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Lipid-based delivery

Lipid-based delivery

Cationic and ionisable lipids condense nucleic acids into lipoplexes that dock onto the anionic cell surface, enter by endocytosis and release their cargo when the lipid chemistry disrupts the endosomal membrane. It is the default route for most adherent cell lines, and the fastest way to a working protocol.

9
Candidates profiled with manufacturer protocols
4
Manufacturers whose documentation was used
13
References listed at the end of this page
How it works

From the tube to the target

  1. 1

    Complexation

    Cationic lipid headgroups neutralise the phosphate backbone, collapsing plasmid DNA into a 100–400 nm particle. Neutral helper lipids such as DOPE or cholesterol are added to control lamellarity and improve fusion with the endosomal membrane.

  2. 2

    Uptake

    The positively charged particle binds proteoglycans and is internalised mainly by clathrin-mediated endocytosis within 30–60 minutes of addition.

  3. 3

    Endosomal escape

    Ionisable lipids are neutral at pH 7.4 and become protonated below pH 6.5, adopting a cone shape that destabilises the endosomal bilayer. This is the step that separates good lipid reagents from mediocre ones.

  4. 4

    Cytoplasmic and nuclear fate

    siRNA and mRNA act directly in the cytosol. Plasmid DNA must reach the nucleus, which happens efficiently only around mitosis — hence the 48–72 h wait for peak expression.

FamilyRepresentative productsFormat bias
Lipopolyamine / cationic lipidLipofectamine 2000, 3000, X-tremeGENE HPPlasmid DNA and general expression
RNA-optimised lipidLipofectamine RNAiMAX, INTERFERinsiRNA and miRNA, reverse transfection
Ionisable LNP lipidLipofectamine MessengerMAX, TransIT-mRNAmRNA, RNP, sensitive cells
Lipid–polymer hybridFuGENE HD, Effectene, ViaFectDifficult-to-transfect adherent lines, serum tolerance
Non-liposomal / histidineEffectene enhancer systemAdherent cells where lipids fail
9 candidates

Reagents and their manufacturer protocols

Each entry below gives the chemistry, the applications the manufacturer validates it for, the dosing basis and the step-by-step protocol. Scale factors follow the manufacturer’s own tables wherever they publish them.

Candidate 01

Lipofectamine 2000

Thermo Fisher / Invitrogen

11668019
Chemistry
Lipopolyamine cationic lipid blend
Best for
General-purpose DNA transfection of HEK293, HeLa, CHO-K1, COS-7; the de facto reference reagent in the literature

Dosing basis: Optimise the lipid:DNA ratio; the vendor's 24-well starting point is 0.8 µg DNA with 2 µL reagent, with two doses tested side by side.

Manufacturer protocol — step by step

  1. 1

    Dilute 0.8 µg plasmid DNA in 50 µL Opti-MEM; dilute 2 µL Lipofectamine 2000 in 50 µL Opti-MEM in a second tube. Run a second condition at 1 µL as well.

  2. 2

    Incubate both dilutions separately at room temperature for 5 min.

  3. 3

    Combine the two dilutions and mix gently by inversion or light tapping — never vortex.

  4. 4

    Incubate the complexes for 20 min at room temperature; the solution should be slightly turbid, not cloudy.

  5. 5

    Add the 100 µL complex to cells in 500 µL medium (fresh or overnight-conditioned) and rock the plate gently.

  6. 6

    Replace with complete medium after 4–6 h if the cells are sensitive to serum-free exposure.

  7. 7

    Assess expression at 24–48 h by fluorescence or reporter assay; for stable lines, split 1:10 at 24 h and start selection at 48 h.

