Non-viral targeted delivery system mediates transfection of thymidine kinase gene of herpes simplex virus into ovarian cancer cells:a comparison between one time and continuous mediation

Objective： To compare the transferring efficiency and killing effects of one time and continuous mediation with GE7, a non-viral targeted delivery system, in transfection of thymidine kinase gene of herpes simplex virus （HSV-tk） into ovarian cancer cells. Methods： GE7 was used to prepare recombinants with β-galactosidase （β-gal） and HSVI-tk; the recombinants were then used to transfect human ovarian cancer line CaOV3 once and continuously. β-gal staining was used to compare the efficiencies of one time and continuous mediation with GE7 system. Ganciclovior （GCV） was introduced into HSVI-tk transfected ovarian cells. Through drawing the cell growth curve and flow cytometry, the killing effects of GCV on once and continuously GE7/HSVI-tk transfected cells were observed. Results： We found that the one time and continuous exogenous gene transfer efficiencies were about 80% and 85%, respectively. When 1 μg/mL GCV was used to treat ovarian cell transfected with HSVI-tk gene, growth inhibiting rates of ovarian cells of one time and continuous transferring were 82% and 90%, respectively; their apoptosis indices were 15 and 30, respectively. Under same GCV concentration, continuous mediation of GE7/pCMV-tk transfection into ovarian cancer cells had more significant inhibitory effect than one time mediation （P 〈 0.05）. Conclusion： Compared with one time mediation, continuous mediation of transfection with GE7 gene delivery system has higher efficiency. Continuous mediation of GE7/HSVI-tk/GCV therapeutic gene system has more powerful killing effect.

Ex vivo non-viral vector-mediated neurotrophin-3 gene transfer to olfactory ensheathing glia: effects on axonal regeneration and functional recovery after implantation in rats with spinal cord injury

Objective Combine olfactory ensheathing glia （OEG） implantation with ex vivo non-viral vector-based neurotrophin- 3 （NT-3） gene therapy in attempting to enhance regeneration after thoracic spinal cord injury （SCI）. Methods Primary OEG were transfected with cationic liposome-mediated recombinant plasmid pcDNA3.1 （＋）-NT3 and subsequently implanted into adult Wistar rats directly after the thoracic spinal cord （T9） contusion by the New York University impactor. The animals in 3 different groups received 4x 1050EG transfected with pcDNA3.1 （＋）-NT3 or pcDNA3.1 （＋） plasmids, or the OEGs without any plasmid transfection, respectively; the fourth group was untreated group, in which no OEG was implanted. Results NT-3 production was seen increased both ex vivo and in vivo in pcDNA3.1 （＋）-NT3 transfected OEGs. Three months after implantation of NT-3-transfected OEGs, behavioral analysis revealed that the hindlimb function of SCI rats was improved. All spinal cords were filled with regenerated neurofilament-positive axons. Retrograde tracing revealed enhanced regenerative axonal sprouting. Conclusion Non-viral vector-mediated genetic engineering of OEG was safe and more effective in producing NT- 3 and promoting axonal outgrowth followed by enhancing SCI recovery in rats.

Non-viral gene delivery carrier of probe ty[pe host molecule--Interactions between DNA and β-cyclodextrin derivative complexes(I)

A host type non-virus gene delivery car- rier, phenanthroline-β-cyclodextrin derivative host molecule, was produced which can be used as mo- lecular probe. Interactions between DZY-1 and DNA were investigated by electrophoresis assay. Hind III enzyme inhibition assay was carried out using DNA condensates induced by host molecules or host- guest molecule complexes to explore their ability to inhibit enzyme digestion. Micro-structure of DNA condensates induced by host molecules and host-guest molecule complexes was observed by scanning electron microscope (SEM). Our work in- dicates the delivery mechanism of DZY-1 used as a gene delivery carrier and also provides a method to design and produce non-virus gene delivery carriers.

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.

Ionizable drug delivery systems for efficient and selective gene therapy

Gene therapy has shown great potential to treat various diseases by repairing the abnormal gene function.However,a great challenge in bringing the nucleic acid formulations to the market is the safe and effective delivery to the specific tissues and cells.To be excited,the development of ionizable drug delivery systems(IDDSs)has promoted a great breakthrough as evidenced by the approval of the BNT162b2 vaccine for prevention of coronavirus disease 2019(COVID-19)in 2021.Compared with conventional cationic gene vectors,IDDSs can decrease the toxicity of carriers to cell membranes,and increase cellular uptake and endosomal escape of nucleic acids by their unique pH-responsive structures.Despite the progress,there remain necessary requirements for designing more efficient IDDSs for precise gene therapy.Herein,we systematically classify the IDDSs and summarize the characteristics and advantages of IDDSs in order to explore the underlying design mechanisms.The delivery mechanisms and therapeutic applications of IDDSs are comprehensively reviewed for the delivery of plasmid DNA(pDNA)and four kinds of RNA.In particular,organ selecting considerations and high-throughput screening are highlighted to explore efficiently multifunctional ionizable nanomaterials with superior gene delivery capacity.We anticipate providing references for researchers to rationally design more efficient and accurate targeted gene delivery systems in the future,and indicate ideas for developing next generation gene vectors.

