A Comprehensive Review of Silymarin Extraction and Liposomal Encapsulation Techniques for Potential Applications in Food

Abstract

This review explores advances in silymarin extraction and liposomal encapsulation techniques, highlighting their potential applications in the food, pharmaceutical, and cosmetic industries. The study evaluates a range of extraction techniques, including supercritical fluid extraction, microwave-assisted extraction, ultrasound-assisted extraction, and enzyme-assisted extraction, emphasising their efficiency and reduced environmental impact. Furthermore, it examines conventional and innovative liposomal encapsulation techniques, including supercritical carbon dioxide encapsulation and microfluidics, which enhance silymarin’s bioavailability and therapeutic effects. The integration of these methods promises more effective, safe, and eco-friendly silymarin products. This comprehensive review aims to inform readers of the latest research and future industrial applications, with a particular focus on the refinement and scaling up of these processes to meet commercial demands.

1. Introduction

Milk thistle, Silybum marianum L. Gaertner, belonging to the Asteraceae family, is an annual or biennial plant that originated in the Mediterranean region and has successfully naturalised in various parts of the world. Known for its distinctive purple flowers and spiny leaves, this plant is not only an integral part of the Mediterranean flora but has also become established in diverse ecosystems worldwide [1,2]. Its adaptability and capacity to flourish outside its native habitat highlight its ecological versatility and importance. Milk thistle has a long history of medicinal use, dating back over two millennia. It has been employed primarily for its efficacy in treating liver diseases such as cirrhosis, hepatitis, and jaundice [3]. Furthermore, it is recognised for its pharmacological effects, including antidiabetic, antifibrotic, cardioprotective, and immunomodulatory properties [4,5]. This robust historical and contemporary evidence emphasises the multifaceted therapeutic potential of milk thistle.

The principal active constituent of standardised Silybum marianum (S. marianum) fruit extracts is silymarin, which represents a complex mixture. This mixture encompasses seven major flavonolignans (silybin A and B, isosilybin A and B, silychristin, isosilychristin, and silydianin) and flavonoids (taxifolin and quercetin), constituting 65–80% w/w (Figure 1). The remaining portion comprises minor and unidentified related congeners. According to the European and American Pharmacopeias, mature fruits are expected to yield no less than 1.5–2% of silymarin-standardised extract [6,7]. Numerous studies have explored the distribution of silymarin within S. marianum seeds with the aim of developing more efficient extraction approaches; however, the data remain inconsistent [8].

The fruit is composed of four principal parts: pericarp, seed integument, albumen, and embryo. The pericarp is formed of three layers: the epidermis, the sub-epidermis, and the membranous layer [9]. Some of the cells of the sub-epidermal layer are filled with a dark brown substance, which is responsible for the mottled appearance of the fruits. The seed integument consists of the integumentary epidermis and a few cell layers that contain calcium oxalate crystals. The albumen consists of a single layer of cells that serve as a storage site for protein. The embryo comprises two large cotyledons, which contain fat as a storage material. The major silymarin components, flavonolignans, have been identified in the outer part of the fruit, encompassing all cell layers from the pericarp epidermis to the albumen [10]. A hypothesis put forth in much of the literature on S. marianum postulates that flavonolignans undergo biosynthesis from taxifolin and coniferyl alcohol. The suboptimal water solubility of silymarin, which has been measured at 0.04 mg/mL, presents a challenge to its bioavailability and contributes to its limited oral absorption, which typically ranges from 23% to 47% [11]. The primary factors limiting the bioavailability of silymarin include extensive phase II metabolism, low permeability across intestinal epithelial cells, insufficient aqueous solubility, and rapid biliary and urinary excretion [12].

A liposome can be defined as a self-assembled vesicle composed of one or more phospholipid bilayers that encapsulate a volume of aqueous media. The mechanism of liposome formation is essentially based on the unfavourable interactions occurring between phospholipids and water molecules. This results in the polar headgroups of phospholipids being exposed to the aqueous phases (inner and outer), while the hydrophobic hydrocarbon tails are forced to face each other in a bilayer [13]. The compound of interest is securely encapsulated in the inner aqueous core or within one or more phospholipid bilayers [14,15]. Liposomes are classified into four types based on their size and lamellarity: small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), multilamellar vesicles (MLVs), and multivesicular vesicles (MVVs) (Table 1). The thickness of a single phospholipid bilayer has been reported to be approximately 4–5 nm [16].

Liposomes have been examined as carriers for therapeutic agents, as analytical tools, and as models of biological membranes [13,17]. In the food industry, liposomes have been investigated to deliver proteins, enzymes, vitamins, antioxidants, and flavours [18,19]. By adding cholesterol, surfactants, or carbohydrates into the phospholipid bilayer, the rigidity, fluidity and permeability can be modified for specific purposes [19,20]. This review outlines various techniques for silymarin extraction and liposomal encapsulation, with the ultimate goal of potential scalability and large-scale integration.

Table 1. Liposome classification by size and number of lamellae.

Type	Size Range	Characteristics
Multilamellar vesicles (MLV) [21]	2–5 µm	Multiple bilayers Better lipophilic drug encapsulation efficiency Common vesicle type produced by Thin Film Hydration in the presence of an organic solvent
Multivesicular vesicles (MVV) [22]	0.5–5 µm	Separate compartments are present in a single MVV
Large unilamellar vesicles (LUV) [23]	≥50 nm	Single bilayer Efficient macromolecule capture Prepared by detergent dialysis, ether injection, reverse-phase evaporation or active loading methods
Small unilamellar vesicles (SUV) [16]	≤50 nm	Smallest size Thermodynamically unstable Prepared by reducing the size of MLV or LUV using probe sonication or gas extruder or by active loading or solvent injection technique

Table 1. Liposome classification by size and number of lamellae.

