Nutraceutical Field: Methods to Distinguish True Liposomes from Pseudoliposomes (Simple Mixtures)

Core Judgment Logic: Three Essential Characteristics of Food-Grade True Liposomes

Hierarchical Distinction Methods (Adapted for Food R&D/Production Scenarios, from Simple to Precise)

(I) Basic Rapid Screening (No Complex Equipment Required, Suitable for Preliminary Workshop Judgment/Raw Material Screening)

1. Visual Observation + Microscopy (Morphological Preliminary Screening)

◦ Operation: Dilute a small amount of sample with purified water, drop onto a glass slide, and observe under an optical microscope (400-1000x); or stain with 0.1% methylene blue (liposome vesicles appear as light blue rings).

◦ Result Judgment:

▪ True Liposomes: The system is a uniform translucent emulsion without obvious precipitation, with dispersed spherical/quasi-spherical particles (particle size 50-1000nm);

▪ Pseudoliposomes: The system is prone to stratification and precipitation, with no fixed morphology, only visible nutritional ingredient particles or phospholipid floccules.

1. Centrifugation Separation Test (EE Preliminary Screening)

◦ Operation: Take 1mL of sample, centrifuge at 10000rpm for 10 minutes, collect the supernatant, and detect the concentration of nutritional ingredients in the supernatant using UV-VIS or HPLC.

◦ Result Judgment:

▪ True Liposomes: Vesicles are not easily broken, and the concentration of free ingredients in the supernatant is low (high EE);

▪ Pseudoliposomes: Nutritional ingredients are easily precipitated or completely dissolved in the supernatant (supernatant concentration is close to total concentration, EE≈0).

1. Dialysis Bag Diffusion Test (Encapsulation Effect Verification)

◦ Operation: Load the sample into a dialysis bag (MWCO=10-50kDa, larger than nutritional ingredient molecules but smaller than liposome particle size), dialyze in a large volume of buffer (simulating body fluid environment) for 2-4 hours, and detect the ingredient concentration in the external dialysate.

◦ Result Judgment:

▪ True Liposomes: Ingredients are encapsulated in vesicles, unable to pass through the dialysis membrane, and the external fluid concentration is low;

▪ Pseudoliposomes: Ingredients diffuse freely, and the external fluid concentration is close to the total concentration of the sample.

(II) Laboratory Precise Verification (Food-Grade Product R&D/Quality Control Confirmation)

1. Particle Size and Zeta Potential Analysis (Structural Uniformity Verification)

◦ Instrument: Dynamic Light Scattering (DLS)

◦ Key Indicators:

▪ True Liposomes: Uniform particle size distribution (PDI .3), zeta potential usually -20~-50mV (natural negative charge of phospholipids), stable values;

▪ Pseudoliposomes: Extremely broad particle size distribution (PDI > 0.5), no fixed zeta potential (uneven dispersion of phospholipids and ingredients, large potential fluctuations).

1. Transmission Electron Microscopy (TEM) Observation (Structural Gold Standard)

◦ Operation: Stain the sample with phosphotungstic acid for negative staining, then observe under TEM (nm-level resolution).

◦ Result Judgment:

▪ True Liposomes: Distinct closed vesicles formed by phospholipid bilayers are clearly visible (black ring-shaped structures with aqueous cores);

▪ Pseudoliposomes: No vesicular structure, only irregular phospholipid aggregates or nutritional ingredient particles (no bilayer characteristics).

◦ Note: Cryo-TEM can be used for food-grade samples to avoid stain residue affecting safety evaluation.

1. Encapsulation Efficiency (EE) Determination (Core Quantitative Indicator)

◦ Principle: Separate free ingredients from encapsulated ingredients and calculate the proportion of encapsulated ingredients to total ingredients (EE% = (Total Ingredients - Free Ingredients)/Total Ingredients × 100%).

◦ Common Food-Grade Adaptable Methods:

▪ Gel Filtration Chromatography (Sephadex G-50/G-100 Column): Separate liposomes from small-molecule free ingredients for quantification;

▪ Ultrafiltration Centrifugation: Retain liposomes with ultrafiltration membranes, allowing free ingredients to pass through for detection;

▪ HPLC Method: Precisely quantify trace active ingredients in food (e.g., polyphenols, vitamins).

