sales@dcchemicals.com
Lipid MK16
Formulation & Performance
Formulation & Performance
Study-specific compositions, preparation conditions and experimental outcomes for MK16.
Publisher supplementary information, main figure pages 3–6 and source-data workbooks for main Figs.3–6 and supplementary figures freshly recovered and checked. Particle statistics were recalculated from deposited replicates. Main-article conditions retained from the prior main/SI review are identified where the complete article could not be newly downloaded; no missing flow or buffer conditions were inferred.
1. Formulations and component amounts
Reported composition and calculated mass are separate. Molecular-weight inputs use catalog values or an explicitly identified reference average; DMG-PEG2000 is fixed initially at 2507. Edit inputs only when using a verified alternative material.
Optimized MK16 BLNP
molar parts (sum 130.75); normalized 45.8891:22.9446:30.5927:0.5736 mol%Main article formulation method (previous main-PDF review pp.2,12); Supplementary Fig.6a, SI printed p.28 / PDF p.29
| Component | Molecular weight | Molar parts | Normalized mol% | Amount (µmol) | Weight (mg) |
|---|---|---|---|---|---|
| MK16 | 45.889 | 60.2788 | 60.1486 | ||
| DOPE | 22.945 | 30.1394 | 22.4246 | ||
| Cholesterol | 30.593 | 40.1858 | 15.5379 | ||
| DMG-PEG2000 | 0.574 | 0.7535 | 1.8890 |
Calculated total: 100.0000 mg lipid. RNA, solvent and process loss are excluded.
DMG-PEG2000 calculations use the website convention 2507. Formulation masses must use normalized molar fractions, while the literature 12.5:1 loading ratio refers only to MK16:mRNA.
How component weights are calculated
For total lipid mass M (mg), molar parts rᵢ and molecular weight MWᵢ: component mass = M × rᵢ × MWᵢ / Σ(r × MW). Component amount (µmol) = component mass (mg) × 1000 / MWᵢ. Molar parts are normalized only for the displayed percentage. The calculation does not infer N/P or RNA dose.
2. Preparation and experimental setup
| Parameter | Reported procedure / limitation | Source |
|---|---|---|
| Loading ratio | MK16:mRNA = 12.5:1 by mass. This applies to MK16 alone and is not N/P or total lipid:mRNA. | Prior main-article review; Supplementary Fig.6a documents MK6E formulation selection |
| Recipe lineage | The formulation screen first varied MK6 molar parts 20, 40 and 60 while DOPE/cholesterol/PEG-lipid remained 30/40/0.75; then MK6:mRNA mass ratios 7.5, 10, 12.5 and 15 were compared. MK16 is the later structurally optimized lipid, not a synonym for MK6 or MK6E. | Supplementary Fig.6a; main Fig.3 |
| Mixing and purification | The earlier article review notes that preparation cross-references a prior method. Exact buffer composition/pH, flow settings and purification parameters for a fully reproducible MK16 preparation have not been recovered here. | Prior main Methods review; newly recovered SI does not supply a complete standalone formulation protocol |
| Particle measurements | Size and PDI, encapsulation, surface charge and cryo-EM are separately reported for MK16. Use MK16 Fig.S9 measurements rather than MK6 Fig.S5 values. | Supplementary Fig.9 and deposited Supplementary Data, sheet Fig.S9 |
3. Particle characterization
| Measurement / batch | Reported result | Evidence context |
|---|---|---|
| Hydrodynamic diameter | 137.0 ± 4.1 nm; recomputed from 133.1, 141.2 and 136.8 nm. Mean±sample SD, n=3. | Supplementary Fig.9a; Supplementary Data, Fig.S9!B2:B4 |
| PDI | 0.108 ± 0.014; recomputed mean±sample SD from 0.099, 0.101 and 0.125, n=3. | Supplementary Fig.9a; Supplementary Data, Fig.S9!C2:C4 |
| mRNA encapsulation | 84.84 ± 1.50%; mean±sample SD, n=3. | Supplementary Fig.9b; Supplementary Data, Fig.S9!B7:B9 |
| Zeta potential | +5.87 ± 1.98 mV; mean±sample SD, n=3. | Supplementary Fig.9b; Supplementary Data, Fig.S9!C7:C9 |