Type	Size Range	Characteristics
Multilamellar vesicles (MLV) [21]	2–5 µm	Multiple bilayers Better lipophilic drug encapsulation efficiency Common vesicle type produced by Thin Film Hydration in the presence of an organic solvent
Multivesicular vesicles (MVV) [22]	0.5–5 µm	Separate compartments are present in a single MVV
Large unilamellar vesicles (LUV) [23]	≥50 nm	Single bilayer Efficient macromolecule capture Prepared by detergent dialysis, ether injection, reverse-phase evaporation or active loading methods
Small unilamellar vesicles (SUV) [16]	≤50 nm	Smallest size Thermodynamically unstable Prepared by reducing the size of MLV or LUV using probe sonication or gas extruder or by active loading or solvent injection technique

2. Silymarin Extraction Methods: From Conventional to Novel

The traditional method for extracting silymarin involves the use of organic solvents, including n-hexane, ethanol, chloroform, and ether. In light of the toxicity concerns associated with organic solvents, a number of alternative techniques have been explored with the aim of reducing or even eliminating the use of such solvents in the extraction process. These include supercritical fluid extraction (SFE), ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), and enzyme-assisted extraction (EAE). A summary of the key characteristics of different silymarin extraction methods is provided in

Table 2. Comparison of conventional and innovative methods of silymarin extraction.

Methods		Organic Solvent	Process Time	Process Type	Conditions	Efficiency
Conventional	SE [24]	Yes	hours to days	batch	up to 100 °C	moderate
Innovative	SFE [25]	no (CO2)	minutes	continuous	31.1 °C, 74 bar	High
	MAE [26]	no (water)	minutes	Batch/semi-continuous	50–100 °C	moderate
	UAE [27]	no (water)	minutes	Batch/semi-continuous	20–60 °C	moderate
	EAE [28]	No	hours	batch	30–50 °C	High

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2.1. Solvent Extraction

Solvent Extraction (SE) stands as a well-established and widely adopted conventional technique for silymarin extraction from milk thistle seeds. This traditional method involves utilising organic solvents to dissolve silymarin from plant material, specifically through the process of grinding or crushing the seeds [24,29]. The crushed seeds are then mixed with a suitable solvent, allowing for interaction between the solvent and the botanical matrix, followed by separating the extract from the residual plant debris. The choice of solvent is crucial, as solvent polarity significantly impacts the efficiency and selectivity of the extraction process. Polar solvents, like water and ethanol, are effective in extracting polar compounds such as flavonoids and phenolics, owing to their ability to form hydrogen bonds. In contrast, non-polar solvents, such as hexane and chloroform, are better suited for extracting lipophilic compounds like oils and terpenes through van der Waals interactions. Additionally, using solvent mixtures can be optimized to target a broader range of compounds [30].

While solvent extraction is praised for its cost-effectiveness, it has a notable drawback: the potential for lower purity due to the co-extraction of unwanted compounds [31]. Milk thistle seeds contain significant amounts of fatty acids (56% polyunsaturated and 21% monounsaturated), which can become undesired by-products in silymarin production [29]. To address this, the European Pharmacopoeia recommends a two-step extraction method using the Soxhlet apparatus, involving a defatting process with lipophilic solvents before silymarin extraction [32]. Despite these measures, achieving complete solvent removal from the final product remains challenging, which can complicate analysis and impact the accuracy of the extracted silymarin. Other factors influencing the effectiveness of the extraction process include temperature and extraction time; higher temperatures can enhance extraction efficiency but risk degrading sensitive compounds, while longer extraction times may increase yields but also extract unwanted materials. Furthermore, the solvent-to-material ratio and pre-treatment of plant materials, such as drying or grinding, play significant roles in determining the overall effectiveness and selectivity of the extraction process.

Silymarin extraction from whole and ground seeds, as well as whole and ground pericarp of S. marianum, was conducted through maceration at room temperature [33]. A 70% v/v hydro-alcohol solution was used with a plant-to-solvent ratio of 1:1 and 1:2 for 1, 3, and 7 days. The mentioned method resulted in silymarin yields of 36.23 ± 6.38 mg/mL, 38.02 ± 1.44 mg/mL, and 40.93 ± 1.23 mg/mL when macerating ground whole seeds, whole pericarps, and ground whole pericarps for 7 days at a plant material to solvent ratio of 1:1.

2.2. Supercritical Fluid Extraction

Supercritical Fluid Extraction (SFE) employs supercritical fluids, typically carbon dioxide, at conditions where they exhibit both liquid and gas properties, thereby enabling the extraction of materials from a variety of sources. The phase diagram for a pure component is illustrated in Figure 2, which depicts the regions of the phase diagram where the liquid, gas, and supercritical fluid states meet at the critical point. Supercritical carbon dioxide (SC-CO₂) is a non-toxic, density-adjustable fluid with solvent behaviour analogous to that of hexane [34]. The moderate critical pressure (7.4 MPa/73.8 bar) and low critical temperature (31.1 °C) of SC-CO₂ render it an optimal candidate for biomaterial processing, offering high selectivity and eliminating the need for toxic solvents.

Silychristin, silydianin, and taxifolin, three of the key flavonolignans found in silymarin, possess higher polarity compared to other components like silybin. This increased polarity enhances their interaction with polar solvents, which traditionally poses a challenge for extraction methods that utilize non-polar or weakly polar solvents. However, in the context of SFE, this polarity issue can be effectively managed by modifying the solvent’s properties. While CO₂ is non-polar under standard conditions, introducing polar co-solvents, such as ethanol or methanol, into supercritical CO₂ allows for the adjustment of polarity. This modification improves the solubility of and selectivity for polar compounds like silychristin, silydianin, and taxifolin, enhancing the extraction efficiency of these constituents and contributing to the overall yield and purity of the silymarin extract [35,36].

Moreover, the tunability of SFE allows for the optimization of extraction parameters (e.g., pressure, temperature, and co-solvent concentration) to selectively target silychristin, silydianin, and taxifolin, ensuring their stability and minimizing degradation. This advantage underscores the suitability of SFE for extracting silymarin components with varying polarities while maintaining a low environmental impact through the use of recyclable solvents.

