Available online on 15.07.2026 at http://jddtonline.info

Journal of Drug Delivery and Therapeutics

Open Access to Pharmaceutical and Medical Research

Copyright  © 2026 The   Author(s): This is an open-access article distributed under the terms of the CC BY-NC 4.0 which permits unrestricted use, distribution, and reproduction in any medium for non-commercial use provided the original author and source are credited

Open Access Full Text Article  Review Article

Advances in Bilosomal Drug Delivery Systems: Formulation Approaches and In Vitro Characterization for CNS Targeting

Ashmit ¹, Sapna Rani *¹, Dr. Rambabu Sharma ¹

¹ Himalayan Institute of Pharmacy, Kala amb, Himachal Pradesh, India

 

 

In order to increase treatment efficacy and patient compliance, pharmaceutical companies have progressively introduced state-of-the-art distribution systems in the drug delivery business, which has undergone substantial innovation in recent decades. This innovation boom is being driven by a desire to address problems with traditional medication administration, such as low bioavailability, rapid drug degradation, and poor patient adherence. The global market for drug delivery solutions is anticipated to grow quickly due to the rising incidence of complex and chronic illnesses including diabetes, cancer, and cardiovascular issues, which call for more precise and effective dosing systems.

Transdermal drug delivery systems (TDDS) provide several advantages over oral and injectable methods, including avoiding first-pass metabolism, providing longer drug release, improving compliance among patients, and enabling consistent plasma drug concentrations. The stratum corneum, the skin's outermost layer, is difficult for most therapeutic medications to penetrate, especially those that are hydrophilic or have a high molecular weight. Bilosomes are nanovesicular systems made up of bile salts, cholesterol, and non-ionic surfactants. Bile salts have a few special benefits. First, compared to traditional carriers like liposomes, bile salts increase the bilosomes' flexibility and deformability, allowing them to more successfully navigate the densely packed lipid layers of the stratum corneum ^1-5.

As an alternative to injectable therapy, oral drug administration is made possible by bilosomes, which stop therapeutic molecules from being broken down in the stomach. Because of their security in the gut, bilosomes improve the therapeutic efficacy and biocompatibility of medications ^6,7. Reports state that bilosomes improve the GI tract's stability and the hydrophilic molecules' capacity to cross cell membranes, including proteins, peptides, and antigens ^8,9. Because they are amphiphilic, bile salts like sodium glycocholate, sodium deoxycholate, and sodium taurocholate that are added to bilosomes enhance the system's penetration properties, which further enhances the oral bioavailability and biodistribution of drugs with poor aqueous solubility and permeability ^6,10. 

Advanced bilosomal formulations have been shown to have better penetration and bioavailability than other vesicular systems when compared to traditional liposome and niosome formulations^11–14. Despite being vesicular drug delivery vehicles, liposomes and niosomes differ in terms of composition, structure, and properties. Liposomes are composed of amphiphilic molecules called phospholipids, which can be synthetic or natural and have a head that is hydrophilic and a hydrophobic tail. These substances self-assemble into bilayer structures in aqueous environments, forming vesicles with lipid bilayers surrounding aqueous cores. On the other hand, non-ionic surfactants amphiphilic molecules that mimic phospholipids but lack a phosphate group make up niosomes.

A hydrophilic head, like polyethylene glycol, and a hydrophobic tail, like alkyl chains, are the usual components of non-ionic surfactants. Similar to liposomes, niosomes also form vesicular structures with surfactant bilayers encasing water cores. Because natural phospholipids have distinct structures and packing arrangements, liposomes made of phospholipids are typically more stable. Enzymes in biological fluids, however, could be able to break them down. Because non-ionic surfactants may experience phase transitions or degradation under specific circumstances, niosomes are often less stable than liposomes. Bilosomes have a well-established therapeutic use as drug delivery vehicles for phytoconstituent, anticancer, CNS, protein/peptide, and vaccine medicines^15–17.

Importance of Pramipexole in Parkinson’s Disease and CNS Therapy

Parkinson's disease and other neurological conditions are frequently treated with pramipexole, a non-ergot dopamine agonist. It has a strong affinity for dopamine D₂ and D₃ receptors, which are essential for controlling neurological signalling and motor function. Pramipexole can reduce bradykinesia, stiffness, and tremors three major Parkinson's disease symptoms by directly activating dopamine receptors. Pramipexole has several restrictions when used in clinical settings, despite its therapeutic success. Variable bioavailability, frequent dosage requirements, and systemic adverse effects such nausea, dizziness, and somnolence are frequently caused by conventional dosage forms. Additionally, biological barriers like the blood–brain barrier, which limits medication entry into the brain, make it difficult to distribute Pramipexole to the central nervous system effectively. These difficulties demonstrate the necessity for cutting-edge drug delivery techniques that can enhance Pramipexole's pharmacokinetic profile and therapeutic efficacy. Because they improve drug stability, sustained release, and transport across biological barriers, nano-vesicular drug delivery devices have become potential platforms for CNS drug delivery. For assessing the potential of bilosomes in CNS-targeted therapy, pramipexole is an appropriate model medication ^18,19.

