The sustained-release characteristics of Nisin under liposome encapsulation

Nisin, a natural antimicrobial peptide, is widely used in food preservation and biomedicine. However, its applications are limited by drawbacks such as poor stability, susceptibility to protease degradation, and short duration of action. Liposomal encapsulation involves enclosing Nisin in phospholipid bilayer vesicles, leveraging the biocompatibility and membrane-controlled release properties of liposomes to achieve sustained release of Nisin, prolong its effective duration, and enhance its stability simultaneously. The following elaborates on the sustained-release mechanism, influencing factors, performance evaluation, and application value:

I. Core Sustained-release Mechanism of Liposome-encapsulated Nisin

As a carrier with a biomembrane-mimetic structure, the sustained-release effect of liposomes on Nisin stems from the controlled-release effect of the carrier structure and the environment-responsive release-triggering mechanism, which is mainly divided into two stages:

Initial Burst Release StageDuring the encapsulation process, part of Nisin does not fully enter the interior of liposomes but adsorbs on the surface of liposomal vesicles or exists in the gaps of the phospholipid bilayer. In the initial stage of contact with the release medium (e.g., food matrix, body fluid), this part of Nisin dissolves and releases rapidly, accounting for 10%–20% of the total encapsulated amount typically. This enables rapid attainment of the initial bacteriostatic concentration, realizing "fast onset of action".

Continuous Sustained-release StageNisin encapsulated in the internal aqueous phase of liposomes or embedded in the phospholipid bilayer is slowly released mainly through two ways:

Passive Diffusion: Molecular gaps exist in the phospholipid bilayer of liposomes, allowing Nisin molecules to diffuse from the interior of liposomes to the external medium by virtue of the concentration gradient. Meanwhile, the Brownian motion of phospholipid molecules generates dynamic pores in the vesicle membrane, further facilitating the slow leakage of Nisin. This process lasts for a long time and serves as the core stage of sustained release.

Environment-responsive Release: Liposomes can achieve targeted sustained release through modification. For example, in the acidic environment of foods (e.g., yogurt, fruit juice), protonation of phospholipid molecules in the liposomal membrane leads to membrane instability and increased permeability, accelerating Nisin release. In the field of biomedicine, the weakly acidic nature and high glutathione concentration of the tumor microenvironment can trigger pH-responsive or reduction-responsive rupture of liposomes, achieving targeted sustained release of Nisin.

In addition, liposomes can protect Nisin from protease hydrolysis and damage by external environmental factors (e.g., high temperature, light), prolong its biological activity half-life, and indirectly enhance the durability of the sustained-release effect.

II. Key Factors Affecting the Sustained-release Properties of Liposome-encapsulated Nisin

The structural parameters, preparation process, and external environment of liposomes directly determine the release rate and release cycle of Nisin. The core influencing factors are as follows:

Liposome Structure and Composition

Phospholipid Type: Liposomes prepared with saturated phospholipids (e.g., dipalmitoylphosphatidylcholine, DPPC) have a dense membrane structure with small molecular gaps, resulting in a slow Nisin diffusion rate and a long sustained-release cycle. Unsaturated phospholipids (e.g., soybean lecithin) form membranes with high fluidity, leading to a faster Nisin release rate.

Vesicle Particle Size: Small-sized liposomes (< 200 nm) have a large specific surface area, exhibiting a slightly higher initial burst release rate but more uniform overall release. Large-sized liposomes (> 500 nm) have an increased membrane thickness and high diffusion resistance, resulting in a longer sustained-release cycle and being suitable for long-term bacteriostatic scenarios.

Membrane Modification Components: Adding cholesterol to the liposomal membrane can enhance membrane rigidity and stability, reduce Nisin leakage, and significantly prolong the sustained-release time. Surface modification with polyethylene glycol (PEG) can decrease the phagocytosis probability of liposomes, delay Nisin release, and improve bioavailability simultaneously.