The SFE method was employed to extract silymarin from milk thistle fruits [32]. SFE enables the use of extractants under elevated pressure and temperatures beyond their boiling points. The increased temperature improves analyte diffusion through cell walls, enhances solubility in the extractant, and reduces the solvent’s viscosity and surface tension. Under optimised extraction conditions (acetone solvent at 50–125 °C and 60 bar for 5 min), SFE yielded a higher recovery of flavonolignans compared to the Soxhlet apparatus.

Palaric et al. [37] demonstrated the efficacy of online sequential extraction for retrieving both triglycerides and flavonolignans from milk thistle seeds using Supercritical Fluid Extraction (SFE). Comprehensive lipid extraction was carried out using pure CO₂ at 40 °C and 25 MPa for 30 min, effectively extracting triglycerides such as LLL (C54:6), PLL (C52:4), and OLL (C54:5). The optimal extraction conditions yielded significant peak areas for these triglycerides, with LLL at 1,210,000, PLL at 986,000, and OLL at 2,200,000. Following lipid extraction, flavonolignans were successfully extracted using a 20% modifier composed of water and ethanol (15/85) over 90 min. This solvent system modification not only enhanced the extraction efficiency but also improved the purity of the flavonolignan extract. Specifically, the purity of silybin, one of the primary flavonolignans, reached over 95% under these optimized conditions. This high level of purity is particularly significant for pharmaceutical applications, where the integrity and concentration of active compounds are critical [37].

2.3. Microwave-Assisted Extraction

Microwave-Assisted Extraction (MAE) utilises microwave irradiation to enhance the extraction process. Microwaves generate heat within the plant material, leading to the disruption of cell walls and improved extraction efficiency. This method is known for its speed and reduced solvent consumption. Compared with conventional methods, MAE offers a number of advantages, including a shorter processing time, the use of reduced quantities of solvents, an increased extraction rate, and superior product quality at a lower cost. The mechanism of MAE is based on the impact of microwaves on molecules by ionic conduction and dipole rotation inside target materials [38]. Ionic conduction is the electrophoretic migration of ions caused by an electromagnetic field applied. Furthermore, the solution is subjected to heating as a consequence of the resistance friction experienced by the solution in relation to the ion flow. Dipole rotation leads to the realignment of polar molecules under an applied electromagnetic field. At the commercial frequency of 2.45 GHz, the dipoles undergo 4.9 × 10⁹ random alignments per second, which results in rapid heating. Thus, the microwaves heat the solvent or solvent mixture directly, and interact directly with the free water molecules present inside the materials, resulting in a rapid rise of pressure within cells and a pressure-driven enhanced mass transfer of target compounds out of the source material, which causes rupture of the plant tissue and release of the active compounds into the organic solvent [38,39].

Zheng et al. [26] conducted research on the extraction of silymarin using MAE, and highlighted the impact of four independent variables—namely extraction time, temperature, ethanol concentration, and solid–liquid ratio—on the yield of silymarin. The study revealed that the independent variables had significant effects on and interactions with silymarin yield, with the exception of extraction time. This was determined through analysis of the data. The optimal extraction parameters were identified as follows: 60 min duration, 112 °C temperature, 81.5% ethanol concentration (v/v), and a solid–liquid ratio of 1:38 (g/mL). Under these conditions, the average experimental silymarin yield was 56.67 ± 1.36 mg/g, closely aligned with the predicted value of 57.40 mg/g [26].

2.4. Ultrasound-Assisted Extraction

Ultrasound-Assisted Extraction (UAE) involves the application of ultrasonic waves to the plant material, showcasing superior performance in mass transfer, cell disruption, solvent penetration and capillary effect [40]. Notably, this technique preserves temperature-sensitive botanical materials while being both straightforward and cost-effective [41]. Furthermore, it is the simplest and most economical technique, and can be easily scaled up for industrial production. The waves create cavitation, leading to the mechanical disruption of cell walls and increased mass transfer. As a result, UAE is recognized as a green extraction technique due to its lower energy consumption and reduced solvent use compared to conventional methods.

In terms of yield, UAE is capable of producing high yields under optimized conditions, particularly when using polar solvents like ethanol, which are effective at solubilizing silymarin’s polar components, such as silychristin and silydianin. However, the yield can be influenced by several factors, including ultrasound power, solvent concentration, and extraction time, necessitating careful optimization to balance yield with purity. The stability of silymarin compounds during UAE is another critical consideration. The high-energy ultrasound waves generate localized heat and free radicals, which can potentially degrade sensitive flavonolignans like silybin. Therefore, controlling the ultrasound parameters (e.g., intensity, duration) is essential to minimize thermal degradation and preserve the bioactivity of the extracted compounds [42].

A study compared the efficiency of traditional ethanol/water maceration with maceration using glycerol/water mixtures, enhanced by ultrasonication, for the extraction of silymarin from Silybum marianum [43]. While glycerol/water maceration initially demonstrated slightly lower efficiency than the ethanol/water method, ultrasonication significantly enhanced the extraction process. Specifically, during a 60-min UAE at 80 °C using a 40% (w/w) glycerol solution, the amount of silymarin extracted reached 107.19 μg/mL, which was comparable to the 116.17 μg/mL obtained via ethanol/water maceration. In terms of extraction yield, the UAE process yielded approximately 10.93 mg/g of silymarin from the glycerol-based solvent, compared to 11.82 mg/g from the ethanol-based maceration. This minor difference underscores the efficiency of glycerol as a solvent when assisted by ultrasonication. Furthermore, the purity of the silymarin extract was maintained at high levels, with silybin A and B being the most abundant components, reflecting the effective extraction of key flavonolignans. Additionally, the antioxidant activity of the extracts, as indicated by radical scavenging activity (RSA), was closely correlated with the silymarin content, reinforcing the viability of glycerol as a green solvent alternative. This method not only reduces solvent toxicity but also enhances the applicability of the extracts in cosmetic formulations.

