Transfersomes: A versatile tool for drug delivery and targeting

 Corresponding author: Rakshitha D M. Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Transfersomes: A versatile tool for drug delivery and targeting Rakshitha D M *, Ganesh N S, J Adlin Jino Nesalin and Vineeth Chandy Department of Pharmaceutics, T. John College of Pharmacy, Bengaluru-83, Karnataka, India. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 358-371 Publication history: Received on 03 January 2025; revised on 13 February 2025; accepted on 16 February 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.21.2.0185 Abstract Transfersomes are an innovative system designed for targeted drug delivery through the skin. They are a type of liposome made from phosphatidylcholine combined with an edge activator. This unique design allows them to penetrate the skin’s outer layer (the stratum corneum) more effectively, either through the intracellular or transcellular pathways. Some of the key advantages of transfersomes include their ability to carry a wide range of drugs, better skin penetration, and their biocompatibility and biodegradability. Transfersomes are adaptable to ambient stress, hence, allowing the ultra-deformable transfersomes to change its membrane composition locally and reversibly, when it is pressed against or attracted into narrow pores. Transfersomes exhibit a flexible structure and higher surface hydrophilicity which play a critical role in the transport of drugs and other solutes using hydration gradients as a driving force to deliver the molecules into and across the skin. Evaluation parameters of transfersomes includes Vesicle size distribution, Zeta potential, Vesicle morphology, Number of vesicles per cubic mm, Entrapment efficiency, Drug content, Turbidity measurement, Degree of deformability, Surface charge, In-vitro drug release, In-vitro skin permeation studies. Transfersomes are incredibly versatile and can be used for controlled drug release, making them ideal for delivering both small and large molecules. They can carry a wide range of drugs, including pain relievers, anesthetics, corticosteroids, sex hormones, anticancer drugs, insulin, gap junction proteins, and albumin. Their ability to transport such diverse compounds makes them a powerful tool in modern medicine for improving drug delivery and effectiveness. Keywords: Transfersomes; Ultra-Deformable Vesicle; Osmotic Gradient; Fexible; Adaptable 1. Introduction New advancements in drug delivery systems are sparking interest in the development of more efficient ways to administer medications. One exciting area is vesicular drug delivery systems, which are part of this wave of innovative approaches1. The increasing demand for more effective treatments with fewer side effects has sparked exciting innovations in the pharmaceutical world, leading to the development of novel drug delivery systems (NDDS). These systems are designed to tackle the challenges of traditional drug delivery, such as short-lasting effects, difficulty in targeting specific areas, and low solubility or absorption of certain medications. Drug delivery systems (DDS) are a groundbreaking innovation with many practical applications. They are designed to release medications at a controlled pace and deliver them directly to specific tissues or cell types, ensuring greater precision. Recent advancements, such as nanoparticles, molecularly imprinted polymers, and 3D printing technology, have become hot topics in this area of research. Drug delivery system is a key approach for achieving targeted, accurate, and more effective drug delivery, making treatments safer and more efficient. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 358-371 359 Drug delivery systems (DDS) are designed to improve how medications work in the body by enhancing their effectiveness and safety. These systems can adjust a drug’s metabolism, strength, toxicity, and how the body recognizes it, creating a better environment for the drug to work and improving its absorption. Compared to traditional drug formulations, Drug delivery system offers several important benefits • Better stability: Drugs are less likely to break down before reaching their target. • Improved targeting: Medications are delivered more precisely, increasing their concentration where needed and reducing side effects. • Controlled release: Drug delivery system ensures drugs are released at the right place and time, even allowing breakthroughs like crossing the blood-brain barrier. • Lower dosages: This minimizes toxicity and improves the overall effectiveness of treatments. DDS doesn’t just deliver drugs to the affected area—it also plays a crucial role in targeting, controlled release, better absorption, and increased stability, making treatments safer and more efficient2. Modern drug delivery systems, like colloidal carriers, are revolutionizing the way we treat various conditions. Compared to traditional methods, these systems offer several benefits, such as better penetration of drugs through the skin, reduced hyperpigmentation, and