Mucus-penetrating nonviral gene vaccine processed in the epithelium for inducing advanced vaginal mucosal immune responses

Establishment of vaginal immune defenses at the mucosal interface layer through gene vaccines promise to prevent infectious diseases among females. Mucosal barriers composed of a flowing mucus hydrogel and tightly conjugated epithelial cells (ECs), which represent the main technical difficulties for vaccine development, reside in the harsh, acidic human vaginal environment. Different from frequently employed viral vectors, two types of nonviral nanocarriers were designed to concurrently overcome the barriers and induce immune responses. Differing design concepts include the charge-reversal property (DRLS) to mimic a virus that uses any cells as factories, as well as the addition of a hyaluronic acid coating (HA/RLS) to directly target dendritic cells (DCs). With a suitable size and electrostatic neutrality, these two nanoparticles penetrate a mucus hydrogel with similar diffusivity. The DRLS system expressed a higher level of the carried human papillomavirus type 16 L1 gene compared to HA/RLS in vivo. Therefore it induced more robust mucosal, cellular, and humoral immune responses. Moreover, the DLRS applied to intravaginal immunization induced high IgA levels compared with intramuscularly injected DNA (naked), indicating timely protection against pathogens at the mucus layer. These findings also offer important approaches for the design and fabrication of nonviral gene vaccines in other mucosal systems.

Reprogrammed siTNFα/neutrophil cytopharmaceuticals targeting inflamed joints for rheumatoid arthritis therapy

Rheumatoid arthritis (RA) is an autoimmune disease characterized by severe synovial inflammation and cartilage damage. Despite great progress in RA therapy, there still lacks the drugs to completely cure RA patients. Herein, we propose a reprogrammed neutrophil cytopharmaceuticals loading with TNFα-targeting-siRNA (siTNFα) as an alternative anti-inflammatory approach for RA treatment. The loaded siTNFα act as not only the gene therapeutics to inhibit TNFα production by macrophages in inflamed synovium, but also the editors to reprogram neutrophils to anti-inflammatory phenotypes. Leveraging the active tendency of neutrophils to inflammation, the reprogrammed siTNFα/neutrophil cytopharmaceuticals (siTNFα/TP/NEs) can rapidly migrate to the inflamed synovium, transfer the loaded siTNFα to macrophages followed by the significant reduction of TNFα expression, and circumvent the pro-inflammatory activity of neutrophils, thus leading to the alleviated synovial inflammation and improved cartilage protection. Our work provides a promising cytopharmaceutical for RA treatment, and puts forward a living neutrophil-based gene delivery platform.

Optimization of a cationic liposomal gene delivery system and study of its endocytic pathway

Abstract A cationic liposomal gene delivery system comprising DOTAP, DOPE, and cholesterol was prepared and optimized. The results showed that the liposome/DNA (LP/DNA) system had spherical morphology, with a particle size of around 150 nm and zeta potential of approximately 30 mV. Cytotoxicity experiments showed that cells treated with all of the liposome carriers- with the exception of LP1-had more than 80% viability even at a weight ratio of 30. The in vitro transfection efficiency was measured using a Promega™ Luciferase Assay System. Of the tested lipoplexes, LP2/DNA showed the highest cell transfection efficiency (at a weight ratio of 10)-which was similar to or slightly lower than that of Lipofectamine® 2000 in HeLa, A549, and SPC-A1 cell lines. After freeze-drying, the cell transfection efficiency decreased slightly (P>0.05). The cell uptake mechanism study showed that LP/DNA lipoplexes mainly entered cells via clathrin-mediated and caveolin-mediated endocytic pathways. The results confirmed that LP2 has potential for use as an effective gene carrier, and provides experimental evidence to support its further development as a safe and effective gene delivery system.

A novel method for DNA delivery into bacteria using cationic copolymers

Amphiphilic copolymers have a wide variety of medical and biotechnological applications, including DNA transfection in eukaryotic cells. Still, no polymer-primed transfection of prokaryotic cells has been described. The reversible addition-fragmentation chain transfer (RAFT) polymer synthesis technique and the reversible deactivation radical polymerization variants allow the design of polymers with well-controlled molar mass, morphology, and hydrophilicity/hydrophobicity ratios. RAFT was used to synthesize two amphiphilic copolymers containing different ratios of the amphiphilic poly[2-(dimethyl-amino) ethyl methacrylate] and the hydrophobic poly [methyl methacrylate]. These copolymers bound to pUC-19 DNA and successfully transfected non-competent Escherichia coli DH5α, with transformation efficiency in the range of 103 colony-forming units per µg of plasmid DNA. These results demonstrate prokaryote transformation using polymers with controlled amphiphilic/hydrophobic ratios.