2.5. Enzyme-Assisted Extraction

Enzyme-Assisted Extraction (EAE) employs enzymes to facilitate the breakdown of plant cell walls, thereby enabling the release of bioactive compounds. Enzymes, including cellulases, hemi-cellulases, pectinases, and a combination of these, have been used for the pretreatment of plant materials. The primary function of cellulase and hemi-cellulase is to act upon cell walls. They act on cell wall components, hydrolysing them and increasing the permeability of the cell wall, thus resulting in a higher yield of the metabolite. However, enzymatic treatment is influenced by a number of variables, including enzyme concentration, temperature, duration, and pH, which collectively affect the enzyme activity.

A study by Liu et al. investigated the optimisation of EAE for the extraction of silybin from the seeds of milk thistle [28]. The researchers optimised conditions for enzymatic hydrolysis, comparing EAE with conventional ethanol extraction. The defatted seeds were treated with a cellulase enzyme under the optimised conditions, and then subjected to ethanol extraction. For comparison, seeds were also extracted using ethanol under reflux conditions. The study revealed that EAE significantly increased silybin yield, achieving 24.81 ± 1.93 mg/g defatted seeds in comparison to 10.42 ± 1.65 mg/g with ethanol extraction, representing a 138% increase. Infrared (IR) spectroscopy confirmed that EAE did not alter the extract’s structure. Scanning Electron Microscopy and Transmission Electron Microscopy (TEM) images indicated that EAE effectively disrupted seed cell walls, enhancing silybin release.

3. Environmental Assessment

In assessing the environmental impact of silymarin extraction methods, factors such as energy consumption and chemical waste generation are key. Supercritical Fluid Extraction (SFE) is advantageous due to its use of recyclable CO₂ and operation at moderate temperatures, consuming approximately 0.1 to 0.3 kWh per gram of silymarin [44]. This efficiency minimizes energy use and chemical waste, making SFE a more sustainable option. In contrast, conventional solvent extraction methods typically require higher energy, around 1.0 to 2.0 kWh per gram of silymarin, and generate significant chemical waste due to large volumes of organic solvents. While Microwave-Assisted Extraction (MAE) and Ultrasound-Assisted Extraction (UAE) reduce energy consumption and process time compared to conventional methods, they still rely on solvents that can affect purity and yield [45,46]. Overall, SFE’s capability to adjust solvent properties and its reduced environmental impact position it as a superior choice for silymarin extraction, balancing efficiency, yield, and sustainability.

4. Liposomal Microencapsulation Using Conventional and Innovative Methods

Conventional and innovative techniques in liposomal encapsulation have been developed to address various challenges in the field, including solvent toxicity, scalability, and encapsulation efficiency. Conventional methods frequently employ organic solvents, which can present toxicity risks, environmental concerns, and challenges in scaling up production. In response, innovative techniques aim to minimise or eliminate the use of these solvents, thereby enhancing the safety and efficacy of liposomal formulations. The key aspects of both conventional and innovative liposomal microencapsulation methods are summarised in

Table 3. Comparison of conventional and innovative methods of liposomal microencapsulation.

Methods		Organic Solvent	Process Time	Process Type	Liposome Size
Conventional	TFH [47]	Yes	>1 h	Batch	Variable
	REV [48]	Yes	2–3 h	Batch	Variable
	Membrane Extrusion [49]	Yes	1–2 h	Batch	dependent on pore size
Innovative	Microfluidics Dense Gas [50]	Yes	15–30 min	semi-continuous	50–100 µm
	Ethanol Injection [51]	Yes	15–30 min	semi-continuous	200–250 nm
	SCRPE [52]	Minimal	2–3 h	Batch	200 nm
	DESAM [53]	Yes	45–60 min	continuous	100–400 nm
	SAS [54]	Minimal	2–3 h	Batch	<50 µm

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4.1. Conventional Methods

4.1.1. Thin Film Hydration

The Thin Film Hydration (TFH) method, also known as the Bangham method, entails the solubilisation of lipid material in a volatile organic solvent, such as hexane, methanol, or chloroform. The solvent is then evaporated to form a thin lipid film, typically using rotary vacuum evaporation or nitrogen blowing. This lipid film is subsequently hydrated to induce liposome formation (Figure 3) [55]. Additional steps, such as extrusion or sonication, are often employed to control the diameter and size distribution of the liposomes [56]. The Bangham method typically produces liposomes with heterogeneous MLVs that exhibit low encapsulation efficiency. However, the duration of hydration and the conditions of agitation are critical in determining the amount of liquid encapsulated within the MLVs. For instance, studies have demonstrated that hydrating the lipid for 20 h with gentle shaking results in greater retention of the aqueous phase in comparison to hydrating for two hours with vigorous shaking, despite the size distribution of the MLVs remaining unchanged [57]. This evidence suggests that a slower hydration process is associated with a higher aqueous volume entrapment.

The study by Elmowafy et al. [58] explored the formulation of silymarin-loaded liposomes aimed at enhancing hepatic targeting, with a particular focus on the influence of formulation parameters on encapsulation efficiency and liver-specific delivery. The research assessed the impact of multiple factors, including lipid composition (specifically phosphatidylcholine and cholesterol), the concentration of cholesterol (ranging from 30% to 50% molar ratio), the ratio of drug to lipid (varying from 1:5 to 1:15), and the incorporation of β-sitosterol β-D-glucoside (Sito-G) as a hepatic targeting ligand in concentrations ranging from 0.17 M to 0.33 M.

