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.

Arginine functionalized hydroxyapatite nanoparticles and its bioactivity for gene delivery

In order to further improve the transfection efficiency of hydroxyapatite nanoparticle （HAp）, arginine functionalized hydroxyapatite （HAp/Arg） was synthesized by hydrothermal synthesis. The morphology, crystallite size and zeta potential of the HAp/Arg were characterized by transmission electron microscopy （TEM）, atomic force microscopy （AFM） and zeta potential analyzer. The loading and protecting properties of HAp/Arg to DNA were tested by electrophoresis. Its cytotoxicity was also measured in Hela cells and HAEC cells by MTT and LDH, and its transfection efficiency was examined by fluorescence microscope and flow cytometry. The results reveal that HAp/Arg is short rod-like and nano single crystal, the mean diameter is 50-90 nm and zeta potential is 35.8 mV at pH 7.4. HAp/Arg to DNA can be condensed by electrostatic effect and protect DNA against degradation in DNase I, and shows high transfection efficiency without cytotoxicity. These results suggest that HAp/Arg can be a promising alternative as a novel gene delivery system.

A novel gene delivery system targeting cells expressing VEGF receptors

Two ligand oligopeptides GV1 and GV2 were designed according to the putative binding region of VEGF to its receptors. GV1, GV2 and endosome releasing oligopeptide HA20 were conjugated with poly-L-lysine or protamine and the resulting conjugates could interact with DNA in a noncovalent bond to form a complex. Using pSV2-β-galactosidase as a reporter gene, it has been demonstrated that exogenous gene was transferred into bovine aortic arch-derived endothelial cells (ABAE) andhuman malignant melanoma cell lines (A375) in vitro. In vivo experiments, exogenous gene was transferred into tumor vascular endothelial cells and tumor cells of subcutaneously transplanted human colon cancer LOVO, human malignant melanoma A375 and human hepatoma graft in nude mice. This system could also target gene to intrahepatically transplanted human hepatoma injected via portal vein in nude mice. These results are correlated with theGene delivery system targeting VEGF receptors relevant receptors (flt-1, flk-1/KDR) expression on the targeted cells and tissues.

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.

Establishing Gene Delivery Systems Based on Small-Sized Chitosan Nanoparticles

Chitosan is a natural cationic polysaccharide, which is often used for preparing biomedical materials because of its high biocompatibility. In this study, chitosan with a molecular weight of 160 kDa was chosen to prepare chitosan nanoparticles （CSNPs） as gene vectors by ionic cross-linking with tripolyphosphate （TPP）. CSNPs were characterized in terms of particle size, zeta potential, and polydispersity index （PDI） using a Zetasizer, and morphology was evaluated by transmission electron microscopy （TEM）. Furthermore, the cytotoxicity and biocompatibility of CSNPs were correspondingly examined by a 3-（4,5-dimethylthiazol-2-yl）-2,5- diphenyltetrazolium bromide （MTT） assay and histological examination. Agarose gel electrophoresis and UV spectrophotometric methods were performed to measure the loading capacity. The cell transfection efficiency of CSNPs loaded with plasmids or siRNA was analyzed by fluorescence microscopy or laser scanning confocal microscopy. The results showed that CSNPs were prepared successfully by the ionic gelation method, which had a smaller partcticle size （100 nm-200 nm）, stable dispersibility, low cytotoxicity, good tissue-biocompatibility, and high gene-loading efficiency. These CSNPs could transfer the plasmids or siRNA to cells. However, CSNPs might have a much higher transfection efficiency for siRNAs than for plasmids, which implies that CSNPs might be a safer and more efficient vector for delivering siRNAs rather than plasmids.

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.

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.