| Apparent particle pKa | 6.86 by the TNS assay; explicitly printed in the fitted-curve inset. | Supplementary Fig.6d, SI printed p.28 / PDF p.29 |
4. Performance by experiment
| Experiment and dose | Readout and comparator | Result and source |
|---|---|---|
| Brain reporter expression Mouse; FLuc mRNA; intravenous; 0.5 mg/kg (dose/time from prior main-article review) | Readout: 6 h Comparator: MC3 and MK6E formulations | MK16 brain signal was approximately 8.3-fold MC3 and 1.7-fold MK6E; deposited Fig.3d has n=3 per group. Separate SI8 comparator experiment gives approximately 7.4-fold ALC-0315 and 6.5-fold SM-102.Fig.3d; Source Data Fig.3, sheet Fig.3d; Supplementary Fig.8 / Data sheet Fig.S8 |
| Brain cell-type expression Mouse; GFP mRNA; intravenous; 1 mg/kg (dose/time from prior main-article review) | Readout: 12 h Comparator: PBS and MC3 at 0.5 or 1 mg/kg | GFP-positive neurons 7.36±0.78%, astrocytes 9.71±0.71%, microglia 2.85±0.44%, and brain capillary endothelial cells 9.18±0.80%. n=3; mean±sample SD recalculated from source data. These are percentages within the respective cell populations.Fig.3f; Source Data Fig.3, Fig.3f!J8:L11 |
| Fluorescent RNA tissue distribution MK16 carrying Alexa-Fluor-647-labelled RNA; intravenous; 0.5 mg/kg in the prior main-article review | Readout: 1 h Comparator: Six measured organs | Brain accounted for 15.33±0.38% of the summed measured-organ fluorescence, n=3. This is not percent injected dose and is not the percentage of functional mRNA expression in the brain.Supplementary Fig.10b; Supplementary Data, Fig.S10b!A2:A4 |
| Functional reporter recombination Ai14 mice; MK16-Cre mRNA; one versus three intravenous administrations | Readout: Endpoint timing and dose require the complete main Fig.4 protocol Comparator: MC3-Cre mRNA and PBS | Deposited Fig.4i shows neuronal tdTomato positivity 8.28±0.65% after one and 16.73±2.15% after three administrations; astrocytes 10.10±1.06% and 19.20±2.98%, respectively (n=3). Recombination accumulates and is not equivalent to instantaneous GFP transfection efficiency.Publisher Fig.4i and caption; Source Data Fig.4, sheet Fig.4i, G8:L9; SI Discussion and Fig.13 |
| Ex vivo human cortical tissue Adult human cortical tissue exposed to MK16 carrying ΔFosb mRNA | Readout: Dose and exposure duration not re-recovered; do not infer from mouse experiments Comparator: PBS | Source Fig.5e records ΔFOSB-positive neurons 4.00±0.99%, astrocytes 6.49±2.33%, and microglia 2.38±0.94%, n=4 tissue slices per group (not a verified four-donor cohort). This is ex vivo expression and does not demonstrate intravenous delivery across a human BBB. Versus PBS: neuron P=0.0004, astrocyte P=0.0020, microglia P=0.0065 by two-tailed Student’s t-test.Publisher Fig.5e and caption; SI Discussion pp.3–4; Source Data Fig.5, Fig.5e!H8:K10 |
| Glioblastoma efficacy Orthotopic U118MG mouse model; Pten mRNA; intravenous 1 mg/kg; three treatments on days 10,13,16 | Readout: Tumour imaging and survival follow-up Comparator: PBS, MK16-control mRNA; separate MC3-Pten comparison | The article reports suppression of tumour growth and approximately 70% survival beyond 120 days for MK16-Pten. The separately deposited SI19 comparison with MC3 uses n=10/group and 1 mg/kg. Exact raw survival counts should not be merged across the two experiments. Main Fig.6 caption gives n=9 PBS, n=9 control mRNA and n=10 Pten mRNA.Publisher Fig.6a,d and caption (fresh visual review); Supplementary Fig.19; Source Data Fig.6 |
| In vitro BBB transport mechanism bEnd.3 monolayer, 0.4-µm Transwell, 1×10^5 cells/well; culture 7–9 d; TEER >60 Ω·cm²; Alexa-Fluor-647 RNA | Readout: Lower-chamber fluorescence at 12 h Comparator: MK16 alone; EIPA 10 µM, MβCD 4.5 mM, CPZ 5 µg/mL or MK-0752 10 nM pretreatment | Transport decreased with MβCD and MK-0752; SI12 source data show relatively little change with EIPA/CPZ. The inhibition experiment supports pathway involvement, not an exclusive transport mechanism.SI Methods 2.3, printed p.21; Supplementary Fig.12c and Fig.S12 source data |