less irritation or burning sensations, even on sensitive or damaged skin. In recent years, extensive research has focused on developing lipid-based carriers, solid lipid nanoparticles, and other advanced colloidal systems. These carriers include Multiple emulsions, microemulsions, nano emulsions, Liposomes, ethosomes, noisomes, transferosomes, Solid lipid nanoparticles, lipid microparticles, nanostructured lipid carriers, designed to improve how drugs diffuse through the skin, enhance their penetration, and target specific areas more effectively. This not only boosts therapeutic outcomes but also makes treatments safer and more efficient3. Transdermal drug delivery systems are medications designed to be applied to the skin in the form of patches or gels. They work by delivering drugs through the skin at a controlled rate, directly into the bloodstream. These systems have evolved significantly in recent years, making it easier to provide consistent, controlled delivery of medications. By targeting specific areas and releasing drugs gradually, transdermal systems can reduce the number and size of doses needed, improving both safety and effectiveness. Over the past couple of decades, these systems have gained popularity because they avoid the liver's first-pass metabolism, which can break down drugs taken orally, and they help improve bioavailability. They also minimize side effects commonly associated with pills or other oral medications. Today, the transdermal route is one of the most exciting areas of drug delivery research, with nearly 40% of new drug candidates in clinical trials focusing on transdermal or skin-based systems4. 2. Transfersomes Transfersome is a trademark owned by the German company IDEA AG, representing its unique drug delivery technology. The name comes from the Latin word "transferre" (meaning "to carry across") and the Greek word "soma" (meaning "body"), symbolizing a "carrying body". Essentially, a Transfersome is a specially designed vesicle, built to act like a natural cell or one involved in releasing substances (exocytosis). This makes it ideal for delivering drugs in a controlled and potentially targeted way. What sets Transfersomes apart is their ultra-flexible, self-adjusting membranes. These allow them to bend and squeeze through tiny pores—much smaller than the vesicle itself—making them highly efficient for crossing barriers in the body5. A Transfersome is a highly flexible and responsive structure designed to adapt under stress. This unique ability allows it to pass through different barriers in the body with ease. As a result, it works as an effective drug carrier, making targeted drug delivery possible without the need for invasive methods while ensuring a slow and steady release of therapeutic agents6. Transfersomes are an exciting breakthrough for improving drug delivery through the skin. What’s fascinating is their ability to squeeze through tiny pores in the stratum corneum that are over ten times smaller than they are. This means that even larger vesicles, around 200–300 nm in size, can easily pass through intact skin7. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 358-371 360 Transfersomes are a highly effective option for transdermal drug delivery, as they can deliver larger amounts of active compounds to the deeper layers of the skin. They work by creating an "osmotic gradient," which allows them to pass through the stratum corneum (SC) via either intracellular or transcellular pathways (Chen et al., 2020). What makes them particularly versatile is their ability to carry both water-loving (hydrophilic) and fat-loving (hydrophobic) molecules. This makes them ideal for delivering multiple drugs or a combination of drugs and natural compounds into the bloodstream at the same time. Their ability to penetrate the skin effectively due to their elasticity has been widely researched and is one of the key reasons they are considered so promising for drug delivery8. 3. Structure Transfersomes are incredibly flexible lipid bilayer vesicles designed to pass through the skin without breaking apart. A novel vesicular drug carrier system called transfersomes, which is composed of phospholipid, surfactant, and water for enhanced transdermal delivery. Each transferosome has at least one inner water-based compartment surrounded by a specially designed lipid bilayer as described in Figure 1. This bilayer’s unique flexibility comes from the addition of "edge activators," which make it less rigid and more adaptable9.Transfersomes are made up of phospholipids and a single-chain surfactant like sodium cholate, deoxycholate, Span 80, or Tween 80. These surfactants, called "edge activators," help loosen the lipid bilayer, making transfersomes much more flexible than liposomes. They can also include up to 10% ethanol and generally have a total lipid concentration of 5–10% in their final aqueous suspension. This unique combination of components gives transfersomes their exceptional flexibility and effectiveness for drug delivery10. Transfersomes are applied