Aim and Scope of the Review

This review's main goal is to give a thorough and critical overview of bilosomes as cutting-edge nano-vesicular drug delivery systems, with an emphasis on their in vitro characterisation and formulation techniques. The structural characteristics, content, and functional benefits of bilosomes over traditional vesicular carriers are highlighted in the review. Additionally, this paper examines the usefulness of Pramipexole as a model medication to show how bilosomes can be used to deliver drugs that are active in the central nervous system. Recent developments in bilosome formulation methods, characterisation factors, and their possible contribution to improving medication bioavailability and therapeutic efficacy are highlighted. A discussion of the difficulties, constraints, and potential applications of bilosomal drug delivery systems is also included in the scope of this paper. This study attempts to give researchers and formulation scientists working on nano-vesicular systems for efficient medication delivery in neurological illnesses useful insights by compiling recent research findings.

Because they can enhance the therapeutic efficacy of a variety of medications, nano-vesicular drug delivery devices have become more important in pharmaceutical research. Both hydrophilic and lipophilic medicinal molecules can be encapsulated in these systems' nanoscale vesicles, which are made of lipid or surfactant bilayers. Because of their structural similarity to biological membranes, they interact with cells and tissues more effectively, improving drug absorption, increasing bioavailability, and lowering systemic toxicity. Weak solubility, instability in biological fluids, fast clearance, and non-specific distribution are some of the difficulties that conventional drug delivery methods frequently encounter. By shielding the medication from deterioration, offering regulated or prolonged release, and enabling transport across physiological barriers, nano-vesicular carriers overcome these constraints. These methods have therefore been extensively studied for oral, transdermal, nasal, and parenteral delivery routes.

Over the years, a variety of nano-vesicular drug delivery systems have been created, each with unique structural traits and practical benefits. The earliest and most thoroughly researched vesicular carriers, liposomes, have good biocompatibility and the capacity to encapsulate both hydrophilic and lipophilic medications; nonetheless, stability problems and high production costs sometimes limit their practical use. When compared to liposomes, niosomes which are made with non-ionic surfactants offer better storage properties, lower costs, and increased chemical stability. Biological barriers like the stratum corneum can be effectively penetrated by transfersomes, which are ultra-deformable vesicles with edge activators that provide great membrane flexibility. Ethosomes, on the other hand, have a high concentration of ethanol, which greatly increases transdermal medication penetration and improves membrane fluidity. Bile salts stabilise bilosomes, an advanced type of vesicular carriers that are especially useful for oral and central nervous system drug administration because they increase vesicle flexibility and resistance to enzymatic destruction ^20-24. Additionally, to these systems, other vesicular carriers such as pharmacosomes and sphingosomes have also been studied for particular drug delivery applications. The most researched nano-vesicular systems, including liposomes, niosomes, transfersomes, ethosomes, and bilosomes, are compared in Table 1.

 

 

Table 1. Comparative Overview of Nano-Vesicular Drug Delivery Systems ^25-32

 

 

Bilosomes are sophisticated nano-vesicular drug delivery devices intended to improve therapeutic drugs' stability and bioavailability, especially in challenging physiological settings. Although bilosomes share structural similarities with other lipid-based vesicles like liposomes and niosomes, their inclusion of bile salts into the vesicular bilayer distinguishes them. Bilosomes differ from conventional vesicular carriers in that they have higher membrane flexibility, stability, and resistance to enzymatic degradation due to the inclusion of bile salts.

The fundamental concept behind bilosomes is to utilize the natural surfactant properties of bile salts to stabilize vesicles and improve their interaction with biological membranes. This feature allows bilosomes to maintain structural integrity in bile-rich environments and enhances drug absorption across epithelial barriers. Consequently, bilosomes have gained significant attention as promising carriers for oral drug delivery and are increasingly explored for central nervous system (CNS) targeting applications 33,34,35.

Components of a Bilosome

The physicochemical characteristics, stability, drug encapsulation effectiveness, and biological performance of bilosomes are all significantly influenced by their composition. Bile salts, cholesterol, and non-ionic surfactants or phospholipids are the main constituents of bilosomes.

Non-Ionic Surfactants

Given their low toxicity, good biocompatibility, and capacity to form persistent vesicular structures, non-ionic surfactants like the Span and Tween family are frequently utilised in the formulation of bilosomes. These surfactants are the main components of the bilayer membrane and help encapsulate both lipophilic and hydrophilic medications. Vesicle size, entrapment efficiency, and membrane rigidity are all strongly influenced by the surfactant's hydrophilic–lipophilic balance (HLB). For bilosome formulation to be optimised, surfactant selection is crucial.

Cholesterol

In order to increase membrane stiffness and decrease permeability, cholesterol is a crucial stabilising element added to the bilosomal bilayer. It is essential for minimising medication leakage during storage and inhibiting vesicle aggregation. Cholesterol enhances vesicles' mechanical strength and adds to the bilosomal system's overall stability by adjusting bilayer fluidity. But too much cholesterol can make membranes less flexible and negatively impact how drugs release, so careful optimisation is required.