Culture vesselDNA per wellReagent per wellDiluent volume
96-well100 ng0.15–0.3 µL2 × 5 µL
24-well0.8 µg1–2 µL2 × 50 µL
6-well4 µg6–10 µL2 × 250 µL
T-75 flask20–30 µg30–50 µL2 × 1.5 mL

Protocol notes

  • Do not add antibiotics during complex formation — aminoglycosides increase apparent toxicity.
  • Serum may be present in the medium during complex addition, but complexes must be formed in serum-free diluent.
  • For maximum potency, remove the medium, rinse once with serum-free medium and overlay the complex in serum-free medium for 4–6 h before topping up with complete medium.

Source: Thermo Fisher — Lipofectamine 2000 reagent protocol

Candidate 02

Lipofectamine 3000

Thermo Fisher / Invitrogen

L3000001
Chemistry
Lipid nanoparticle formulation plus P3000 enhancer
Best for
High-efficiency plasmid DNA delivery across hard-to-transfect adherent lines; also the standard system for CRISPR plasmid work

Dosing basis: Test two lipid doses per experiment; the P3000 enhancer is fixed at 2 µL per µg DNA.

Manufacturer protocol — step by step

  1. 1

    Dilute 500 ng plasmid DNA in 25 µL Opti-MEM, then add 1 µL P3000 enhancer and mix.

  2. 2

    Dilute 0.75 µL and 1.5 µL Lipofectamine 3000 reagent separately in 25 µL Opti-MEM each — two doses for optimisation.

  3. 3

    Combine diluted DNA with each diluted lipid (1:1), mix gently and incubate 10–15 min at room temperature.

  4. 4

    Add 50 µL complex drop-wise to cells in 500 µL complete medium; serum and antibiotics may be present.

  5. 5

    Incubate at 37 °C and assay at 1–3 days. No medium change is required.

Culture vesselDNAP3000Reagent (low / high)
96-well100 ng0.2 µL0.15 / 0.3 µL
24-well500 ng1 µL0.75 / 1.5 µL
6-well2500 ng5 µL3.75 / 7.5 µL

Protocol notes

  • Cell-line variants published by the vendor: A549 uses 0.5 µg DNA with 1.5 µL reagent at 6-well scale, HepG2 and MCF7 prefer the lower reagent dose.
  • For co-transfection of two plasmids, keep the total DNA constant and split it between constructs.
  • Complexes are stable for several hours but should be used within 30 min for consistency.

Source: Thermo Fisher — Lipofectamine 3000 reagent protocol

Candidate 03

Lipofectamine RNAiMAX

Thermo Fisher / Invitrogen

13778150
Chemistry
RNA-optimised cationic lipid formulation
Best for
siRNA and miRNA knockdown in nearly all cell types, including primary cells; designed for reverse transfection

Dosing basis: Very tolerant ratio — 1 µL reagent per 10 pmol siRNA in 24-well format is a typical starting point.

Manufacturer protocol — step by step

  1. 1

    Prepare the plate for reverse transfection: add 5 pmol siRNA to 50 µL Opti-MEM per 24-well.

  2. 2

    Dilute 0.5 µL RNAiMAX in 50 µL Opti-MEM and combine with the siRNA dilution; mix gently.

  3. 3

    Incubate 10–20 min at room temperature to form the RNA–lipid complex.

  4. 4

    Add trypsinised cells in 500 µL complete medium directly onto the complex; do not remove the complex.

  5. 5

    Assess transcript knockdown at 24–48 h and protein knockdown at 48–72 h.

Protocol notes

  • The high tolerance to reagent excess makes RNAiMAX the most forgiving lipid reagent in the catalogue for RNA.
  • Freeze–thaw cycles degrade performance; aliquot on first use.
  • For suspension cells, forward transfection with 1.5 µL reagent and 20 pmol siRNA per 24-well is more reliable.

Source: Thermo Fisher — Lipofectamine RNAiMAX protocol

Candidate 04

Lipofectamine MessengerMAX

Thermo Fisher / Invitrogen

LMRNA003
Chemistry
Ionisable lipid nanoparticle formulation for RNA
Best for
mRNA and Cas9 mRNA delivery, including primary neurons and hard-to-transfect cells; also supports RNP formats

Dosing basis: Scale by surface area; a 24-well starting point is 500 ng mRNA with 1.5 µL reagent.

