Efficient DNA Delivery Research Articles

Optimizing Transfection Efficiency of Spermine Polar Head Cholesterol-Based Cationic Lipids with Amino Acid Linker.

In this work, a series of spermine polar head cholesterol-based cationic lipids with various amino acid spacers were synthesized and evaluated as non-viral gene delivery systems. The physicochemical properties of the resulting lipoplexes, formed from these lipids and DOPE, were assessed, including zeta-potential, DNA binding and DNA protection from serum. Transfection efficiency and cytotoxicity were examined under serum-free and 10-40 % serum-containing conditions. The results showed that the physicochemical properties of cationic lipids, both with and without amino acid spacers, were not significantly different. Cationic liposomes composed of lipid Sper-Ahx-Chol, which has a 6-aminohexanoic acid spacer, and DOPE exhibited greater transfection efficiency in HeLa cells compared to Lipofectamine3000, both in the absence and presence of 10-40 % serum. Additionally, lipid Sper-His-Chol with a histidine spacer and Sper-Ahx-Chol showed higher efficiency than Lipofectamine3000 against HEK293T under 40 % serum conditions. These results suggest that the incorporation of amino acids into the cationic lipids can significantly enhance their DNA delivery efficiency. Specifically, certain amino acid modifications improved transfection efficiency while maintaining low cytotoxicity. Our findings highlight the potential of amino acid-tailored cationic lipids as promising vectors for enhanced DNA delivery.

Efficient Polymeric Nanoparticle Gene Delivery Enabled Via Tri- and Tetrafunctional Branching.

Poly(β-amino ester) (PBAE) nanoparticles (NPs) show great promise for nonviral gene delivery. Recent studies suggest branched PBAEs (BPBAEs) offer advantages over linear counterparts, but the effect of polymer structure has not been well investigated across many chemical constituents. Here, a library of BPBAEs was synthesized with tri- and tetrafunctional branching. These polymers self-assemble with DNA to form highly cationic, monodisperse NPs with notably small size (∼50 nm). Optimal transfection occurred with polymer structures that featured moderate PBAE branching, enabling complete DNA encapsulation, rapid NP uptake, and robust expression at low DNA doses and polymer amounts. Optimized NPs enabled efficient DNA delivery to diverse cell types in vitro while maintaining high cellular viability, demonstrating significant improvements over a well-performing linear PBAE counterpart. BPBAEs also facilitated efficient mRNA and siRNA delivery, highlighting the versatility of these structures and demonstrating the broad utility of BPBAE NPs as vectors for nucleic acid delivery.

Utilization of chitosan nanocomposites loaded with quantum dots enables efficient and traceable DNA delivery

Chitosan is widely employed in gene carriers due to its excellent gene loading capacity, ease of modification, and exceptional biodegradability. However, low gene delivery efficiency, high cytotoxicity, and lack of tracer biomimetic properties limit its clinical use. To address these issues, a novel biomimetic tracking gene delivery carrier, RBCm-C50kQT, was constructed by using the design scheme of cell membrane coated carbon quantum dots/chitosan. This carrier improves stability and tracking performance while embedding the cell membrane enhances biosafety. RBCm-C50kQT effectively carries and protects DNA, improving uptake and transfection efficiency with reduced cytotoxicity. It maintains strong fluorescence tracking and shows high uptake efficiencies of 83.62 % and 77.45 % in 293 T and HeLa cells, respectively, with maximum transfection efficiencies of 68.80 % and 45.47 %. This advancement supports gene therapy improvements and paves the way for future clinical applications.

Potential applications of PEI loaded graphene oxide quantum dots in safe and efficient DNA delivery

In order to improve the safety and efficacy of gene delivery using polyethyleneimine (PEI), this work focused on developing PEI quantum dots and incorporating them onto graphene to improve gene loading capacity. The successful creation of graphene-polyethyleneimine quantum dots (GPQDs) was confirmed through fluorescence emission spectrum and infrared spectroscopy. Both polyethyleneimine quantum dots (PQDs) and GPQDs exhibited blue fluorescence when excited between 310 and 430 nm, indicating that they retained the fluorescence characteristics of broad excitation and narrow emission. Furthermore, GPQDs displayed smaller particle sizes, increased hydrophilicity, and better dispersibility compared to PQDs. GPQDs were able to efficiently load DNA with lower cytotoxicity than PQDs and PEI. The enhanced fluorescence properties of GPQDs allowed for the observation that GPQDs/DNA complexes were effectively internalized by cells with higher uptake efficiency than PEI/DNA and PQDs/DNA complexes. The study demonstrated that GPQDs/DNA complexes successfully transfected 293T and HeLa cells, with GPQDs prepared using PEI25k showing improved cell uptake and transfection efficiency compared to those prepared with PEI10k. Overall, the findings suggest that GPQDs are a more effective and safer gene delivery vector than PEI, with promising potential for future gene delivery applications.