◦ Result Judgment: EE ≥20% (for water-soluble ingredients such as vitamin C) or ≥30% (for fat-soluble ingredients such as DHA) indicates true liposomes; EE % indicates a simple mixture system.

1. Differential Scanning Calorimetry (DSC) (Phospholipid Phase Transition Verification)

◦ Principle: Interaction between nutritional ingredients and phospholipid bilayers in true liposomes changes the characteristic phase transition temperature (Tc) of phospholipids; the Tc of phospholipids in simple mixtures is consistent with pure phospholipids.

◦ Result Judgment:

▪ True Liposomes: Phase transition peak broadening and Tc shift (e.g., Tc decreases after vitamin E embedding);

▪ Pseudoliposomes: Phase transition peak is identical to pure phospholipids (no ingredient-phospholipid interaction).

1. In Vitro Release Curve Determination (Absorption Efficiency Verification)

◦ Operation: Use the dialysis bag method to simulate the gastrointestinal environment (pH=1.2 hydrochloric acid solution → pH=7.4 buffer), and determine the ingredient release amount at different time points.

◦ Result Judgment:

▪ True Liposomes: Gentle release curve, cumulative release rate  48 hours (sustained release protection, reducing gastric acid destruction);

▪ Pseudoliposomes: Cumulative release rate >90% within 12 hours (rapid exposure, prone to degradation).

(III) Advanced Verification (High-Demand Food/Dietary Supplement Scenarios)

1. Confocal Laser Scanning Microscopy (CLSM) Observation

◦ Operation: Label phospholipids with fluorescent probes (e.g., DiI for bilayer labeling) and nutritional ingredients (e.g., FITC for peptide labeling), then observe fluorescence distribution.

◦ Result Judgment:

▪ True Liposomes: Overlapping of two fluorescence signals (ingredients encapsulated in liposomes);

▪ Pseudoliposomes: Separation of two fluorescence signals (no fixed binding between ingredients and phospholipids).

1. Small-Angle X-Ray Scattering (SAXS)

◦ Principle: Phospholipid bilayers of true liposomes produce characteristic diffraction peaks (around 2θ=20°), which are absent in simple mixtures.

◦ Result Judgment: Presence of characteristic diffraction peaks → True liposomes; Absence of characteristic peaks → Pseudoliposomes.

Common Misunderstandings and Key Reminders in the Food Field

1. Misunderstanding 1: "Emulsion = Liposomes"

Pseudoliposomes may also form emulsions due to phospholipid aggregation, but they have a broad particle size distribution (PDI >0.5) and no vesicular structure, which can be quickly distinguished by TEM or EE determination.

2. Misunderstanding 2: "Contains Phospholipids = Liposomes"

The core of liposomes is "bilayer vesicles", not just the addition of phospholipids. Phospholipids in simple mixed food systems only act as emulsifiers and cannot encapsulate or protect nutritional ingredients.

3. Key Reminder: EE is the Core Quantitative Indicator

Regardless of morphology, systems with EE  basically be judged as pseudoliposomes (or failed food-grade liposome preparation), which cannot achieve sustained release and bioavailability improvement of nutritional ingredients.

4. Rapid Detection Scheme for Food Production Workshops

Adopt the "Centrifugation + UV Detection" combination: After centrifugation at 10000rpm for 10 minutes, if the ratio of nutritional ingredient concentration in the supernatant to total concentration is <10% and the system has no stratification, it can be initially judged as qualified food-grade liposomes; otherwise, it is a simple mixture system.

Summary

The core logic for distinguishing true/false liposomes in the nutraceutical field is: First quantify the encapsulation and protection effect of nutritional ingredients through EE, then verify the vesicular structure through morphology (TEM/microscopy), and finally confirm the practical application value through processing stability and in vitro release curves. It is suitable for the R&D and quality control of dietary supplements and functional foods (e.g., DHA liposomes, probiotic liposomes, plant polyphenol liposomes). The combination of "EE (HPLC method) + TEM observation" is preferred, balancing accuracy and food-grade safety requirements.