Encapsulation efficiency was observed to decrease as the concentration of Sito-G increased, with values dropping from approximately 70% to around 60%. The study also examined the drug release profile, noting that the presence of Sito-G modified the release in non-PEGylated liposomes, whereas in PEGylated liposomes, the release profile remained largely unaffected by Sito-G. The mean particle size of the silymarin-loaded liposomes was optimized to enhance cellular uptake and drug delivery efficacy, typically falling within a range suitable for efficient hepatic targeting. In vitro experiments using HepG2 cells highlighted the differences in drug uptake efficiency between non-PEGylated and PEGylated liposomes. Non-PEGylated liposomes containing 0.17 M Sito-G exhibited the highest cellular drug uptake at 37.5%, while PEGylated liposomes showed a maximum uptake of 18% at both 0.17 and 0.33 M Sito-G ratios. The liposomes, with a controlled size of 100–150 nm after extrusion, demonstrated enhanced targeting efficiency and consistent in vitro performance [58].

4.1.2. Reverse-Phase Evaporation

Szoka and Papahadjopoulos introduced the creation of lipid vesicles using the Reverse-Phase Evaporation (REV) technique [59]. It entails the rapid injection of an aqueous drug solution into an organic solvent containing dissolved lipids, while simultaneously subjecting the mixture to bath sonication. This process results in the formation of water droplets within the organic solvent, leading to the creation of a “water-in-oil” emulsion. The emulsion is then reduced to a semi-solid gel using a rotary evaporator. Subsequently, vigorous mechanical agitation is applied to the gel, causing a phase reversal from a water-in-oil emulsion to an oil-in-water dispersion, resulting in an aqueous suspension of the vesicles. During this agitation, some water droplets coalesce to form the external phase, while the remaining droplets constitute the entrapped aqueous volume (Figure 4). This process produces large unilamellar vesicles with diameters ranging from 0.1 to 1 μm.

A study investigated the use of the REV method to encapsulate silymarin in hybrid liposomes to improve its bioavailability [60]. The liposomes, composed of lecithin, cholesterol, stearyl amine, and Tween 20 in a 9:1:1:0.5 molar ratio, achieved a notable encapsulation efficiency of 69.22%. Stability studies indicated that liposomes stored at 4 °C retained better stability, with minimal changes in encapsulation efficiency and particle size over 90 days, compared to those stored at room temperature.

The drug release profile showed sustained release over 6 h, indicating prolonged therapeutic availability. In vivo experiments using a rat model of carbon tetrachloride (CCl4)-induced liver damage demonstrated significant hepatoprotective effects, with reductions in serum glutamic oxalacetate transaminase (SGOT) and serum glutamic pyruvate transaminase (SGPT) levels.

4.1.3. Membrane Extrusion

Vesicle extrusion is a technique that is widely employed in the preparation of liposomes [55]. This method involves forcing pre-formed MLVs through nanoporous membranes to produce monodisperse unilamellar liposomes [61]. Initially, natural or synthetic lipids are dried into a thin film, which is then hydrated to form MLVs. The typical size of these MLVs is between 0.5 and 10 µm, and they undergo 10 freeze/thaw cycles to enhance lamellarity. Subsequently, the MLVs are extruded through double-stacked nanoporous membranes with defined pore sizes (100 or 200 nm). During the extrusion process, the MLVs’ lipid membranes rupture, forming unilamellar liposomes within the nanopore channels (Figure 5). This process is repeated five to ten times to achieve the desired size distribution, with the liposome size generally matching the nanopore diameter. It is essential that extrusion be performed at temperatures above the lipids’ phase transition temperature (T_C) to ensure that the lipids are in a liquid crystalline phase, thereby providing the necessary membrane flexibility. Track-etched polycarbonate (PCTE) membranes are commonly used for this application [49].

The membrane extrusion method was utilized to encapsulate silibinin within liposomes to enhance its potential as a treatment for breast cancer [62]. Liposome-encapsulated silibinin (LES) nanoparticles were produced with a controlled size distribution. The resulting liposomes were spherical and had an average size of 60 nm, which is suitable for enhanced cellular uptake and therapeutic efficacy.

The encapsulation efficiency of the LES nanoparticles was found to be 85%, demonstrating the effectiveness of the membrane extrusion method in achieving high drug loading. The in vitro drug release profile exhibited an initial burst release of silibinin within the first 12 h, followed by a sustained release over a period of 12 days. These characteristics are beneficial for maintaining therapeutic drug levels over an extended period. In terms of anticancer efficacy, the LES nanoparticles significantly reduced the viability of MCF-7 breast cancer cells, with an IC50 value of 20 μM, compared to 38 μM for free silibinin, indicating enhanced potency. Furthermore, the study showed that the LES formulation modulated key apoptotic genes, increasing the expression of pro-apoptotic genes like Caspase-3 and Bax, while decreasing the expression of anti-apoptotic genes like Bcl-2.

4.1.4. Sonication

The sonication method is commonly used to create SUVs [63]. This technique involves the sonication of MLVs produced through conventional methods (TFH, REV), utilising either a bath-type or probe-type sonicator under an inert atmosphere, such as nitrogen or argon (Figure 6a,b). The process of sonication employs the use of pulsed, high-frequency sound waves to disrupt the MLVs, resulting in the formation of small SUVs with diameters ranging from 15 to 50 nm. The objective of sonication is to achieve a uniform dispersion of SUVs, which can enhance tissue penetration. The key factors influencing the size distribution of the final emulsion product are the frequency, power input, and sonication time. While probe-type sonicators deliver high levels of sonic energy to lipid suspensions, they can cause overheating and degradation, and may also introduce titanium particles into the suspension, necessitating their removal by centrifugation. Consequently, bath sonication is the more commonly preferred method. In this approach, the suspension is placed in a test tube within the bath sonicator and sonicated for a period of 5–10 min above the lipid’s T_C, which is the temperature at which the lipid melts [64].

