Promote Gene Transfection Research Articles

Micropattern‐Controlled Cell Density and Its Effect on Gene Transfection of Mesenchymal Stem Cells

AbstractInfluence of cell density on gene transfection and the underlying mechanism remain unclear. In this study, micropatterned surfaces are prepared to control cell density, cell morphology, and cell–cell interactions to investigate how and why cell density affects gene transfection of mesenchymal stem cells. Cell density is controlled by adjusting the ratio of cell adhesive area to nonadhesive area. Gene transfection is dependent on cell density. A moderate cell density is beneficial for gene transfection, while low (2.5 ± 0.7 × 103 cells cm−2) and high (112.6 ± 9.6 × 103 cells cm−2) cell densities have negative effects on transfection. Influence of cell density on gene transfection is strongly correlated with its influence on cellular uptake capacity and DNA synthesis. High cellular uptake and DNA synthesis promote gene transfection. Variations in cell morphology and cell–cell interaction levels of populations with different cell densities could explain the influence of cell density. Large cell size (low cell density) and strong cell–cell interaction (high cell density) could increase gene transfection. Cell protrusion of large cells could slightly promote gene transfection. Balance of these factors is important for maximal gene transfection. The results should be useful for understanding the effect of cell density on gene transfection.

Promoting gene transfection by ROS responsive silicon nanowire arrays.

The development of a fast and safe reactive oxygen species (ROS)-responsive vector is generally limited by the intracellular unstable ROS concentration, and a relatively long time is still needed for the complete intracellular release of drugs or genes induced by ROS. In this work, a gene transfection platform based on ROS-responsive silicon nanowire arrays (SN) is developed, to promote the gene transfection efficiency for several cell lines. Briefly, the surface of the ROS generating system, gold nanoparticle modified SN (SN-Au), is grafted with poly[(2-acryloyl)ethyl(p-boronic acid benzyl)diethylammonium bromide] (B-PDEAEA), an oxidation-responsive charge-reversal cationic polymer. Plasmid DNA (pDNA) bound on the surface through electrostatic interactions was directly delivered into the cells by the time the nanowires penetrate the cells. SN-Au can generate ROS under light treatment, which has an influence on the surface charge change of B-PDEAEA grafted on gold nanoparticles, realizing effective pDNA release in the cytosol for transfection. Nearly 80% of DNA released from the surface of the platform after treated with 1 mM ROS for 10 min. The transfection efficiency of the platform for several cell types was significantly enhanced after a short period of light exposure (3.2-fold for HeLa cells, 7.6-fold for L929 cells, 2.3-fold for BMSC cells and 6.2-fold for mESC cells). The platform also has good biocompatibility. Overall, our results suggest that ROS-responsive SN is a novel, efficient and safe platform for drug and gene transfection.

Microencapsulated Chitosan-Based Nanocapsules: A New Platform for Pulmonary Gene Delivery.

In this work, we propose chitosan (CS)-based nanocapsules (NCs) for pulmonary gene delivery. Hyaluronic acid (HA) was incorporated in the NCs composition (HA/CS NCs) aiming to promote gene transfection in the lung epithelium. NCs were loaded with a model plasmid (pCMV-βGal) to easily evaluate their transfection capacity. The plasmid encapsulation efficiencies were of approx. 90%. To facilitate their administration to the lungs, the plasmid-loaded NCs were microencapsulated in mannitol (Ma) microspheres (MS) using a simple spray-drying technique, obtaining dry powders of adequate properties. In vivo, the MS reached the deep lung, where the plasmid-loaded CS-based NCs were released and transfected the alveolar cells more homogeneously than the control formulation of plasmid directly microencapsulated in Ma MS. The HA-containing formulation achieved the highest transfection efficiency, in a more extended area and more homogeneously distributed than the rest of tested formulations. The new micro-nanostructured platform proposed in this work represents an efficient strategy for the delivery of genetic material to the lung, with great potential for the treatment of genetic lung diseases.

