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Transfection technology is one of the core technologies in modern molecular biology, cell engineering, and gene therapy research. Its development has accompanied continuous advances in nucleic acid delivery materials, cell biology research, and gene regulation technologies. From the initial use of simple chemical reagents to facilitate the entry of exogenous DNA into cells, to the development of non-viral delivery systems based on lipids and polymers, and further to RNA delivery, gene editing, and nanodelivery platforms, transfection technology has continuously overcome limitations in nucleic acid delivery efficiency, cellular compatibility, and application scope. Technological innovations at each stage have focused on improving the efficiency of exogenous genetic material entry into cells, promoting intracellular release, reducing cytotoxicity, and meeting the requirements of different cell types and research applications. Today, transfection technology has evolved from a traditional laboratory tool for gene expression into a key technological platform supporting functional genomics, protein expression, cell engineering, gene therapy, and precision medicine.
Early Chemical Transfection Stage (1960s–1970s)
Development of DEAE-Dextran and Calcium Phosphate Precipitation Methods
The development of in vitro transfection technology can be traced back to the 1960s, when researchers began exploring ways to introduce exogenous genetic material into mammalian cells to investigate gene expression regulation and changes in cellular functions. Because naturally occurring nucleic acid molecules carry numerous negative charges and the cell membrane surface is also negatively charged, free DNA or RNA cannot readily cross the cell membrane. Therefore, early studies primarily focused on altering the surface properties of nucleic acids or enhancing interactions between nucleic acids and the cell membrane to improve delivery efficiency.
DEAE-dextran (diethylaminoethyl dextran) was one of the earliest chemical transfection materials used for nucleic acid delivery into eukaryotic cells. This method utilizes positively charged DEAE-dextran molecules to interact electrostatically with negatively charged DNA, forming complexes that facilitate the adsorption of nucleic acids onto the cell membrane and their subsequent entry into cells through endocytosis. Because DEAE-dextran is simple to use, inexpensive, and enables rapid experimental procedures, it played an important role in early gene expression studies, viral gene function analysis, and reporter gene assays. However, the method has limitations, including cell type-dependent transfection efficiency, a relatively narrow range of applications, and potential cytotoxicity at high concentrations. Therefore, it has primarily been used for transient transfection rather than as an ideal method for long-term stable expression.
Subsequently, in 1973, Graham and van der Eb established the classical calcium phosphate precipitation transfection method, further advancing gene delivery into eukaryotic cells. This method involves mixing DNA with calcium chloride and phosphate, causing DNA molecules to form fine calcium phosphate precipitates. These particles can be taken up by cells and release DNA intracellularly, thereby enabling exogenous gene expression. Compared with the DEAE-dextran method, calcium phosphate transfection can support more stable DNA delivery and can be used to generate stably expressing cell lines. Consequently, it became a classical method in molecular cloning and gene expression research during the 1970s and 1980s. Numerous studies of gene function, promoter activity, and recombinant protein expression employed this technique. However, the efficiency of calcium phosphate transfection is affected by multiple experimental factors, including DNA concentration, precipitate particle size, culture conditions, pH, and cell status. Therefore, optimization of experimental conditions is critical for achieving consistent results. Although more efficient delivery systems have subsequently been developed, calcium phosphate transfection continues to be used in certain cellular systems and viral vector production because of its low cost and minimal equipment requirements.
Development of Lipid-Mediated Transfection (1980s)
Lipofection Drives Advances in Non-Viral Transfection
In the 1980s, advances in lipid materials drove transfection technology into a period of rapid development of non-viral delivery systems. Although traditional chemical transfection methods could introduce exogenous DNA into cells, they generally suffered from limited efficiency and a relatively narrow range of compatible cell types. Researchers therefore began searching for delivery materials that were more efficient, safe, and broadly applicable.
In 1987, Felgner et al. first reported the liposome-mediated transfection technology known as Lipofection. This approach uses the synthetic cationic lipid DOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) to interact electrostatically with DNA and form lipid–DNA complexes known as lipoplexes. Through interactions between the lipid structures and the cell membrane, these complexes facilitate nucleic acid entry into cells. This breakthrough enabled non-viral delivery systems to achieve relatively high transfection efficiency and good experimental reproducibility for the first time, establishing an important foundation for the development of modern transfection reagents.
