Plasmid DNA Research Articles

The development of multifunctional carriers for gene delivery is a critical challenge in modern therapeutics, particularly in the context of multi-drug therapy (MDT). In this study, we report the synthesis and characterization of fluorinated guanidino-polyamine conjugates based on low-generation polyamidoamine (PAMAM) dendrimers and low molecular weight polyethyleneimine (PEI) polymers. These conjugates are designed to act as both efficient transfection agents and artificial ribonucleases, providing a dual-function approach to gene therapy. The functionalization with fluorinated guanidino groups enhances DNA condensation, facilitates intracellular delivery, and enables tracking via 19F MRI. Potentiometric and kinetic studies demonstrate their phosphodiesterase activity on a model compound, with PAMAM G4 derivatives exhibiting the highest catalytic efficiency. Biolayer interferometry and transfection experiments confirm mRNA cleavage activity, leading to reduced gene expression. Additionally, transfection studies with plasmid DNA (pDNA) indicate high gene delivery efficiency, surpassing conventional PEI-based systems while maintaining low cytotoxicity. These findings suggest that the conjugates presented herein, and in particular those derived from low-generation PAMAM dendrimers, can serve as promising multifunctional carriers for a combined diagnostic and MDT, offering a new strategy for synergistic gene delivery and RNA degradation.

Cell-based therapies have transformed the treatment landscape for a range of diseases, leveraging both genome modification and cell reprogramming to create targeted treatments. Such therapies rely on the efficient internalization of biomolecules into living cells. Unfortunately, existing cargo delivery methods, such as those based on viral vectors and electroporation, are often compromised by cytotoxicity, poor delivery efficiencies, and low throughput. To overcome these limitations, a viscoelastic squeezing methodology is presented that uses viscoelastic microfluidics to perform mechanoporation in a rapid and contact-free manner. Through the control of the flow rates of a sample stream containing cells and cargo and a surrounding viscoelastic sheath flow, the width of a "virtual channel" formed between the two streams can be regulated. Elastic forces generated within this virtual channel are then used to deform contained cells and internalize user-defined payloads. The effectiveness and utility of the platform are assessed through the delivery of mRNA, plasmid DNA, and clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) ribonucleoprotein complexes into a variety of cell lines. Data confirms that viscoelastic squeezing provides for enhanced delivery efficiencies when compared to conventional poration techniques, whilst maintaining high cell viabilities and throughputs of 20 million cells per minute, and thus represents a powerful tool for cellular engineering.

BackgroundAmoxicillin is among the most frequently prescribed antibiotics globally, either as monotherapy or in combination with clavulanic acid as amoxicillin-clavulanic acid (AMC). However, the prolonged use of AMC and other antibiotics has intensified selection pressure, accelerating the emergence of AMC-resistant and multidrug-resistant (MDR) strains. Klebsiella, a member of the ESKAPE pathogens, employs diverse resistance mechanisms against multiple classes of antibiotics. This study was aimed to identify environmental Klebsiella isolates resistant to AMC with MDR phenotype and to investigate the underlying genetic determinants contributing to their resistance and virulence.MethodologyWater samples were collected from 14 sites, encompassing both wastewater and natural aquatic environments, and screened for AMC resistance on AMC supplemented Klebsiella-Selective agar base media. Antibiotic profiling of AMC resistant isolates was done by Kirby-Bauer’s disc diffusion test. Phenotypically positive MDR isolates were identified by MALDI-ToF MS. Furthermore, Klebsiella pneumoniae isolates were selected for PCR based detection of antibiotic resistance and virulence factor associated genes using plasmid and genomic DNA as a template respectively. Horizontal gene transfer experiment was carried out using K. pneumoniae isolates as donor and plasmid-free and antibiotic sensitive Escherichia coli J53R strain as a recipient. Biofilm formation was detected by crystal violet assay and visualised in SEM. The hypermucoviscosity of K. pneumoniae (hmvKp) was confirmed by string test.ResultsOf the total 178 AMC resistant bacterial isolates, 119 displayed MDR phenotype. Among 63 putative AMC-resistant, MDR isolates exhibiting a non-metallic sheen on EMB agar, MALDI-TOF MS-based identification confirmed 33 to be Klebsiella pneumoniae. PCR based screening for resistance determinants revealed the presence of blaTEM (100%), blaSHV (75.75%), blaCTX−M (54.54%), blaNDM (27.27%), blaOXA−48 (39.39%), blaCMY (48.48%), qnrA (6.06%), qnrB (87.87%), qnrS (93.93%), tetA (81.81%), and tetB (27.27%), alongside sul1 (90.90%) and dfrA12 (9.09%) genes. Additionally, virulence-associated genes viz., fimH (33.33%), mrkD (78.78%), ecpA (54.54%), iucC (54.54%), and rmpA (6.06%) were also detected. Furthermore, biofilm formation assay demonstrated that 24 (72.72%) isolates were strong biofilm-formers, indicating their potential for pathogenicity.ConclusionOccurrence of hypervirulent, AMC resistant and MDR Klebsiella pneumoniae in aquatic environment is a concern and further studies are required to explore their potential threat in dissemination of resistance and clinical implications.Graphical abstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s12879-025-11806-5.