A comparative study was conducted to evaluate the efficacy of encapsulating silymarin in liposomes and bilosomes, using the sonication method to optimize particle size and drug delivery properties [65]. Both formulations were initially prepared using the thin-film hydration method, followed by sonication at 200 W for 5 min. The sonicated liposomes achieved an average particle size of 142 nm with a polydispersity index (PDI) of 0.245 and an encapsulation efficiency of 85%. In contrast, bilosomes, which incorporated bile salts to enhance stability and absorption, had a slightly larger average size of 150 nm and a higher encapsulation efficiency of 90%. Both formulations exhibited similar in vitro release profiles, with an initial burst release of 40% of silymarin within the first 4 h, followed by sustained release over 24 h.

In vivo studies using a rat model of paracetamol-induced hepatotoxicity demonstrated that both silymarin-loaded liposomes and bilosomes effectively reduced liver enzyme levels, including alanine transaminase (ALT) and aspartate transaminase (AST), compared to free silymarin. However, bilosomes showed a more pronounced reduction in these enzyme levels, indicating superior hepatoprotective effects.

4.2. Innovative Methods

4.2.1. Microfluidic Microencapsulation

The utilisation of microfluidics affords the opportunity for more precise control over fluid streams, circumventing the disordered flows that are characteristic of conventional methodologies. This is achieved through the use of microscale processes that are governed by distinct principles [66]. One notable microfluidics technique is the microfluidic hydrodynamic focusing (MHF) method, which was developed by Jahn et al. [67] for the production of liposomes. In this approach, a lipid solution in alcohol is directed through the central channel of the microfluidic device, while aqueous solutions are directed through the adjacent channels. The lateral aqueous streams act to focus the lipid stream into a narrow junction within the device. The formation of liposomes occurs as a result of the diffusion of different molecular species at the interface between the alcohol and aqueous phases. Initially, micelles form, followed by the formation of liposomes (Figure 7). By precisely adjusting the flow rate ratio (FRR) between the lipid and aqueous streams and the total flow rate (TFR), it is possible to achieve monodisperse liposomes with diameters typically ranging from 50 to 200 µm. This precise size control is critical for ensuring uniformity in liposomal applications. The mean diameter of the liposomes was shown to be directly related to lipid concentration and inversely related to FRR. Additionally, device geometry was identified as a factor influencing liposome characteristics [50,68]. In summary, microfluidic techniques offer superior control over the physical properties of nanoparticles. The MHF method produces uniform vesicles in terms of size and lamellarity in a single step, in contrast to the TFH method, which necessitates additional steps, such as extrusion or sonication to achieve uniformity. Furthermore, this technique exhibits enhanced encapsulation efficiency (up to 80%) and reproducibility, even at large scales, rendering it well-suited for the production of personalised products [50]. The interaction between the encapsulated compound and the lipids can influence the overall stability of the liposome. Hydrophilic drugs that interact strongly with the lipid headgroups may be more stably encapsulated, while hydrophobic drugs that partition the lipid bilayer can have their release rates modulated by altering the lipid acyl chain length and saturation.

An example of the application of microfluidic encapsulation is the fabrication of iohexol-containing liposomes for medical imaging purposes. Delama et al. [69] utilized the microfluidic technique, which involves precise mixing of lipid and aqueous solutions under controlled flow conditions, to create stable liposomes encapsulating iohexol, a contrast agent used in imaging. The study investigated various phosphocholine lipids, including DMPC, DOPC, DPPC, and DSPC, combined with cholesterol at a fixed 2:1 molar ratio.

The microfluidic method achieved a high encapsulation efficiency of over 70% across all formulations. In terms of release profiles, the study found that the majority of formulations exhibited an iohexol release of approximately 0.12 mg/mL after two hours, which was non-toxic to kidney cells, highlighting its potential safety for biomedical applications. Notably, the release profile was influenced by the specific lipid composition, with DOPC emerging as the optimal formulation.

Stability studies showed that the DOPC-based liposomes maintained their stability for four weeks, as indicated by consistent particle size, polydispersity index (PDI), and zeta potential, at both 5 °C and 37 °C. This long-term stability, coupled with the high encapsulation efficiency and controlled release, underscores the effectiveness of the microfluidic technique in producing liposomes suitable for medical imaging, particularly in CT imaging where iohexol serves as a key contrast agent [69].

4.2.2. Ethanol Injection Method

The ethanol injection method, first developed by Batzri and Korn in 1973, is a widely used technique for producing SUVs in a straightforward and rapid manner. This method was developed to overcome the limitations of the TFH method, which include difficulty in scaling up and the need for sonication to achieve homogeneity. The ethanol injection method involves the injection of an ethanolic lipid solution into an aqueous medium, resulting in the spontaneous formation of single bilayer liposomes without the need for sonication (Figure 8). This method has several advantages: it protects phospholipids from oxidative degradation and prevents potential damage to solute molecules or toxicity associated with sonication probes [70]. Unlike the TFH method, which relies primarily on hazardous organic solvents such as chloroform and methanol, the ethanol injection method uses ethanol, which is safer for workers [71]. Liposomes produced using ethanol injection are typically smaller and more monodisperse, which makes them suitable for applications requiring high transfection efficiency, such as gene delivery [72]. Additionally, this method is more suitable for industrial scale production due to its simplicity, safety, and reproducibility [73,74].

The ethanol injection method is widely used in the pharmaceutical, cosmetic, and food industries for its efficacy in encapsulating both hydrophilic and lipophilic drugs, thereby enhancing their stability, bioavailability, and controlled release profiles [75]. In pharmaceutical applications, this method has been particularly effective in encapsulating quercetin, a potent antioxidant flavonoid. By injecting an ethanolic solution of lipids and quercetin into an aqueous phase, researchers have consistently produced quercetin-loaded liposomes with a uniform size of approximately 120 nm and high encapsulation efficiencies ranging from 70% to 75% [51].