Research progress of poly (β-amino ester)s-based gene vector / 药学学报

Poly (β-amino ester)s (PβAEs) contain tertiary amine backbones and biodegradable ester bonds, making them highly biocompatible and pH-responsive. Meanwhile, originated from combinatorial libraries, PβAEs are simple to synthesize, easy to obtain raw materials and can be easily adapted to meet the different performance needs of gene carriers by adjusting the monomer type, monomer ratio and reaction time. Therefore, PβAEs are promising material for non-viral gene carriers. This paper provides a comprehensive overview of the properties and synthesis of PβAEs gene carriers and summarizes the progress of research on the gene delivery of each type of PβAEs.

Construction and application of gene delivery systems for primary dendritic cells / 中国药科大学学报

@#Nowadays, there is still no mature gene delivery system for safe and effective transfection on primary dendritic cells (DC). Herein, we constructed a liposome-based gene delivery system for primary DCs and optimized the preparation method to improve the transfection efficiency of siRNA on primary DCs. In this study, different methods, including co-incubation method, ethanol injection method, and protamine compound method, were used to prepare liposome/siRNA complexes based on different cationic lipids. Moreover, particle size, zeta potential, siRNA loading capacity, safety, stability, uptake efficiency and gene silencing efficiency of various liposome/siRNA complexes were detected to screen the optimal cationic lipid as well as its preparation method. We demonstrated that the OA2/siRNA delivery system prepared by the co-incubation method exhibited the best safety, uptake efficiency and gene silencing effect, compared to other siRNA delivery systems including the commercial Lipo2000. In summary, we provide a safe and effective gene delivery vector for primary DC cells through simple preparation method, which could also offer a gene delivery platform for other immune cells.

Self-assembling dendrimers for biomedical applications / 中国药科大学学报

@#Dendrimers, a special class of synthetic polymers known for their well-defined ramified structures and unique multivalent cooperativity, hold great promise for various biomedical applications. However, preparation of defect-free dendrimers of high-generation on a large scale remains challenging because of the tedious and time-consuming synthesis as well as difficult purification. To overcome these limitations, an alternative strategy based on self-assembling approach has been developed to construct supramolecular dendrimers using small amphiphilic dendrimer-building units. By virtue of the amphiphilic nature, these small dendrimer-building units self-assemble and form large non-covalent supramolecular dendritic structures that mimic high-generation covalent dendrimers. Here, we present a brief overview of the supramolecular dendrimers developed in our group for the delivery of nucleic acid therapeutics, anticancer drug and imaging agents.

Synchronous conjugation of i-motif DNA and therapeutic siRNA on the vertexes of tetrahedral DNA nanocages for efficient gene silence

The functionality of DNA biomacromolecules has been widely excavated, as therapeutic drugs, carriers, and functionalized modification derivatives. In this study, we developed a series of DNA tetrahedron nanocages (Td),

Construction and in vitro evaluation of pH-responsive and tumor-targeted PTEN/PLGA-(HE)10-MAP nanoparticles / 中国药科大学学报