Manufacturer protocol — step by step

  1. 1

    Dilute 500 ng mRNA in 25 µL Opti-MEM.

  2. 2

    Dilute 1.5 µL MessengerMAX in 25 µL Opti-MEM, then combine and mix gently.

  3. 3

    Incubate 10–15 min at room temperature — do not exceed 30 min.

  4. 4

    Add 50 µL drop-wise to cells in 500 µL complete medium; serum is compatible.

  5. 5

    Detect protein expression at 6–24 h; wash and refresh medium after 4 h if toxicity is observed.

Protocol notes

  • Tune the mRNA dose first, then the reagent. Above ~1 µg per 24-well, innate immune sensing in primary cells rises sharply.
  • Use capped, poly(A)-tailed mRNA with a 5′ untranslated region validated for the cell type; base-modified nucleotides reduce innate activation.
  • For RNP: complex Cas9 protein with sgRNA first, then mix with the lipid reagent.

Source: Thermo Fisher — Lipofectamine MessengerMAX

Candidate 05

X-tremeGENE HP DNA

Roche / Sigma-Aldrich

6366236001
Chemistry
Non-liposomal lipid blend in a proprietary formulation
Best for
Broad mammalian host range including CHO and HEK293 bioproduction lines; serum- and antibiotic-compatible

Dosing basis: Ratio of reagent (µL) to DNA (µg) between 1:1 and 6:1. Test 2:1 and 3:1 for most lines.

Manufacturer protocol — step by step

  1. 1

    Dilute the chosen DNA amount in 100 µL serum-free medium per 24-well.

  2. 2

    Add X-tremeGENE HP at the chosen ratio (start 3 µL per µg DNA) directly into the medium — do not touch the tube wall.

  3. 3

    Vortex briefly or mix by inversion, then incubate 15–20 min at 15–25 °C.

  4. 4

    Add the complex drop-wise to cells in complete medium; no medium change is needed.

  5. 5

    Analyse expression 24–72 h after transfection; for stable pools, add selection antibiotic 24–48 h post-transfection.

Culture vesselDNASerum-free diluentReagent at 3:1
96-well100 ng10 µL0.3 µL
24-well1 µg100 µL3 µL
6-well2 µg200 µL6 µL

Protocol notes

  • Sensitive to over-dosing: above ~6:1 the complex becomes visibly toxic in most lines.
  • Works reliably in CHO-K1 and CHO-S, where many liposomal reagents underperform.

Source: Roche / Sigma-Aldrich — X-tremeGENE HP DNA transfection protocol

Candidate 06

X-tremeGENE 9 DNA

Roche / Sigma-Aldrich

6365779001
Chemistry
Multi-component lipid–polymer blend
Best for
One-reagent compatibility with DNA, siRNA and co-transfection; a good default when only a single reagent budget is available

Dosing basis: 1:1 to 6:1 reagent-to-DNA; 3:1 is the vendor's recommended start.

Manufacturer protocol — step by step

  1. 1

    Dilute DNA in 100 µL Opti-MEM per 24-well (1 µg is typical).

  2. 2

    Add X-tremeGENE 9 at a 3:1 ratio directly into the medium.

  3. 3

    Incubate 15–20 min at room temperature.

  4. 4

    Add drop-wise to cells in complete medium; antibiotics and serum may be present.

  5. 5

    For siRNA co-delivery with DNA, keep the reagent fixed and add siRNA to the same dilution.

Protocol notes

  • Less efficient than a dedicated DNA reagent for stable-line work, but simpler to schedule.
  • Compatible with the same dilution protocol for DNA and siRNA, so dual experiments need only one optimisation.

