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Lipofectamine
Lipofectamine
Lipofectamine
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Lipofectamine or Lipofectamine 2000 is a common transfection reagent, produced and sold by Invitrogen, used in molecular and cellular biology.[1] It is used to increase the transfection efficiency of RNA (including mRNA and siRNA) or plasmid DNA into in vitro cell cultures by lipofection.[1] Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the transfection payload, e.g. DNA plasmids.

Lipofectamine consists of a 3:1 mixture of DOSPA (2,3‐dioleoyloxy‐N‐ [2(sperminecarboxamido)ethyl]‐N,N‐dimethyl‐1‐propaniminium trifluoroacetate) and DOPE,[2] which complexes with negatively charged nucleic acid molecules to allow them to overcome the electrostatic repulsion of the cell membrane.[3] Lipofectamine's cationic lipid molecules are formulated with a neutral co-lipid (helper lipid).[3] The DNA-containing liposomes (positively charged on their surface) can fuse with the negatively charged plasma membrane of living cells, due to the neutral co-lipid mediating fusion of the liposome with the cell membrane, allowing nucleic acid cargo molecules to cross into the cytoplasm for replication or expression.[3]

In order for a cell to express a transgene, the nucleic acid must reach the nucleus of the cell to begin transcription. However, the transfected genetic material may never reach the nucleus in the first place, instead being disrupted somewhere along the delivery process.[3] In dividing cells, the material may reach the nucleus by being trapped in the reassembling nuclear envelope following mitosis.[3] But also in non-dividing cells, research has shown that Lipofectamine improves the efficiency of transfection, which suggests that it additionally helps the transfected genetic material penetrate the intact nuclear envelope.[3]

This method of transfection was invented by Dr. Yongliang Chu.[4]

See also

[edit]

References

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Lipofectamine

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from Grokipedia
Lipofectamine is a family of proprietary cationic lipid-based reagents developed and commercialized by (now ) for the efficient delivery of nucleic acids, such as DNA, siRNA, and mRNA, into eukaryotic cells. Launched in 1993 as a first-generation product, it builds on the foundational lipofection technology pioneered by Philip L. Felgner and colleagues in 1987, which introduced synthetic cationic lipids for non-viral gene transfer. The core mechanism of Lipofectamine involves the formation of lipoplexes—electrostatic complexes between the positively charged and negatively charged nucleic acids—that promote cellular uptake primarily through , followed by endosomal escape and release into the for subsequent nuclear translocation or . The original formulation consists of a 3:1 (w/w) mixture of the polycationic lipid 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and the helper lipid dioleoyl (DOPE), enabling high efficiency in a variety of cell lines with relatively low . Over time, the Lipofectamine portfolio has evolved to address broader applications and challenging cell types. Lipofectamine 2000, an improved second-generation reagent, enhances compatibility with both adherent and suspension cells, supports co-transfection of siRNA and DNA, and performs effectively in serum-containing media without requiring media changes, making it ideal for high-throughput RNAi and studies. Lipofectamine 3000 represents the latest advancement, incorporating lipid nanoparticle technology and an optional P3000 enhancer reagent to achieve superior rates (up to 75% in difficult cells like primary neurons), reduced toxicity, and versatility for tools such as /, while maintaining reproducibility across established cell lines, stem cells, and hard-to-transfect primary cells. Specialized variants, like Lipofectamine Stem, further optimize delivery for human pluripotent stem cells in workflows. Widely adopted in molecular and , Lipofectamine reagents have been cited in tens of thousands of publications for applications including transient and stable , , , and therapeutic delivery research. Their non-viral nature offers advantages over viral vectors, such as ease of use, scalability, and avoidance of , though optimization is often required for specific cell types and cargos.

Development

Invention

Lipofectamine was developed by researchers at , Inc., in the early 1990s as a cationic lipid-based system designed for the delivery of nucleic acids into eukaryotic cells. It was first described by P. Hawley-Nelson, V. Ciccarone, G. Gebeyehu, J. Jessee, and P.L. Felgner in a 1993 publication. This innovation emerged at Life Technologies, Inc., where researchers developed formulations using polycationic lipids to form liposomes that complex with DNA or RNA, enabling efficient cellular uptake without viral vectors. The development of Lipofectamine addressed key limitations of earlier non-viral transfection methods, such as calcium phosphate co-precipitation, which was introduced in the 1970s but suffered from inconsistent efficiency, high variability due to pH sensitivity, and potential cytotoxicity in sensitive cell types. Unlike electroporation, which required expensive equipment and often caused cell membrane damage, Lipofectamine offered a simpler, reagent-based approach for broad applicability in eukaryotic cells, prioritizing non-viral safety and reproducibility. The composition of Lipofectamine, including the 3:1 (w/w) of DOSPA and DOPE, was first detailed in the 1993 publication. Early demonstrations in the highlighted Lipofectamine's superior performance compared to prior liposomal systems like Lipofectin, as described in initial studies and technical reports. For instance, the 1993 technical report described its formulation as a 3:1 (w/w) providing markedly improved gene expression levels over prior liposomal systems like Lipofectin.