to the skin without using an occlusive dressing, which allows them to pass through the stratum corneum’s lipid layers due to the skin's natural hydration and osmotic forces. They've been used successfully to deliver a wide range of substances, including small molecules, peptides, proteins, and vaccines. According to IDEA AG, transfersomes are capable of penetrating the stratum corneum and deeper skin layers, eventually reaching the bloodstream to deliver their cargo11. Figure 1 Structure of Transfersome 3.1. Penetration through skin When something is applied to the skin, it can enter the body through three main pathways:as mentioned in Table 1. Table 1 Pathways for penetration through skin Hair Follicles and Sebaceous Glands The substance can travel down the hair follicles and interact with the sebaceous glands, providing an indirect route into the deeper layers of the skin. Sweat Ducts It can also pass through sweat ducts, which act as small natural channels into the skin. Stratum Corneum The most common route is directly across the stratum corneum, the skin's outermost layer. This layer is a highly organized, lipid-rich barrier that most substances need to overcome to penetrate deeper. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 358-371 361 Each pathway plays a role in how substances are absorbed through the skin and can be used strategically for effective drug delivery11. The intercellular lipid matrix, which plays a vital role in the skin's protective barrier, is created by keratinocytes in the middle to upper layers of the stratum granulosum. These cells release lamellar structures into the spaces between them. Once in the stratum corneum, this material forms organized lipid bilayers, where the hydrocarbon chains line up tightly, and the polar head groups are surrounded by a thin water layer. This unique structure helps the skin retain moisture and block harmful substances from entering12. The way molecules move through the stratum corneum has been extensively studied, with ongoing discussions about whether the intercellular or transcellular pathways play a bigger role and is represented in the Figure 2. However, it's now generally accepted that the intercellular lipid route is the main pathway for most small, uncharged molecules to penetrate the skin13. For highly hydrophilic molecules, the transcellular route may play a larger role in penetration. However, the lipid bilayers between the keratinocytes remain the main barrier to how quickly these molecules can pass through. Interestingly, using solvents to strip away some of the lipids from the stratum corneum has been shown to increase drug absorption, even for very water-soluble molecules14. Figure 2 Diagrammatic representation of the stratum corneum and the intercellular and transcellular routes of penetration 4. Advantages Transfersomes can deform and pass through narrow constriction (from 5 to 10 times less than their own diameter) without measurable loss. • They have high entrapment efficiency, in case of lipophilic drug near to 90%. • This high deformability gives better penetration of intact vesicles. • They can act as a carrier for low as well as high molecular weight drugs e.g. analgesic, anesthetic, corticosteroids, sex hormone, anticancer, insulin, gap junction protein, and albumin. • Transfersomes possess an infrastructure consisting of hydrophobic and hydrophilic moieties together and as a result can accommodate drug molecules with wide range of solubility. • They act as depot, releasing their contents slowly and gradually. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 358-371 362 • They can be used for both systemic as well as topical delivery of drug. • They are biocompatible and biodegradable as they are made from natural phospholipids similar to liposomes. • They protect the encapsulated drug from metabolic degradation. • Easy to scale up, as procedure is simple, do not involve lengthy procedure and unnecessary use or pharmaceutically unacceptable additives15. 4.1. Limitations • Transfersomes are prone to oxidative degradation, which makes them chemically unstable. However, this issue can be greatly minimized by removing oxygen from the solution and replacing it with inert gases like nitrogen or argon16. Keeping transfersomes in a cool place and protecting them from light can also help prevent oxidation. Techniques like freeze-drying or spray-drying after preparation can make transfersomes more stable for storage. • One of the challenges with using transfersomes for drug delivery is the difficulty of getting pure natural phospholipids. A practical solution is to use synthetic phospholipids instead17. • Transfersomal formulations tend to be costly because of the expensive raw materials used in lipid excipients and the specialized equipment needed for production. To keep costs down, phosphatidylcholine is often used as it’s a more budget-friendly option15. 