Contrast-Enhanced Ultrasound in the Diagnosis of Ocular Pathology: Application Prospects

Contrast-enhanced ultrasound is a highly informative method for the differential diagnosis of focal changes in various organs and tissues, which allows to visualize vascular structures and obtain new previously unavailable information when using standard b-scan. At the beginning of the 21st century after the development of a new generation of contrast agents consisting of microbubbles with inert gas contrast-enhanced ultrasound became widely used to improve visual assessment of small-caliber vessels in the diagnosis of the abdominal cavity and retroperitoneal space pathology: benign and malignant liver formations, spleen pathology, inflammatory diseases and tumors of the pancreas, defects in renal perfusion, tumor and cystic lesions of the kidneys. Assessment of intraocular tumors microcirculation and diagnosis of vitreoretinal pathology are still two main directions of using contrast-enhanced ultrasound in ophthalmology. In recent years, there has been a growing interest in the use of contrast-enhanced ultrasound in the study of perfusion of different types of uveal melanoma in an animal experiment. A promising direction in the field of genetic engineering is the use of local contrast-enhanced ultrasound exposure on retinal cells (retinal ganglion cells, retinal pigment epithelium) to promote gene transfection (non-viral nucleic acid delivery into the DNA of a cell), which is an important part of gene therapy. Despite the successes achieved in the study of contrast-enhanced ultrasound effectiveness in medicine, further research is needed to increase the informative value of this method in the diagnosis of ophthalmopathology.

Low-frequency ultrasound combined with microbubbles improves gene transfection in prostate cancer cells in vitro and in vivo.

The aim of this study is to explore whether low-frequency ultrasound combined with microbubbles improves pEGFP genes transfection into human prostate cancer cells. Ultrasound with frequency of 80kHz and duty cycle of 50% was adopted in the study; in in vitro experiments, cell lysis, and membrane damage were evaluated after ultrasound exposure; and the membrane continuity and transfection efficiency were observed by transmission electron microscope and laser scanner, respectively. Human prostate cancer xenograft models were exposed to ultrasound and transfection efficiency and histological examination were analyzed. Compared with the control group, ultrasound combined with microbubbles significantly improves gene transfection efficiency (P<.05). In in vitro study, ultrasound combined with microbubbles resulted in cell lysis and the interruption of cell membrane continuity, and its average transfection efficiency was 9.9%; the green fluorescence intensity was 15.2% in the ultrasound combined with microbubbles group in vivo; both values were higher than that in the control group (P<.05). Low-frequency ultrasound combined with microbubbles could be used as a method to promote gene transfection in prostate cancer cells.

Influence of Cell Morphology on Mesenchymal Stem Cell Transfection.

Gene transfection has broad applications in bioengineering and biomedical fields. Although many gene carrier materials and transfection methods have been developed, it remains unclear how cell morphology including cell spreading and elongation affects gene transfection. In this study, human bone marrow-derived mesenchymal stem cells (hMSCs) were cultured on micropatterns and transfected with cationic pAcGFP1-N1 plasmid complexes. The relationship between the cell morphology of hMSCs and gene transfection was investigated using micropatterning techniques. Spreading and elongation of hMSCs were precisely controlled by micropatterned surfaces. The results showed that well-spread and elongated hMSCs had high transfection efficiency. Analysis of the uptake of exogenous genes and DNA synthesis activity indicated that the well-spread and elongated cell morphology promoted gene transfection through enhanced uptake of the cationic complexes and accelerated DNA synthesis. The results should provide useful information for understanding of cell morphology on gene transfection and development of efficient gene transfection methods.

Co-transfection of star-shaped PDMAEMAs enhance transfection efficiency of protamine/pDNA complexes in the presence of serum

In previous studies, we have found that un-complexed free cationic polymers greatly promote gene transfection efficiency of pDNA complexes. Dividing the gene transfection system into two parts, bound chains and free chains, we exploited mixing different biomaterials as the second component of our designed system to improve gene transfection outcome. In this study, we investigated the effectiveness of star-shaped poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) with different arm numbers as free chains (i.e., co-transfection agent) while using natural salmon protamine as bound chains. We explored the gene transfer ability of the system in both serum free and complete cell culture medium. Our results reveal that the star-shaped PDMAEMAs enhance gene transfection efficiency of protamine/pDNA complexes, in the same range and even better than the gold standard polyethyleneimine (PEI, branched type with 25 kg/mol molar mass) in serum-free and serum containing medium conditions, respectively. From 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and Lactate Dehydrogenase (LDH) release data, it was found that the PDMAEMA stars have tolerable cytotoxicity but with significant membrane disruption level, especially when increasing the arm number. Flow cytometry experiments showed that PDMAEMA stars clearly improve the uptake rate as well as the number of protamine/pDNA complexes taken up by the target cells. Using Taqman Real-time Polymerase Chain Reaction (PCR), further exploration of the effect of PDMAEMAs during different gene transfection stages reflects that these un-bound star-shaped polymers mainly act upon the cellular internalization step. Our results indicate that the star-shaped PDMAEMAs are promising vectors for gene delivery.