Moreover, the ethanol injection method plays a crucial role in the preparation of silymarin-loaded liposomes aimed at enhancing antitumor efficacy [76]. This method was optimized using response surface methodology (RSM) to achieve high entrapment efficiency (EE%) of silymarin. The optimized formulation involved 7.8 mg/mL of lecithin, with a silymarin-to-lecithin mass ratio of 1:26 and a lecithin-to-cholesterol ratio of 10:1. The resulting liposomes showed a remarkable entrapment efficiency of 96.58% ± 3.06%, with a particle size of 290.3 ± 10.5 nm and a zeta potential of +22.98 ± 1.73 mV. Sustained release of silymarin was observed in vitro, primarily driven by a diffusion mechanism. Furthermore, cytotoxicity studies on A549 lung cancer cells indicated that the silymarin-loaded liposomes exhibited a stronger inhibitory effect compared to free silymarin, highlighting their potential for intravenous cancer therapy.

4.2.3. Supercritical Reverse Phase Evaporation

The Supercritical Reverse Phase Evaporation (SCRPE) method entails the mixing of a phospholipid solution in SC-CO₂ with an aqueous solution under pressure, resulting in the formation of a CO₂/water emulsion. Upon depressurisation, this emulsion undergoes a conversion process, ultimately yielding a liposomal suspension [77].

In the early 2000s, Otake et al. developed the SCRPE method. Cholesterol, a hydrophobic compound, is dissolved with other lipids in SC-CO₂ and ethanol to form a homogeneous supercritical solution. Concurrently, glucose, which was selected as the hydrophilic compound, is introduced into the supercritical solution at a slow rate while stirring. The subsequent reduction in pressure releases CO₂ and results in the formation of a liposomal suspension (Figure 9) [77]. The size and morphology of the liposomes can be controlled by adjusting the pressure and the amount of ethanol [78]. Aburai et al. (2011) employed this technique to encapsulate hydrophilic compounds, including glucose and bovine serum albumin, as well as hydrophobic compounds, such as cholesterol. The results of their studies demonstrated that the SCRPE process could achieve encapsulation efficiencies up to four times higher than those produced by the TFH method, particularly for bovine serum albumin. Furthermore, a stability study indicated that liposomes produced by the SCRPE method retained 60% encapsulation efficiency after 48 h, whereas those prepared by the TFH method demonstrated complete release [79]. Additionally, Okada et al. (2010) used the SCRPE method to prepare liposomes encapsulating a contrast agent for in vivo studies in rabbits [80]. The SCRPE process is versatile, enabling the preparation of nano-sized liposomes with controllable physicochemical properties [52].

4.2.4. Depressurisation of an Expanded Solution into Aqueous Media

The Depressurisation of an Expanded Solution into Aqueous Media (DESAM) method was developed by Meure et al. [81]. This technique involves the hydration of lipids by depressurising an expanded solution into an aqueous medium through a nozzle, thereby rendering it suitable for the large-scale production of unilamellar liposomes (Figure 10) [81]. The process yields liposomes with a diameter ranging from 50 to 200 nm and a polydispersity index below 0.29. Transmission Electron Microscopy (TEM) images confirm that the produced liposomes are unilamellar. The DESAM method has been demonstrated to be effective for the encapsulation of both hydrophilic and hydrophobic compounds. For instance, encapsulation efficiencies of up to approximately 10% for isoniazid and up to 25% for cholesterol have been achieved. Despite the use of organic solvents in this method, the final product contains residual solvent levels of less than 4% (v/v), which can be further reduced to 2.2% (v/v) by bubbling CO₂ through the liposomal dispersion [82]. Furthermore, this method provides good physical stability for the liposomes, as they exhibited minimal changes in size and size distribution when stored at 5 °C over an eight-month period.

4.2.5. Supercritical Antisolvent

The Supercritical Antisolvent (SAS) technique operates on the principle of injecting an organic solution through a capillary tube into a vessel filled with supercritical fluid (Figure 11). Lesoin et al. [54] employed this method to produce proliposomes by introducing a calcein solution into the high-pressure vessel and mixing it with an Ultraturax T25 high-speed mixer to create liposomes. For comparison, calcein-loaded liposomes were also prepared using the TFH method. The results indicated that the liposomes produced via SAS exhibited a more consistent and narrower particle size distribution (PSD), ranging from 0.1 to 100 μm, compared to those made with the TFH method. Furthermore, the SAS method resulted in a reduction in residual solvent levels in the final product. The liposomes prepared by SAS were smaller and displayed spherical morphology, in contrast to those produced by TFH. Despite these advantages, the encapsulation efficiency was higher with the TFH method, achieving approximately 20%, while SAS-prepared liposomes had an efficiency between 10% and 20%. Both methods demonstrated that liposomes were unstable after one month of storage at 4 °C.

Bridson et al. [83] also utilised the SAS approach to create proliposomes by mixing a phospholipid solution with supercritical fluid in a heated T-piece. They performed a 15-min washing step with pure supercritical fluid to eliminate residual solvent, and infrared spectroscopy confirmed that the proliposomes were solvent-free. Subsequent hydration of these proliposomes produced liposomes with an average diameter of about 5 μm [83].

5. The Challenges Related to Industrial Scalability and Regulatory Considerations

The transition from laboratory-scale techniques to industrial-scale production poses several challenges, particularly in terms of scalability and regulatory compliance. Among the methods discussed, microfluidics and ethanol injection are identified as semi-continuous processes that offer promising potential for industrial scalability. Microfluidics allows for precise control over the size and uniformity of liposomes, which is critical for maintaining product consistency at a larger scale. Its ability to produce monodisperse liposomes with high encapsulation efficiency makes it suitable for commercial applications with relatively minor adjustments. Similarly, the ethanol injection method, known for its simplicity and rapid processing time, is another semi-continuous technique that is well-suited for scaling up. This method can produce liposomes with consistent characteristics, which is advantageous for industrial production. The straightforward nature of the process also facilitates easier integration into existing production lines, enhancing its viability for large-scale manufacturing. In contrast, methods such as Supercritical Reverse Phase Evaporation (SCRPE) and Supercritical Anti-Solvent (SAS) are batch processes, which present inherent challenges for scalability. The batch nature of these techniques limits their throughput, making them less suitable for industrial-scale production without significant modifications. To address these challenges, further development and optimization of these methods are required to enhance their scalability and efficiency in large-scale operations. The Depressurization of an Expanded Solution into Aqueous Media (DESAM) method, on the other hand, is a continuous process that offers greater potential for industrial scale-up. Its ability to produce unilamellar liposomes with consistent size distribution makes it a strong candidate for commercial implementation. The continuous nature of DESAM allows for higher throughput, making it more practical for large-scale production.