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.

Surface Modification of Biomimetic PLGA-(ASP-PEG) Matrix with RGD-Containing Peptide:a New Non-Viral Vector for Gene Transfer and Tissue Engineering

RGD-containing peptide （ K16-GRGDSPC） , characterized as non-viral gene vectors, was fabricated to modify the surface of PLGA-[ASP- PEG] matrix, which offered the foundation for gene transfer with porous matrix of gene activated later. Peptide was synthesized and matrix was executed into chips A, B and chip C. Chip C was regarded as control. Chips A and B were reacted with cross-linker. Then chip A was reacted with peptide. MS and HPLC were ased to detect the .14W and purity of peptide. Sulphur, existing on the surface of biomaterials, was detected by XPS. The purity of un-reacted peptide in residual solution was detected by a spectrophotometer. HPLC shows that the peptide purity was 94%- 95% , and MS shows that the MW was 2 741. 3307. XPS reveals that the binding energy of sulphur was 164 eV and the ratio of carbon to sulphur （C/S） was 99. 746 ：0. 1014 in reacted chip A. The binding energy of sulphur in reacted chip B was 164 eV and 162 eV, C/ S was 99.574：0.4255, aM there was no sulphur in chip C. Peptide was manufactured and linked to the surface of biomimetic and 3-D matrix, which offered the possibilities for gene transfer and tissue engineering with this new kind of non-viral gene vector.

Localized,non-viral delivery of nucleic acids:Opportunities,challenges and current strategies

Localized delivery of drugs is an emerging field both with regards to drug delivery during disease as well as in tissue engineering.Despite significant achievements made in the last decades,the efficient delivery of proteins and peptides remains challenging,especially in cases requiring long-term release of proteins after application.The localized delivery of nucleic acids(NA)represents an interesting alternative due to higher physicochemical stability of NA,increased efficiency by harnessing cells as bioreactors for the production of required proteins and improved versatility with regards to expression of specific proteins through plasmid DNA or repression of gene products through siRNA.However,unlike most proteins and peptides,NA must be delivered to the cytoplasm or nucleus to be efficacious,resulting in significant delivery challenges.We herein describe frequently used non-viral vectors for the delivery of NA including polyplexes,lipoplexes and lipopolyplexes and summarize recent developments in the field of nucleic acid delivery systems for local application based on hydrogels,solid scaffolds and physical delivery methods.The challenges associated with the different approaches are identified and options to address these challenges are discussed.

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.

Non-viral gene coating modified IOL delivering PDGFR-αshRNA interferes with the fibrogenic process to prevent posterior capsular opacification

Posterior capsule opacification(PCO),the most common complication after cataract surgery,is caused by the proliferation,migration and epithelial-mesenchymal transition(EMT)of residual lens epithelial cells in the capsule bag.Although the surface modification and drug loading of intraocular lens(IOLs)have been effective in preventing PCO to some extent,the intraocular safety of anti-proliferative drug application is still a major limitation in clinical application.In this study,we used non-viral gene delivery systems in combination with layer-by-layer(LBL)self-assembly technology,and the modified IOL could effectively prevent the development of PCO by interfering with the EMT process mediated by the platelet-derived growth factor receptor-α(PDGFR-α).Herein,the gene fragments were wrapped by electrostatic conjugation using polyethyleneimine-graft-poly(ethylene glycol)to form gene complexes.Gene complexes were characterized by dynamic light scattering,transmission electron microscopy(TEM)and agarose gel electrophoresis,and evaluated for storage and serum stability.The layer assembly behavior of the IOL surface,changes in optical properties and the release behavior of the gene complexes were characterized using quartz crystal microbalance,UV-vis,contact angle and TEM.In vitro experiments showed that the IOL coating has good bio-compatibility and can achieve the corresponding transfection effect,and the released gene complexes exhibited excellent cell internalization and lysosomal escape behaviors,as well as effective inhibition of PDGFR-αexpression and its mediated EMT process.The early PCO prevention effect and bio-compatibility evaluation of the modified IOL in vivo were evaluated by implantation into animal eyes.This study provides a new strategy for the development of surface modifications of small nucleic acid drugs and non-toxic EMT interference therapies for PCO.