| Functional delivery after Transwell crossing bEnd.3 upper chamber and N2a lower chamber; N2a seeded at 1×10^5/well one day earlier; MK16-FLuc mRNA; MβCD 4.5 mM or nirogacestat 10 nM pretreatment | Readout: 18 h; Bright-Glo readout after 5 min Comparator: MK16 without inhibitor and PBS | Inhibitor-sensitive luciferase expression in both compartments supports transport followed by functional RNA delivery. Dose not specified in the recovered SI method.SI Methods 2.3; main Fig.3i source data and Supplementary Fig.12d |
| In vivo transport inhibition Mice pretreated with MβCD 100 mg/kg or nirogacestat 30 mg/kg 1 h before labelled-RNA MK16 BLNPs | Readout: Brain IVIS at 1 h after BLNP administration Comparator: MK16 without inhibitor | Both pretreatments reduced brain fluorescence. The inhibitors affect biological pathways and this experiment alone does not establish molecular specificity.SI Methods 2.3; Source Data Fig.3, Fig.3j |
| Single-dose systemic tolerability MK16-FLuc mRNA versus MC3-FLuc mRNA; intravenous 1 mg/kg | Readout: 6,24,48 h Comparator: PBS and MC3 | 32-plex cytokine/chemokine analysis, ALT, AST, BUN and H&E were assessed. The authors report transient responses with recovery and no detected hepatic/renal injury under these tested conditions; n=3, not a chronic or human safety assessment.SI Methods 2.4, pp.21–22; Supplementary Figs.14–16 |
| Repeated-dose tolerability Ai14 Cre-reporter study; single or triple systemic administrations | Readout: 5 d after final administration Comparator: PBS and MC3 | Cytokines, ALT/AST/BUN and histology were evaluated; Supplementary Fig.17 marks group comparisons nonsignificant (n=3). This later time point does not exclude earlier transient responses.SI Methods 2.4; Supplementary Fig.17, printed pp.40–41 |
Mechanistic evidence
The lipid contains an MK-0752-derived BBB-crossing headgroup. Transwell inhibition, functional reporter transport and in vivo inhibitor experiments support involvement of caveolae-associated and γ-secretase-associated pathways. These results should be distinguished from a claim of a unique receptor mechanism or clinical CNS targeting.
5. Interpretation and unresolved details
- 60:30:40:0.75 are molar parts, not percentages. Normalize before calculating component masses.
- MK6, MK6E and MK16 are distinct stages of lipid/formulation optimization; particle properties and biological results must remain assigned to the correct material.
- Brain fluorescence distribution, luciferase output, GFP-positive cell fractions and irreversible Cre reporter activation measure different phenomena and cannot be substituted for one another.
- Human cortical-slice expression is an ex vivo result. It does not establish human systemic BBB crossing.
- Complete preparation conditions and the dose/timing of the expanded Cre and human-slice experiments remain incomplete in this re-review. Results are displayed with those explicit gaps rather than filled from related experiments.
Results apply to the stated LNP composition, cargo, process, dose, route and model. LNP experimental measurements are not batch-release specifications for the isolated lipid.
6. References and data access
Wang C, Xue Y, Markovic T, et al. Blood-brain-barrier-crossing lipid nanoparticles for mRNA delivery to the central nervous system. Nature Materials. 2025;24:1653-1663.
DOI: 10.1038/s41563-024-02114-5
Download experiment table (CSV) · Open printable Formulation & Performance guide
Revision FP-20261024-b2 · Source review: 24 October 2026 · DC Chemicals