A Novel Approach of Low-frequency Ultrasonic Naked Plasmid Gene Delivery and Its Assessment

Objective To deliver the naked genes into cells through the bioeffects of cell membrane porous produced by low-frequency ultrasound (US) and to investigate the safety by determining the threshold of cell damage and membrane permeability. Methods The suspension of red cells from chickens, rabbits, rats, and S180 cells was exposed to calibrated US field with different parameters in still and flowing state. Laser scanning confocal microscopy, fluorescent microscopy, scanning electron microscopy, flow cytometry and spectrophotometry were used to examine cell morphology, membrane permeability, enzymes, free radicals, naked gene expression efficiency, threshold of cell damage and cell viability. Results The plasmid of green fluorescent protein (GFP) as a reporter gene was delivered into S180 cells under optimal conditions without cell damage and cytotoxicity. The transfection rate was (35.83±2.53)% (n=6) in viable cells, and the cell viability was (90.17±1.47)% (n=6). Also, malondialdehyde, hydroxyl free radical, alkaline phosphatase, and acid phosphatase showed a S-shaped growth model (r=0.98±0.01) in response to the permeability change and alteration of cell morphology. The constant E of energy accumulation in US delivery at 90% cell viability was an optimal control factor, and at 80% cell viability was the damage threshold. Conclusion US under optimal conditions is a versatile gene therapy tool. The intensity of GFP expression in US group has a higher fluorescent peak than that in AVV-GFP group and control group (P<0.001). The optimal gene uptakes, expression of gene and safety depend on E, which can be applied to control gene delivery efficiency in combination with other parameters. The results are helpful for development of a novel clinical naked gene therapeutic system and non-hyperthermia cancer therapeutic system.

Receptor-mediated gene delivery using polyethylenimine (PEI) coupled with polypeptides targeting FGF receptors on cells surface

Objective: To construct a novel kind of nonviral gene delivery vector based on polyethylenimine (PEI) conjugated with polypeptides derived from ligand FGF with high transfection efficiency and according to tumor targeting ability. Methods: The synthetic polypeptides CR16 for binding FGF receptors was conjugated to PEI and the characters of the polypeptides in-cluding DNA condensing and particle size were determined. Enhanced efficiency and the targeting specificity of the synthesized vector were investigated in vitro and in vivo. Results: The polypeptides were successfully coupled to PEI. The new vectors PEI-CR16 could efficiently condense pDNA into particles with around 200 nm diameter. The PEI-CR16/pDNA polyplexes showed significantly greater transgene activity than PEI/pDNA in FGF receptors positive tumor cells in vitro and in vivo gene transfer, while no difference was observed in FGF receptors negative tumor cells. The enhanced transfection efficiency of PEI-CR16 could be blocked by excess free polypeptides. Conclusion: The synthesized vector could improve the efficiency of gene transfer and targeting specificity in FGF receptors positive cells. The vector had good prospect for use in cancer 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.

Gaussia Luciferase Reporter Assay for Assessment of Gene Delivery Systems in Vivo

Objective To develop an alternative method for assessment of gene delivery systems in vivo.Methods Mouse primary spleen lymphocytes were genetically modified in vitro by a retroviral vector harboring a Gaussia luciferase(Gluc) expression cassette.After implantation of these cells into recipient mice,the expression of Gluc was detected in whole blood or plasma collected.Results As little as 10 μL whole blood drawn from the recipient mice could guarantee prompt reading of Gluc activity with a luminometer.And the reading was found in good correlation with the number of genetically modified spleen lymphocytes implanted to the mice.Conclusions Gluc may be useful as an in vivo reporter for gene therapy researches,and Gluc blood assay could provide an alternative method for assessment of gene delivery systems in vivo.