An Amphiphilic Cell-Penetrating Macrocycle for Efficient Cytosolic Delivery of Proteins, DNA, and CRISPR Cas9.

The discovery of safe platforms that can circumvent the endocytic pathway is of great significance for biological therapeutics that are usually degraded during endocytosis. Here we show that a self-assembled and dynamic macrocycle can passively diffuse through the cell membrane and deliver a broad range of biologics, including proteins, CRISPR Cas9, and ssDNA, directly to the cytosol while retaining their bioactivity. Cell-penetrating macrocycle CPM can be easily prepared from the room temperature condensation of diketopyrrolopyrrole lactams with diamines. We attribute the high cellular permeability of CPM to its amphiphilic nature and chameleonic properties. It adopts conformations that partially bury polar groups and expose hydrophobic side chains, thus self-assembling into micellar-like structures. Its superior fluorescence makes CPM trackable inside cells where it follows the endomembrane system. CPM outperformed commercial reagents for biologics delivery and showed high RNA knockdown efficiency of CRISPR Cas9. We envisage that this macrocycle will be an ideal starting point to design and synthesize biomimetic macrocyclic tags that can readily facilitate the interaction and uptake of biomolecules and overcome endosomal digestion.

Comparative analysis of lipid-peptide nanoparticles prepared via microfluidics, reverse phase evaporation, and ouzo techniques for efficient plasmid DNA delivery

In the current “era of lipid carriers,” numerous strategies have been developed to manufacture lipid nanoparticles (LNPs). Nevertheless, the potential impact of various preparation methods on the characteristics, use, and/or stability of these LNPs remains unclear. In this work, we attempted to compare the effects of three different preparation methods: microfluidics (MF), reverse phase evaporation (RV), and ouzo (OZ) on lipid-peptide NPs (LPNPs) as plasmid DNA delivery carriers. These LPNPs had the same components, namely DOTMA cationic lipid, DSPC, cholesterol, and protamine. Subsequently, we compared the LPNPs in terms of their physicochemical features, functionality as gene delivery vehicles in two distinct cell lines (NT2 and D1-MSCs), and finally, their storage stability over a six-month period.It was clear that all three LPNP formulations worked to deliver EGFP-pDNA while keeping cells alive, and their physicochemical stability was high for 6 months. However, the preparation technique had a significant impact on their physicochemical characteristics. The MF produced LPNPs with a lesser size, polydispersity index, and zeta potential than the other synthesis methods. Additionally, their DNA entrapment efficiency, cell viability, and functional stability profiles were generally superior. These findings provide new insights for comparing different manufacturing methods to create LPNPs with the desired characteristics for effective and safe gene delivery.

A gene delivery system with autophagy blockade for enhanced anti-angiogenic therapy against Fusobacterium nucleatum-associated colorectal cancer

