1 Naked Plasmid DNA Delivery
DNA exhibits far higher stability than RNA in biological systems. Direct intravenous injection of naked plasmid DNA yields robust transgene expression in mice, rats and non-human primates. Targeted intravascular administration to the liver enables efficient delivery of naked plasmid DNA into hepatocytes and potent exogenous gene expression.
Naked plasmid DNA can be delivered via injection and electroporation. Notably, injection achieves 10,000-fold higher in vivo expression than electroporation. Formulated with stabilizers such as hyaluronic acid, naked plasmid DNA can be processed into inhalable powder. Hyaluronic acid extends the shelf life of lyophilized plasmid preparations and facilitates pulmonary delivery in mouse models. The naked plasmid product Neovasculgen™ has been approved by the EU and FDA for clinical use.
Nevertheless, naked plasmid DNA delivery has inherent limitations. Only a minor fraction of administered plasmids internalize into target cells, and nucleic acids are prone to degradation by interstitial DNase, thereby requiring elevated dosing regimens.
2 Vector-Mediated Delivery
Cationic lipids, polymers and peptides can condense DNA into nanocomplexes to address the drawbacks of naked DNA delivery. Common chemical vectors are categorized into lipid, polymeric and inorganic materials. Exosomes also serve as promising natural carriers for DNA transportation.
2.1 Lipid Vectors
Lipid molecules consist of hydrophilic heads and hydrophobic tails, capable of encapsulating lipophilic and hydrophilic therapeutics and releasing nucleic acids via membrane fusion. Lipid nanoparticles are widely adopted and commercialized in DNA delivery owing to favorable targeting capability, superior biocompatibility, drug resistance and endosomal escape efficiency. Multiple lipid formulations including DOTMA and DOTAP are applied alone or in combination for carrier construction.
2.2 Polymeric Vectors
Polymers are macromolecules assembled from repetitive monomeric units, classified as natural and synthetic polymers. Natural polysaccharides, particularly chitosan and its derivatives, are extensively utilized due to exceptional biosafety and biodegradability. Gelatin and albumin are predominant protein-based carriers, while hydrogels also function as feasible DNA delivery platforms.
Most polymeric carriers possess excellent biocompatibility, low toxicity and degradability. Modification optimization is required for natural polymers, and scalable manufacturing remains challenging for synthetic counterparts, which may trigger excessive immune responses. Further research is essential to broaden their clinical applicability.
2.3 Inorganic Material Vectors
Inorganic nanoparticles are synthesized from diverse raw materials, grouped into metallic materials (iron oxide and metallic compounds) and non-metallic materials (quantum dots, carbon nanotubes, graphene and silica-based composites).
2.3.1 Metallic Nanoparticles
Metallic nanoparticles adopt core-shell structures with favorable biocompatibility, stable storage performance, facile synthesis, universal applicability and low cytotoxicity. Gold nanoparticles feature negligible toxicity, silver nanoparticles exert antibacterial effects, and iron oxide nanoparticles gain popularity for low cost, biosafety and inherent targeting potential, standing among the most thoroughly investigated nanomaterials for oncotherapy. Their limited in vivo degradability restricts clinical translation, and relevant research remains in the early exploratory stage.
2.3.2 Non-metallic Nanoparticles
Non-metallic nanoparticles present superior biosafety and potential high transfection efficiency compared with metallic counterparts. Carbon nanotubes derived from graphene sheets possess outstanding drug loading capacity, chemical inertness and controllable drug release profile, allowing real-time delivery monitoring via molecular probes. Poor aqueous solubility constitutes their major defect. Silica nanoparticles are favored for simple synthesis, convenient surface modification and high transfection efficacy. Suboptimal biodegradability hinders their advancement in DNA delivery applications.
2.4 Exosomes
Exosomes are bilayer lipid vesicles ranging from 40 to 160 nm in diameter, circulating in extracellular fluids and secreted by all cell types through late endosome inward budding. As natural intercellular communication mediators, exosomes possess prominent advantages:
Capability to encapsulate and transport diverse biomacromolecules;
Natural penetration across biological barriers including the blood-brain barrier, enabling access to avascular tissues;
Long-term retention in target tissues with low clearance rate;
Feasibility of genetic engineering modification.