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

Chitosan DNA nanoparticles for oral gene delivery

Gene therapy is a promising technology with potential applications in the treatment of medical conditions, both congenital and acquired. Despite its label as breakthrough technology for the 21st century, the simple concept of gene therapy - the introduction of a functional copy of desired genes in affected individuals - is proving to be more challenging than expected. Oral gene delivery has shown intriguing results and warrants further exploration. In particular, oral administration of chitosan DNA nano-particles, one the most commonly used formulations of therapeutic DNA, has repeatedly demonstrated successful in vitro and in vivo gene transfection. While oral gene therapy has shown immense promise as treatment options in a variety of diseases, there are still signifcant barriers to overcome before it can be considered for clinical applications. In this review we provide an over-view of the physiologic challenges facing the use of chitosan DNA nanoparticles for oral gene delivery at both the extracellular and intracellular level. From administration at the oral cavity, chitosan nanoparticles must traverse the gastrointestinal tract and protect its DNA contents from signifcant jumps in pH levels, various intestinal digestive enzymes, thick mucus layers with high turnover, and a proteinaceous glycocalyx meshwork. Once these extracellular barriers are overcome, chitosan DNA nanoparticles must enter intestinal cells, escape endolysosomes, and disassociate from genetic material at the appropriate time allowing transport of genetic material into the nucleus to deliver a therapeutic ef-fect. The properties of chitosan nanoparticles and modified nanoparticles are discussed in this review. An understanding of the barriers to oral gene delivery and how to overcome them would be invaluable for future gene therapy development.

Enhanced pyroptosis induction with pore-forming gene delivery for osteosarcoma microenvironment reshaping

Osteosarcoma is the most common malignant bone tumor without efficient management for improving 5-year event-free survival.Immunotherapy is also limited due to its highly immunosuppressive tumor microenvironment(TME).Pore-forming gasdermins(GSDMs)-mediated pyroptosis has gained increasing concern in reshaping TME,however,the expressions and relationships of GSDMs with osteosarcoma remain unclear.Herein,gasdermin E(GSDME)expression is found to be positively correlated with the prognosis and immune infiltration of osteosarcoma patients,and low GSDME expression was observed.A vector termed as LPAD contains abundant hydroxyl groups for hydrating layer formation was then prepared to deliver the GSDME gene to upregulate protein expression in osteosarcoma for efficient TME reshaping via enhanced pyroptosis induction.Atomistic molecular dynamics simulations analysis proved that the hydroxyl groups increased LPAD hydration abilities by enhancing coulombic interaction.The upregulated GSDME expression together with cleaved caspase-3 provided impressive pyroptosis induction.The pyroptosis further initiated proinflammatory cytokines release,increased immune cell infiltration,activated adaptive immune responses and create a favorable immunogenic hot TME.The study not only confirms the role of GSDME in the immune infiltration and prognosis of osteosarcoma,but also provides a promising strategy for the inhibition of osteosarcoma by pore-forming GSDME gene delivery induced enhanced pyroptosis to reshape the TME of osteosarcoma.

Modulating the Oxidation Degree of Linear Polyethyleneimine for Preparation of Highly Efficient and Low-cytotoxicity Degradable Gene Delivery Carriers

Polyethyleneimine(PEI),as a widely used polymer material in the field of gene delivery,has been extensively studied for modification and shielding to reduce its cytotoxicity.However,research aimed at preparing degradable PEI is scarce.In this work,the hydrogen peroxide(H_(2)O_(2))oxidation method was used to introduce degradable amide groups in the PEI and a series of oxidized PEI22k(oxPEI22k)with different degrees of oxidation were synthesized by regulating the dosage of H_(2)O_(2).The relationship between the oxidation degree of oxPEI22k and the gene transfection efficiency of oxPEI22k was studied in detail,confirming that the oxPEI22k with oxidation degrees of 16.7%and 28.6%achieved improved transfection efficiency compared to unmodified PEI.These oxPEI22k also proved reduced cytotoxicity and improved degradability.Further,this strategy was extended to the synthesis of low-molecular-weight oxPEI1.8k.The oxPEI1.8k with suitable oxidation degree also achieved improved transfection efficiency and reduced cytotoxicity.In brief,this work provided high-efficiency and low-cytotoxicity degradable gene delivery carriers by regulating the oxidation degree of PEI,which was of great significance for promoting clinical applications of PEI.

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.