@#To construct PTEN/PLGA-(HE)10-MAP nanoparticles, which encapsulated PTEN plasmid DNA and combined with the pH-responsive cell-penetrating peptides (CPPs), and to investigate their effects of gene delivery and anti-tumor targets in vitro. Poly (lactic-co-glycolic acid) (PLGA) nanoparticles loaded with PTEN plasmid DNA were prepared by double emulsification-solvent evaporation method. PTEN/PLGA-(HE)10-MAP nanoparticles were prepared by coupling the histidine-glutamic acid-model amphipathic peptide nanocomplex [(HE)10-MAP] to the surface through amide condensation reaction. Particle size, Zeta potential, encapsulation rate and drug loading were tested to characterize the nanoparticles. By analyzing the cytotoxicity, cellular uptake, targeted transfection of eukaryotic expression plasmids and anti-tumor cell proliferation, the feasibility as a targeted gene delivery system were evaluated. The particle size of PTEN/PLGA-(HE)10-MAP nanoparticles was (266.5 ± 2.86) nm, with the encapsulation efficiency (80.6 ± 6.11)%. Zeta potentials were -(6.7 ± 0.26) mV, +(0.7 ± 0.22) mV and +(37.5 ± 0.85) mV at pH 7.4, 7.0 and 6.5, respectively. In the cytotoxicity test, the cell survival rates of tumor and normal cells were above 80%.Non-loading PLGA-(HE)10-MAP nanoparticles showed no obvious cytotoxicity. The results of cellular uptake experiments showed that PTEN/PLGA-(HE)10-MAP nanoparticles were more readily taken up by cells.The results of CCK-8 showed that the nanoparticles could pH-specifically inhibit proliferation of tumor cell in vitro.And PTEN/PLGA-(HE)10-MAP nanoparticles may be applied in tumor gene therapy.

Target-specific gene delivery in plant systems and their expression: Insights into recent developments

High-yielding Indian cotton varieties are not amenable for regeneration and transformation because they arerecalcitrant in nature. In this work, we have developed Narasimha (NA1325) cotton variety by introducing threeCrygenes driven by three different promoters conferring insect resistance. The meristematic region of embryo axisexplants were infected and co-cultivated with Agrobacterium tumefacience (LBA4404) harbouring pMDC100vector with three Cry gene cassettes (a-globulin : Cry2Ab, DECaMV35s : Cry1F and nodulin : Cry1Ac) with Npt IIas a selectable marker gene. Out of 1010 embryo axes explants infected, 121 (T0) regenerated under two rounds ofkanamycin selection medium. About 2551 T1 seeds were collected from 111 T0 plants and these seeds screened againwith kanamycin at seedling stage. The transgenic plants were characterized by PCR, real time quantitative PCR,lateral flow strip protein assay and insect bioassay. Out of 145 kanamycin resistant plants (T1), twelve showedamplification of all four transgenes: Npt II, Cry2Ab, Cry1F and Cry1Ac through PCR with expected amplicons as395, 870, 840 and 618 bp, respectively. Further, lateral flow strip test revealed Cry1F and Cry1Ac proteinsaccumulated in 12 plants, whereas Cry2Ab protein was detected in eight only. The transcripts of all three Cry geneswere accumulated significantly higher in transgenic plants at T2 generation. The transgenic lines showed effectiveresistance against Helicoverpa armigera and Spodoptera litura larvae. The T2 line L-3 exhibited highest percentageof insect mortality, in which transcripts of all cry genes were accumulated higher than other plants. The transgeniccotton plants carrying triple Cry genes could be an excellent germplasm resource for the breeders for introgressions.

Recent progress of non-viral vector polyethylenimine in the application of gene delivery / 药学学报

In recent years, non-viral gene vectors have attracted great attention for efficient gene delivery due to the advantages, including low toxicity, low immunogenicity and simple preparation. Polyethylenimine (PEI) is one of the typical non-viral gene carriers that have been widely utilized for gene delivery owing to its superior capabilities in gene compression and buffering capacity. This article discusses the processes of gene delivery and the barriers of PEI-based carrier during the gene delivery, such as low biocompatibility, cytotoxicity, lack of specific targeting and insufficient gene release, etc. Therefore, we summarize the multiple approaches for the modifications of PEI in terms of improved biocompatibility, degradability, specific targeting and buffering capacity. Furthermore, we also review on the recent impressive progresses of smart stimuli-responsive PEI carriers, including endogenous stimuli (pH, reactive oxygen species, glutathione, biomolecular, etc), exogenous stimuli (light, temperature, magnetic field, etc) and dual-responsive strategies, which might provide guidance for the development of more efficient and safer non-viral gene vectors.

Biopolymers augment viral vectors based gene delivery

The success of viral vectors mediated gene therapy is still hampered by immunogenicity and insufficient transgeneexpression. Alternatively, non-viral vectors mediated gene delivery has the advantage of low immunogenicity despiteshowing low transgene expression. By carefully considering the advantages of each approach, hybrid vectors are currentlybeing developed by modifying the viral vectors using non-viral biopolymers. This review provides an overview of thehybrid vectors currently being developed.