4.2. Transfersomes v/s other carrier systems: At first glance, transfersomes might seem a lot like liposomes, which are lipid bilayer vesicles. But in reality, they’re completely different—transfersomes are far more flexible and adaptable than typical liposomes. Table 2 represents the comparison of different approaches. Table 2 Comparison of different approaches for permeation enhancement18. Method Advantage Disadvantage Liposomes Phospholipid vesicle, biocompatible, biodegradable Less skin penetration less stable Proliposome Phospholipid vesicle, more stable than liposomes Less penetration, cause aggregation and fusion of vesicles Physical methods e.g.iontophoresis Increase penetration of intermediate size charged molecule Only for charged drugs, transfer efficiency is low (less than 10%) Niosomes Non-ionic surfactants vesicles Less skin penetration easy handling But will not reach up to deeper skin layer Proniosomes Greater stability, Will convert into noisome in situ, stable Less skin penetration easy handling But will not reach up to deeper skin layer Transfersomes and Protransfersomes More stable, high penetration due to high deformability, biocompatible and biodegradable, suitable for both low and high molecular weight and also for lipophilic as well as hydrophilic drugs and reach up to deeper skin layers None, but for some limitations Transfersomes are different from mixed micelles in two key ways. First, they’re much larger—usually 10 to 100 times bigger than typical lipid micelles. More importantly, transfersomes have a water-filled core, while micelles are just tiny fatty droplets. This unique structure allows transfersomes to carry both water-soluble and fat-soluble substances, unlike micelles, which can only hold fat-soluble ones. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 358-371 363 Researchers used Confocal Scanning Laser Microscopy (CSLM) to explore how different carrier systems—like mixed lipid micelles, liposomes, and transfersomes—penetrate intact murine skin. Transfersomes stood out as the most effective, thanks to their flexibility. They were able to move through the stratum corneum and reach the deeper, living layers of the epidermis in significant amounts6,19,20. 4.3. Mechanism of action Vesicles are tiny colloidal particles with a water-based core surrounded by a bilayer made of amphiphilic molecules. They’re incredibly useful for drug delivery, as they can hold water-soluble drugs inside their core and fat-soluble drugs within their lipid bilayer. Mechanism of action of Transfersomes is as represented in the Figure 3. Figure 3 Mechanism of action of Transfersomes They can penetrate intact skin effectively, but only when applied without covering the skin. This uncovered state is important because it creates an osmotic gradient across the skin, which helps the process21. A study by Cevc and Blume found that transfersomes penetrate the skin through a process called hydrotaxis (or xerophobia). This means they’re drawn to the deeper, moisture-rich layers of the skin instead of the dry outer surface. This moisture-seeking behavior happens because, after the formulation is applied to the skin without a cover, moisture starts to evaporate, guiding the transfersomes inward22. The difference in water activity across the skin, created by the natural transdermal gradient, generates a strong force that acts on transfersomes. This force helps widen the spaces between skin cells, forming tiny channels about 20–30 nm wide. These channels allow the highly flexible transfersomes to pass through the skin, following the hydration gradient23. An osmotic gradient forms as water evaporates from the skin's surface due to body heat. This gradient drives the transfersomes to move across the skin, delivering therapeutic agents from the application site to the target area. This process ensures effective treatment with minimal systemic toxicity2. 4.3.1. Methods of preperation • Thin film hydration • Ethanol injection method • Modified hand shaking method • Reverse phase evaporation method • Vortexing sonication method • High pressure homogenization method 4.4. Thin film hydration / rotary evaporation-sonication method Transfersomes can be prepared by the thin film hydration method. Required quantities of phosphatidylcholine and surfactant will be taken in a round bottom flask and dissolved in chloroformand ethanol byshaking. The thin film will be formed by rotary evaporation using a rotary evaporator for 15 minutes at 25°C, 600 mm/hg pressure, and 100 rpm. Vacuumwill be applied for one hour to dry the film. The drug will be dissolved in10ml of 7.4 pH phosphate buffer, which will be heated to 55°C. Then the film will be hydrated with the heated buffer by hand shaking for half an hour. The mixture will then be stirred for half an hour in an orbital shaker. Afterward, the transferosomes will be observed under World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 358-371 364 a microscope. The transfersomal suspension will be stored in a refrigerator at 4°C24.The diagrammatic representation is shown in Figure 4. Figure 4 Thin film hydration method 5. Ethanol injection method Transfersomes will be prepared by the ethanol injection method. Three different steps will be followed: • Membrane compounds (PC, non-ionic surfactants, and/or cholesterol) will bedissolved in ethanol and injected at 60 °C into a heated phosphate buffer salinity(PBS) at 60 °C and gently stirred at 400 rpm with a magnetic stirrer. 