Pre-Treatment with Either L-Carnitine or Piracetam Increases Ultrasound-Mediated Gene Transfection by Reducing Sonoporation-Associated Apoptosis

Sonoporation, the use of ultrasound to alter the permeability of cell membranes, is a non-viral technique used to facilitate gene delivery, possibly by opening transient pores in the cell membrane. However, sonoporation may have negative bio-effects on cells, such as causing apoptosis, which limits its efficacy in gene delivery. In this study, we investigated whether pre-treatment with either L-carnitine or piracetam could protect cells from undergoing apoptosis after sonoporation and the possible mechanisms. We found that either L-carnitine or piracetam can promote gene transfection without reducing cell viability, possibly by reducing cavitation-induced reactive oxygen species generation, reversing alterations of mitochondrial membrane potential, preventing caspase-3/7 activity and facilitating mitochondrial ATP production. In conclusion, pre-treatment with either L-carnitine or piracetam could protect cells from sonoporation-associated apoptosis by preserving mitochondrial function.

Enhanced gene transfection efficiency by low-dose 25 kDa polyethylenimine by the assistance of 1.8 kDa polyethylenimine

Gene therapy is a promising strategy for treatments of various diseases. Efficient and safe introduction of therapeutic genes into targeted cells is essential to realize functions of the genes. High-molecular-weight polyethylenimines (HMW PEIs) including 25 kDa branched PEI and 22 kDa linear PEI are widely used for in vitro gene transfection. However, high-gene transfection efficiency is usually accompanied with high cytotoxicity, which hampers their further clinical study. On the contrary, low-molecular-weight polyethylenimines (LMW PEIs) such as 1.8 kDa PEI and 800 Da PEI show good biocompatibility but their applications are limited by the poor DNA condensation capability. In this study, we find that 1.8 kDa PEI, but not 800 Da PEI combined with low-dose 25 kDa PEI could significantly promote gene transfection with low cytotoxicity. Plasmids encoding enhanced green fluorescence protein (EGFP) were delivered by the combined PEI and gene transfection efficiency was evaluated by microscopic observation and flow cytometry. Parameters including concentrations of 25 kDa PEI and 1.8 kDa PEI and preparation ways were further optimized. This study presents an efficient and safe combined PEI-based non-viral gene delivery strategy with potential for in vivo applications.

Polyplex Micelles with Phenylboronate/Gluconamide Cross-Linking in the Core Exerting Promoted Gene Transfection through Spatiotemporal Responsivity to Intracellular pH and ATP Concentration.

Polyplexes as gene delivery carriers require integrated functionalities to modulate intracellular trafficking for efficient gene transfection. Herein, we developed plasmid DNA (pDNA)-loaded polyplex micelles (PMs) from poly(ethylene glycol)-based block catiomers derivatized with 4-carboxy-3-fluorophenylboronic acid (FPBA) and d-gluconamide to form pH- and ATP-responsive cross-linking in the core. These PMs exhibited robustness in the extracellular milieu and smooth endosomal escape after cellular uptake, and they facilitated pDNA decondensation triggered by increased ATP concentration inside of the cell. Laser confocal microscopic observation revealed that FPBA installation enhanced the endosomal escapability of the PMs; presumably, this effect resulted from the facilitated endo-/lysosomal membrane disruption triggered by the released block catiomers with hydrophobic FPBA moieties in the side chain from the PM at lower pH condition of endo-/lysosomes. Furthermore, the profile of intracellular pDNA decondensation from the PMs was monitored using Förster resonance energy transfer measurement by flow cytometry; these observations confirmed that PMs optimized for ATP-responsivity exerted effective intracellular decondensation of loaded pDNA to attain promoted gene transfection.

Lipid-Coated Gold Nanoparticles Functionalized by Folic Acid as Gene Vectors for Targeted Gene Delivery in vitro and in vivo.

