Xiaoyun Zhao

Guanidinylated bioresponsive poly(amido amine)s designed for intranuclear gene delivery

Guanidinylated poly(amido amine)s with multiple disulfide linkages (Gua-SS-PAAs) were designed and constructed as nonviral gene carriers. The main chains of these novel carriers were synthesized based on monomers containing guanidino groups (guanidine hydrochloride and chlorhexidine), which could avoid complicated side-chain-modification reactions while introducing the guanidino groups. The synthesized Gua-SS-PAAs polymers were characterized by 1H nuclear magnetic resonance, molecular weight, and polydispersity. Furthermore, Gua-SS-PAAs polymers were complexed with pDNA, and the properties of the complexes were determined, including entrapment efficiency, particle size, ζ-potential, atomic force microscopy images, stability, DNA complexation ability, reduction sensitivity, cytotoxicity, and transfection efficiency. The new Gua-SS-PAAs carriers exhibited higher transfection efficiency and lower cytotoxicity compared with two widely used gene delivery carriers, polyethylenimine and lipofectamine 2000. Furthermore, the relationship between the side-chain structure and morphological/biological properties was extrapolated, and the results showed that guanidine in the side chain aids in the improvement of transfection efficiency. In addition, the introduction of guanidino group might confer the new carriers with nuclear localization function compared to carriers without it.

Recent advances on extracellular vesicles in therapeutic delivery: Challenges, solutions, and opportunities

Graphical abstract Figure. No Caption available. Abstract Extracellular vesicles (EVs) are intrinsic mediators of intercellular communication in our body, allowing functional transfer of biomolecules (lipids, proteins, and nucleic acid) between diverse locations. Such an instrumental role evokes a surge of interest within the drug delivery community in tailoring EVs for therapeutic delivery. These vesicles represent a novel generation of drug delivery systems, providing high delivery efficiency, intrinsic targeting properties, and low immunogenicity. In the recent years, considerable research efforts have been directed toward developing safe and efficient EV‐based delivery vehicles. Although EVs are shown to harbor great promise in therapeutic delivery, substantial improvements in exploring standardized isolation techniques with high efficiency and robust yield, scalable production, standard procedures for EV storage, efficient loading methods without damaging EV integrity, understanding their in vivo trafficking, and developing novel EV‐based nanocarriers are still required before their clinical transformation. In this review, we seek to summarize the recent advance on harnessing EVs for drug delivery with focus on state‐of‐the‐art solutions for overcoming major challenges.

Novel guanidinylated bioresponsive poly(amidoamine)s designed for short hairpin RNA delivery

Two different disulfide (SS)-containing poly(amidoamine) (PAA) polymers were constructed using guanidino (Gua)-containing monomers (ie, arginine [Arg] and agmatine [Agm]) and N,N′-cystamine bisacrylamide (CBA) by Michael-addition polymerization. In order to characterize these two Gua-SS-PAA polymers and investigate their potentials as short hairpin RNA (shRNA)-delivery carriers, pSilencer 4.1-CMV FANCF shRNA was chosen as a model plasmid DNA to form complexes with these two polymers. The Gua-SS-PAAs and plasmid DNA complexes were determined with particle sizes less than 90 nm and positive ζ-potentials under 20 mV at nucleic acid:polymer weight ratios lower than 1:24. Bioresponsive release of plasmid DNA was observed from both newly constructed complexes. Significantly lower cytotoxicity was observed for both polymer complexes compared with polyethylenimine and Lipofectamine 2000, two widely used transfection reagents as reference carriers. Arg-CBA showed higher transfection efficiency and gene-silencing efficiency in MCF7 cells than Agm-CBA and the reference carriers. In addition, the cellular uptake of Arg-CBA in MCF7 cells was found to be higher and faster than Agm-CBA and the reference carriers. Similarly, plasmid DNA transport into the nucleus mediated by Arg-CBA was more than that by Agm-CBA and the reference carriers. The study suggested that guanidine and carboxyl introduced into Gua-SS-PAAs polymers resulted in a better nuclear localization effect, which played a key role in the observed enhancement of transfection efficiency and low cytotoxicity. Overall, two newly synthesized Gua-SS-PAAs polymers demonstrated great potential to be used as shRNA carriers for gene-therapy applications.

