Co-dissolve the drug with lipids in an organic solvent, remove solvent completely, then form and size liposomes, and purify to remove free/crystalline drug.

Typical starting screen: drug 1–5 mol% of total lipid; hydrate ≥10 °C above the highest lipid Tm; cholesterol 20–45 mol% (optimize for stability vs loading); finish with SEC/TFF/dialysis to remove free drug/solvent.

Common methods

· Thin-film hydration: lipids + drug in CHCl₃:MeOH (2:1 v/v) → rotavap to dryness → high-vac ≥1–2 h above Tm → hydrate above Tm (optionally add 5–10 freeze–thaw cycles) → extrude (e.g., 100 nm, ≥10 passes) → purify.

· Ethanol injection / microfluidics: lipids + drug in ethanol (or IPA) mixed into buffer above Tm. At the mixing point, ethanol is typically ~20–40% v/v; immediately dilute and remove solvent by TFF/dialysis to ≤1% v/v; purify.

· Passive incubation (co-solvent-assisted): add drug stock in ethanol or DMSO to preformed liposomes above Tm; keep final co-solvent ≤2–5% v/v; incubate 15–60 min; remove solvent/free drug by SEC/dialysis/TFF.

Key checks

· Size/PDI (DLS): after sizing and after purification (target PDI ≤0.15–0.2).

· Drug-to-lipid ratio: disrupt vesicles (e.g., MeOH/IPA or ACN), quantify drug by HPLC/UV or LC-MS, and measure lipid (phosphate assay or HPLC-ELSD). Report mol drug per mol lipid and overall recovery.

· Free drug/crystals: visual/microscopy check; verify removal by SEC profile or filtration.

· Zeta potential: optional but helpful for batch consistency.

· Residual solvent: confirm removal (e.g., GC), per your regulatory limits.

· Stability: 2–8 °C, protected from light; consider 37 °C serum release tests.

Pitfalls & fixes

· Precipitation during formation: lower drug mol%; raise temperature (≥Tm + 10 °C); extend incubation; add ≤2% ethanol during hydration (then remove); verify complete drying of the film.

· Poor retention / rapid release: increase cholesterol (within 20–45 mol%); switch to higher-Tm lipids (e.g., DSPC vs DOPC); reduce drug mol%; evaluate serum release.

· Batch variability: standardize vacuum time (≥1–2 h), hydration temperature, freeze-thaw count, extrusion pore size & passes, and (for microfluidics) FRR/TFR.

· High PDI/aggregation: include 1–5 mol% PEG-lipid; ensure moderate ionic strength (10–50 mM buffer); re-extrude; avoid prolonged room-temp holds.

Notes

· Remote loading via ion gradients is mainly for amphipathic weak bases; it rarely helps strictly hydrophobic drugs.

· Some hydrophobes bind plastics — use glassware and low-binding tips where possible.

· Cholesterol improves stability but can reduce loading at high % — balance empirically.

Aladdin：https://www.aladdinsci.com/