Despite the tremendous potential of the CRISPR/Cas9 gene-editing technology in precision therapeutics, intracellular delivery remains a major challenge. High cytoplasmic viscosity and lysosomal entrapment significantly impair the cytosolic transport and gene-editing efficiency. In this study, we demonstrate that both the size and magnetic responsiveness of Fe3O4 nanoclusters can be finely tuned by modulating ionic strength, enabling their rapid propulsion under external magnetic fields. Leveraging this property, we develop magnetic nanoparticle cluster nanorobots (MagCbots) of approximately 200 nm in size by electrostatically assembling Fe3O4 nanoclusters with CRISPR-Cas9 plasmids. Under magnetic actuation, MagCbots exhibit rapid rotation in highly viscous intracellular environments, achieving a linear velocity of ∼0.41 μm/s. MagCbots reduce intracellular viscosity by approximately 50% and enhance lysosomal escape efficiency by 3-fold compared to nonactuated counterparts. Their porous architecture not only offers high payload capacity but also protects plasmid DNA from enzymatic degradation. Notably, MagCbots enable efficient genome editing of both PD1 and PLK1 genes across various cell lines including hard-to-transfect Jurkat T cells. This magnetically driven nanorobot platform presents a promising strategy for active intracellular delivery and holds significant potential for advancing gene therapy and related biomedical applications.

The popularity of rAAV vectors in gene therapy are placing a burden on current production systems. To improve the accessibility of these life changing treatments, increases in production yields and a reduction in the cost-of-goods are needed. Transient transfection is the most common way to introduce rAAV-encoding plasmids to producer cells but it suffers from significant drawbacks such as low and inconsistent yields as well as high cost due to its need for plasmid DNA. This study aims to address the low yield of transient transfection-based rAAV production through advanced methods in process characterization. Adherent and suspension cultures of a HEK293T cell line were triple-transfected for rAAV9 production using polyethylenimine (PEI). Samples were taken at various times post-transfection for analysis with bulk and single-cell transcriptomics. It was revealed that 46% of the cells lacked transcripts of genes from at least one plasmid, indicating that a significant proportion of the cells did not have the genes necessary for rAAV9 production. Among the remaining 54% of the cells expressing genes from all three plasmids, only 8% showed high plasmid gene expression. Flow cytometric analysis of intracellular rAAV9 confirmed these results by showing that only ~ 3% of cells contained assembled rAAV9 capsids. Titre analysis by qPCR of the supernatant and lysate of the producer cells indicated an average culture performance of 1013 vg/L. Analysis of the single-cell transcriptomic data showed that a significant proportion of cells that had high plasmid gene expression were in the S-phase. Trajectory inference highlighted that genes involved in the G2-M phase transition, immune response, and protein unfolding were differentially expressed at the branch point between high and low plasmid expression. This study reveals a significant bottleneck in the transient transfection-based production of rAAV. With less than 5% of cells producing rAAV, significant improvements in titres can be achieved if this fraction can be increased. Moreover, regulation of the cell-cycle, inhibition of the immune response, and alleviating protein misfolding all potentially offer the key to enabling these life changing treatments to reach a wider audience.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-26261-0.