Commercialization

Lipofectamine was introduced by in 1993 as a ready-to-use cationic lipid-based designed to simplify delivery into eukaryotic cells. This launch marked a significant advancement in providing researchers with an efficient, off-the-shelf solution for , eliminating the need for custom preparation. Initial marketing efforts by Invitrogen positioned Lipofectamine as a gold standard for in vitro transfection in molecular biology laboratories, with detailed protocols released in the mid-1990s to guide its use in DNA and RNA delivery experiments. These protocols emphasized its broad applicability across cell types, contributing to its rapid integration into standard lab workflows. Invitrogen's product portfolio, including Lipofectamine, underwent significant corporate changes through mergers and acquisitions. In 2000, Invitrogen acquired the original Life Technologies, though the Lipofectamine brand remained under Invitrogen's development. A pivotal merger occurred in 2008 when Invitrogen combined with Applied Biosystems to form Life Technologies Corporation, bringing Lipofectamine under the new entity's umbrella. In 2014, Thermo Fisher Scientific acquired Life Technologies for $13.6 billion, integrating Lipofectamine into its broader life sciences solutions and rebranding the parent company while retaining the product's legacy. By the late , Lipofectamine had achieved widespread adoption in academic and research, becoming a staple cited in thousands of publications for its reliable performance in studies. This early solidified its role as an essential tool, with over tens of thousands of citations accumulated since its introduction, reflecting its enduring influence on methodologies.

Composition

Original Formulation

The original formulation of Lipofectamine consists of a 3:1 (w/w) liposome mixture of the polycationic DOSPA, chemically known as 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate, and the neutral co-lipid DOPE, or dioleoylphosphatidylethanolamine. This reagent is supplied as a 2 mg/mL solution in membrane-filtered . Upon dilution for use, the solution forms milky suspensions characteristic of liposome aggregation into multilamellar vesicles. For optimal stability, the original Lipofectamine is recommended to be stored at 4°C in a sealed , where it remains viable for up to 12 months without freezing.

Key Components

Lipofectamine's formulation primarily consists of the cationic lipid DOSPA (2,3-dioleyloxy-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium) and the helper lipid DOPE (dioleoylphosphatidylethanolamine). DOSPA serves as the key cationic component, imparting a positive charge that electrostatically binds to negatively charged nucleic acids, facilitating their condensation and packaging into lipoplexes; its headgroup enhances efficiency due to the multivalent interactions mimicking natural polyamine-DNA binding. DOPE, a neutral fusogenic , promotes destabilization and fusion by adopting an inverted hexagonal (HII) phase structure, which aids in the disruption of endosomal membranes during intracellular delivery. The between DOSPA and DOPE is critical: DOSPA enables the initial electrostatic complexation with nucleic acids, while DOPE supports subsequent endosomal escape through its phase behavior, optimizing overall performance. This combination exhibits a low cytotoxicity profile relative to earlier cationic lipids, with typical cell viability exceeding 80% in standard transfection protocols across various cell types.