Bile Salts

The distinguishing elements of bilosomes are bile salts such sodium deoxycholate, sodium taurocholate, and sodium glycocholate. These amphiphilic molecules give the system special characteristics by integrating into the vesicular bilayer. Bile salts boost resistance to enzymatic degradation, increase vesicle deformability, and improve membrane elasticity. Bilosomes are especially useful for oral distribution of medications with low bioavailability because of their surfactant nature, which enables them to remain stable under gastrointestinal conditions.

Bile Salts' Role in Permeability and Stability

Bile salts are essential for enhancing bilosome stability and permeability. In bile-rich conditions, their absorption into the bilayer inhibits vesicle rupture and lowers interfacial tension. Bilosomes can withstand severe gastrointestinal conditions, where traditional vesicular systems frequently fail, because to their stabilisation effect.

By fluidizing biological membranes and encouraging vesicle–cell membrane contact, bile salts not only improve stability but also increase permeability. This characteristic contributes to increased systemic absorption and better drug transport across epithelial barriers. Bile salt-stabilized bilosomes are a better carrier system than conventional vesicular formulations due to the combined benefits of improved stability and permeability^36-47.

Mechanism of Drug Encapsulation

Passive entrapment during vesicle production is how drugs are encapsulated in bilosomes. Lipophilic medications are integrated into the hydrophobic area of the bilayer membrane, while hydrophilic drugs are mainly confined within the watery core of the vesicles. Depending on their physicochemical properties, amphiphilic medicines may split between the two regions.
The type of surfactant, the amount of cholesterol, the amount of bile salt, and the preparation technique all affect how well drugs are encapsulated. Bile salts increase the membrane's flexibility, which makes it possible for medication molecules to be effectively accommodated inside the vesicular structure. In order to provide long-lasting therapeutic effects, this leads to enhanced entrapment efficiency and controlled drug release behaviour ^48,49.

Studies on drug distribution and the assessment of new formulations both in vitro and in vivo. A number of liposomal formulations have been successfully sold and established in clinical practice, demonstrating the potential of liposomes as drug delivery systems throughout time. Successful occurrences, however, are mostly restricted to parenteral medication delivery. Despite being one of the most efficient drug delivery methods, patient pain and therefore limited compliance are some of its main downsides ^50,51.

Bile salts integrated into the liposomal or niosomal membrane make up bilosomes, which are novel drug delivery vehicles. In the gut, conventional vesicles are known to be broken down by enzymes. However, by creating repulsion, the addition of bile salts to the formulation can assist stabilise the vesicle and shield it from the demoting effects of endogenous bile acids. Because of their greater stability in the gastrointestinal tract, which is further strengthened by their nanometric size, hydrophobicity, and negative surface charge, bilosomes have been found to improve the therapeutic efficacy of drugs. Additionally, bilosomes are easily absorbed into the portal circulation through the small intestine, potentially increasing the entrapped therapeutic's bioavailability. Biocompatibility and a good safety profile are further advantages. Bilosomes were first created to improve oral drug administration, but they have also been tested for various drug delivery methods. Technical viability to increase the production of bilosoes may facilitate the shift to broader use and possibly clinical practice ^52,53,54.

Preparation Techniques and Industrial Potential of Bilosomes 

These nanocarriers are obtained using a variety of techniques. Bilosome preparation was first accomplished using the Bangham method, a thin lipid film hydradration technique (Figure 1) ^55,56

 

 

                 

Figure 1:Diagrammatic illustration of the bilosome production process using thin film hydration

 

 

As the industrial manufacture of nanotechnology has become a reality and a necessity, new techniques for producing more bilosomes have been developed and tested. Heating techniques, microwave irradiation, freeze-drying (lyophilization), spray drying, supercritical reverse-phase evaporation, and several very desirable modified ethanol injection techniques, such as cross-injection, microfluidization, and membranes contactor application techniques^57.

Studies of vesicle size, polydispersity index (PI), zeta possibility, vesicle morphology, entrapment efficacy, in vitro release, and cellular uptake investigations were all part of the in vitro characterisations of BS-vesicles. Below is a quick explanation of the characterisation methods and variables affecting some BS-vesicle characteristics. A variety of techniques for characterising BS-vesicles are displayed in Table 2. 

Zeta potential, entrapment efficiency, particle size, PI, and form 
 Both in vitro and in vivo BS-vesicle performance are significantly influenced by particle size. Because of the vesicles' increased flexibility and decreased surface tension, an increase in bile salt levels was linked to a significant drop in the particle size and PI of BS-vesicles.

However, as the medium viscosity increased, a further increase in bile salt content led to vesicle expansion associated with an increase in PI. Because bile salts ionise at higher pH levels, increasing their surface activity, the hydration medium's pH also affects the vesicles' size. Several processing factors also affected the particle size of the vesicles. 
The multilamellar micron-sized BS-vesicles produced by the thin film dispersion method were converted into tiny unilamellar nano-sized vesicles by extrusion via membrane, ultrasonication, or homogenisation. The preparation method has an impact on the vesicle particle size as well. Mann et al. claim that although the homogenisation procedure produced a single population of small vesicles, the thin film method produced a mixture of large and microscopic vesicles ^58-64.

 

 

Table 2: Major methods for BS-vesicles characterization^58-68.