Anti-angiogenesis has emerged a promising strategy against colorectal cancer (CRC). However, the efficacy of anti-angiogenic therapy is greatly compromised by the up-regulated autophagy levels resulting from the evolutionary resistance mechanism and the presence of Fusobacterium nucleatum (F. nucleatum) in CRC. Herein, we report a cationic polymer capable of blocking autophagic flux to deliver plasmid DNA (pDNA) encoding soluble FMS-like tyrosine kinase-1 (sFlt-1) for enhanced anti-angiogenic therapy against F. nucleatum-associated CRC. The autophagy-inhibiting cationic polymer, referred to as PNHCQ, is synthesized by conjugating hydroxychloroquine (HCQ) into 3,3′-diaminodipropylamine-pendant poly(β-benzyl-L-aspartate) (PAsp(Nors)), which can be assembled and electrostatically interacted with sFlt-1 plasmid to form PNHCQ/sFlt-1 polyplexes. Hydrophobic HCQ modification not only boosts transfection efficiency but confers autophagy inhibition activity to the polymer. Hyaluronic acid (HA) coating is further introduced to afford PNHCQ/sFlt-1@HA for improved tumor targeting without compromising on transfection. Consequently, PNHCQ/sFlt-1@HA demonstrates significant anti-tumor efficacy in F. nucleatum-colocalized HT29 mouse xenograft model by simultaneously exerting anti-angiogenic effects through sFlt-1 expression and down-regulating autophagy levels exacerbated by F. nucleatum challenge. The combination of anti-angiogenic gene delivery and overall autophagy blockade effectively sensitizes CRC tumors to anti-angiogenesis, providing an innovative approach for enhanced anti-angiogenic therapy against F. nucleatum-resident CRC. Statement of significanceUp-regulated autophagy level within tumors is considered responsible for the impaired efficacy of clinic antiangiogenic therapy against CRC colonized with pathogenic F. nucleatum. To tackle this problem, an autophagy-inhibiting cationic polymer is developed to enable efficient intracellular delivery of plasmid DNA encoding soluble FMS-like tyrosine kinase-1 (sFlt-1) and enhance anti-angiogenic therapy against F. nucleatum-associated CRC. HA coating that can be degraded by tumor-enriching hyaluronidase is further introduced for improved tumor targeting without compromising transfection efficiency. The well-orchestrated polyplexes achieve considerable tumor accumulation, efficient in vivo transfection, and effectively reinforce the sensitivity of CRC to the sFlt-1-derived anti-angiogenic effects by significantly blocking overall autophagy flux exacerbated by F. nucleatum challenge, thus harvesting robust antitumor outcomes against F. nucleatum-resident CRC.

Convenient synthesis and delivery of a megabase-scale designer accessory chromosome empower biosynthetic capacity

Synthetic biology confers new functions to hosts by introducing exogenous genetic elements, yet rebuilding complex traits that are based on large-scale genetic information remains challenging. Here, we developed a CRISPR/Cas9-mediated haploidization method that bypasses the natural process of meiosis. Based on the programmed haploidization in yeast, we further developed an easy-to-use method designated HAnDy (Haploidization-based DNA Assembly and Delivery in yeast) that enables efficient assembly and delivery of large DNA, with no need for any fussy in vitro manipulations. Using HAnDy, a de novo designed 1.024 Mb synthetic accessory chromosome (synAC) encoding 542 exogenous genes was parallelly assembled and then directly transferred to six phylogenetically diverse yeasts. The synAC significantly promotes hosts’ adaptations and increases the scope of the metabolic network, which allows the emergence of valuable compounds. Our approach should facilitate the assembly and delivery of large-scale DNA for expanding and deciphering complex biological functions.

Enhancing the safety and effectiveness of polyethylenimine gene delivery through cell membrane encapsulation

Due to the powerful DNA loading capacity and excellent proton sponge effect, the cationic polymer polyethyleneimine (PEI) has been demonstrated as a versatile, efficient DNA delivery tool both in vitro and in vivo. However, the excess surface positive charge of PEI and the common capture/clearance of PEI/DNA complex by the immune system limit its further clinical applications. To reduce the cytotoxicity resulting from surface charge and avoid the clearance of nanoparticles interacting with proteins and circulating immune cells in the blood, a bionic gene delivery system was developed by coating PEI/DNA complexes with natural cell membranes. The obtained RAW Cell membrane-coated PEI/DNA capsules (RAWm-PDc) and Red blood Cell membrane-coated PEI/DNA capsules (RBCm-PDc) complex showed excellent dispersibility and stability with an average particle size of 200–300 nm. Moreover, the RAWm-PDc/RBCm-PDc complex had great DNA loading capacity and protected the bound DNA effectively from nuclease hydrolysis. Most importantly, the RAWm-PDc/RBCm-PDc complex exhibited low protein adsorption, reduced cytotoxicity and high gene delivery efficiency. All the results demonstrated the potential of RAWm-PDc/RBCm-PDc as a promising non-viral gene delivery vector.

DNA-Based Nonviral Gene Therapy─Challenging but Promising.