Mechanism of Action

Lipoplex Formation

Lipofectamine, a cationic lipid formulation, forms lipoplexes through electrostatic interactions between its positively charged liposomes and the negatively charged phosphate backbone of anionic nucleic acids such as DNA or RNA. This binding drives the spontaneous condensation of nucleic acids into compact complexes, where the cationic headgroups of lipids like DOSPA neutralize the polyanionic charges, resulting in stable lipid-nucleic acid assemblies known as lipoplexes. These interactions are the primary force behind complex formation, enabling efficient packaging without covalent linkages. Optimal complexation requires specific ratios of Lipofectamine reagent to nucleic acid to balance charge neutralization and avoid excess free lipid, which can lead to toxicity or instability. Standard protocols recommend 2-3 µL of Lipofectamine 2000 per 1 µg of DNA for most mammalian cell lines, though this can vary from 1.5-4 µL per µg depending on cell type and nucleic acid amount; for example, in a 24-well plate, 1.0-2.5 µL is used with 0.5 µg DNA. These ratios ensure a net positive charge on the lipoplex surface, facilitating subsequent cellular interactions while promoting efficient encapsulation. The resulting lipoplexes typically exhibit a multilamellar vesicle structure, consisting of concentrically arranged bilayers sandwiching layers of nucleic acids, with overall sizes ranging from 100-300 nm in diameter. This architecture protects the encapsulated nucleic acids from degradation by extracellular nucleases, enhancing stability during delivery. analyses confirm these nanoscale dimensions, which are influenced by the lipid-to-nucleic acid ratio and preparation conditions, contributing to the complexes' ability to evade rapid clearance in biological media. In practice, lipoplex formation follows a straightforward protocol: the is diluted in serum-free medium such as Opti-MEM, combined with the solution at the optimal ratio, and incubated for 5-20 minutes at to allow complete complexation. This brief assembly step occurs in the absence of serum to prevent interference from proteins, yielding ready-to-use lipoplexes that can then be applied directly to cells. Variations in incubation time, such as up to 30 minutes in optimized formulations, further refine size uniformity and potential.

Cellular Uptake and Delivery

Lipoplexes formed by Lipofectamine are primarily internalized by cells through endocytic pathways, including clathrin-mediated and caveolae-mediated , although direct fusion with the plasma can also occur in certain cell types. This uptake process allows the lipoplexes to enter the cell without causing significant disruption initially, facilitating their to early endosomes. Unlike some other delivery systems, Lipofectamine-based lipoplexes exhibit efficient avoidance of microtubule-dependent intracellular trafficking, which may contribute to their rapid progression through the endosomal pathway. Once in the s, the acidic environment triggers endosomal escape, a critical step for releasing the into the . The helper dioleoylphosphatidylethanolamine (DOPE) in Lipofectamine formulations plays a key role here; its in the low-pH endosome promotes a transition to a hexagonal phase, destabilizing the endosomal and enabling the lipoplex to disrupt it for release. This mechanism ensures that the nucleic acids are liberated efficiently from the endolysosomal compartment, minimizing degradation by lysosomal enzymes. For DNA delivery, the released plasmid must further traffic to the nucleus for transgene expression, which typically requires cell division to allow nuclear envelope breakdown and entry during mitosis. In highly transfectable cell lines such as HEK293, this process can achieve transfection efficiencies up to 80%, highlighting the effectiveness of Lipofectamine in supportive cellular contexts. In contrast, RNA delivery—such as mRNA for translation or siRNA for gene silencing—relies solely on cytoplasmic release, as these molecules do not require nuclear entry to exert their effects.

Applications

DNA Transfection

Lipofectamine is primarily utilized for the transient or stable expression of plasmid DNA encoding reporter genes, such as green fluorescent protein (GFP) or luciferase, in mammalian cell lines to facilitate gene function analysis and protein production. This non-viral method has become a cornerstone in cell biology research, enabling the introduction of exogenous DNA without the need for viral vectors. The technique supports both short-term expression for immediate phenotypic observation and long-term integration for stable cell line generation, marking a significant advancement in non-viral gene delivery since its foundational development. Transfection efficiency with is optimized for adherent mammalian cells, such as and Chinese hamster ovary (CHO) lines, where rates typically range from 20% to 70% depending on cell type, DNA quality, and optimization parameters. High cell density at 70-90% confluence enhances uptake, with protocols recommending plating 0.5-2 × 10^5 cells per well in a 24-well plate. A standard procedure involves diluting 0.5-2 µg of plasmid DNA in serum-free medium like Opti-MEM, mixing with Lipofectamine at a 1:2 to 1:3 DNA (µg):reagent (µl) ratio, and incubating for 20 minutes to form lipoplexes before adding to cells. The complexes are typically incubated with cells for 4-6 hours in serum-free conditions, after which complete can be added to minimize while maintaining expression. Successful DNA transfection via Lipofectamine enables diverse applications, including the study of gene regulation, signaling pathways, and recombinant protein expression for biochemical assays. For instance, transient expression of luciferase reporters allows quantification of promoter activity, while stable integration in CHO cells supports high-yield protein production in biomanufacturing. This approach, pioneered in the late 1980s, revolutionized non-viral gene therapy research by providing a reproducible alternative to earlier methods like calcium phosphate precipitation, achieving 5- to over 100-fold higher efficiency in various mammalian lines.