Photochemical internalization and its application in gene delivery / 上海交通大学学报（医学版）

The translation of gene therapy from bench to bedside depends on efficient intracellular gene delivery. The macromolecular biologics such as gene combined with vectors tend to enter into the cells by means of endocytosis, where the biologics may encounter the risk of degradation in endolysosome. Recently, photochemical internalization (PCI) has emerged as a promising technique to overcome endo-lysosomal sequestration, which utilizes photosensitizer and light resulting in reactive oxygen species at sub-lethal level to destruct biofilm and facilitate intracellular drug delivery. In this article, the mechanism of PCI technology and its development for gene delivery were reviewed, which can provide the scientific basis for the possible utilization of PCI to solve the problem of endo-lysosomal escape in gene delivery.

Photochemical internalization and its application in gene delivery / 上海交通大学学报（医学版）

The translation of gene therapy from bench to bedside depends on efficient intracellular gene delivery. The macromolecular biologics such as gene combined with vectors tend to enter into the cells by means of endocytosis, where the biologics may encounter the risk of degradation in endo-lysosome. Recently, photochemical internalization (PCI) has emerged as a promising technique to overcome endo-lysosomal sequestration, which utilizes photosensitizer and light resulting in reactive oxygen species at sub-lethal level to destruct biofilm and facilitate intracellular drug delivery. In this article, the mechanism of PCI technology and its development for gene delivery were reviewed, which can provide the scientific basis for the possible utilization of PCI to solve the problem of endo-lysosomal escape in gene delivery.

Potential of Calcium Carbonate Nanoparticles for Therapeutic Applications

@#The application of nanoparticles (NPs) has attracted considerable attention as targeted delivery systems. CaCO3 has become the focus due to its advantages including affordability, low toxicity, biocompatibility, cytocompatibility, pH sensitivity and sedate biodegradability and environment friendly materials. In this article, we will discuss the potential roles of CaCO3-NPs in three major therapeutic applications; as antimicrobial, for drug delivery, and as gene delivery nanocarrier.

The status and prospect of ultrasound and microbubble-mediated gene delivery in cardiovascular disease gene therapy / 中国胸心血管外科临床杂志

@#With the development of molecular and cellar cardiology, gene therapy to cardiovascular disease has become the hot spot and the direction of study. Now, preclinical studies on ultrasound-mediated gene delivery (UMGD) in cardiovascular disease have achieved some success, but it is still hindered by a series of practical challenges for clinical translation. Even so, UMGD still holds the promise to cardiovascular disease in gene therapy for its non-invasiveness, accuracy, safety and ability to deliver multiple genes with repeated deliveries. In this review, we will focus on the basic principle, the current development, the future prospect and drawbacks of UMGD in the therapeutic applications of cardiovascular disease.

Cholesterol improves the transfection efficiency of polyallylamine as a non-viral gene delivery vector

ABSTRACT Cationic polymers such as polyallylamine (PAA) having primary amino groups are poor transfection agents and possess a high cytotoxicity index when used without any chemical modification. In this study, PAA was modified with cholesterol in order to improve transfection efficiency and to reduce cytotoxicity. PAA polymers with molecular weights of 15 and 65 kDa were selected and grafted with cholesterol at percentages of 5, 10, 15, 30, and 50. After purification, the efficacy of the synthetic vectors was evaluated in terms of DNA condensation using the ethidium bromide test, buffering capacity, particle size, zeta potential, transfection efficiency, and cytotoxicity assay in Neuro2A cell lines. According to the ethidium bromide test, these vectors can condense DNA at moderate and high carrier to plasmid (C/P) ratios. The buffering capacity of the prepared vector in both molecular weights was less than unmodified PAA. Particle size measurements demonstrated that modified PAAs were able to form nanoparticles ranging in size from 125 to 530 nm. The vectors based on PAA 15 kDa demonstrated a better transfection efficiency than the vectors made of PAA 65 kDa. Cytotoxicity studies showed that toxicity of all vectors was less than PAA. Some cholesterol modified polymers composed of PAA (15 kDa) were suitable vectors for gene delivery with low cytotoxicity.