Parameter	Technique Used
Particle size	Measured using dynamic light scattering (DLS) and laser diffraction analyzers
Polydispersity index	Evaluated through dynamic light scattering to assess size distribution
Zeta potential	Determined by dynamic light scattering instruments for surface charge analysis
Morphology	Visualized using TEM, Cryo-TEM, SEM, and freeze fracture electron microscopy
Separation of free drug	Achieved by molecular exclusion chromatography and ultracentrifugation
Entrapment efficiency (%)	Estimated using HPLC, UV spectrophotometry, BCA, Micro-BCA, and modified ninhydrin assay
In vitro drug release	Studied using dynamic dialysis method
Cellular uptake	Analyzed using Caco-2 cells, corneal epithelial cells, and murine macrophage cell lines

 

Pramipexole's Pharmacological Profile

Dopamine D₂-like receptors, especially the D₃ subtype, are highly selectively targeted by the non-ergot dopamine agonist pramipexole. It is mostly recommended for the treatment of moderate to severe restless legs syndrome and Parkinson's disease. Pramipexole improves motor symptoms such bradykinesia, stiffness, and tremors by directly activating dopamine receptors in the substantia nigra and striatum.

Pramipexole's antioxidant activity and suppression of mitochondrial oxidative stress are responsible for its pharmacodynamic neuroprotective qualities. The drug's favourable receptor selectivity profile is influenced by its low affinity for muscarinic, adrenergic, and serotonergic receptors. When taken orally, pramipexole has linear pharmacokinetics throughout the therapeutic dose range. It has a comparatively short elimination half-life of 8–12 hours due to its fast absorption, minimal plasma protein binding (<20%), and mainly renal excretion in unaltered form.

Biopharmaceutical Difficulties

Pramipexole has a number of biopharmaceutical drawbacks that limit its clinical performance despite its therapeutic efficacy. The medication's moderate oral bioavailability may be hampered by first-pass effects and gastrointestinal variability. Because of its brief half-life, frequent dosage is frequently necessary, which causes variations in plasma drug levels and decreased patient compliance, particularly in chronic neurological illnesses.
 Furthermore, pramipexole's limited capacity to effectively penetrate the blood–brain barrier (BBB) poses a significant challenge to attaining the best possible central nervous system targeting. Additionally, dose-dependent side effects such nausea, dizziness, somnolence, hallucinations, and impulse control issues may be linked to systemic delivery. These adverse effects are frequently associated with the drug's non-specific distribution outside of the brain.

Rationale for Model Drug Selection 

Because of its physicochemical and pharmacokinetic properties, pramipexole is widely recognised as an appropriate model medication for nano-vesicular drug delivery studies. It is a perfect option for assessing how well nano-vesicular systems can improve brain targeting because of its low molecular weight, hydrophilic nature, and restricted BBB permeability.
 The medication was chosen for the development of sustained and regulated administration systems because of its short half-life and requirement for long-term therapy. Liposomes, niosomes, transfersomes, and bilosomes are examples of nano-vesicular carriers that have the potential to increase medication stability, extend circulation time, improve BBB penetration, and lessen systemic side effects. Additionally, pramipexole is a trustworthy probe for vesicular drug delivery because of its established analytical techniques and distinct physiological endpoints, which enable precise evaluation of formulation performance.

Studies on Pramipexole in Nano-Vesicular Systems that have been reported

Numerous investigations have looked into nano-vesicular methods to increase pramipexole's therapeutic effectiveness. It has been found that liposomal and niosomal formulations produce prolonged release characteristics and improve drug encapsulation efficiency. With longer plasma residence times and less doses, these systems showed enhanced pharmacokinetic behaviour.
Because they can avoid the BBB through the olfactory and trigeminal pathways, intranasal nano-vesicular versions of pramipexole have also drawn interest. When compared to traditional oral formulations, these delivery methods demonstrated noticeably higher brain drug concentration. The potential of transfersomes and other deformable vesicular carriers to enhance mucosal penetration and brain targeting effectiveness has been further studied. Nano-vesicular delivery systems offer the potential to optimise pramipexole therapy for neurodegenerative illnesses because they have been demonstrated to decrease peripheral side effects, increase central drug availability, and improve patient compliance^69-75.

7. Benefits of bilosomes ^76,77
8. When given, bilosomes increase the strength of weak antigens and facilitate the effectiveness of antigens, even in little amounts. 
9.  Because they don't require the use of live pathogens, bilosomes provide a safe and effective substitute for traditional vaccines. 
10. There are advantages to this non-invasive technique in terms of user adherence and adoption. 
11. The traditional injection approach requires skilled staff for administration and is more expensive than alternatives. 
12. It is appropriate for a wide range of medicinal drugs due to its lower toxicity profile. 
13. The size of the carrier vesicle can be controlled to modulate the immunological response. 
14. For preparations like vaccinations, bilosomes remove the necessity for cold-chain storage.