RNA Delivery

Lipofectamine reagents facilitate the delivery of molecules into cells, enabling transient functional studies without altering the . These cationic lipid-based formulations form complexes with , promoting cellular uptake and subsequent biological activity. For (RNAi) applications, Lipofectamine RNAiMAX is particularly optimized, providing high efficiency with reduced optimization needs across diverse cell types. In siRNA and miRNA delivery, Lipofectamine enables by incorporating the RNA into the (RISC), which targets and degrades complementary mRNA. Typical protocols use siRNA concentrations of 10-50 nM, achieving 70-90% target within 24-48 hours post-transfection in mammalian cell lines. This efficiency supports precise , with minimal off-target effects when using low doses and validated siRNA designs. For mRNA delivery, Lipofectamine MessengerMAX promotes rapid in the , yielding detectable protein expression within hours and avoiding genomic integration risks associated with DNA vectors. Doses of 0.5-2 μg mRNA per well in 96-well plates often result in robust, homogeneous expression suitable for protein function studies. This approach has been instrumental in development, where mRNA in antigen-presenting cells simulates immune responses for preclinical evaluation. RNA delivery with Lipofectamine generally exhibits lower toxicity compared to DNA transfection, as it bypasses nuclear entry and reduces cellular stress, with cell viability often exceeding 80-90% at optimal conditions. Reagents like RNAiMAX are formulated for minimal , making them suitable for sensitive applications. They are particularly effective in hard-to-transfect cells, such as primary neurons, where efficiencies reach up to 83% for siRNA uptake with low neuronal damage. Key applications include for function, where Lipofectamine supports automated RNAi assays in 96- or 384-well formats to identify essential or drug targets. Additionally, it enables CRISPR-Cas9 ribonucleoprotein (RNP) delivery, with Lipofectamine CRISPRMAX achieving editing efficiencies of 55-85% in various cell lines, facilitating precise modifications without persistent expression.

Variants

Lipofectamine 2000

Lipofectamine 2000 was introduced in the early 2000s by (now part of ) as an advanced cationic lipid-based reagent, offering enhanced efficiency and reduced compared to the original Lipofectamine formulation. This variant was designed to address limitations in transfecting challenging cell types, such as primary and post-mitotic cells, while maintaining a straightforward, serum-compatible protocol that minimizes the need for specialized media or additives. Its development marked a significant step forward in non-viral tools, enabling broader applicability in research. The formulation of Lipofectamine 2000 consists of a proprietary blend of cationic lipids, which carry positively charged head groups and hydrophobic hydrocarbon tails, combined with neutral helper lipids that promote fusogenic properties to facilitate endosomal escape. These components self-assemble into unilamellar liposomes approximately 100 nm in diameter when prepared in aqueous solutions. The reagent is supplied as a 1 mg/mL solution in water, stable at 2–8°C, and forms lipoplexes with nucleic acids through electrostatic interactions between the cationic lipids and the negatively charged phosphate backbone of DNA or RNA. This composition enhances stability and cellular interaction while reducing aggregation issues seen in earlier reagents. In terms of performance, Lipofectamine 2000 demonstrates up to 10-fold higher transfection efficiency than the original Lipofectamine in primary cells, including post-mitotic neurons, where it achieves 20–30% transfection rates compared to less than 3% previously. It is particularly effective for delivering both DNA and siRNA, supporting gene expression studies and RNA interference applications with low toxicity across a wide range of adherent mammalian cell types. Additionally, its compatibility with high-throughput formats, such as 96-well plates, allows for scalable transfections in multi-well assays without compromising efficiency or cell viability. A seminal 2004 study validated these enhancements, demonstrating Lipofectamine 2000's efficacy in primary rat hippocampal and cortical neurons, where it enabled robust expression and siRNA-mediated knockdown in 96-well formats suitable for . The research highlighted its utility in challenging models like primary hepatocytes, underscoring improvements in nuclear delivery and overall success rates.