In addition to scalability, regulatory considerations are critical for the commercial implementation of these techniques. Ensuring that the processes are consistent, reproducible, and compliant with safety standards is essential. This includes adhering to guidelines set by regulatory bodies such as the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) [84]. Rigorous validation and quality control measures must be implemented to ensure that the final products meet the necessary safety and efficacy standards required for food, pharmaceutical, and cosmetic applications.

By addressing these scalability and regulatory challenges, the reviewed techniques can be more effectively translated into commercial products, thereby maximizing their potential impact in various industries.

6. Liposome Applications

Liposomes, versatile and biocompatible vesicles, have been employed in a multitude of fields due to their distinctive capacity to encapsulate both hydrophilic and lipophilic substances. Their applications encompass the pharmaceutical, cosmetic, and food industries, underscoring their multifunctional capabilities.

6.1. Pharmaceutical Applications

Liposomes have revolutionised the field of drug delivery systems. These versatile nanoparticles can be formulated in a range of forms, including liquids (suspensions), solids (powders), and semi-solids (gels and creams), and can be administered topically or via parenteral routes. Their primary advantage in pharmaceuticals lies in their ability to enhance the efficacy of drugs while reducing their toxicity.

6.1.1. Systemic Liposomal Drugs

The encapsulation of drugs within liposomes affords protection from enzymatic degradation while the drugs are in circulation. To illustrate, penicillin and cephalosporin that have been encapsulated in liposomes are protected from β-lactamase enzymes, thereby extending their therapeutic effectiveness [85].

The use of liposomes facilitates targeted drug delivery, which is particularly useful in the context of cancer treatment. By modulating the distribution of the drug within the body, liposomes facilitate the enhancement of drug efficacy at reduced doses while simultaneously reducing the incidence of adverse effects [86]. This targeted delivery is particularly important in the context of chemotherapy, where the reduction of toxicity represents a fundamental objective.

6.1.2. Topical Liposomal Drugs

The use of liposomes in topical applications allows for the targeted delivery of drugs to specific sites. This reduces systemic exposure and improves efficacy, making them a valuable tool in the treatment of skin conditions and localised infections. The effectiveness of liposomes in skin treatment applications is based on the similarity between the lipid vesicles’ bilayer structure and natural membranes, facilitating alterations in cell membrane fluidity and the fusion of liposomes with them [87].

6.2. Cosmetic Applications

In the field of cosmetics, liposomes are employed to facilitate the penetration of active ingredients into the deeper layers of the skin, thereby enhancing the efficacy of skincare products. They assist in the sustained release of active compounds, which in turn improves dermal hydration and reduces the appearance of fine lines and wrinkles [88].

6.3. Food Applications

Liposomes are emerging as a promising tool in the food industry due to their ability to encapsulate and protect sensitive ingredients, rendering them an attractive option for use in a variety of food products.

The encapsulation of nutrients is a potential application of this technology. The encapsulation of vitamins, antioxidants and flavours by liposomes improves their stability and bioavailability. This application is particularly beneficial in dairy products, where liposomes can enhance the nutritional profile and extend the shelf life of the product.

The utilisation of liposomes in fermentation processes is another potential application of this technology. The encapsulation of enzymes in liposomes has been demonstrated to markedly reduce fermentation times in cheese production, thereby enhancing productivity and product quality. For example, the use of liposome-encapsulated enzymes has been demonstrated to reduce the ripening time of cheddar cheese by up to 50%, resulting in significant economic benefits [89].

Liposomes are also being investigated for their potential in food preservation, wherein antimicrobial agents are encapsulated. This approach has the potential to effectively control the growth of spoilage microorganisms, thereby ensuring food safety and extending shelf life.

In the agricultural sector, liposome-encapsulated biocides, such as fungicides, herbicides, and pesticides, offer prolonged action with reduced environmental impact. Furthermore, liposomes can be designed to adhere to plant surfaces, ensuring longer retention and effectiveness.

7. Conclusions

Silymarin, composed of seven flavonolignans with varying polarities, presents unique challenges for efficient extraction and encapsulation. Among the various extraction methods, supercritical fluid extraction (SFE) stands out due to its ability to adjust polarity characteristics. This makes SFE capable of extracting both polar and non-polar compounds effectively while minimizing the use of toxic solvents. Additionally, SFE is known for its relatively low energy consumption compared to other extraction techniques, making it not only efficient but also environmentally friendly.

While traditional and some advanced encapsulation methods have been studied for silymarin, novel techniques such as Supercritical Reverse Phase Evaporation (SCRPE), Deep Eutectic Solvents Assisted Method (DESAM), and Supercritical Anti-Solvent (SAS) have not yet been explored. These innovative methods offer significant potential by reducing or even eliminating the need for toxic solvents, enhancing the safety and environmental sustainability of the encapsulation process. Moreover, these methods could lead to the production of liposomes with higher encapsulation efficiency, smaller vesicular sizes, and improved long-term stability. The scalability of these techniques also makes them promising candidates for large-scale liposome production, facilitating their integration into various industrial applications.

Exploring these advanced encapsulation techniques represents an untapped field for further research. Such developments could significantly improve the bioavailability and stability of silymarin, thereby broadening its effective applications across medical, cosmetic, and food industries. Future research efforts should aim to integrate these novel methods to optimize silymarin delivery systems, providing sustainable and efficient solutions that leverage the full therapeutic potential of this valuable natural compound.