Stimuli-Responsive Gene Delivery Nanocarriers for Cancer Therapy

Gene therapy provides a promising approach in treating cancers with high efficacy and selectivity and few adverse effects.Currently,the development of functional vectors with safety and effectiveness is the intense focus for improving the delivery of nucleic acid drugs for gene therapy.For this purpose,stimuli-responsive nanocarriers displayed strong potential in improving the overall efficiencies of gene therapy and reducing adverse effects via effective protection,prolonged blood circulation,specific tumor accumulation,and controlled release profile of nucleic acid drugs.Besides,synergistic therapy could be achieved when combined with other therapeutic regimens.This review summarizes recent advances in various stimuliresponsive nanocarriers for gene delivery.Particularly,the nanocarriers responding to endogenous stimuli including pH,reactive oxygen species,glutathione,and enzyme,etc.,and exogenous stimuli including light,thermo,ultrasound,magnetic field,etc.,are introduced.Finally,the future challenges and prospects of stimuli-responsive gene delivery nanocarriers toward potential clinical translation are well discussed.The major objective of this review is to present the biomedical potential of stimuli-responsive gene delivery nanocarriers for cancer therapy and provide guidance for developing novel nanoplatforms that are clinically applicable.

Insights on drug and gene delivery systems in liver fibrosis

Complications of the liver are amongst the world’s worst diseases.Liver fibrosis is the first stage of liver problems,while cirrhosis is the last stage,which can lead to death.The creation of effective anti-fibrotic drug delivery methods appears critical due to the liver’s metabolic capacity for drugs and the presence of insurmountable physiological impediments in the way of targeting.Recent breakthroughs in anti-fibrotic agents have substantially assisted in fibrosis;nevertheless,the working mechanism of anti-fibrotic medications is not fully understood,and there is a need to design delivery systems that are well-understood and can aid in cirrhosis.Nanotechnology-based delivery systems are regarded to be effective but they have not been adequately researched for liver delivery.As a result,the capability of nanoparticles in hepatic delivery was explored.Another approach is targeted drug delivery,which can considerably improve efficacy if delivery systems are designed to target hepatic stellate cells(HSCs).We have addressed numerous delivery strategies that target HSCs,which can eventually aid in fibrosis.Recently genetics have proved to be useful,and methods for delivering genetic material to the target place have also been investigated where different techniques are depicted.To summarize,this review paper sheds light on themost recent breakthroughs in drug and gene-based nano and targeted delivery systems that have lately shown useful for the treatment of liver fibrosis and cirrhosis.

Efficient Gene Delivery to Myocardium with Ultrasound Targeted Microbubble Destruction and Polyethylenimine

The aim of present study was to evaluate the feasibility and efficiency of enhanced green fluorescent protein （EGFP） gene delivery to myocardium in vivo by ultrasound targeted microbubble destruction （UTMD） and polyethylenimine （PEI）. SonoVue/DNA and PEI/DNA/SonoVue complexes were prepared. Gel electrophoresis analysis was performed to determine the structural integrity of plasmid DNA or PEI/DNA after UTMD. Solutions of plasmid DNA, SonoVue/DNA, PEI/DNA complexes or PEI/DNA/SonoVue complexes were respectively transduced into BALB/c mice hearts by means of transthoracic ultrasound irradiation. Mice undergoing PBS injection, plasmid injection or PEI/DNA complexes injection without ultrasound irradiation served as controls. Gene expression in myocardium was detected 4 days after treatment. Cryosections and histological examinations were conducted. Electrophoresis gel assay showed no damage to DNA or PEI/DNA complexes after UTMD. When the heart was not exposed to ultrasound, the expression of EGFP was observed in the subendocardial myocardium obviously. The strongest expression was detected in the anterior wall of the left ventricle when the heart was exposed to ultrasound alone. Injection of PEI/DNA complexes and UTMD resulted in the highest transfection efficiency and the distributional difference of EGFP was not obvious. No tissue damage was seen histologically. In conclusion, a combination of UTMD and PEI was highly effective in transfecting mice hearts without causing any apparently adverse effect. It provides an alternative to current clinical gene therapy and opens a new concept of non-viral gene delivery for the treatment of cardiac disease.

Synthesis of a novel multivalent galactoside with high hepatocyte targeting for gene delivery

A novel bifunctional glycolipid which carded a cluster of thiogalactosides as the hepatocyte targeting ligand for gene delivery was prepared. Hexa-antennary alcohol 1 was used as the core scaffold to attach a cholesterol molecule by a poly（ethylene glycol） chain, while its remaining branches were linked with five acetylgalactosides, which would be deacetylated later to produce pentaantennary galactoside. Liposome containing the galactoside showed high affinity and transfection activity in hepatoma cells HepG2.