Over the past decades, significant progress has been made in utilizing nucleic acids, including DNA and RNA molecules, for therapeutic purposes. For DNA molecules, although various DNA delivery systems have been established, viral vector systems are the go-to choice for large-scale commercial applications. However, viral systems have certain disadvantages such as immune response, limited payload capacity, insertional mutagenesis and pre-existing immunity. In contrast, nonviral systems are less immunogenic, not size limited, safer, and easier for manufacturing compared with viral systems. What's more, nonviral DNA vectors have demonstrated their capacity to mediate specific protein expression in vivo for diverse therapeutic objectives containing a wide range of diseases such as cancer, rare diseases, neurodegenerative diseases, and infectious diseases, yielding promising therapeutic outcomes. However, exogenous plasmid DNA is prone to degrade and has poor immunogenicity in vivo. Thus, various strategies have been developed: (i) designing novel plasmids with special structures, (ii) optimizing plasmid sequences for higher expression, and (iii) developing more efficient nonviral DNA delivery systems. Based on these strategies, many interesting clinical results have been reported. This Review discusses the development of DNA-based nonviral gene therapy, including novel plasmids, nonviral delivery systems, clinical advances, and prospects. These developments hold great potential for enhancing the efficacy and safety of nonviral gene therapy and expanding its applications in the treatment of various diseases.

Lactosylated lipid calcium phosphate-based nanoparticles: A promising approach for efficient DNA delivery to hepatocytes.

For safe and effective gene therapy, the ability to deliver the therapeutic nucleic acid to the target sites is crucial. In this study, lactosylated lipid phosphate calcium nanoparticles (lac-LCP) were developed for targeted delivery of pDNA to the hepatocyte cells. The lac-LCP formulation contained lactose-modified cholesterol (CHL), a ligand that binds to the asialoglycoprotein receptor (ASGR) expressed on hepatocytes, and polyethyleneimine (PEI) in the core. Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) were used to monitor the chemical modification, and the physicochemical properties of NPs were studied using dynamic light scattering (DLS) and transmission electron microscopy (TEM). To evaluate transfection efficiency, cellular uptake and GFP expression were assessed using fluorescence microscopy and flow cytometry. The results revealed that lactose-targeted particles (lac-LCP) had a significant increase in cellular uptake by hepatocytes. The inclusion of a low molecular weight PEI (1.8 KDa) with a low PEI/pDNA ratio of 1 in the core of LCP, elicited high degrees of GFP protein expression (by 5 and 6-fold), which exhibited significantly higher efficiency than PEI 1.8 KDa and Lipofectamine. The successful functionalization and nuclear delivery of LCP NPs described here indicate its promise as an efficient delivery vector to hepatocyte nuclei.

Nebulized inhalation of LPAE-HDAC10 inhibits acetylation-mediated ROS/NF-κB pathway for silicosis treatment

Silicosis is a serious silica-induced respiratory disease for which there is currently no effective treatment. Irreversible pulmonary fibrosis caused by persistent inflammation is the main feature of silicosis. As an underlying mechanism, acetylation regulated by histone deacetylases (HDACs) are believed to be closely associated with persistent inflammation and pulmonary fibrosis. However, details of the mechanisms associated with the regulation of acetylated modification in silicosis have yet to be sufficiently established. Furthermore, studies on the efficient delivery of DNA to lung tissues by nebulized inhalation for the treatment of silicosis are limited. In this study, we established a mouse model of silicosis successfully. Differentially expressed genes (DEGs) between the lung tissues of silicosis and control mice were identified based on transcriptomic analysis, and HDAC10 was the only DEG among the HDACs. Acetylomic and combined acetylomic/proteomic analysis were performed and found that the differentially expressed acetylated proteins have diverse biological functions, among which 12 proteins were identified as the main targets of HDAC10. Subsequently, HDAC10 expression levels were confirmed to increase following nebulized inhalation of linear poly(β-amino ester) (LPAE)-HDAC10 nanocomplexes. The levels of oxidative stress, the phosphorylation of IKKβ, IκBα and p65, as well as inflammation were inhibited by HDAC10. Pulmonary fibrosis, and lung function in silicosis showed significant improvements in response to the upregulation of HDAC10. Similar results were obtained for the silica-treated macrophages in vitro. In conclusion, HDAC10 was identified as the main mediator of acetylation in silicosis. Nebulized inhalation of LPAE-HDAC10 nanocomplexes was confirmed to be a promising treatment option for silicosis. The ROS/NF-κB pathway was identified as an essential signaling pathway through which HDAC10 attenuates oxidative stress, inflammation, and pulmonary fibrosis in silicosis. This study provides a new theoretical basis for the treatment of silicosis.