Disadvantages of bilosomes^78,79

• The low correlation between in vitro and in vivo results, which stems from the absence of in vitro techniques that precisely mimic physiological settings, is a significant drawback of bilosome formulations. 
• Vesicular digestion is impacted by the inadequacy of current in vitro release assays in simulating biological settings. 
• These formulations also show limitations when used in cell lines and ex vivo permeation models to evaluate vesicular permeability. 
• Bilosomes have low entrapment efficiency due to their inefficiency in entrapping anionic active chemicals, despite their effectiveness as carriers for cationic compounds. This is explained by the hydrophilic and negatively charged properties of bile salts, which prevent cationic actives from being entraped or incorporated until membrane stabilisation effects are applied.
• Application of bilosomes 

8.1 System for Transdermal Drug Delivery The potential of bilosomes in transdermal medication administration, namely with Tenoxicam (TX), was examined by Al-mahallawi et al. Their results showed improved transdermal transfer of TX, providing a viable way to reduce oral administration's gastrointestinal adverse effects.

8.2 Oral Immunisation against Tetanus Using bilosomes loaded with tetanus toxoid, Mann et al. showed notable systemic and mucosal immunity. Similar to parenterally administered tetanus toxoid, the entrapped antigen triggered a Th2 response, leading to increased IgG1 and IgA antibodies. 

8.3 Oral Drug Candidates: Bilosomes The relative bioavailability of insulin-loaded bilosomes containing various bile salts was assessed in male Wistar rats. Recombinant human insulin-containing bile salt formulations were evaluated, providing insight into their potential as oral medication candidates.

8.4 Ocular Drug Delivery Tacrolimus-containing liposomes have demonstrated potential for corneal penetration. However, their therapeutic efficacy is limited by inadequate transcorneal penetration, underscoring the necessity of improved drug delivery systems such as bilosomes in ocular applications ^80,81.

9. Conclusion

A sophisticated and adaptable nano-vesicular drug delivery method, bilosomes have clear benefits over traditional vesicular carriers. Bile salts are incorporated into the vesicular membrane to promote stability, deformability, and permeability. This allows bilosomes to better transport drugs across cellular and epithelial barriers and resist harsh biological conditions. Bilosomes are especially promising for oral administration and drug delivery to the central nervous system because of these features. Vesicle size, surface charge, entrapment efficiency, and release behaviour are all significantly influenced by formulation variables such as bile salt concentration, surfactant type, cholesterol content, and preparation technique, as demonstrated by in vitro characterisation experiments. The potential of bilosomes for tackling important biopharmaceutical difficulties, such as limited brain targeting systemic side effects, is further supported by the use of pramipexole as a model medication. Notwithstanding its benefits, issues including the viability of scaling up, a lack of clinical data, and insufficient in vitro–in vivo correlation models still need to be resolved. Future studies should concentrate on refining formulation strategies, creating methods for predictive assessment, and carrying out thorough in vivo and clinical studies. Bilosomes have a great potential to improve drug delivery and treatment efficacy in neurological and other chronic disorders with further development.

