate, glutathione, cysteine and arginine, respectively, aiming to enhance the interactions of complexes with cells, which were quantified utilizing a quartz crystal microbalance (QCM) biosensor, circumventing the tedious material screening process of cell transfection. Upon incorporation with PLL and DNA, the multifunctional oligomers endow the formulated ternary complexes with great properties suitable for transfection, such as anti-aggregation in serum, destabilized endosome membrane, numerous functional sites for promoted endocytosis and therefore robust transfection activity. Furthermore, different from the conventional strategy of decreasing cytotoxicity by reducing the charge density, the multifunctional oligomer incorporation strategy maintains the highly positive charge density, which is essential for efficient cellular uptake. This system develops a new platform to modify natural polycations towards clinical gene therapy.

N,N,N-trimethylchitosan modified with well defined multifunctional polymer modules used as pDNA delivery vector.

A novel non-viral gene carrier based on N,N,N-trimethylchitosan (TMC) has been fabricated. First, well-defined copolymer P(PEGMA-co-DMAEMA) was synthesized through reversible addition fragmentation chain transfer (RAFT) polymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and N,N-(2-dimethylamino)ethyl methacrylamide (DMAEMA). Then allyl group grafting N,N,N-trimethylchitosan (Allyl-TMC) was synthesized via the reaction between allyl bromide and hydroxyl of TMC. Finally, P(PEGMA-co-DMAEMA) and folate were ordinally grafted onto Allyl-TMC to obtain TMC-g-P(PEGMA-co-DMAEMA)-FA. In comparison with pristine chitosan, TMC-g-P(PEGMA-co-DMAEMA)-FA has achieved both better water solubility and stronger pDNA packaging ability, which can contribute to improving gene transfection. Gene delivery efficiency of a series of TMC based functional polymers with different chitosan molecular weights has been tested. The results show that 20k-TMC-g-P(PEGMA-co-DMAEMA)-FA/pDNA complex at the weight ratio of 20 achieve the highest transfection efficiency in 293 T cells. This work presents a new strategy to modify chitosan efficiently as gene carrier material.

Polycation-Based Ternary Gene Delivery System.

Recent progress in gene therapy has opened the door for various human diseases. The greatest challenge that gene vectors still face is the ability to sufficiently deliver nucleic acid into target cells. To overcome various barriers, plenty of researches have been undertaken utilizing diverse strategies, among which a wide variety of polycation/pDNA vectors have been developed and explored frequently. For enhanced transfection efficiency, polycations are constantly utilized with covalent modifications, which however lead to reduced positive charge density and changed properties of polycation/pDNA complexes. Accordingly, non-covalent or ternary strategy is proposed. The cationic properties of polycations can be retained and the transfection efficiency can be enhanced by introducing additional polymers with functional groups via non-covalent assembly. This review will discuss the construction and advantages of ternary complexes gene delivery system, including low toxicity and enhanced gene expression both in vitro and in vivo. Recent progress and expectations with promising results that may have some reference for clinical application are also discussed.

Evaluation of the effects of amphiphilic oligomers in PEI based ternary complexes on the improvement of pDNA delivery.

Two kinds of novel oligomers were prepared by reversible addition fragmentation chain transfer (RAFT) polymerization and incorporated into the polyethyleneimine (PEI) gene delivery system through non-electrostatic assembly to improve gene transfection efficiency. The non-electrostatic assembly process was first investigated via probing the interactions of the oligomers with plasmid DNA (pDNA), PEI and AD-293 cells using a quartz crystal microbalance (QCM). The results show that the prepared oligomers almost had no interaction with pDNA while they had much stronger interactions with PEI and AD-293 cells. Meanwhile, we found that the two kinds of oligomers had different interactions with AD-193 cells, which caused different effects on gene transfection. The data of QCM tests combined with the in vitro transfection results can be used to explain what effects the oligomers have on improving gene transfection. The results also indicate that the strategy of detecting the interactions of oligomers with pDNA, polycations and cells will contribute to predetermine whether the prepared oligomer is efficient in improving gene transfection.

Gene delivery of PEI incorporating with functional block copolymer via non-covalent assembly strategy.

A novel functional diblock polymer P(PEGMA-b-MAH) is prepared and incorporated to improve the gene delivery efficiency of poly(ethyleneimine) PEI via non-covalent assembly strategy. First, P(PEGMA-b-MAH) is prepared from l-methacrylamidohistidine methyl ester (MAH) by reversible addition fragmentation chain transfer polymerization, with poly[poly(ethylene glycol) methyl ether methacrylate] (P(PEGMA)) as the macroinitiator. Then P(PEGMA-b-MAH) is assembled with plasmid DNA (pDNA) and PEI (M(w)=10kDa) to form PEI/P(PEGMA-b-MAH)/pDNA ternary complexes. The agarose gel retardation assay shows that the presence of P(PEGMA-b-MAH) does not interfere with DNA condensation by the PEI. Dynamic light scattering tests show that PEI/P(PEGMA-b-MAH)/pDNA ternary complexes have excellent serum stability. In vitro transfection indicates that, compared to the P(PEGMA-b-MAH) free PEI-25k/pDNA binary complexes, PEI-10k/P(PEGMA-b-MAH)/pDNA ternary complexes have lower cytotoxicity and higher gene transfection efficiency, especially under serum conditions. The ternary complexes proposed here can inspire a new strategy for the development of gene and drug delivery vectors.