5.6mLof the organic phase will be injected into 50 mL of the aqueous phase (PBS). • The organic phase will be eliminated using a rotary evaporator in a heatingbath at 50 °C. • Finally, the prepared transferosomes will be sonicated, and the samples will be ice-cooled to avoid excessive heat from sonication25. 5.1. Vortexing -sonication method: The phospholipids, edge activator and the drug are mixed in a phosphate buffer. The mixture is then vortexed until a milky transfersomal suspension is obtained. It is then sonicated, using a bath sonicator, for a respective time at room temperature and then extruded through polycarbonate membranes.(example: 450 and 220 nm)26. 5.2. Modified hand shaking method The modified handshaking method has the same basic principle as the rotary evaporation-sonication method. The preparation is shown in flow chart in Figure 5.In the modified handshaking process, the organic solvent, the lipophilic drug, the phospholipids and edge activator are added in a round-bottom flask. All the excipients should completely dissolve in the solvent and obtain a clear transparent solution. Then, the organic solvent is removed by evaporation while handshaking instead of using the rotary vacuum evaporator.In the meantime, the round-bottom flask is partially World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 358-371 365 immersed in the water bath maintained at a high temperature (example: 40–60 ◦C). A thin lipid film is then formed inside the flask wall. The flask is kept overnight for complete evaporation of the solvent. The formed film is then hydrated with the appropriate buffer solution with gentle shaking at a temperature above its phase transition temperature. The hydrophilic drug incorporation can be done in this stage.The resultant vesicles formed are bath sonicated, then sonicated vesicles extruded through a bed of polycarbonate membrane(100-200nm) and finally transfersomal suspension is formed10. Figure 5 Modified hand shaking mathod 5.3. Reverse phase evaporation method The phospholipids and edge activator are added to a round-bottom flask and dissolved in the organic solvent mixture (example: diethyl ether and chloroform). The lipophilic drug can be incorporated in this step. Then, the solvent is evaporated using rotary evaporator to obtain the lipid films. The lipid films are redissolved in the organic phase mostly composed of isopropyl ether and/or diethyl ether. Subsequently, the aqueous phase is added to the organic phase, leading to a two-phase system. The hydrophilic drug incorporation can be done in this stage. This system is then subjected to sonication using a bath sonicator until a homogeneous w/o (water in oil) emulsion is formed. The organic solvent is slowly evaporated using rotary evaporator to form a viscous gel, which then becomes a vesicular suspension27 as shown in Figure 6. World Journal of Biology Pharmacy and Health Sciences, 2025, 21(02), 358-371 366 Figure 6 Reverse phase evaporation method 5.4. High pressure homogenization method The phospholipids, edge activator and the drug are uniformly dispersed in PBS or distilled water containing alcohol and followed by ultrasonic shaking and stirred simultaneously. The mixture is then subjected to intermittent ultrasonic shaking. The resulting mixture is then homogenized using a high-pressure homogenizer. Finally, the transfersomes are stored in appropriate conditions28. 6. Evaluation of transfersomes 6.1. Vesicle size distribution and zeta potential Vesicle size, size distribution and zeta potential were determined by Dynamic Light Scattering system by Malvern Zeta sizer. 6.2. Vesicle morphology29 The diameter of vesicles can be measured using photon correlation spectroscopy or dynamic light scattering (DLS). To prepare the samples, they were suspended in distilled water, filtered through a 0.2 mm membrane, diluted with filtered saline, and then analyzed for size using DLS. Transfersome vesicles can also be visualized using techniques like electron microscopy (EM) or phase-contrast microscopy. To assess the stability of the vesicles, their size and structure were monitored over time. Mean size was measured with DLS, while any structural changes were observed using transmission electron microscopy (TEM). 6.3. No. of vesicles per cubic mm30 This is an essential step in optimizing the composition and refining process variables. Non-sonicated transfersome formulations are diluted 1:5 with 0.9% sodium chloride solution. A haemocytometer and an optical microscope are then used for analysis (19). The number of Transfersomes in 80 small squares is counted and calculated using the following formula: • Total number of transfersomes per cubic mm = • (Total number of transfersomes counted × dilution factor × 4000) /Total number of squares counted.