Breast cancer (BC) continues to be the most common cancer affecting women globally, posing significant therapeutic challenges due to limited therapeutic efficacy, non-specific drug targeting, systemic toxicity, and the emergence of chemoresistance. These hurdles emphasize the critical need for novel and effective treatment strategies. Nanomedicine has revolutionized cancer therapy, especially D-alpha-tocopheryl polyethylene glycol succinate (TPGS)-based nanoparticles (TPGS-NPs), which have emerged as a promising strategy due to their enhanced therapeutic potential. TPGS, an amphiphilic derivative of Vitamin E, not only enhances the solubility, stability, and bioavailability of lipophilic drugs but also exhibits intrinsic anti-BC properties. The incorporation of TPGS into NPs significantly enhances their physicochemical properties. Further, the engineering of TPGS-NPs with targeting ligands significantly improves their specificity towards cancer cells. Also, TPGS-NPs show great potential to improve photothermal and photodynamic therapies due to their excellent physicochemical properties. Moreover, TPGS-NPs demonstrate an excellent ability for gene (plasmid DNA, siRNA, and miRNA) delivery by enhancing stability and transfection efficiency. This review explores the multifaceted role of TPGS and the role of different TPGS-NPs in circumventing the limitations of conventional chemotherapy in BC treatment. Overall, developing TPGS-NPs offers a versatile and multifaceted approach to achieve better therapeutic outcomes against BC. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Introduction: The present work aims to synthesize, characterize, and DNA binding properties of copper, nickel, zinc, and iron complexes of a well-known polyphenol drug curcumin. The metal ion complexes of curcumin's synthesized transition metal ion complexes were characterized by UV-vis spectroscopy and FTIR. Method: The complexation of curcumin with transition metal ion complexes changes the color of the curcumin based on the metal incorporated. Moreover, the shift in the absorption maxima of curcumin metal ion complexes may confirm the formation of coordination complexes. Then, FTIR results indicated that the coordination of curcumin with metal ions in the ketone group of curcumin confirms the formation of 1:2 (M: L) complexes. Results: Then, we evaluated the DNA binding properties of synthesized metal ion complexes of curcumin using electronic absorption spectra and agarose gel electrophoresis. The results indicated that the curcumin zinc complex exhibited less DNA binding constant among the four metal ion complexes than all the other compounds tested, confirming its weak interaction. Conclusion: Moreover, all the compounds degrade plasmid DNA, confirming their DNA cleavage activity. From our findings, these compounds will pave the way for developing new anticancer drugs with less toxicity.

A microfluidic based stem-loop probes mediated isothermal amplification for multiplex detection of Vibrio parahaemolyticus.

It is estimated that ~2 billion people worldwide cannot afford even basic medicines. Bioreactor-produced recombinant protein therapies (BRPTs), among the world’s most-expensive medicines, have revolutionized treatment of a wide-spectrum of human-diseases, particularly the ~ 7,000 incurable, rare human single-protein, monogenic-deficiency diseases. Currently, BRPT are limited by toxicity, immunogenicity, short protein T1/2s and high-cost, creating a worldwide access-gap between those who can afford BRPTs versus those who cannot. Fabry disease (FD) is a monogenic deficiency disease caused by pathogenic-variants of the galactosidase-a (GLA) gene. FD damages heart, kidneys, CNS, gastrointestinal tract, and eyes. State-of-the-art anti-FD therapy, hGLA enzyme-replacement-therapy (ERT), requires bi-weekly IV infusions lifelong, costing ~ $200,000 per-patient per-year. High-lifetime costs can cause significant numbers of FD patients to permanently discontinue hGLA-ERT, thereby accelerating FD progression, which can lead to premature-death. Subcutaneously administered plasmid DNA alone has not been previously reported to transfect subcutaneous fat. Here we show one Subcutaneous Administration of HEDGES DNA vectors Alone (SAHDA) encoding the wildtype hGLA protein safely produces durable hGLA serum protein levels in the 1–10 ng/mL normal human hGLA range in immunocompetent mice. Unexpectedly, one administration of a highly-optimized SAHDA version encoding hGLA produced durable, ~ 100-fold higher hGLA serum protein levels than the 10 ng/mL high-normal human level. Importantly, components of the SADHA platform can be simply-modified to control the level and duration of hGLA serum protein-levels produced over a broad temporal-range in mice. Furthermore, one SAHDA-based administration of a HEDGES DNA-vector encoding a highly-neutralizing anti-SARS-CoV-2 monoclonal antibody (mAb) safely produces long-term protective serum mAb levels in immunocompetent-mice. SAHDA offers multiple advantages over BRPT, including not requiring an intact cold-chain and being readily freeze-dried. This combination enables SAHDA’s rapid deployment, then prolonged storage at ambient temperatures, even in equatorial-areas worldwide. SAHDA can readily be self-administered worldwide. It also obviates severe intravenous-infusion reactions. Taken-together, SAHDA may more effectively-, safely-, durably-, and cost-effectively-treat a spectrum of now poorly-treatable diseases worldwide.