Lipid-based nanoparticles as gene vectors have attracted considerable attention for their high gene transfection efficiency and low cytotoxicity. In our previous work, we synthesized gold nanoparticles/dimethyldioctadecylammonium bromide (DODAB)/dioleoylphosphatidylethanolamine (DOPE) (GDD) as anionic lipid- and pH-sensitive gene vectors. To further realize targeted gene transfection, a series of gold nanoparticles/DODAB/DOPE/DOPE-folic acid (DOPE-FA) with various ratios of DOPE-FA were prepared and termed as GFn (for which n=1.0, 2.5, 5.0, 7.5, or 10.0 %). The gene transfection efficiency mediated by GF2.5 can reach about 85 % for MCF-7 (FA-receptor-positive cells), higher than those of the negative control (GDD, 35 %) and positive control (Lipofectamine 2000, 65 %). However, GF2.5 does not further promote gene transfection into A549 (FA-receptor-negative cells). The higher gene transfection efficiency for MCF-7 cells can be attributed to enhanced cellular uptake efficiency mediated by the FA targeting ability. Furthermore, GF2.5 was also found to accumulate at the specific tumor site and showed enhanced in vivo gene delivery ability. In addition, no significant harm was observed for the main tissues of the mice after treatment with GF2.5. Therefore, GF2.5, with the targeting ability and improved transfection efficiency, shows promise for its utility in gene therapy for tumor cells that overexpress FA receptors. We believe the results of this study will find more broad applications in gene therapy.

Tetraphenylethylene-based gemini surfactant as nonviral gene delivery system: DNA complexation, gene transfection and cellular tracking

Gemini surfactant (GS) has been proved to be a powerful system for nonviral gene delivery. Herein, a family of tetraphenylethylene (TPE)-based GS possessing different lengths of hydrophobic tails and different linkages between the TPE and [12]aneN3 moieties (1a, 2a, 1b and 2b) were synthesized for facilitating DNA condensation, promoting gene transfection and tracing cellular fate of GS/DNA complex. These GS can readily self-assemble into micelles in aqueous solution with ultralow critical micelle concentrations (CMC, 1.2−1.3μM), and have good pH-/photo-stabilities and strong DNA binding capacities. DNA nanoparticles condensed by the above GSs are in a range of 90−110nm and in the form of spherical morphology showing great stability in the absence and presence of fetal bovine serum. Biological activities including cytotoxicity, cell transfection and cellular internalization were systematically investigated. Resultsrevealed that good biocompatibility (e.g., all the tested cells showed more than 80% of cell viabilities at 30μM) and high transfection efficiency (e.g., 2a/DOPE showed 4.5-fold TE of Lipo2000 in HeLa cell line) were conferred to these GSs. Moreover, the spatiotemporal messages regarding the cellular uptake, location and release of the DNA condensates were clearly captured by the fluorescence changes. Particularly, the impacts of the hydrophobic length and linkage on the physicochemical properties and biological activities were discussed in detail, shedding light on the corresponding structure-activity relationship. The results have demonstrated that TPE-based GSs are very promising as new gene delivery carrier and instructive for rational expansion of novel nonviral gene system.

Low molecular weight polyethylenimine-conjugated gold nanospheres: a platform for selective gene therapy controlled by near-infrared light.

Whether PEI2k-HAuNS could promote gene transfection efficiency controlled by near-infrared (NIR) light. This safe nonviral gene delivery system was obtained by conjugating low molecular weight (2 kDa) polyethylenimine (PEI) onto hollow gold nanospheres (PEI2k-HAuNS). Upon NIR laser irradiation, there was a conspicuous increase both in the in vitro and in vivo transfection achieved by the nanocomplexes. Furthermore, a plasmid encoding the tumor suppressor TP53 (pTP53) was applied to test antitumor activity. The enhanced gene transfection efficiency and therapy of PEI2k-HAuNS were achieved via the mediation of an NIR laser compared with the other treatments in vitro and in vivo. The application of NIR laser irradiated PEI2k-HAuNS can be used as a promising gene delivery systems in vitro and in vivo.

Polyplex Micelles with Double-Protective Compartments of Hydrophilic Shell and Thermoswitchable Palisade of Poly(oxazoline)-Based Block Copolymers for Promoted Gene Transfection.