Exosomes hold immense prospects in plasmid DNA delivery. Major loading strategies include electroporation, ultrasonication, exosome-liposome hybridization and genetic manipulation of exosome-secreting cells.
3 Physical Delivery Methods
Physical approaches constitute fundamental strategies for intracellular DNA delivery. Conventional needle injection covers intramuscular, intravenous, intradermal and intratumoral administration. Emerging techniques such as DNA tattooing, microneedle injection, gene gun, jet injection, electroporation, ultrasound-mediated delivery and mucosal administration attract growing research attention.
3.1 Conventional Injection
Vaccination research has gradually shifted from intramuscular to intradermal injection over the past decade. The skin acts as a natural pathogen barrier with potent immunological activity. Subcutaneous administration enhances antigen expression and improves the immunogenicity of DNA vaccines. Intravenous injection remains the mainstream physical route for chemically formulated DNA delivery, whereas intratumoral injection is predominantly applied in cancer therapy to alleviate systemic toxicity and elicit tumor-specific immune responses.
3.2 Needle-Free Injection
Needle-free injection devices penetrate tissue barriers via high-pressure fluid or pulsed energy without conventional syringes. This modality alleviates pain, eliminates needle-associated injuries and improves patient compliance. Precise control over delivery depth and dosage achieves accurate targeting. Certified needle-free injection systems are clinically available for gene therapy.
3.3 Microneedles
Microneedles are micro-projection arrays with heights of 25–2000 μm, widths of 50–250 μm and tip thicknesses of 1–25 μm, categorized into solid, coated, hollow, dissolvable and hydrogel microneedles. Microneedles enhance DNA stability and facilitate matrix-based delivery. Potential risks include nucleic acid contamination and degradation on metallic microneedle surfaces. Insufficient disease models confine relevant studies to laboratory scale.
3.4 DNA Tattooing
DNA tattooing is an innovative transdermal delivery technique. The first human clinical trial (NTR4607/NL4474) conducted in 2017 verified the safety and biological efficacy of HPV16 DNA tattoo vaccination for vulvar intraepithelial neoplasia. This method is also compatible with cationic liposome-encapsulated DNA vaccines.
3.5 Gene Gun
The gene gun physically delivers DNA into target cells by bombarding tissues with heavy metal microcarriers coated with plasmids. This technique bypasses cell membranes and drastically reduces required plasmid dosage. Nevertheless, high-velocity particle impact impairs cell viability, and high operational costs limit large-scale application.
3.6 Electroporation
Electroporation ranks among the most efficient DNA delivery technologies. Electric pulses generate transient membrane pores to facilitate plasmid internalization, applicable to both naked and vector-loaded DNA. Optimal parameters including electrode configuration, tissue characteristics, plasmid size and pulse protocols determine transfection efficiency. High electric field induces thermal damage and cell apoptosis, accompanied by painful sensations.
3.7 Ultrasound-Mediated Delivery
Ultrasound triggers transient membrane pores via microbubble oscillation for DNA internalization, featuring non-invasiveness, safety and flexible operation. This approach effectively suppresses tumor invasion and metastasis. Low-frequency ultrasound also serves as a potent adjuvant to activate immune cells.
3.8 Mucosal Immunization
Mucosal immunization prevents pathogen invasion across mucosal surfaces without injection. Mucosal tissues include ocular, nasal, respiratory, gastrointestinal and genital tracts. Oral administration induces systemic and mucosal immune responses to block superficial infection. Vaginal delivery of DRLS elicits higher IgA levels than intramuscular injection, providing rapid mucosal defense. These findings lay a foundation for novel mucosal non-viral genetic vaccine development.
Conclusion
Plasmid DNA gains extensive research interest for inherent merits including high safety, convenient production, transportation and storage, emerging as a promising candidate for cancer therapy.
Low transfection efficiency and insufficient protein expression hinder industrial transformation. Diversified delivery strategies are developed to overcome intrinsic limitations and achieve remarkable progress. This review systematically summarizes mainstream carriers and delivery modalities, providing comprehensive references for frontier research on plasmid DNA transportation.