Koi herpesvirus disease (KHVD), caused by Cyprinid herpesvirus-3 (CyHV-3), poses a significant threat to global aquaculture due to its high mortality rates and economic impact. Current diagnostic methods, such as PCR, are limited by equipment dependency and procedural complexity, hindering point-of-care (POC) applications. To address this, we developed an integrated assay combining recombinase-aided amplification (RAA) with CRISPR-Cas13a-mediated SHERLOCK technology and lateral flow detection (LFD) for rapid and visual detection of CyHV-3 in clinical samples. The KHV-SHERLOCK assay targets a conserved region of the CyHV-3 thymidine kinase (TK) gene, demonstrating exceptional specificity with no cross-reactivity to related pathogens or host DNA. Sensitivity evaluations revealed a detection limit of 100 ag/μL for CyHV-3 plasmid DNA, tenfold more sensitive than the conventional PCR (1 fg/μL) assay, even in the presence of 100 ng of carp genomic DNA as background interference. Clinical validation using 50 archived samples showed 100% concordance with reference PCR results, confirming diagnostic reliability. The assay's isothermal RAA step (37°C, 40 min) and CRISPR-Cas13a detection (37°C, 1 h) enable equipment-free operation, while LFD provides unambiguous visual results within minutes. This platform merges high sensitivity with POC practicality, offering a transformative tool for field-based KHVD surveillance.

The evolution of the virulence and antibiotic resistance of staphylococci, important opportunistic pathogens, is strongly determined by their mobilome, which can spread by phage virions or small-headed particles resulting from the hijacking of helper phage machinery by phage satellites named phage-inducible chromosomal islands (PICIs). Despite known mechanisms of the formation of transducing particles, it has not yet been possible to analyze their DNA content at the single-virion level. Using the Staphylococcus epidermidis model and long-read nanopore sequencing, we determined the sequence structure and ratio of phage and PICI genophores, plasmid, and bacterial DNA packaged in normal and small-headed virions. It was shown that the ratios vary mainly depending on the helper phage and the antimicrobial used for induction. When the effect of a strictly lytic phage and its combination with ciprofloxacin on a packaged mobilome was analyzed, no significant increase in mobilome dissemination was observed compared to antibiotics alone. Here, we demonstrate a novel approach for the analysis of transduced bacterial mobilome and show in vitro that lytic phage-based therapeutic strategies do not increase the risk of mobile genetic element transfer.

Melanoma is a type of cancer that originates in melanocytes with high malignancy, mainly because the cancer cells can spread through the body and cause metastasis. It is projected that the annual incidence of melanoma will increase by over 50% between 2020 and 2040. DNA vaccines have been explored for the treatment and prevention of various globally important diseases and are a promising resource for melanoma. Generally, DNA vaccines are classified as third-generation vaccines, which consist of DNA plasmids encoding target antigens. Therefore, their mechanism of action involves delivering one or more genes of interest into host cells, triggering an immune response against the target antigen. In summary, the DNA vaccine must enter the cell cytoplasm and migrate to the nucleus to initiate replication, transcription, and production of the target antigen. Despite their many advantages, DNA constructs produce low levels of antigens in vivo due to the challenges in effectively activating an immune response. This review addresses general aspects relevant to the application of DNA vaccines in the landscape of melanoma treatment, focusing mainly on physical and chemical methods used to enhance DNA-based vaccines, considering key aspects of technology, advantages, limitations, applications, and the evolution of clinical translation.

Comparison of anion-exchange chromatography matrices for purification of linear and supercoiled plasmid in a direct lysate workflow.

IntroductionTauopathy models are essential in vitro systems for investigating tau-targeted therapies and advancing Alzheimer’s disease research. Extracellular vesicles (EVs), owing to their high biocompatibility, low toxicity, and reduced immunogenicity, represent promising carriers for gene delivery and disease modeling.MethodsWe investigated the potential of EVs as a delivery system for the human four-repeat tau isoform lacking N-terminal sequences (4R0N) and enhanced green fluorescent protein (EGFP) into Neuro-2a cells. EV-mediated transfection efficiency was compared with conventional methods, including lentiviral and chemical (lipofectamine and polyethyleneimine, PEI) approaches. Response surface methodology (RSM) was used to optimize EV-mediated delivery parameters.ResultsEVs successfully delivered large plasmid DNA into Neuro-2a cells, resulting in detectable tau and EGFP expression. Optimization via RSM further improved gene delivery efficiency and reproducibility compared to unoptimized EV preparations and conventional transfection methods.DiscussionThese findings demonstrate that EVs can serve as a robust and biocompatible platform for tau gene delivery, providing a promising alternative to traditional transfection strategies for generating physiologically relevant tauopathy models.