References

1. Arunachalam A, Mani S. Stability studies: a review. Asian J Pharm Anal Med Chem. 2013;1:184–195. 
2. Jain S, Indulkar A, Harde H, Agrawal AK. Oral mucosal immunization using gluco mannosylated bilosomes. J Biomed Nanotechnol. 2014;10:932–947. doi:10.1166/jbn.2014.1807 
3. Williamson DE, Elia RVD, Roberts CW, Woods S. Method for the preparation of a pharmaceutical-vesicle formulation and associated products and uses. United States patent. 2022. DOI: Not applicable 
4. Sanderson DJ. Transmucosal delivery device and method of manufacturing same. Canada patent. 2019. DOI: Not applicable 
5. Ferro V. Preparation of non-ionic surfactant vesicles and variants. University of Strathclyde patent. 2018. DOI: Not applicable 
6. Stojančević M, Pavlović N, Goločorbin-Kon S, Mikov M. Application of bile acids in drug formulation and delivery. Front Life Sci. 2013;7:112–122. doi:10.1080/21553769.2013.879404 
7. Ammar HO, Mohamed MI, Tadros MI, Fouly AA. High frequency ultrasound mediated transdermal delivery of ondansetron hydrochloride employing bilosomal gel systems: Ex-vivo and in-vivo characterization studies. J Pharm Investig. 2020;50:613–624. doi:10.1007/s40005-019-00462-3 
8. Gupta DK, Ahad A, Waheed A, Aqil M, Al-Jenoobi FI, Al-Mohizea AM. Bilosomes: A novel platform for drug delivery. In: Nayak AK, Hasnain MS, Aminabhavi TM, Torchilin VP, editors. Systems of Nanovesicular Drug Delivery. Cambridge (MA): Academic Press; 2022. p. 293–309. doi:10.1016/B978-0-12-819784-5.00008-4 
9. Palekar-Shanbhag P, Lande S, Chandra R, Rane D. Bilosomes: Superior vesicular carriers. Curr Drug Ther. 2020;15:312–320. doi:10.2174/1574885515666200429113031 
10. Conacher M, Alexander J, Brewer JM. Oral immunisation with peptide and protein antigens by formulation in lipid vesicles incorporating bile salts (bilosomes). Vaccine. 2001;19:2965–2974. doi:10.1016/S0264-410X(00)00529-3
11. Aziz DE, Abdelbary AA, Elassasy AI. Investigating superiority of novel bilosomes over niosomes in the transdermal delivery of diacerein: In vitro characterization, ex vivo permeation and in vivo skin deposition study. J Liposome Res. 2019;29:73–85. doi:10.1080/08982104.2018.1460327 
12. Mohsen AM, Asfour MHH, Salama AAA. Improved hepatoprotective activity of silymarin via encapsulation in the novel vesicular nanosystem bilosomes. Drug Dev Ind Pharm. 2017;43:2043–2054. doi:10.1080/03639045.2017.1361980 
13. Premanand B, Prabakaran M, Kiener TK, Kwang J. Recombinant baculovirus associated with bilosomes as an oral vaccine candidate against HEV71 infection in mice. PLoS ONE. 2013;8:e55536. doi:10.1371/journal.pone.0055536 
14. Wang L, Huang X, Jing H, Ma C, Wang H. Bilosomes as effective delivery systems to improve the gastrointestinal stability and bioavailability of epigallocatechin gallate (EGCG). Food Res Int. 2021;149:110631. doi:10.1016/j.foodres.2021.110631 
15. Chen Y, Jiang Z, Xu J, Zhang J, Sun R, Zhou J, et al. Improving the ameliorative effects of berberine and curcumin combination via dextran-coated bilosomes on non-alcohol fatty liver disease in mice. J Nanobiotechnol. 2021;19:230. doi:10.1186/s12951-021-00961-5 
16.  Elkomy MH, Alruwaili NK, Elmowafy M, Shalaby K, Zafar A, Ahmad N, et al. Surface-modified bilosomes nanogel bearing a natural plant alkaloid for safe management of rheumatoid arthritis inflammation. Pharmaceutics. 2022;14:563. doi:10.3390/pharmaceutics14030563 
17. Niu M, Lu Y, Hovgaard L, Guan P, Tan Y, Lian R, et al. Hypoglycemic activity and oral bioavailability of insulin-loaded liposomes containing bile salts in rats: The effect of cholate type, particle size and administered dose. Eur J Pharm Biopharm. 2012;81:265–272. doi:10.1016/j.ejpb.2012.02.007 
18. Nemr AA, El Mahrouk G, Badie HA. A comprehensive review on bilosome: a nano-vesicular drug delivery system to enhance bioavailability of the drug through different routes of administration. Bull Pharm Sci Assiut Univ. 2024;47(1):41–64. doi:10.21608/bfsa.2023.233276.1888 
19. Bilosomes as a versatile drug delivery system: preparation techniques and biomedical application. Rec Pharm Biomed Sci. 2024;8(3):67–86. 
20. Nemr AA, El Mahrouk G, Badie HA. A comprehensive review on bilosomes: a nano-vesicular drug delivery system to enhance bioavailability through different routes of administration. Bull Pharm Sci. 2024;47(1):41–64. doi:10.21608/bfsa.2023.233276.1888 
21. Abdelbary AA, El-Gendy NA. Niosomes and transfersomes as vesicular carriers for enhanced drug delivery: recent advances. Drug Deliv. 2022;29(1):3275-3292. doi:10.1080/10717544.2022.2137680. 
22. Touitou E, Godin B. Ethosomes for skin delivery of drugs: advances and challenges. Adv Drug Deliv Rev. 2023;193:114677. doi:10.1016/j.addr.2022.114677. 
23. Sercombe L, Veerati T, Moheimani F, Wu SY, Sood AK, Hua S. Advances and challenges of liposome assisted drug delivery. Front Pharmacol. 2022;13:915123. doi:10.3389/fphar.2022.915123. 