Mouse genome rewriting and tailoring of three important disease loci

Genetically engineered mouse models (GEMMs) help us to understand human pathologies and develop new therapies, yet faithfully recapitulating human diseases in mice is challenging. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences, which control spatiotemporal gene expression patterns and splicing in many human diseases1,2. Including regulatory extensive genomic regions, which requires large-scale genome engineering, should enhance the quality of disease modelling. Existing methods set limits on the size and efficiency of DNA delivery, hampering the routine creation of highly informative models that we call genomically rewritten and tailored GEMMs (GREAT-GEMMs). Here we describe ‘mammalian switching antibiotic resistance markers progressively for integration’ (mSwAP-In), a method for efficient genome rewriting in mouse embryonic stem cells. We demonstrate the use of mSwAP-In for iterative genome rewriting of up to 115 kb of a tailored Trp53 locus, as well as for humanization of mice using 116 kb and 180 kb human ACE2 loci. The ACE2 model recapitulated human ACE2 expression patterns and splicing, and notably, presented milder symptoms when challenged with SARS-CoV-2 compared with the existing K18-hACE2 model, thus representing a more human-like model of infection. Finally, we demonstrated serial genome writing by humanizing mouse Tmprss2 biallelically in the ACE2 GREAT-GEMM, highlighting the versatility of mSwAP-In in genome writing.

Branch Unit Distribution Matters for Gene Delivery.

As a key nonviral gene therapy vector, poly(β-amino ester) (PAE) has demonstrated great potential for clinical application after two decades of development. However, even after extensive efforts in structural optimizations, including screening chemical composition, molecular weight (MW), terminal groups, and topology, their DNA delivery efficiency still lags behind that of viral vectors. To break through this bottleneck, in this work, a thorough investigation of highly branched PAEs (HPAEs) was conducted to correlate their fundamental internal structure with their gene transfection performance. We show that an essential structural factor, branch unit distribution (BUD), plays an important role for HPAE transfection capability and that HPAEs with a more uniform distribution of branch units display better transfection efficacy. By optimizing BUD, a high-efficiency HPAE that surpasses well-known commercial reagents (e.g., Lipofectamine 3000 (Lipo3000), jetPEI, and Xfect) can be generated. This work opens an avenue for the structural control and molecular design of high-performance PAE gene delivery vectors.

Synthesis and Transfection Efficiency of Disulfide Policationic Amphiphiles

The synthesis of new polycationic amphiphiles containing a disulfide group in their structure has been carried out. Cationic liposomes were formed on the basis of the obtained compounds and helper lipid 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine, which demonstrated the absence of toxicity to HEK293 and HeLa cells and high delivery efficiency of fluorescently labeled oligodeoxyribonucleotide. The efficiency of plasmid DNA delivery depended on the cell line and structure of the amphiphile, with liposomes based on tetracationic amphiphiles being the most effective transfectants. These liposomes may be used for in vitro transfection of eukaryotic cells as well as for further in vivo biological tests.

Peptide-DNA Co-Assembled Nanoparticles as a Nonviral Vector for Gene Delivery with High Transgene Expression

Nonviral vectors for gene therapy are a safer alternative to viral vectors that elicit a harmful immune response. Artificial viruses combine the strengths of viral and nonviral vectors and have emerged to be safe and efficient gene delivery vectors. Herein, we report the development of peptide–DNA coassembled nanoparticles as a new efficient vector for gene delivery with high transgene efficiency. The key to our success is to introduce several rationally designed functional short peptide modules: (1) a pH-sensitive segment, (2) an endosomal interruption segment, (3) a cell penetration peptide with high positive charges, (4) a nuclear localization signal peptide with positive charges, (5) aromatic groups to tune the hydrophobic/hydrophilic ratio, and (6) redox-sensitive residues. These peptide–DNA coassembled nanoparticles achieved efficient DNA delivery with transfection efficiency over 90% in a serum-containing medium. This work demonstrates the effectiveness of the module-based design approach and lays the groundwork for potential biomedical applications of peptide-based biomaterials.

Tumor microenvironment responsive nanocarriers for efficient antisense DNA delivery and enhanced chemodynamic therapy

A supramolecular strategy for the crafting of tumour microenvironment sensitive NPs composed of artemisinin, ferrocene and antisense DNA for combination cancer therapy is demonstrated.

One-pot synthesis of dynamically cross-linked polymers for serum-resistant nucleic acid delivery.