Local intramuscular administration of synthetic plasmid DNA (pDNA) encoding monoclonal antibodies (mAb) offers an alternative to recombinant protein-based mAb delivery. In this phase 1 dose-escalation study, we evaluated the safety, tolerability and pharmacokinetics of a pDNA cocktail encoding AZD5396 and AZD8076, modified versions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralizing mAb cocktail tixagevimab/cilgavimab in healthy adults. Participants received up to four intramuscular doses of pDNA encoding both DNA-based mAbs (DMAbs), administered using CELLECTRA electroporation. The primary endpoints were safety and pharmacokinetics. All 44 participants received at least one dose; DMAbs were detected in 100% of evaluable participants (n = 39), with serum concentrations reaching a peak of 1.61 µg ml-1. Sustained expression was observed in all participants during the 72 weeks of follow-up. The study product was well tolerated, with no product-related serious adverse events reported. Exploratory analyses demonstrated binding to multiple SARS-CoV-2 Spike protein variants and neutralizing activity in a standard pseudovirus assay. No antidrug antibodies were detected across approximately 1,000 serum samples using validated tiered assays. To our knowledge, these data represent the first-in-human proof-of-concept that synthetic pDNA DMAb technology permits the durable in vivo production of a functional mAb cocktail. This study further underscores the collective importance of synthetic design, formulation and delivery to achieve biologically relevant expression of gene-encoded biologics. DMAb delivery may represent a long-acting, scalable, cold-chain-independent platform against a wide range of diseases that can be targeted with mAbs and their derivatives. ClinicalTrials.gov registration: NCT05293249.

CRISPR/Cas9 gene editing strategy for cancer therapy: non-viral nanocarrier-mediated delivery of plasmids, RNA and ribonucleoprotein complexes.

Background8-Oxo-7,8-dihydroguanine (8-hydroxyguanine, GO) is a major damaged base caused by oxidation. Misincorporation of dATP opposite GO by DNA polymerases leads to a G:C→T:A transversion at the damaged site via GO:A intermediate formation. The GO:A pair is also formed by 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate incorporation opposite A. The GO:C and GO:A pairs are both repaired through the base excision repair (BER) pathway to suppress the G:C→T:A mutations. GO:C also induces action-at-a-distance mutations around the damaged base. These untargeted mutations seem to be induced through the excision of GO from GO:C by DNA glycosylases, such as OGG1 and NEIL1, in the BER pathway. The adenine base of GO:A is excised by a specific adenine DNA glycosylase, MUTYH, and this excision potentially induces action-at-a-distance mutations.ResultsIn this study, plasmid DNA bearing a GO:A pair was introduced into human U2OS cells to investigate the untargeted mutations by the GO:A pair. The GO:A pair induced action-at-a-distance mutations at C bases in 5'-TpC-3' of the GO-strand, in contrast to those by GO:C, which elicit mutations at G bases of 5'-GpA-3'. Furthermore, the untargeted mutations were suppressed by the MUTYH knockdown.ConclusionThe GO:A pair induced the action-at-a-distance mutations through base excision by the MUTYH glycosylase.Supplementary InformationThe online version contains supplementary material available at 10.1186/s41021-025-00340-0.