Source: Roche / Sigma-Aldrich — X-tremeGENE 9 DNA transfection reagent

Candidate 07

Effectene

Qiagen

301425
Chemistry
Non-liposomal lipid (Effectene) with Enhancer and DNA-condensation buffer
Best for
Adherent cells including primary fibroblasts and endothelial cells; high efficiency with low lipid dosage

Dosing basis: Vendor recommends 1 µg DNA per 60 mm dish with 25 µL Enhancer and 10 µL Effectene — a better ratio than most liposomal reagents.

Manufacturer protocol — step by step

  1. 1

    Mix 1 µg DNA with DNA-condensation buffer to 100 µL total, then add 8 µL Enhancer and vortex 10 s.

  2. 2

    Incubate 2–5 min at room temperature.

  3. 3

    Add 25 µL Effectene and mix by vortexing 10 s.

  4. 4

    Incubate 5–10 min at room temperature to form the lipoplex.

  5. 5

    Add 600 µL complete medium to the tube, mix by pipetting, and transfer the whole volume onto cells that have been washed once with PBS.

  6. 6

    Incubate 5–6 h, then rinse and feed with complete medium; assay at 24–72 h.

Culture vesselDNAEnhancerEffectene
24-well0.2 µg1.6 µL2 µL
12-well0.4 µg3.2 µL4 µL
60 mm dish1 µg8 µL10 µL
6-well1 µg8 µL25 µL

Protocol notes

  • Effectene is a non-liposomal lipid, so it performs differently from Lipofectamine: expect lower peak expression but far lower toxicity.
  • The enhancer step is essential — omitting it collapses efficiency by an order of magnitude.
  • Wash cells before adding the complex; residual serum markedly inhibits the Effectene lipoplex.

Source: Qiagen — Effectene transfection reagent protocol

Candidate 08

FuGENE HD

Promega

E2311
Chemistry
Non-liposomal multi-component lipid formulation
Best for
Difficult-to-transfect adherent lines, primary cells and stem cells; excellent serum and antibiotic tolerance

Dosing basis: 3:1 reagent-to-DNA is the vendor's default; range 2:1 to 6:1.

Manufacturer protocol — step by step

  1. 1

    Equilibrate nucleic acid and reagent to room temperature.

  2. 2

    Add 1 µg DNA to 100 µL serum-free medium per 24-well.

  3. 3

    Add 3 µL FuGENE HD directly into the liquid — avoid the tube wall.

  4. 4

    Mix briefly (vortex is acceptable) and incubate 10–15 min at room temperature.

  5. 5

    Add drop-wise to cells; serum and antibiotics may be present throughout.

  6. 6

    Assay at 24–48 h; replace medium after 24 h for long-term experiments.

Protocol notes

  • The most forgiving lipid-class reagent for serum-sensitive primary cultures.
  • Its low toxicity suits multi-day protocols such as differentiation experiments.

Source: Promega — FuGENE HD transfection reagent protocol

Candidate 09

ViaFect

Promega

E4981
Chemistry
Lipid formulation optimised for high-throughput and 96-well work
Best for
Automated and small-volume transfection where robustness across plates matters more than peak expression

Dosing basis: 3:1 reagent-to-DNA with the same dilution scheme as FuGENE HD.

Manufacturer protocol — step by step

  1. 1

    Dilute 100 ng DNA in 10 µL serum-free medium per 96-well.

  2. 2

    Add 0.3 µL ViaFect and mix.

  3. 3

    Incubate 10–15 min at room temperature.

  4. 4

    Add 10 µL drop-wise to cells in 100 µL medium; no medium removal required.

  5. 5

    Assay at 24–48 h.

Protocol notes

  • Manufactured without animal-derived components, which simplifies documentation for bioproduction workflows.

Source: Promega — ViaFect transfection reagent protocol

Cross-cutting rules

What applies to every candidate on this page

  • Complex formation is chemistry, not plumbing: hold the incubation time and temperature constant across every condition.
  • Never vortex lipoplexes unless the vendor's protocol explicitly allows it; shear destroys the particle.
  • Serum-free diluent is required for complex formation even when serum is present during exposure.

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