Preparation and Characterization of Cationic PLGA-PEG-Lf/DOPE Nanoparticles for HO-1 Gene Delivery

Cationic nanoparticles （NPs） for gene delivery were successfully prepared by assembling earboxylation poly（lactic-co-glycolic acid） （PLGA）, polyethylene glycol （PEG）, L-ct-Phosphatidylethanolamine （DOPE） and octadecyl quaternized carboxymethyl chitosans （OQCMC）. Lactoferrin （Lf） was selected as a targeting ligand conjugated to PLGA via bifunctional PEG, yielding PLGA-PEG-Lf/DOPE NPs to be used for gene vectors. Fourier transform infrared spectroscopy （FTIR）, UV and nuclear magnetic resonance （NMR） spectroscopy were performed to evaluate the synthesis of the vectors. The characteristics of the vectors loaded heine oxygenase （HO-1） gene were evaluated by transmission electron microscope （TEM）, particle size analyser and fluorescent microscopy. The experimental results showed that the obtained vectors were spherical in shape with average particle size of 142.2 nm and zeta potentials of ＋16.4 inV. The vectors could protect the loaded gene from the degradation by nuclease. For 293T cells, there is high transfection efficiency of the vectors similar to liposome-2000. It can be concluded that the established cationic PLGA-PEG-Lf/DOPE NPs have potential gene delivery ability for gene therapy.

Design of Dendrimer Modified Carbon Nanotubes for Gene Delivery

To investigate the efficiency of polyamidoamine dendrimer grafted carbon nanotube （dendrimer-CNT） mediated entrance of anti-survivin oligonucleotide into MCF-7 cells, and its effects on the growth of MCF-7 cells. Methods： Antisense survivin oligonucleotide was anchored onto polyamidoamine dendrimer grafted carbon nanotubes to form dendrimer-CNT-asODN complex and the complex was characterized by Zeta potential, AFM, TEM, and 1% agarose gel electrophoresis analysis. Dendrimer-CNT-asODN complexes were added into the medium and incubated with MCF-7 cells. MTT method was used to detect the effects of asODN and dendrimer-CNT-asODN on the growth of MCF-7 cells. TEM was used to observe the distribution of dendrimer-CNT-asODN complex within MCF-7 cells. Results： Successful synthesis of dendrimer-CNT-asODN complexes was proved by TEM, AFM and agarose gel electrophoresis. TEM showed that the complexes were located in the cytoplasm, endosome, and lysosome within MCF-7 cells. When dendrimer-CNT-asODN （1.0 μmol/L） and asODN （1.0 μmol/L） were used for 120 h incubation, the inhibitory rates of MCF-7 cells were （28.22±3.5）% for dendrimer-CNT-asODN complex group, （9.23±0.56）% for only asODN group, and （3.44±0.25）% for dendrimer-CNT group. Dendrimer-CNT-asODN complex at 3.0 μmol/L inhibited MCF-7 cells by （30.30±10.62）%, and the inhibitory effects were in a time- and concentration-dependent manner. Conclusion： Dendrimer-CNT nanoparticles may serve as a gene delivery vector with high efficiency, which can bring foreign gene into cancer cells, inhibiting cancer cell proliferation and markedly enhancing the cancer therapy effects.

Enhanced lysosome escape mediated by 1,2-dicarboxylic-cyclohexene anhydride-modified poly-L-lysine dendrimer as a gene delivery system

Antisense oligodeoxynucleotide(ASODN)can directly interfere a series of biological events of the target RNA derived from tumor cells through Watson-Crick base pairing,in turn,plays antitumor therapeutic roles.In the study,a novel HIF-1αASODN-loaded nanocomposite was formulated to efficiently deliver gene to the target RNA.The physicochemical properties of nanocomposite were characterized using TEM,FTIR,DLS and zeta potentials.The mean diameter of resulting GEL-DGL-FA-ASODN-DCA nanocomposite was about 170–192 nm,and according to the agarose gel retardation assay,the loading amount of ASODN accounted for 166.7 mg/g.The results of cellular uptake showed that the nanocomposite could specifically target to HepG2 and Hela cells.The cytotoxicity assay demonstrated that the toxicity of vectors was greatly reduced by using DCA to reversibly block the cationic DGL.The subcellular distribution images clearly displayed the lysosomal escape ability of the DCA-modified nanocomposite.In vitro exploration of molecular mechanism indicated that the nanocomposite could inhibit m RNA expression and HIF-1αprotein translation at different levels.In vivo optical images and quantitative assay testified that the formulation accumulated preferentially in the tumor tissue.In vivo antitumor efficacy research confirmed that this nanocomposite had significant antitumor activity and the tumor inhibitory rate was 77.99%.These results manifested that the GEL-DGL-FA-ASODNDCA nanocomposite was promising in gene therapeutics for antitumor by interacting directly with target RNA.