Chili leaf curl virus (ChiLCV) is a highly destructive begomovirus that causes significant economic losses in chili production across the Indian subcontinent. Accurate detection of the virus is crucial for effective disease management. This study presents a Recombinase Polymerase Amplification (RPA)-assisted DNA endonuclease-targeted CRISPR trans reporter (DETECTR) system for the rapid, highly sensitive, and specific detection of ChiLCV, specifically targeting the AC1 gene sequence. A crRNA specific to AC1 gene of ChiLCV- was designed, and the RPA conditions were optimized. The detection method involves cleaving a tagged oligo reporter (Fluorophore-quencher or Biotin-FAM), allowing results to be visualized via either a fluorescence read-out-based assay or a Lateral Flow Assay (LFA) with gold nanoparticles conjugated to FAM antibody. We standardized the critical concentration of the biotin-FAM oligo reporter such that, in the presence of the viral genome, the activated CRISPR-Cas12a cleaves all reporters, resulting in a dark test line on the lateral flow strip. This RPA-assisted fluorescence or LFA readout-based DETECTR system demonstrates exceptional specificity and sensitivity, detecting ChiLCV at a concentration as low as 7 femtograms when using cloned plasmid DNA, comparable to the gold standard detection method, like real-time PCR. The system successfully detected the virus in crude leaf extracts from infected plants while distinguishing ChiLCV from related begomoviruses and damage caused by common pests like mites and thrips. The DETECTR system was finally validated with field infected samples collected from major chili-growing states of India. To the best of our knowledge, this is the first demonstration of a CRISPR-based assay for ChiLCV that can be applied directly to crude leaf extracts, thereby enhancing its potential utility in point-of-care diagnostics. A key advantage of this diagnostic approach is its rapid processing time and field applicability, making it an accessible and practical tool for farmers and agricultural specialists to implement timely virus disease management strategies for chili crops.

Immunomodulatory molecules play a crucial role in the establishment and maintenance of chronic pain. Among these, anti-inflammatory interleukin-10 (IL-10) has emerged as one of therapeutic options to ameliorate pain state. Some components reduced pain through stimulating endogenous IL-10. However, IL-10 has a short half-life, which limits its long-term treatment of chronic pain. Gene therapy targeting il10 gene expression has shown promise in preclinical studies. There are mainly two approaches to achieving successful transfection of target genes into cells, viral vectors and non-viral methods. Both Watkin and Milligan groups have well investigated and reviewed the non-viral mediated IL-10 delivery for chronic pain treatment, especially focusing on plasmid DNA encoding IL-10 including phase I clinical trial (XT-150) (see Articles: Gene Therapy (2009) 16, 470-475; Neuromodulation (2012) 15: 520-526; and Front. Immunol.(2019)10:3009). Viral-vector-mediated gene therapy is a desirable route of administration to require local and long-term expression for chronic pain management. Therefore, in this review we focused on the utility of viral vector-mediated IL-10 expressions in preclinical pain models. These studies consistently demonstrated the potential of IL-10-based gene therapy as a novel and sustained therapeutic approach for chronic pain.

The blood–brain barrier (BBB) restricts the entry of therapeutic agents into the brain cardiovascular regulatory region, potentially contributing to drug-resistant hypertension. Objective: The objective of this study was to overcome this limitation by modifying PEGylated liposomes with transferrin (Tf) to facilitate Tf receptor binding at the BBB and penetratin (Pen), a cell-penetrating peptide, to enhance neuronal uptake. Methods: This study evaluated the efficacy of Tf-Pen-liposomes in delivering angiotensin-converting enzyme 2 (ACE2) or EGFP (control) genes across the BBB in rats. In addition, the therapeutic effect of intravenous administration of Tf-Pen-Lip carrying plasmid DNA encoding ACE2 (Tf-Pen-Lip-pACE2) was tested in a neurogenic hypertension model induced by intracerebroventricular (ICV) infusion of angiotensin II (Ang II) via osmotic pump implantation and brain cannulation. Results: Conjugation with Tf and Pen significantly enhanced liposome-mediated gene transfection in cultured cells and increased transport across an in vitro BBB model. In vivo, intravenous administration of Tf-Pen-Lip-pACE2 or Tf-Pen-Lip-pGFP successfully elevated ACE2 or EGFP expression, respectively, in the hypothalamic paraventricular nucleus (PVN). Chronic ICV infusion of Ang II produced a sustained increase in blood pressure and heart rate, accompanied by sympathetic overactivation and elevated arginine vasopressin (AVP) secretion, hallmarks of neurogenic hypertension. Notably, intravenous Tf-Pen-Lip-pACE2 treatment dramatically attenuated Ang II–induced neurogenic hypertension, whereas Tf-Pen-Lip-pGFP had no effect on pressor responses, sympathetic activity, or AVP secretion. Conclusions: This dual-functionalized liposomal delivery system effectively transported the ACE2 gene across the BBB into the brain, increased ACE2 expression, and markedly attenuated neurogenic hypertension following systemic administration.