As lipid chemistry continued to advance, researchers further optimized cationic lipid structures and introduced helper lipids, such as DOPE (dioleoyl phosphatidylethanolamine) and DOTAP, to enhance nucleic acid loading, promote cellular uptake, and improve endosomal escape. These developments drove the emergence of commercial lipid-based transfection reagents, making lipid-mediated transfection one of the most widely used non-viral nucleic acid delivery methods in research laboratories. Compared with early chemical methods, lipid transfection offers simple procedures, relatively high transfection efficiency, broad cell-type compatibility, and the ability to deliver both DNA and RNA. It is therefore widely used for plasmid DNA expression, reporter gene analysis, protein expression, gene function studies, and RNA interference experiments. The development of lipid-mediated delivery not only changed the way basic life science research was conducted but also laid an important foundation for subsequent mRNA delivery and lipid nanoparticle (LNP) technologies.
Polymer-Mediated Transfection Stage (1990s)
Development of PEI and Other High-Efficiency Delivery Materials
With increasing demand for gene function studies, recombinant protein expression, and gene therapy research, researchers began exploring nucleic acid delivery materials that could provide greater stability, efficiency, and cost-effectiveness than lipid-based systems. During the 1990s, polymer-mediated transfection technologies developed rapidly, with polyethylenimine (PEI) becoming one of the most representative non-viral delivery materials.
In 1995, Boussif et al. reported that PEI could efficiently mediate DNA delivery into mammalian cells, marking an important milestone in the rapid development of polymer-based delivery systems. PEI contains numerous cationic amine groups that can interact electrostatically with DNA to form stable complexes, thereby improving nucleic acid stability and promoting cellular uptake. PEI can also utilize the so-called “proton sponge effect” to promote proton absorption during endosomal acidification, causing changes in osmotic pressure and thereby facilitating endosomal escape and release of nucleic acid complexes into the cytoplasm, ultimately enhancing gene expression efficiency.
Compared with lipid-based transfection systems, PEI offers advantages including simple preparation, low cost, strong nucleic acid-binding capacity, and suitability for large-scale production. It has therefore been widely used for plasmid DNA transfection, recombinant protein expression, viral vector production, and gene function studies. Particularly in highly expressing cell systems such as HEK293 cells, PEI has become a commonly used DNA delivery material in both laboratory research and industrial production. However, polymer-based materials also present challenges. For example, high-molecular-weight PEI can cause membrane damage, increased cytotoxicity, and insufficient long-term biocompatibility. Subsequent research therefore focused on developing lower-toxicity polymers, biodegradable polymers, and surface modification strategies to improve delivery efficiency and safety. The development of polymer-mediated transfection helped transform non-viral delivery systems from simple nucleic acid-binding materials into designable, high-performance gene delivery platforms.
RNA Transfection Technology Development Stage (2000s)
Expansion from DNA Delivery to RNA Regulation
Entering the 21st century, rapid advances in RNA biology drove transfection technology beyond traditional DNA delivery toward the delivery of RNA molecules. Early transfection technologies primarily focused on plasmid DNA introduction and exogenous protein expression. However, RNA molecules are relatively unstable, are susceptible to degradation by intracellular nucleases, and have limited ability to cross the cell membrane, making their delivery a major challenge in gene regulation research.
With the discovery of the RNA interference (RNAi) mechanism and the development of small interfering RNA (siRNA) technology, researchers began using transfection systems to directly deliver functional RNA molecules into cells, enabling precise regulation of specific gene expression levels.
Around 2001, siRNA-mediated gene silencing technology developed rapidly and became an important tool for functional genomics research. By delivering synthetic siRNA molecules into cells, siRNA can be recognized by the intracellular RNA-induced silencing complex (RISC) and guide the degradation of target mRNA, thereby suppressing the expression of specific genes. This technological breakthrough enabled researchers to rapidly investigate gene function and validate disease-related targets, while also driving the development of RNA interference-based therapeutics. Meanwhile, shRNA (short hairpin RNA) delivery technologies also gradually matured. Through plasmid or viral vectors, specific RNA sequences can be continuously expressed to achieve longer-term and more stable gene silencing.
With the development of mRNA technologies, mRNA transfection became a new research focus. Unlike DNA delivery, mRNA does not need to enter the nucleus and can directly utilize the cytoplasmic translation machinery to express target proteins. Therefore, mRNA offers rapid expression, no risk of genomic integration, and a relatively short regulatory window. mRNA transfection has gradually been applied to protein expression studies, cellular reprogramming, immune cell engineering, and RNA drug development. However, RNA molecules are inherently unstable and cells possess multiple innate immune recognition mechanisms. Therefore, RNA delivery systems need to further improve RNA stability, reduce immune stimulation, and enhance intracellular release efficiency.
This stage drove the development of new RNA transfection reagents, lipid delivery materials, and nanodelivery systems, gradually transforming transfection technology from a traditional “gene expression tool” into an important technological platform supporting RNA regulation and RNA therapeutic research. Advances in RNA delivery also laid a critical foundation for subsequent mRNA vaccines, RNA therapeutics, and gene-editing delivery systems.