Construction of serum-resistant cationic polymer α-CD-PAMAM and evaluation of its performances as gene delivery vector / 药学学报

Polyamidoamine (PAMAM) dendrimers as synthetic gene vectors are efficient gene delivery systems. In this study, a kind of α-cyclodextrin-PAMAM conjugates polymer (CyD-G1) was synthesized as a gene delivery vector. Based on 1H NMR detectation, about 6.4 PAMAM-G1 molecules was grafted onto an α-CD core. Agarose gel electrophoresis revealed that CyD-G1 could efficiently bind with DNA to condense them into nano-scale particles, which showed a similar binding capacity of PEI-25K. Besides, it could protect DNA from DNase I degradation in a low N/P ratio. When N/P ratio in the CyD-G1/DNA polyplex was 40, the average particle size of CyD-G1/DNA polyplex was about 120 nm, and zeta potential was +21 mV. This polyplex could maintain its particle size in serum-containing solution within 360 min. In comparison with PEI-25K carrier, CyD-G1 showed low cytotoxicity in various cell lines. Cell transfection results showed that CyD-G1 efficiently delivered DNA into cells at N/P=80 compared with Lipofectamine 2000 and PEI-25K.Unlike Lipofectamine 2000 and PEI-25K, in serum-containing test condition, CyD-G1/DNA polyplex could maintain the transgene activities. The results of confocal laser scanning microscopy indicated that most DNA entered into cell nuclei within 4 h, and this phenomenon was consistent with the results calculated by flow cytometry. Taken together, CyD-G1 showed good transgene activities and the gene delivery vector could be used not only in vitro but also in vivo.

GGI as a gene carrier delivering MDR1 siRNA to A549/DDP cells for reversal of multidrug resistance / 药学学报

This study was designed to reverse multidrug resistance of lung cancer cells by downregulating MDR1 genes through RNA interference (RNAi) technology. A novel biodegradable cationic polymer (PEG-bPLG-g-PEIs, GGI) was synthesized and characterized by 1H NMR. The particle size and zeta potential were measured by dynamic light scattering (DLS). The cell viability profile of GGI was tested by MTT method with both A549 and A549/DDP cell lines. Flow cytometry (FCM) technology was used to investigate the efficiency and intensity of delivering siRNA to cells by GGI polymer. RT-PCR and Western blot were used to detect the mRNA and P-gp expression after GGI/MDR1 siRNA transfection assay. The sensitivity of cisplatin administration after transfecting GGI/MDR1 siRNA polyplexs was performed with MTT and Annexin V-FITC/PI methods. The results suggest that the particle size and zeta potential of GGI/siRNA were 150-200 nm and 16-28 mV. GGI exhibited a lower cell cytotoxity than PEI 25K and higher efficiency of delivering siRNA, which dramatically decreased the expression of MDR1 mRNA and P-gp of A549/DDP cells and increased much sensitivity to cisplatin in A549/DDP cells. GGI holds a great potential in gene delivery as a novel cationic polymer for further investigation.

Advances in ultrasound-targeted microbubble-mediated gene therapy for liver fibrosis

Hepatic fibrosis develops as a wound-healing scar in response to acute and chronic liver inflammation and can lead to cirrhosis in patients with chronic hepatitis B and C. The condition arises due to increased synthesis and reduced degradation of extracellular matrix (ECM) and is a common pathological sequela of chronic liver disease. Excessive deposition of ECM in the liver causes liver dysfunction, ascites, and eventually upper gastrointestinal bleeding as well as a series of complications. However, fibrosis can be reversed before developing into cirrhosis and has thus been the subject of extensive researches particularly at the gene level. Currently, therapeutic genes are imported into the damaged liver to delay or prevent the development of liver fibrosis by regulating the expression of exogenous genes. One technique of gene delivery uses ultrasound targeting of microbubbles combined with therapeutic genes where the time and intensity of the ultrasound can control the release process. Ultrasound irradiation of microbubbles in the vicinity of cells changes the permeability of the cell membrane by its cavitation effect and enhances gene transfection. In this paper, recent progress in the field is reviewed with emphasis on the following aspects: the types of ultrasound microbubbles, the construction of an ultrasound-mediated gene delivery system, the mechanism of ultrasound microbubble-mediated gene transfer and the application of ultrasound microbubbles in the treatment of liver fibrosis.