Fusion Wheat Histone H4 Protein Increases Transfection Efficiency of Non-viral DNA Vector

The lack of efficient and non-toxic gene delivery, preferably with non-viral DNA vectors, is generally regarded as a major limitation for gene therapy. In this study, a wheat histone H4 gene was cloned from Triticum aestivum, sequenced, modified and expressed in E. coli. The wheat histone H4 gene and reconstructed H4TL gene encoded wheat histone H4 and a recombinant protein of 141 amino acids with an approximate molecular weight of 15500. Gel electrophoresis mobility shift assays demonstrated that the purified protein had high affinity for DNA. Most significantly, the complex of plasmid pEGFP/C1 with H4TL was transfected with increased efficiency into MCF-7, HO8910, LNCap, A549 and HeLa cells in vitro. These results demonstrate that the targeting of non-viral vectors to tumor-specific receptors provides a cheap, simple and highly efficient tool for gene delivery.

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.

Applications and developments of gene therapy drug delivery systems for genetic diseases

Genetic diseases seriously threaten human health and have always been one of the refractory conditions facing humanity.Currently,gene therapy drugs such as siRNA,shRNA,antisense oligonucleotide,CRISPR/Cas9 system,plasmid DNA and miRNA have shown great potential in biomedical applications.To avoid the degradation of gene therapy drugs in the body and effectively deliver them to target tissues,cells and organelles,the development of excellent drug delivery vehicles is of utmost importance.Viral vectors are the most widely used delivery vehicles for gene therapy in vivo and in vitro due to their high transfection efficiency and stable transgene expression.With the development of nanotechnology,novel nanocarriers are gradually replacing viral vectors,emerging superior performance.This review mainly illuminates the current widely used gene therapy drugs,summarizes the viral vectors and non-viral vectors that deliver gene therapy drugs,and sums up the application of gene therapy to treat genetic diseases.Additionally,the challenges and opportunities of the field are discussed from the perspective of developing an effective nano-delivery system.

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.

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.

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.

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.

Synthesis and Evaluation of Novel Chitosan Derivatives for Gene Delivery

A series of novel water soluble chitosan derivatives as gene vectors was synthesized. The delivery systems were tested for their ability to form complexes with plasmid DNA by utilizing agarose gel electrophoresis, particle size analysis, zeta potential measurement and scanning electron microscopy. Furthermore, cytotoxicity of chitosan derivatives and transfection efficiency of polyplexes were evaluated in vitro. The experimental results showed that the novel chitosan derivatives had lower cytotoxicity, good DNA condensation, and higher transfection efficiencies compared to chitosan in both 293T and HeLa cell lines. It was indicated that these chitosan derivatives were promising candidates for non-viral gene vectors.

Effects of lipid shell microbubble on ultrasound mediated EGFP gene delivery to transplanted tumors:initial experience

Objective： To investigate the feasibility of ultrasound （US） mediated enhanced green fluorescent protein （EGFP） gene delivery in subcutaneous transplanted tumors of human cervical carcinoma （He/a） and the contribution of lipid shell microbubble （LSMB） on gene transfection. Methods： LSMB and plasmid were injected into nude mice by tail vein followed local US irradiation （P ＋ LSMB ＋ US group）. US exposure parameter was set at 2.0 W/cm2, 2 rain, duty cycle 20%. EGFP expression was evaluated by imaging for 7 days. Nude mice undergoing plasmid injection alone （P group）, plasmid injection and US exposure （P ＋ US group）, plasmid and LSMB injection （P ＋ LSMB group） were used as controls. Frozen section and histological examinations were conducted. Expression of EGFP was scored. Kinetics of protein expression post transfection and localization in vivo were evaluated. Results： Plasmid injection with LSMB plus US exposure strongly increased gene transfer efficiency. Strong EGFP expression was mainly seen in LSMB ＋ P ＋ US group. It was significantly higher than any of the following groups, P group, US ＋ P group, or LSMB ＋ P group （P 〈 0.01）./n vivo expression level of post-US 3 days was significantly higher than any other time points （P 〈 0.01）. There was not significant expression level of EGFP in other organs or tissues regardless of US exposure. No tissue damage was seen histologically. Conclusion： The combination of LSMB and US exposure could effectively transfer plasmid DNA to transplanted tumors without causing any apparently adverse effect. LSMB could be effective as a non-viral vector system in in vivo gene delivery. It would be a safe gene delivery method and provide an alternative to current clinical gene therapy.