Below I’ll give practical, literature-backed guidance you can use right away: suggested ratios (relative to total lipid/niosomal lipid), pros/cons of pre- vs post-insertion, and a short stepwise protocol you can try and optimize.

Quick recommendations (what to try first)

DSPE-PEG2000-Folate (DSPE-PEG-FA): start with 0.5–1.0 mol% of the total lipid (i.e., 0.5–1.0 mole percent of all lipids/niosomal surfactant+cholesterol). This is widely used for effective targeting without excessive steric hindrance.

Background PEG (stealth) if used: if you want a “stealth” PEG layer and also include folate-PEG, use 2–5 mol% DSPE-PEG2000 (MeO) as the main PEG, and keep DSPE-PEG-FA lower (0.2–1 mol%) so folate is exposed but not overcrowded.

Mass % patents / reports vary (some report very low weight ratios like 0.05–0.15% w/w vs lecithin); those are mass ratios and not mol% — convert carefully if comparing. For reproducibility use mol% of total lipids.

Pre-insertion vs Post-insertion — which to use?

Post-insertion (recommended for niosomes/liposomes): prepare DSPE-PEG-FA micelles and incubate with preformed niosomes at a temperature above the membrane’s fluidity transition. Advantages: ligand inserts mainly into the outer leaflet (efficient surface display), preserves internal cargo, you control outer-surface ligand density, and you waste less ligand than pre-insertion. Many reports show post-insertion gives better control and similar or better targeting.

Pre-insertion (co-formulation): add DSPE-PEG-FA into the organic phase with other lipids/surfactants during film formation or micelle assembly. Simpler (one-step) and acceptable for some protocols, but part of DSPE-PEG-FA can end up on the inner leaflet or sequestered inside the vesicle bilayer (so you often need more ligand to achieve the same outer density).

Bottom line: for precise control of surface folate density and to conserve ligand, use post-insertion. If you need a single-step manufacturing method and are less concerned about ligand efficiency, pre-insertion is acceptable.

Practical stepwise protocol (post-insertion) — a starting point

1. Make your niosomes by your chosen method (thin film hydration, reverse phase, microfluidics, etc.). Characterize size & zeta.

2. Prepare DSPE-PEG2000-FA micelles: dissolve DSPE-PEG-FA in a small volume of ethanol or PBS (warm) to form micelles; typical concentration: make a micellar solution with DSPE-PEG-FA at e.g. 1–5 mM.

3. Incubation conditions: mix micelles with preformed niosomes at a ratio calculated to give 0.5 mol% DSPE-PEG-FA relative to total lipid (start 0.5% and also test 1.0%). Incubate at ~40–60°C (above your surfactant/lipid transition) for 30–60 min with gentle shaking. Temperature choice depends on the phase behavior of your niosomal components — aim for a temp that increases membrane fluidity but won’t damage cargo.

4. Remove free micelles/ligand: Purify (size-exclusion chromatography or dialysis) to remove uninserted DSPE-PEG-FA.

5. Characterize: measure size, PDI, zeta potential. Quantify folate on surface (UV assay for folate, HPLC, or colorimetric assays) and run binding/internalization assays on folate receptor positive vs negative cells. Expect small size increase and modest zeta potential changes.

Design notes & tips

Don’t overload with ligand. High ligand density often reduces targeting due to steric hindrance or receptor blocking; low densities (0.2–1 mol%) are often optimal.

If you already PEGylate with MeO-PEG: keep folate-PEG as a minor fraction (e.g., DSPE-PEG-MeO 2–5 mol% + DSPE-PEG-FA 0.2–1 mol%). Some studies show folate binding is best when no extra mPEG competes sterically — you’ll need to optimize.

Niosomes vs liposomes: niosomes (non-ionic surfactant vesicles) accept PEG-lipids similarly to liposomes; insertion kinetics may differ slightly because of surfactant chain composition — keep temperature and incubation time as optimization variables.

Suggested experiments to optimize

1. Compare 0.2%, 0.5%, 1.0% mol% DSPE-PEG-FA (post-insertion) and measure binding/internalization.

2. Compare post-insertion vs pre-insertion at the same nominal mol% (measure actual surface folate, not just input amount).

3. If using therapeutic cargo, assess encapsulation efficiency and cargo leakage after modification.

4. Characterize receptor specificity with folate-receptor positive and negative cell lines and do a competition assay with free folic acid.

thank you H.A Adewuyi but I want a reference on the ratio that I use without optimizing?

A common starting ratio is 5–10% DSPE-PEG2000-folate mixed with 90–95% DSPE-PEG2000 (without folate), based on total lipid content. Optimize based on targeting efficiency and nanoparticle stability.

Best,

Davide

Davide Frumento , thank you your assistance, it is really appreciated