Improving the stability of polyplex micelles under physiological conditions is a critical issue for promoting gene transfection efficiencies. To this end, hydrophobic palisade was installed between the inner core of packaged plasmid DNA (pDNA) and the hydrophilic shell of polyplex micelles using a triblock copolymer consisting of hydrophilic poly(2-ethyl-2-oxazoline), thermoswitchable amphiphilic poly(2-n-propyl-2-oxazoline) (PnPrOx) and cationic poly(L-lysine). The two-step preparation procedure, mixing the triblock copolymer with pDNA below the lower critical solution temperature (LCST) of PnPrOx, followed by incubation above the LCST to form a hydrophobic palisade of the collapsed PnPrOx segment, induced the formation of spatially aligned hydrophilic-hydrophobic double-protected polyplex micelles. The prepared polyplex micelles exhibited significant tolerance against attacks from nuclease and polyanions compared to those without hydrophobic palisades, thereby promoting gene transfection. These results corroborated the utility of amphiphilic poly(oxazoline) as a molecular thermal switch to improve the stability of polyplex gene carriers relevant for physiological applications.

A controlled release system for simultaneous promotion of gene transfection and antitumor effects

Chemotherapy combining gene therapy may offer new opportunities in the cancer treatments, so co-delivery of drugs and genes become more and more popular. Among these reports, greatest advantage is the comprehensive effects of genes and drugs. However, it is rarely reports that gene transfection and antitumor effect are promoted simultaneously. In this work, the hyperbranched glycoconjugated polymer is modified with the chlorambucil (CHB) and its chemical structures are well analyzed by Fourier transform infrared spectroscopy (FTIR), 1H nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). The biophysical properties are assessed by transmission electron microscope (TEM) and agarose gel electrophoresis (AGE). Confocal laser scanning microscope (CLSM) and flow cytometry have verified the cell internalization between the carriers and cells. In vitro drug release shows that the drug can slowly release from carrier. Antitumor effect of covalent drug is better than the CHB because the polycations can efficient transport drug into the nucleus. Owning to the hydrophobic modification and CHB controlled released from polycations, grafted polymers show high transfection efficiency than hyperbranched glycoconjugated polymers. All profiles demonstrate that a controlled release carrier of drug can promote gene transfection and antitumor effect, giving a new expectation for the best comprehensive treatment of cancer patients.

Revisiting complexation between DNA and polyethylenimine: does the disulfide linkage play a critical role in promoting gene delivery?

Despite its cytotoxicity, polyethylenimine (PEI) is still used as a golden reference in gene transfection. Long PEI chains are more effective but also more cytotoxic. To solve this problem, an alternative strategy is to link short PEI chains into a longer PEI with disulfide bonds because they are degradable in the cell. However, how PEI promotes gene transfection is still unclear. Also, the chain length of PEI is also increased as disulfide bonds are formed. Therefore, it is important to investigate whether the increase in transfection efficiency is attributable to the disulfide linkage, chain size, or both. To distinguish between such factors, a novel method is developed here to make longer linear PEI with disulfide bonds (lPEI(s-s)) by linking the mercapto groups of short linear PEI (lPEI(i)). By comparing the physiochemical properties and the transfection efficiencies of short lPEI(i), long lPEI(s-s), and un-degradable long PEI, it is found that introducing disulfide bonds instead of directly using longer PEI chains has less effect on gene transfection, and it is the chain length that plays a key role in promoting gene transfection.

Polyplex micelles with thermoresponsive heterogeneous coronas for prolonged blood retention and promoted gene transfection.

Adequate retention in blood circulation is a prerequisite for construction of gene delivery carriers for systemic applications. The stability of gene carriers in the bloodstream requires them to effectively resist protein adsorption and maintain small size in the bloodstream avoiding dissociation, aggregation, and nuclease digestion under salty and proteinous medium. Herein, a mixture of two block catiomers consisting of the same cationic block, poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PAsp(DET)), but varying shell-forming blocks, poly[2-(2-methoxyethoxy) ethyl methacrylate] (PMEO2MA), and poly[oligo(ethylene glycol) methyl ether methacrylate] (POEGMA), was used to complex with plasmid DNA (pDNA) to fabricate polyplex micelles with mixed shells (MPMs) at 20 °C. The thermoresponsive property of PMEO2MA allows distinct phase transition from hydrophilic to hydrophobic by increasing incubation temperature from 20 to 37 °C, which results in a distinct heterogeneous corona containing hydrophilic and hydrophobic regions at the surface of the MPMs. Subsequent study verified that this transition promoted further condensation of pDNA, thereby giving rise to improved complex and colloidal stability. The proposed system has shown remarkable stability in salty and proteinous solution and superior tolerance to nuclease degradation. As compared with polyplex micelles formed from single POEGMA-b-PAsp(DET) block copolymer, in vivo circulation experiments in the bloodstream further confirmed that the retention time of MPMs was prolonged significantly. Moreover, the proposed system exhibited remarkably high cell transfection activity especially at low N/P ratios and negligible cytotoxicity and thus portends promising utility for systemic gene therapy applications.