Physical Transfection Technology Development Stage (1990s–Present)
Electroporation Expands Applications in Hard-to-Transfect Cells
In addition to chemical delivery systems, physical methods for nucleic acid delivery also developed rapidly during the late 20th century. Among these, electroporation is one of the most widely used and well-established physical transfection methods.
Electroporation technology originated in the 1980s. Its core principle is to apply external electric fields to generate short electrical pulses that cause transient rearrangement of the lipid bilayer of the cell membrane, creating reversible nanoscale pores in the membrane. These transient pores allow macromolecules such as DNA, RNA, proteins, and nucleic acid–protein complexes to enter cells.
Compared with chemical carrier-based transfection methods, electroporation does not require nucleic acids to form complexes with delivery materials. Therefore, it is not subject to carrier-specific structural limitations and can be applied to many cell types for which conventional chemical transfection methods provide relatively low efficiency. Electroporation is particularly advantageous for primary cells, hematopoietic cells, immune cells, and stem cells.
For example, during CAR-T cell production, researchers commonly use electroporation to deliver CAR gene constructs, CRISPR/Cas gene-editing components, or mRNA molecules to engineer immune cell functions. Electroporation is also widely used in gene knockout, gene knock-in, and cell therapy-related research.
With advances in electrophysiology and micro/nanofabrication technologies, conventional electroporation has continued to evolve. Modern electroporation platforms precisely control voltage, current, pulse duration, and buffer composition to improve cell viability and nucleic acid delivery efficiency. Meanwhile, emerging technologies such as single-cell electroporation, microfluidic electroporation, and nanoelectroporation have enabled more precise and controllable nucleic acid delivery.
However, electroporation still has certain limitations, including relatively high equipment costs, the need to optimize experimental parameters for different cell types, and the possibility that some cells may experience stress responses induced by electrical pulses. Current research therefore focuses primarily on improving delivery efficiency, reducing cellular damage, and further expanding applications in cell therapy and gene editing.
Gene Editing and Precision Nucleic Acid Delivery Stage (2010–Present)
CRISPR, mRNA, and LNP Drive the Evolution of Transfection Technology
Since 2010, rapid advances in CRISPR/Cas gene editing, mRNA technologies, and nanodelivery systems have brought transfection technology into a new stage characterized by precision, multifunctionality, and clinical translation. Traditional transfection technologies primarily focused on introducing DNA or RNA into cells and achieving gene expression, whereas modern gene delivery systems need to meet more complex requirements, including efficient delivery of CRISPR/Cas editing components, precise regulation of RNA expression, and safe delivery of therapeutic nucleic acid molecules.
The emergence of CRISPR/Cas systems has greatly accelerated the development of gene editing. Compared with traditional gene-editing methods, CRISPR technology offers simple design, high editing efficiency, and broad applicability. However, its application is highly dependent on delivery technologies capable of accurately introducing Cas nucleases, guide RNA (single guide RNA, sgRNA), Cas9 mRNA, or Cas9 protein–RNA complexes, also known as ribonucleoprotein (RNP) complexes, into target cells. Therefore, transfection technology has become one of the key factors limiting the efficiency and safety of gene editing. Currently, lipid-based delivery, electroporation, and viral vector delivery are all widely used in CRISPR-related research.
Meanwhile, the development of lipid nanoparticles (LNPs) has become an important breakthrough in the field of nucleic acid delivery. Unlike conventional cationic lipid systems, LNPs typically use ionizable lipids as core components. These lipids exhibit relatively low toxicity under physiological conditions while facilitating nucleic acid encapsulation and release under acidic formulation or endosomal conditions. Typical LNP systems consist of ionizable lipids, helper lipids, cholesterol, and polyethylene glycol (PEG) lipids. By optimizing the ratios of different components, researchers can improve nucleic acid stability, circulation properties, and intracellular release efficiency.
The development of LNP technology has helped advance RNA delivery from laboratory research toward clinical applications. mRNA vaccines and RNA therapeutics based on LNP delivery platforms have demonstrated the clinical feasibility of nucleic acid delivery technologies and have further accelerated the development of gene therapy, immunotherapy, and personalized medicine.
In the future, transfection technology will continue to evolve toward targeted delivery, intelligent stimulus-responsive delivery, low-immunogenicity materials, and AI-assisted design, providing more efficient and safer next-generation gene delivery solutions for precision medicine and cell engineering.
References
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[3] Boussif O, Lezoualc’h F, Zanta MA, et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proceedings of the National Academy of Sciences USA. 1995;92(16):7297-7301.
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