Lipofectamine 3000 and Later Versions

Lipofectamine 3000, introduced in 2014 by , represents a significant advancement in reagents through its formulation of advanced nanoparticles combined with the P3000 enhancer . This combination enables superior delivery of plasmid DNA, achieving over 70% efficiency in challenging cell types, including induced pluripotent stem cells (iPSCs) and other hard-to-transfect lines, while maintaining high cell viability. Lipofectamine RNAiMAX is a specialized variant optimized for applications, particularly the delivery of (siRNA) and (miRNA) mimics or inhibitors. It provides high efficiency across a wide range of cell types with minimal , which supports the use of lower concentrations to reduce potential off-target effects associated with RNA knockdown experiments. Lipofectamine MessengerMAX, launched in late 2014, is tailored for mRNA and delivers exceptional protein expression levels in primary cells and neurons, often outperforming traditional DNA-based methods by up to fivefold in these sensitive cell types. This reagent facilitates rapid, transient without genomic integration, making it ideal for studying protein function in hard-to-transfect primary cultures. Among later variants, Lipofectamine CRISPRMAX, developed around 2016, enhances the delivery of ribonucleoprotein (RNP) complexes for CRISPR-Cas9 , offering improved efficiency in diverse mammalian cell lines compared to earlier lipid formulations. Lipofectamine Stem, introduced in 2018, is optimized for transfecting pluripotent stem cells with minimal differentiation, supporting workflows in . Overall, these post-2014 iterations reflect a broader trend in Lipofectamine development toward formulations with lower toxicity profiles and expanded compatibility across cell types, including primary and stem cells, to support advanced genomic and transcriptomic .

Advantages and Limitations

Benefits

Lipofectamine reagents demonstrate high transfection efficiency, typically achieving 50-90% in standard cell lines such as HEK 293 and , with Lipofectamine 3000 reaching over 70% in many cases. This efficiency is particularly notable in hard-to-transfect cells, where it can be up to 10-fold higher than earlier formulations like Lipofectamine 2000, while maintaining superior cell viability compared to methods that often compromise survival due to physical stress on cells. The versatility of Lipofectamine extends to a broad spectrum of nucleic acids, including plasmid DNA, mRNA, siRNA, and genome-editing tools like CRISPR-Cas9 and TALENs, enabling effective delivery across diverse applications. It performs reliably in a wide array of cell types, from common immortalized lines like CHO and Jurkat to challenging primary cells such as neurons, stem cells, and hepatocytes, without requiring cell-specific optimizations in many protocols. Lipofectamine offers exceptional ease of use through a straightforward, non-viral protocol that involves simple mixing of with nucleic acids and addition to cells, requiring no specialized beyond standard lab incubators. This approach ensures high reproducibility and scalability, supporting in multi-well formats for rapid experimental workflows. As a commercially available product from , Lipofectamine is cost-effective, with reaction costs as low as pennies and a of up to 12 months when stored properly at 4°C, facilitating accessible and efficient in research settings worldwide. Its widespread adoption underscores its role in enabling quick, reliable s without the need for custom synthesis or extensive preparation.

Drawbacks

Lipofectamine exhibits significant cell-type dependency, with efficiencies often below 20% in challenging cell types such as non-adherent cells, primary immune cells, and mesenchymal stem cells (MSCs), necessitating extensive protocol optimization for each application. For instance, in adipose-derived MSCs, optimized conditions achieved only 23.75% , while unoptimized protocols yield even lower results, highlighting the reagent's variable performance across cell lines and primary cultures. This dependency arises from differences in properties and uptake mechanisms, limiting its broad applicability without tailored adjustments. Toxicity represents a key limitation of Lipofectamine, manifesting as dose-dependent that compromises cell viability, particularly at higher concentrations required for efficient . In various cell types, including cells and endothelial cells, elevated doses (e.g., >2 µL of Lipofectamine LTX or 6 µL of Lipofectamine 2000) lead to reduced viability ranging from 20-55%, with effects including membrane disruption and that persist post-transfection. This can confound experimental outcomes by altering cell health and profiles, often requiring careful dose to balance delivery efficacy and survival. The transient nature of Lipofectamine-mediated further restricts its utility, as expression typically peaks within 12-48 hours and declines significantly over days to weeks, rendering it unsuitable for applications demanding , long-term genetic modifications without additional selection strategies. In primary cells like MSCs, expression levels drop markedly by 48 hours post-, limiting studies to short-term effects unless integrated with integration methods. Due to these drawbacks, alternatives such as viral vectors are preferred for applications owing to their higher and stability, while or nucleofection offer superior performance in primary and hard-to-transfect cells, achieving up to 90% with reduced chemical despite some (35-45%). Later variants like Lipofectamine 3000 have partially addressed through formulation improvements, but cell-type challenges persist.

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