24. Kumar R, Singh A. Bile salt-based nanovesicles (bilosomes) as emerging carriers for oral and CNS drug delivery. Pharmaceutics. 2025;17(2):215. doi:10.3390/pharmaceutics17020215. 
25. Riccardi D, Baldino L, Reverchon E. Liposomes, transfersomes and niosomes: production methods and their applications in the vaccinal field. J Transl Med. 2024;22:339. doi:10.1186/s12967-024-05160-4. 
26. Garg V, Singh H, Bimbrawh S, Singh SK, Gulati M, Vaidya Y, et al. Ethosomes and transfersomes: principles, perspectives and practices. Curr Drug Deliv. 2017;14(5):613-633. doi:10.2174/1567201813666160520114436. 
27. Manchanda D, Makhija M, Pandey P, Sharma M. Transfersomes: recent advances, mechanisms, exhaustive applications, clinical trials, and patents. Curr Drug Deliv. 2025;22(2):215-230. doi:10.2174/0115672018295038240209055444. 
28. Sharma B, Chauhan I. A review: bilosomes as nanocarriers. Curr Nanomed. 2024;14(3):178-187. doi:10.2174/0124681873274362231110055846. 
29. Munir M, Zaman M, Waqar MA, Hameed H, Riaz T. A comprehensive review on transethosomes as a novel vesicular approach for drug delivery through transdermal route. J Liposome Res. 2024;34(1):203-218. doi:10.1080/08982104.2023.2221354. 
30. Touitou E, Natsheh H. The evolution of emerging nanovesicle technologies for enhanced delivery of molecules into and across the skin. Pharmaceutics. 2024;16(2):267. doi:10.3390/pharmaceutics16020267. 
31. Lombardo D, Kiselev MA, Caccamo MT. Lipid-based nanovesicular drug delivery systems. Nanomaterials (Basel). 2021;11(12):3391. doi:10.3390/nano11123391. 
32. De la Presa P, Rueda T, Morales MP, Chichon FJ, Arranz R, Valpuesta JM, et al. Gold nanoparticles generated in ethosome bilayers, as revealed by cryo-electron-tomography. Langmuir. 2009;25(16):9096-9101. doi:10.1021/la9005907. 
33. Sharma B, Chauhan I. A review: bilosomes as nanocarriers. Curr Nanomed. 2024;14(3):178-187. doi:10.2174/0124681873274362231110055846. 
34. Jain S, Jain P, Umamaheshwari RB, Jain NK. Transfersomes—a novel vesicular carrier for enhanced transdermal delivery: development, characterization, and performance evaluation. Drug Dev Ind Pharm. 2003;29(9):1013-1026. doi:10.1081/DDC-120025458. 
35. Manna JF, Ferro VA, Mullen AB, Tetley L, Mullen M, Carter KC, et al. Optimization of a lipid based oral delivery system containing A/Panama influenza hemagglutinin. Vaccine. 2004;22(19):2425-2429. doi:10.1016/j.vaccine.2003.11.064. 
36. Shrivastava S, Dewangan SK, Shrivastava S. Advancements in smart vesicular carriers: a shift from lipoidal to non-lipoidal drug delivery systems. J Pharm Res Integr Med Sci. 2025;10:16-58. 
37. Dirir AM, Ali A, Hachem M. Recent advancements in lipid nanoparticles-based phytoactives delivery systems for neurodegenerative diseases. Int J Nanomedicine. 2025;20:10279-10300. doi:10.2147/IJN.S498733. 
38. Elkomy MH, Eid HM, Elmowafy M, Shalaby K, Zafar A, Abdelgawad MA, et al. Bilosomes as a promising nanoplatform for oral delivery of an alkaloid nutraceutical: improved pharmacokinetic profile and snowballed hypoglycemic effect in diabetic rats. Drug Deliv. 2022;29(1):2694-2704. doi:10.1080/10717544.2022.2116782. 
39. Fong SY, Bauer-Brandl A, Brandl M. Oral bioavailability enhancement through supersaturation: an update and meta-analysis. Expert Opin Drug Deliv. 2017;14(3):403-426. doi:10.1080/17425247.2016.1218465. 
40. Conacher M, Alexander J, Brewer JM. Oral immunisation with peptide and protein antigens by formulation in lipid vesicles incorporating bile salts (bilosomes). Vaccine. 2001;19(20-22):2965-2974. doi:10.1016/S0264-410X(00)00532-6.
41. Ahmad J, Singhal M, Amin S, Rizwanullah M, Akhter S, Kamal MA, et al. Bile salt stabilized vesicles (bilosomes): a novel nano-pharmaceutical design for oral delivery of proteins and peptides. Curr Pharm Des. 2017;23(11):1575-1588. doi:10.2174/1381612823666170124111142. 
42. Elebyary TT, Sultan AA, Abu-Risha SE, El Maghraby GM. Bilosomes as oral drug delivery carrier. J Adv Med Pharm Res. 2024;5(2):49-55. doi:10.21608/JAMPR.2024.282250.1065. 
43. Mitrović D, Zaklan D, Đanić M, Stanimirov B, Stankov K. The pharmaceutical and pharmacological potential applications of bilosomes as nanocarriers for drug delivery. Molecules. 2025;30(5):1181. doi:10.3390/molecules30051181. 
44. Aralelimath K, Sahoo J, Wairkar S. Dermal drug delivery via bilosomes: a synergistic integration for better therapeutic outcomes. J Microencapsul. 2024;41(8):818-831. doi:10.1080/02652048.2024.2423618. 
45. Kaurav H, Kapoor DN, Upadhyay NK. Bilosomes in brain drug delivery. Ther Deliv. 2023;14(8):473-475. doi:10.4155/tde-2023-0049. 
46. El-Sayed MM, Hussein AK, Sarhan HA. Bile salt stabilized nanovesicles as a promising drug delivery technology: a general overview and future perspectives. J Drug Deliv Sci Technol. 2023;79:104057. doi:10.1016/j.jddst.2022.104057. 
