Formulation & Performance
The 2020 study progressed from a 24-lipid Jurkat reporter screen to primary human T-cell delivery and functional leukemia-cell coculture experiments.
C14-4 in Billingsley et al., Nano Letters (2020). Crude and purified materials and luciferase versus CAR cargos are kept separate.
Formulation components and molecular-weight conversion
| Component | DC Chemicals product | Molecular weight | Molar ratio | Mass at 1 µmol total lipid (mg) |
|---|---|---|---|---|
| C14-4 | DC59010 | 1365.3 | 35 | Not calculable from disclosed data |
| DOPE (as abbreviated in the source) | Not uniquely mapped | Not established | 16 | Not calculable from disclosed data |
| Cholesterol | DCD-041 | 386.65 | 46.5 | Not calculable from disclosed data |
| C14-PEG (exact anchor unverified) | Not uniquely mapped | Not established | 2.5 | Not calculable from disclosed data |
Reported molar composition 35:16:46.5:2.5. The source expands DOPE inconsistently: dioleoyl in Results and distearoyl in Methods. The exact PEG anchor is not verified. N/P and lipid:RNA mass ratio were not verified; do not assume 10:1 or substitute DMG-PEG2000.
Component mass calculator
The source does not disclose all inputs; missing fields are intentionally blank. Enter your own values to calculate a research scenario. Molecular weights and ratios are editable. Results are calculated, not measured study data. No N/P or RNA dose is inferred.
| Component | MW | Molar ratio | Mass (mg) | Mass % |
|---|---|---|---|---|
| C14-4 | ||||
| DOPE (as abbreviated in the source) | ||||
| Cholesterol | ||||
| C14-PEG (exact anchor unverified) |
Cargo-specific particle properties
| Luciferase LNP | Diameter (nm) | PDI | Encapsulation (%) | Apparent pKa | mRNA (ng/µL) |
|---|---|---|---|---|---|
| Crude C14-4 | 70.17 ± 0.41 | 0.176 ± 0.006 | 92.53 | 6.143 | 35.6 ± 2.12 |
| Purified C14-4 | 65.19 ± 0.83 | 0.189 ± 0.014 | 86.3 | 6.505 | 29.8 ± 2.31 |
SI Table S3. Diameter, PDI and mRNA concentration are reported mean ± SD. DLS measurements were performed in triplicate. These are luciferase formulations; the EE/pKa values must not be relabelled as CAR formulation measurements.
CAR-mRNA particle properties
| CAR mRNA formulation | Diameter (nm) | PDI | mRNA (ng/µL) |
|---|---|---|---|
| Crude C14-4 | 70.17 ± 0.41 | 0.176 ± 0.006 | 35.38 ± 1.94 |
| Purified C14-4 | 65.19 ± 0.83 | 0.189 ± 0.014 | 29.28 ± 1.14 |
Main Figure 5C, visually reviewed. Values are reported mean ± SD. Encapsulation efficiency and apparent pKa are not provided in this CAR-specific table.
Primary human T-cell reporter response
| Experiment | Observation | Source |
|---|---|---|
| Crude C14-4 luciferase mRNA; 75–450 ng per 60,000 activated T cells; 24 h; n=3 biological replicates | Dose-dependent reporter expression; normalized to the 75 ng group. Viability declines at higher exposures. | Figure 4A |
| Crude versus purified C14-4; 300 and 450 ng per 60,000 activated T cells; n=3 biological replicates | Purified C14-4 increased reporter expression at both tested doses (paired t-test, p<0.05), without a significant additional loss of viability. | Figure 4C |
| Jurkat luciferase kinetics monitored through 96 h | Main text reports a 23% decline after 48 h, supporting transient rather than stable expression. | Figure 3D and main text |
Doses read directly from Figure 4 axes; response bar heights have not been digitized into new numerical fold-change claims. These reporter doses do not resolve the separately printed CAR-dose denominator.
Cell delivery and CAR-T activity
| Model / cargo | Context | Finding | Source |
|---|---|---|---|
| Jurkat cells / luciferase mRNA | 30 ng per 60,000 cells; 48 h; n=4 biological replicates | C14-4 led the 24-lipid screen; reporter expression exceeded MessengerMAX under the matched assay. | Fig.3 |
| Primary human T cells / CD19 CAR mRNA | Activated CD4:CD8 cells, 1:1; ex vivo delivery | Purified C14-4 produced CAR expression comparable to electroporation; approximate normalized MFI increases 9.4-fold and 10-fold, respectively. Crude C14-4 gave 4.5-fold. | Fig.5A–B and main text |
| Primary human T-cell viability | CD19 CAR delivery; n=3 biological replicates | Purified C14-4 76%; crude C14-4 78%; electroporation 31%. | Fig.5D and text |
| CAR-T / Nalm-6 coculture | 48 h; E:T 0.5, 0.25, 0.125, 0.0625 and 0; n=3 wells | Purified C14-4 and electroporation generated comparable cancer-cell killing in this coculture model. | Fig.5E |
Representative flow-plot MFIs are not replicate means. The paper’s CAR dose is printed as 450 ng/µL with an unclear denominator; no converted per-cell dose is provided.
Interpretation and tolerability
| Evidence | Interpretation |
|---|---|
| Human cells ex vivo | The main strength is retained CAR-mediated killing with less loss of cell viability than the electroporation comparator. |
| Animal evidence | No animal biodistribution, tumor-control or tolerability results were established in this 2020 study. |