Effect of Polyethylenimine Content on Liposome/Polyethylenimine Complexes-Mediated Gene Delivery

Cationic liposome(Lipo) and polyethylenimine(PEI) are widely applied for nonviral gene transfection.In this study,in order to combine the favorable properties of Lipo and PEI systems for gene delivery,Lipo/PEI complexes with different contents of PEI(5%,10%,20% and 40% relative to phosphatidyl choline in reaction system) were prepared.The physicochemical properties of Lipo/PEI complexes,as well as the influences of PEI content on the storage stability,cytotoxicity and transfection efficiency were investigated.The transmission electron microscopy(TEM) images showed that Lipo/PEI complexes had smaller size compared to pure Lipo.The zeta potential values decreased with the increasing content of PEI.After storaged for 3 months at 4 ℃,obvious aggregation was observed when the addition of PEI content was up to 20%.In vitro cytotoxicity assay showed that Lipo/PEI complexes had decreased cytotoxicity over pure PEI,while the cytotoxicity was enhanced as the PEI content increased.Importantly,the luciferase activity assay and confocal microscope observation revealed that Lipo/PEI complexes prepared with the lowest amount of PEI(Lipo/PEI-5%)possessed the highest transfection efficiency.Thus,these results suggest that feeding the appropriate content of PEI in Lipo/PEI complexes allows them to be excellent vehicle for gene delivery.

A 3-D in vitro tumor model for investigation of bacteria-mediated gene delivery

Introduction Cancer is an attractive target of gene therapy and currently represents the disease in most clinical trials[1]. Strategies for cancer gene therapy include: (1) stimulation of immune responses to tumor cells,(2) delivery of specific enzymes

MSC based gene delivery methods and strategies improve the therapeutic efficacy of neurological diseases

Mesenchymal stem cells(MSCs)are promising seed cells for neural regeneration therapy owing to their plasticity and accessibility.They possess several inherent characteristics advantageous for the transplantation-based treatment of neurological disorders,including neural differentiation,immunosuppression,neurotrophy,and safety.However,the therapeutic efficacy of MSCs alone remains unsatisfactory in most cases.To improve some of their abilities,many studies have employed genetic engineering to transfer key genes into MSCs.Both viral and nonviral methods can be used to overexpress therapeutic proteins that complement the inherent properties.However,to date,different modes of gene transfer have specific drawbacks and advantages.In addition,MSCs can be functionalized through targeted gene modification to facilitate neural repair by promoting neural differentiation,enhancing neurotrophic and neuroprotective functions,and increasing survival and homing abilities.The methods of gene transfer and selection of delivered genes still need to be optimized for improved therapeutic and targeting efficacies while minimizing the loss of MSC function.In this review,we focus on gene transport technologies for engineering MSCs and the application of strategies for selecting optimal delivery genes.Further,we describe the prospects and challenges of their application in animal models of different neurological lesions to broaden treatment alternatives for neurological diseases.

Mesoporous silica nanoparticles for drug and gene delivery

Mesoporous silica nanoparticles(MSNs) are attracting increasing interest for potential biomedical applications. With tailored mesoporous structure, huge surface area and pore volume,selective surface functionality, as well as morphology control, MSNs exhibit high loading capacity for therapeutic agents and controlled release properties if modified with stimuli-responsive groups, polymers or proteins. In this review article, the applications of MSNs in pharmaceutics to improve drug bioavailability, reduce drug toxicity, and deliver with cellular targetability are summarized. Particularly,the exciting progress in the development of MSNs-based effective delivery systems for poorly soluble drugs, anticancer agents, and therapeutic genes are highlighted.