Encapsulation Process Parameters

Encapsulation Efficiency: In liposomes with high encapsulation efficiency (> 80%), most Nisin is present in the internal aqueous phase, leading to a low initial burst release rate and optimal sustained-release properties. When the encapsulation efficiency is too low, the proportion of Nisin adsorbed on the surface is high, which tends to cause the problem of "excessively fast burst release and insufficient subsequent release".

Preparation Method: Liposomes prepared by the thin-film hydration method have a regular membrane structure and stable sustained-release effect. Liposomes prepared by the reverse-phase evaporation method have higher encapsulation efficiency, making them suitable for the encapsulation of water-soluble Nisin. Liposomes prepared by the ultrasonic emulsification method have a smaller particle size and more uniform release rate.

External Environmental Conditions

pH Value: The acidic environment (pH 3–5) can disrupt the charge balance of the liposomal membrane, accelerate membrane rupture, and increase the Nisin release rate by 2–3 times. In neutral or weakly alkaline environments, the liposomal membrane remains stable, resulting in a longer sustained-release cycle.

Temperature: Elevated temperature (e.g., pasteurization temperature of 60–70℃ in food processing) can enhance the fluidity of phospholipid molecules, increase membrane pores, and accelerate Nisin release. Low-temperature storage (4℃) can maintain the structural stability of liposomes and delay release.

Medium Components: Proteases and surfactants in the food matrix can destroy the liposomal membrane structure and promote Nisin release. Serum proteins in body fluids can bind to liposomes, reduce their permeability, and prolong the sustained-release time.

III. Evaluation Methods for Sustained-release Properties of Liposome-encapsulated Nisin

To quantify the sustained-release effect, a combination of in vitro and in vivo experiments is required for evaluation. The core indicators and methods are as follows:

In Vitro Release Experiment

Experimental Model: The dialysis bag method or diffusion cell method is adopted. Liposome-encapsulated Nisin is placed in a simulated release medium (e.g., phosphate-buffered saline, PBS; simulated gastric fluid, SGF) and shaken under set temperature and pH conditions.

Core Indicators:

Cumulative Release Rate: Samples are taken at different time points, and the Nisin concentration in the release medium is determined by high-performance liquid chromatography (HPLC) or the inhibition zone method to calculate the cumulative release rate and draw the release curve.

Sustained-release Cycle: The time required for the cumulative release rate to reach 80% is used as the evaluation standard for the sustained-release cycle.

Burst Release Rate: The cumulative release rate within the initial 2 hours, which should ideally be controlled within 20%.

In Vivo / Practical Application Effect Evaluation

Food Field: Liposome-encapsulated Nisin is added to meat products and dairy products, and the viable count in the food is detected regularly to evaluate the long-term bacteriostatic effect of Nisin. Compared with free Nisin, liposome-encapsulated Nisin can double or triple the shelf life of food.

Biomedical Field: Through animal experiments, the Nisin concentration-time curve in blood or target organs is determined, and pharmacokinetic parameters (e.g., half-life t₁/₂, bioavailability F) are calculated. Liposomal encapsulation can prolong the in vivo half-life of Nisin by 3–5 times.

IV. Application Value and Optimization Directions

Application Value

Food Preservation: Liposome-encapsulated Nisin can achieve long-term bacteriostasis, reduce the dosage of preservatives, and is suitable for low-temperature meat products, fermented dairy products, etc. Meanwhile, it avoids the activity loss of Nisin caused by direct contact with food components.

Biomedicine: As an antibacterial preparation, sustained-release liposomes can deliver Nisin to the infection site in a targeted manner, increase local drug concentration, and reduce systemic side effects, making them suitable for the treatment of skin infections, oral inflammation, etc.

Optimization Directions

Targeted Sustained-release Modification: Modify the liposome surface with specific ligands (e.g., antibodies, polysaccharides) to achieve targeted release of Nisin to specific bacteria or target tissues.

Composite Carrier Construction: Compound liposomes with polysaccharides such as chitosan and sodium alginate to form "liposome-polysaccharide" composite microspheres, further improving sustained-release stability and encapsulation efficiency.

Industrial Preparation Process Optimization: Develop continuous and large-scale liposome preparation technologies (e.g., microfluidic preparation) to reduce production costs and promote practical applications.