Disulfide-bond-containing agamatine-cystaminebisacrylamide polymer demonstrates better transfection efficiency and lower cytotoxicity than polyethylenimine in NIH/3T3 cells

Previously, we synthesized a non‐viral vector containing disulfide bond by polymerization of agamatine (AGM) and N,N′‐cystaminebisacrylamide (CBA). In this study, we investigated the transfection efficiency of disulfide bond (SS) containing AGM‐CBA polymer in gene delivery into NIH/3T3 cells, and examined the factors affecting its transfection efficiency by comparing with polyethylenimine (PEI). In addition, experiments were carried out to determine the mechanisms of cell entry pathways and intracellular behavior of AGM‐CBA/pDNA polyplexes. The transfection efficiency of AGM‐CBA/pDNA with different weight ratios and different amounts of pDNA was measured and the pathways mediated transfection processes were studied by using various endocytosis inhibitors. To determine the intracellular behavior of AGM‐CBA/pDNA polyplexes, the transfection efficiencies of AGM‐CBA/pDNA and PEI/pDNA polyplexes with different combination structures were determined by using reporter gene and fake plasmid DNA. The transfection efficiency of AGM‐CBA/pDNA polyplexes was correlated with its weight ratio of AGM‐CBA and pDNA, and the amount of pDNA. Both AGM‐CBA/pDNA and PEI/pDNA polyplexes enter into cell by clathrin‐ and caveolae‐mediated endocytic pathways. However, AGM‐CBA/pDNA showed different intracellular behavior in NIH/3T3 cells compared to PEI/pDNA polyplexes. It was hypothesized that disulfide bond in AGM‐CBA could be an important factor contributing to its intracellular behavior and better transfection efficiency. Overall, AGM‐CBA demonstrated better transfection efficiency and lower cytotoxicity than PEI in NIH/3T3 cells as a gene delivery vector.

Comparison of exosome-mimicking liposomes with conventional liposomes for intracellular delivery of siRNA

Graphical abstract Figure. No Caption available. Abstract Exosomes have been extensively explored as delivery vehicles due to low immunogenicity, efficient cargo delivery, and possibly intrinsic homing capacity. However, therapeutic application of exosomes is hampered by structural complexity and lack of efficient techniques for isolation and drug loading. Liposomes represent one of the most successful therapeutic nanocarriers, but are frequently criticized by short blood circulation and inefficient intracellular drug delivery. In this circumstance, a promising strategy is to facilitate a positive feedback between two fields. Herein, exosome‐mimicking liposomes were formulated with DOPC/SM/Chol/DOPS/DOPE (21/17.5/30/14/17.5, mol/mol), and harnessed for delivery of VEGF siRNA to A549 and HUVEC cells. Compared with Lipo 2000 and DOTAP liposomes, exosome‐mimicking liposomes exhibited less than four‐fold cytotoxicity but higher storage stability and anti‐serum aggregation effect. Exosome‐mimicking liposomes appeared to enter A549 cells through membrane fusion, caveolae‐mediated endocytosis, and macropinocytosis, while enter HUVEC through caveolae‐mediated endocytosis, which revealed that the uptake pathway was dependent on cell types. Notably, exosome‐mimicking liposomes exhibited significantly higher cellular uptake and silencing efficiency than PC‐Chol liposomes (>three‐fold), suggesting the unique lipid composition did enhance the intracellular delivery efficiency of exosome‐mimicking liposomes to a significantly greater extent. However, it still remained far from satisfactory delivery as compared to cationic Lipo 2000 and DOTAP liposomes, which warranted further improvement in future research. This study may encourage further pursuit of more exosome‐mimicking delivery vehicles with higher efficiency and biocompatibility.

Functionalized extracellular vesicles as advanced therapeutic nanodelivery systems

&NA; Extracellular vesicles (EVs) are membrane enclosed vesicles that are shed by almost all cell types, and play a fundamental role in cell‐to‐cell communication. The discovery that EVs are capable of functionally transporting nucleic acid‐ and protein‐based cargoes between cells, rapidly promotes the idea of employing them as drug delivery systems. These endogenous vesicles indeed hold tremendous promise for therapeutic delivery. However, issues associated with exogenously administered EVs, including rapid clearance by the immune system, apparent lack of targeting cell specificity, and insufficient cytoplasmic delivery efficiency, may limit their therapeutic applicability. In this review, we discuss recent research avenues in EV‐based therapeutic nanodelivery systems. Furthermore, we narrow our focus on the development of modification strategies to enhance the delivery properties of EVs, and elaborate on how to rationally harness these functionalized vesicles for therapeutic delivery. Graphical abstract Figure. No caption available.