Cationic polymers used for nucleic acid delivery often suffer from complicated syntheses, undesired intracellular cargo release and low serum stability. Herein, a series of ternary polymers were synthesized via facile green chemistry to achieve efficient plasmid DNA and mRNA delivery in serum. During the one-pot synthesis of the ternary polymer, acetylphenylboric acid (APBA), polyphenol and low-molecular weight polyethyleneimine (PEI 1.8k) were dynamically cross-linked with each other due to formation of an imine between PEI 1.8k and APBA and formation of a boronate ester between APBA and polyphenol. Series of polyphenols, including ellagic acid (EA), epigallocatechin gallate (EGCG), nordihydroguaiaretic acid (NDGA), rutin (RT) and rosmarinic acid (RA), and APBA molecules, including 2-acetylphenylboric acid (2-APBA), 3-acetylphenylboric acid (3-APBA) and 4-acetylphenylboric acid (4-APBA), were screened and the best-performing ternary polymer, 2-PEI-RT, constructed from RT and 2-APBA, was identified. The ternary polymer featured efficient DNA condensation to favor cellular internalization, and the acidic environment in endolysosomes triggered effective degradation of the polymer to promote cargo release. Thus, 2-PEI-RT showed robust plasmid DNA transfection efficiencies in various tumor cells in serum, outperforming the commercial reagent PEI 25k by 1-3 orders of magnitude. Moreover, 2-PEI-RT mediated efficient cytosolic delivery of Cas9-mRNA/sgRNA to enable pronounced CRISPR-Cas9 genome editing in vitro. Such a facile and robust platform holds great potential for non-viral nucleic acid delivery and gene therapy.

Mechanistic Insights into the Superior DNA Delivery Efficiency of Multicomponent Lipid Nanoparticles: An In Vitro and In Vivo Study.

Lipid nanoparticles (LNPs) are currently having an increasing impact on nanomedicines as delivery agents, among others, of RNA molecules (e.g., short interfering RNA for the treatment of hereditary diseases or messenger RNA for the development of COVID-19 vaccines). Despite this, the delivery of plasmid DNA (pDNA) by LNPs in preclinical studies is still unsatisfactory, mainly due to the lack of systematic structural and functional studies on DNA-loaded LNPs. To tackle this issue, we developed, characterized, and tested a library of 16 multicomponent DNA-loaded LNPs which were prepared by microfluidics and differed in lipid composition, surface functionalization, and manufacturing factors. 8 out of 16 formulations exhibited proper size and zeta potential and passed to the validation step, that is, the simultaneous quantification of transfection efficiency and cell viability in human embryonic kidney cells (HEK-293). The most efficient formulation (LNP15) was then successfully validated both in vitro, in an immortalized adult keratinocyte cell line (HaCaT) and in an epidermoid cervical cancer cell line (CaSki), and in vivo as a nanocarrier to deliver a cancer vaccine against the benchmark target tyrosine-kinase receptor HER2 in C57BL/6 mice. Finally, by a combination of confocal microscopy, transmission electron microscopy and synchrotron small-angle X-ray scattering, we were able to show that the superior efficiency of LNP15 can be linked to its disordered nanostructure consisting of small-size unoriented layers of pDNA sandwiched between closely apposed lipid membranes that undergo massive destabilization upon interaction with cellular lipids. Our results provide new insights into the structure-activity relationship of pDNA-loaded LNPs and pave the way to the clinical translation of this gene delivery technology.

Safe and efficient DNA delivery based on tannic acid-ion coordination encapsulation

ABSTRACT Polyethyleneimine (PEI) is a non-viral gene vector of frequent concern because of its high transfection efficiency. However, its clinical application is limited by the cytotoxicity caused by high positive charge density. Therefore, reducing toxicity and maintaining high transfection efficiency through appropriate strategies are of great significance to promote the development of PEI as gene vector. In order to reduce the toxicity of PEI, this work studied the feasibility of encapsulating PEI/DNA complexes by using the coordination between tannic acid (TA) and metal ions (Mn+). Firstly, TA formed a coating layer on the surface of PEI/DNA complex through electrostatic action, and then the quaternary complex (Mn+(TA/PEI/DNA)) system was prepared by the coordination between metal ions and TA. In order to better construct a gene delivery system with high transfection efficiency and low toxicity, the effects of PEI molecular weight, mass ratio of PEI/DNA, mass ratio of TA, and types of metal ions on the transfection efficiency, cytotoxicity, particle size, zeta potential, and cell uptake pathway of the quaternary complex were studied. The results showed that the quaternary complex based on PEI70k and encapsulated by coordination between TA and potassium ions showed better safety than PEI70k/DNA while maintaining high transfection efficiency. This showed that the strategy of coordination encapsulation of TA and ions to improve the safety of PEI as gene carrier is feasible, which provides a new reference for the design of gene delivery system based on PEI.