Gene Delivery of SOCS3 Protects Mice from Lethal Endotoxic Shock

Suppressor of cytokine signaling 3 （SOCS3） was reported as a feedback inhibitor of cytokine receptor signaling by inhibiting the JAK-STAT signal transduction pathway. We sought to test the anti-endotoxic septic shock effect of liposome mediated gene delivery of SOCS3 in a lethal endotoxic shock mouse model. BALB/c mice were injected intraperitoneally with 200μg pcDNA3.1-SOCS3 cationic liposomes, while pcDNA3.1-IL-10 and empty vector as positive and negative control respectively. Forty-eight hours after gene delivery, mice were challenged with 4 μg of E.coli 0127：B8 LPS and 18 mg D-GaIN administered i.p. 90 min later, serum TNF-α level was determined. Survival over the next 48 h was evaluated. Peritoneal macrophages from survival mice were stimulated in vitro with 1 μg/ml LPS for 18 h, and the supernatants were harvested for determination of the amount of TNF-α. We found that gene delivery of SOCS3 significantly increase the mouse survival rate from 27.8 ± 9.6% of control group to 61.1 ± 9.6% （p 〈 0.01）. In comparison with control group （218 ± 13 pg/ml） and sham delivery group （219 ± 22 pg/ml）, gene delivery of SOCS3 reduced the level of serum TNF-α （68 ± 9 pg/ml） significantly （p 〈 0.01）. Furthermore, gene delivery of SOCS3 displayed the capacity of prevention of tolerance of peritoneal macrophages to LPS. These findings suggest that gene delivery of SOCS3 mediated by liposome is a promising approach for endotoxic septic shock treatment. Cellular ＆ Molecular Immunology.

Gene delivery in peritoneal dialysis related peritoneal fibrosis research

Objective To summarize the development of gene delivery vectors in peritoneal fibrosis research and discuss the feasibility and superiority of lentiviral vectors. Data sources The data in this article were collected from PubMed database with relevant English articles published from 1995 to 2011. Study selection Articles regarding the gene therapy in peritoneal fibrosis research using non-viral vectors, adenoviral vectors, retroviral vectors, and lentiviral vectors were selected. Data were mainly extracted from 60 articles, which are listed in the reference section of this review. Results Non-viral vector-mediated gene delivery （including naked DNA for ex vivo, oligonucleotides, ultrasound- contrast agent mediated naked gene delivery, etc.） and viral vector-mediated gene delivery （including adenovirus, helper-dependant adenovirus, and retrovirus vectors） have been successfully applied both in the mechanistic investigation and the potential prevention and treatment of peritoneal fibrosis. Conclusions Peritoneal fibrosis is a major complication of peritoneal dialysis （PD）. Recently, the wide use of the gene delivery technique made it possible to access and further research peritoneal fibrosis. The use of lentiviral vector is expected to be widely used in PD research in the future due to its advantages in gene delivery.

Gene delivery into isolated Arabidopsis thaliana protoplasts and intact leaves using cationic, a-helical polypeptide

The application of gene delivery materials has been mainly focused on mammalian cells while rarely extended to plant engineering. Cationic polymers and lipids have been widely utilized to efficiently deliver DNA and siRNA into mammalian cells. However, their applica- tion in plant cells is limited due to the different membrane structures and the presence of plant cell walls. In this study, we developed the cationic, a-helical polypeptide that can effectively deliver DNA into both isolated Arabidopsis thaliana protoplasts and intact leaves. The PPABLG was able to condense DNA to form nanocomplexes, and they exhibited significantly improved transfection efficiencies compared with commercial transfection reagent Lipofec- tamine 2000 and classical cell penetrating peptides such as poly（L-lysine）, HIV-TAT, arginine9, and poly（L-arginine）. This study therefore widens the utilities of helical polypeptide as a unique category of gene delivery materials, and may find their promising applications toward plant gene delivery.

Guanidinoamidized Linear Polyethyleneimine for Gene Delivery

Guanidine was introduced to low molecular weight linear polyethyleneimine （LPEI） via amide groups, to explore the effect of both guanidine degree and pendant chain length on its transfection behavior. The resulting guanidinoamidized LPEIs （GLPEIs） could dramatically reduce LPEI＇s toxicity, enhance its DNA-packaging capability, cellular uptake and therefore transfection efficiency. These polyplexes were taken up very efficiently via caveolae-mediated endocytosis and their transfection efficiencies in ovarian cancer cells were significantly improved compared to native LPEIlok polyplexes. Among these GLPEIs, LPEI-C3-G100 showed higher DNA affinity even than LPEI25k and the highest transfection efficiency, probably due to the optimization of polymer chain flexibility. Of notice, LPEI-C3-G100 polyplexes could more effectively accumulate into cytoplasm than LPEI25k, although the transfection efficiency of LPEI-C3-G100 polyplexes was not superior to that of LPEI25k polyplexes, which would be probably attributed to the more efficient release of LPEI25k polyplexes than LPEI-C3-G100 polyplexes in the cytoplasm.