Exosome-based small RNA delivery: Progress and prospects

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Liposome-chaperoned cell-free synthesis for the design of proteoliposomes: Implications for therapeutic delivery

Cell-free (CF) protein synthesis has emerged as a powerful technique platform for efficient protein production in vitro. Liposomes have been widely studied as therapeutic carriers due to their biocompatibility, biodegradability, low toxicity, flexible surface manipulation, easy preparation, and higher cargo encapsulation capability. However, rapid immune clearance, insufficient targeting capacity, and poor cytoplasmic delivery efficiency substantially restrict their clinical application. The incorporation of functional membrane proteins (MPs) or peptides allows the transfer of biological properties to liposomes and imparts them with improved circulation, increased targeting, and efficient intracellular delivery. Liposome-chaperoned CF synthesis enables production of proteoliposomes in one-step reaction, which not only substantially simplifies the production procedure but also keeps protein functionality intact. Building off these observations, proteoliposomes with integrated MPs represent an excellent candidate for therapeutic delivery. In this review, we describe recent advances in CF synthesis with emphasis on detailing key factors for improving CF expression efficiency. Furthermore, we provide insights into strategies for rational design of proteoliposomal nanodelivery systems via CF synthesis. STATEMENT OF SIGNIFICANCE Liposome-chaperoned CF synthesis has emerged as a powerful approach for the design of recombinant proteoliposomes in one-step reaction. The incorporation of bioactive MPs or peptides into liposomes via CF synthesis can facilitate the development of proteoliposomal nanodelivery systems with improved circulation, increased targeting, and enhanced cellular delivery capacity. Moreover, by adapting lessons learned from natural delivery vehicles, novel bio-inspired proteoliposomes with enhanced delivery properties could be produced in CF systems. In this review, we first give an overview of CF synthesis with focus on enhancing protein expression in liposome-chaperoned CF systems. Furthermore, we intend to provide insight into harnessing CF-synthesized proteoliposomes for efficient therapeutic delivery.

Uptake Pathways of Guandinylated Disulfide Containing Polymers as Nonviral Gene Carrier Delivering DNA to Cells

Polymers of guanidinylated disulfide containing poly(amido amine)s (Gua‐SS‐PAAs), have shown high transfection efficiency and low cytotoxicity. Previously, we synthesized two Gua‐SS‐PAA polymers, using guanidino containing monomers (i.e., arginine and agmatine, denoted as ARG and AGM, respectively) and N,N’‐cystaminebisacrylamide (CBA). In this study, these two polymers, AGM‐CBA and ARG‐CBA were complexed with plasmid DNA, and their uptake pathway was investigated. Complexes distribution in MCF‐7 cells, and changes on cell endosomes/lysosomes and membrane after the cells were exposed to complexes were tested. In addition, how the transfection efficiency changed with the cell cycle status as well as endocytosis inhibitors were studied. The polymers of AGM‐CBA and ARG‐CBA can avoid endosomal/lysosomal trap, therefore, greatly delivering plasmid DNA (pDNA) to the cell nucleoli. It is the guanidine groups in the polymers that enhanced complexes’ permeation through cell membrane with slight membrane damage, and targeting to the nucleoli. J. Cell. Biochem. 118: 903–913, 2017.

Intracellular distribution and internalization pathways of guanidinylated bioresponsive poly(amido amine)s in gene delivery

Guanidinylated bioresponsive poly(amido amine)s polymers, CAR-CBA and CHL-CBA, were synthesized by Michael-type addition reaction between guanidine hydrochloride (CAR) or chlorhexidine (CHL) and N,N′-cystaminebisacrylamide (CBA). Previous studies have shown that both polymers had high transfection efficiencies as gene delivery carriers. In this study, we investigated the nucleolus localization abilities and cellular internalization pathways of these two polymers in gene delivery. Each polymer condensed plasmid DNA (pDNA) and formed nanoparticle complexes, and then their transfection studies were performed in MCF-7 cells. Both complexes were found enriched in nucleolus after cellular transfection, and their transfection efficiencies were significantly improved when transfection was performed on MCF-7 cells arrested at M phase. The transfection efficiency of CAR-CBA-pDNA was inhibited by chlorpromazine, and cell endosomes were disrupted after being exposed to CAR-CBA-pDNA. In regards to CHL-CBA-pDNA, its transfection efficiency was not affected by three types of endocytosis inhibitors used in the study, and CHL-CBA-pDNA showed no effect on endosomes. Cellular lactate dehydrogenase release and membrane morphology were changed after cells were transfected by the two complexes. The results indicated that both CAR-CBA and CHL-CBA polymers demonstrated good nucleolus localization abilities. It was beneficial for transfection when cells were arrested at M phase. CAR-CBA-pDNA cellular internalization was involved with clathrin-mediated endocytosis pathway, and escaping from endosomal entrapment, while the cellular uptake of CHL-CBA-pDNA occurs via clathrin- and caveolae-independent mechanism.