Synergistic effect of a biosurfactant and protamine on gene transfection efficiency

Several barriers need to be overcome to ensure successful gene transfection, including passing of the foreign gene through the plasma membrane, escape of this material from lysosomal degradation, and its translocation into the nucleus. We previously showed that the biosurfactant mannosylerythritol lipid-A (MEL-A) enhanced the efficiency of gene transfection mediated by cationic liposomes by facilitating rapid delivery of foreign genes into target cells through membrane fusion between liposomes and the plasma membrane. Moreover, using MEL-A-containing cationic liposomes, the foreign gene was efficiently delivered into the nucleus because it was released directly into the cytosol and thus escaped lysosomal degradation. Here we investigated the effect of pre-condensation of plasmid DNA by a cationic polymer, protamine, on gene transfection. We found that the efficiency of pre-condensed DNA transfection mediated by MEL-A-containing OH liposomes was >10 times higher than that of non-condensed DNA transfection. In contrast, the efficiency of pre-condensed DNA transfection mediated by OH liposomes was only 1.5 times higher than that of non-condensed DNA transfection. MEL-A did not influence plasmid DNA encapsulation by cationic liposomes, but it greatly accelerated the nuclear delivery of pre-condensed plasmid DNA. Our findings indicate that MEL-A and protamine synergistically accelerate the nuclear delivery of foreign gene and consequently promote gene transfection efficiency.

Progress and perspectives in developing polymeric vectors for in vitro gene delivery

The development of safe, efficient and controllable gene-delivery vectors has become a bottleneck to human gene therapy. Synthetic polymeric vectors, although safer than viral carriers, generally do not possess the required efficacy, apparently due to a lack of functionality to overcome at least one of many intracellular gene-delivery obstacles. Currently, the exact mechanisms of how these polymeric vectors navigate each intracellular obstacle ("slit"), as well as their particular physical/chemical properties that contribute to efficient intracellular trafficking remain largely unknown, making it rather difficult to further improve the efficacy of non-viral polymeric vectors in vitro and in vivo. In this review, we first give a brief overview of synthetic polymeric vectors that have been designed and developed for gene delivery and highlight some promising candidates for clinical applications. Our main focus is on discussing the intracellular trafficking mechanisms of the DNA-polymer complexes ("polyplexes"), with less effort on the DNA-polymer complexation in the extracellular space as well as the in vivo systemic administration of genes in animal models and human clinical trials. In particular, we identified and discussed four critical, but often over-looked issues for successful DNA-polymer intracellular trafficking, especially our recent confirmation that it is free cationic polymer chains in the solution mixture of DNA and polymer that actually promote gene transfection and the polycationic chains within the polyplexes mainly play a protective role. Instead of the previously proposed and widely used escape model from late endolysosomes, the current hypothesis is that free polycationic chains with a sufficient length (∼20 nm) can block the initial endocytic-vesicle-to-endolysosome pathway.

Homo-catiomer integration into PEGylated polyplex micelle from block-catiomer for systemic anti-angiogenic gene therapy for fibrotic pancreatic tumors

Homo-poly{N′-[N-(2-aminoethyl)-2-aminoehtyl]aspartamide} [PAsp(DET), H] was attempted to integrate into poly (ethylene glycol) (PEG)-b-PAsp(DET)] (B) formulated polyplex micelle with the aim of enhancing cell transfection efficiency for PEGylated polyplex micelle via H integration. In vitro evaluations verified H integration of potent stimulation in enhancing cell-transfecting activity of PEGylated polyplex micelles via promoted cellular uptake and facilitated endosome escape. In vivo anti-angiogenic tumor suppression evaluations validated the feasibility of H integration in promoting gene transfection to the affected cells via systemic administration, where loaded anti-angiogenic gene remarkably expressed in the tumor site, thereby imparting significant inhibitory effect on the growth of vascular endothelial cells, ultimately leading to potent tumor growth suppression. These results demonstrated potency of H integration for enhanced transfection activity and potential usage in systemic applications, which could have important implications on the strategic use of H integration in the non-viral gene carrier design.