Highly efficient retinal gene delivery with helper-dependent adenoviral vectors

There have been significant advancements in the field of retinal gene therapy in the past several years.In particular,therapeutic efficacy has been achieved in three separate human clinical trials conducted to assess the ability of adeno-associated viruses(AAV)to treat of a type of Leber’s congenital amaurosis caused by RPE65 mutations.However,despite the success of retinal gene therapy with AAV,challenges remain for delivering large therapeutic genes or genes requiring long DNA regulatory elements for controlling their expression.For example,Stargardt’s disease,a form of juvenile macular degeneration,is caused by defects in ABCA4,a gene that is too large to be packaged in AAV.Therefore,we investigated the ability of helper dependent adenovirus(HD-Ad)to deliver genes to the retina as it has a much larger transgene capacity.Using an EGFP reporter,our results showed that HD-Ad can transduce the entire retinal epithelium of a mouse using a dose of only 1
105 infectious units and maintain transgene expression for at least 4 months.The results demonstrate that HD-Ad has the potential to be an effective vector for the gene therapy of the retina.

The proper strategy to compress and protect plasmid DNA in the Pluronic L64-electropulse system for enhanced intramuscular gene delivery

Intramuscular expression of functional proteins is a promising strategy for therapeutic purposes.Previously,we developed an intramuscular gene delivery method by combining Pluronic L64 and optimized electropulse,which is among the most efficient methods to date.However,plasmid DNAs(pDNAs)in this method were not compressed,making them unstable and inefficient in vivo.We considered that a proper compression of pDNAs by an appropriate material should facilitate gene expression in this L64-electropulse system.Here,we reported our finding of such a material,Epigallocatechin gallate(EGCG),a natural compound in green teas,which could compress and protect pDNAs and significantly increase intramuscular gene expression in the L64-electropulse system.Meanwhile,we found that polyethylenimine(PEI)could also slightly improve exogenous gene expression in the optimal procedure.By analysing the characteristic differences between EGCG and PEI,we concluded that negatively charged materials with strong affinity to nucleic acids and/or other properties suitable for gene delivery,such as EGCG,are better alternatives than cationic materials(like PEI)for muscle-based gene delivery.The results revealed that a critical principle for material/pDNA complex benefitting intramuscular gene delivery/expression is to keep the complex negatively charged.This proof-of-concept study displays the breakthrough in compressing pDNAs and provides a principle and strategy to develop more efficient intramuscular gene delivery systems for therapeutic applications.

Effects of Chirality on Gene Delivery Efficiency of Polylysine

Chirality is a key factor in the biological activity of many biomolecules. Poly（L-lysine）（PLL）, a polypeptide synthesized from L-lysine, is one of the mostly used cationic polymers for gene delivery. The effect of chirality of polylysine（PL） on its gene delivery remains unknown. Herein, we prepared three polylysines（PLs） with the similar molecular weight but different backbone chiralities including poly（L-lysine）（PLL）, poly（D-lysine）（PDL） and poly（DL-lysine）（PDLL）. The side chains of each PL were modified with propylene oxide（PO） of different chiralities including（R）PO,（S）PO and（R,S）PO. These PL-POs with distinct chirality in main and side chains could condense p DNA into polyplexes. The polyplexes had approximately the same size, zeta potential and binding ability, but showed distinct gene transfection efficiency. We found that the PLs of L-configuration in the main chain had higher transfection efficiency than that of D or DL configuration due to their faster cellular uptake, while the side chain chirality had no effect on transfection efficiency.

Size-dependent gene delivery of amine-modified silica nanoparticles

Silica-based nanoparticles are promising carriers for gene delivery applications. To gain insights into the effect of particle size on gene transfection efficiency, amine-modified monodisperse St6ber spheres （NH2-SS） with diameters of 125, 230, 330, 440, and 570 nm were synthesized. The in vitro transfection efficiencies of NH2-SS for delivering plasmid DNA encoding green fluorescent protein （GFP） （pcDNA3-EGFP, abbreviated as pcDNA, 6.1 kbp） were studied in HEK293T cells. NH2-SS with a diameter of 330 nm （NH2-SS330） showed the highest GFP transfection level compared to NH2-SS particles with other sizes. The transfection efficiency was found as a compromise between the binding capacity and cellular uptake performance of NH2-SS330 and pcDNA conjugates. NH2-SS330 also demonstrated the highest transfection efficiency for plasmid DNA （pDNA） with a bigger size of 8.9 kbp. To our knowledge, this study is the first to demonstrate the significance of particle size for gene transfection efficiency in silica-based gene delivery systems. Our findings are crucial to the rational design of synthetic vectors for gene therapy.