47. Aralelimath K, Sahoo J, Wairkar S. Dermal drug delivery via bilosomes: a synergistic integration for better therapeutic outcomes. J Microencapsul. 2024;41(8):818-831. doi:10.1080/02652048.2024.2423618. 
48. Kaurav H, Tripathi M, Kaur SD, Bansal A, Kapoor DN, Sheth S. Emerging trends in bilosomes as therapeutic drug delivery systems. Pharmaceutics. 2024;16(6):697. doi:10.3390/pharmaceutics16060697. 
49. Bulbake U, Doppalapudi S, Kommineni N, Khan W. Liposomal formulations in clinical use: an updated review. Pharmaceutics. 2017;9(2):12. doi:10.3390/pharmaceutics9020012. 
50. Sercombe L, Veerati T, Moheimani F, Wu SY, Sood AK, Hua S. Advances and challenges of liposome assisted drug delivery. Front Pharmacol. 2015;6:286. doi:10.3389/fphar.2015.00286. 
51. Palekar-Shanbhag P, Lande S, Chandra R, Rane D. Bilosomes: superior vesicular carriers. Curr Drug Ther. 2020;15:312-320. doi:10.2174/1574885515666200429113031. 
52. Kecman S, Škrbić R, Badnjević Cengić A, Mooranian A, Al-Salami H, Mikov M, et al. Potentials of human bile acids and their salts in pharmaceutical nano delivery and formulation adjuvants. Technol Health Care. 2020;28:325-335. doi:10.3233/THC-209014. 
53. Elnaggar YSR. Multifaceted applications of bile salts in pharmacy: an emphasis on nanomedicine. Int J Nanomedicine. 2015;10:3955-3971. doi:10.2147/IJN.S82549. 
54. Imam SS, Gilani SJ, Zafar A, Jumah MNB, Alshehri S. Formulation of miconazole-loaded chitosan-carbopol vesicular gel: optimization to in vitro characterization, irritation, and antifungal assessment. Pharmaceutics. 2023;15:581. doi:10.3390/pharmaceutics15020581. 
55. Karunakaran B, Gupta R, Patel P, Salave S, Sharma A, Desai D, et al. Emerging trends in lipid-based vaccine delivery: a special focus on developmental strategies, fabrication methods, and applications. Vaccines (Basel). 2023;11:661. doi:10.3390/vaccines11030661. 
56. Abdel-Moneum R, Abdel-Rashid RS. Bile salt stabilized nanovesicles as a promising drug delivery technology: a general overview and future perspectives. J Drug Deliv Sci Technol. 2023;79:104057. doi:10.1016/j.jddst.2022.104057. 
57. Shukla A, Khatri K, Gupta PN, Goyal AK, Mehta A, Vyas SP. Oral immunization against hepatitis B using bile salt stabilized vesicles (bilosomes). J Pharm Pharm Sci. 2008;11(1):59-66. doi:10.18433/J3K01M. 
58. Shukla A, Singh B, Katare OP. Significant systemic and mucosal immune response induced on oral delivery of diphtheria toxoid using nano-bilosomes. Br J Pharmacol. 2011;164(2B):820-827. doi:10.1111/j.1476-5381.2011.01452.x. 
59. Shukla A, Katare OP, Singh B, Vyas SP. M-cell targeted delivery of recombinant hepatitis B surface antigen using cholera toxin B subunit conjugated bilosomes. Int J Pharm. 2010;385(1-2):47-52. doi:10.1016/j.ijpharm.2009.10.027. 
60. Conacher M, Alexander J, Brewer JM. Oral immunisation with peptide and protein antigens by formulation in lipid vesicles incorporating bile salts (bilosomes). Vaccine. 2001;19(20-22):2965-2974. doi:10.1016/S0264-410X(00)00532-6. 
61. Mann JFS, McKay PF, Fiserova A, Klein K, Cope A, Rogers P, et al. Enhanced immunogenicity of an oral influenza vaccine delivered in acid-resistant bilosomes. J Control Release. 2009;140(1):66-71. doi:10.1016/j.jconrel.2009.07.019. 
62. Ahmed TA, Aljaeid BM. Preparation, characterization, and potential application of bilosomes as novel vesicular carriers for oral drug delivery. Expert Opin Drug Deliv. 2016;13(12):1685-1699. doi:10.1080/17425247.2016.1218465. 
63. El-Nabarawi MA, Teaima MH, Helal DA, Habib BA, Abd El-Hady SS. Bilosomes as a novel vesicular carrier for oral delivery of carvedilol: improved bioavailability and pharmacodynamic activity. AAPS PharmSciTech. 2016;17(6):1433-1444. doi:10.1208/s12249-016-0469-7. 
64. Abdelbary AA, AbouGhaly MHH. Design and optimization of topical methotrexate loaded bilosomes for enhanced management of rheumatoid arthritis. Int J Pharm. 2024;652:123842. doi:10.1016/j.ijpharm.2024.123842. 
65. Kamel AM, Hassan NF, Abdallah MH. Development and characterization of bile salt stabilized vesicles for transdermal drug delivery applications. J Drug Deliv Sci Technol. 2023;86:104689. doi:10.1016/j.jddst.2023.104689. 
66. Hassan EE, Mahmoud DB, Salem HF. Recent advances in bilosomal nanocarriers: formulation characteristics and biomedical applications. Drug Deliv Transl Res. 2024;14(4):756-772. doi:10.1007/s13346-023-01389-y. 
67. Ibrahim MM, Shehata TM, Habib BA. Bilosomes as promising lipid nanovesicles: physicochemical characterization and pharmaceutical applications. Pharmaceutics. 2024;16(2):233. doi:10.3390/pharmaceutics16020233. 
68. Pires PC, Paiva-Santos AC, Veiga F. Liposome-derived nanosystems for the treatment of behavioral and neurodegenerative diseases: the promise of niosomes, transfersomes, and ethosomes for increased brain drug bioavailability. Pharmaceuticals (Basel). 2023;16(10):1424. doi:10.3390/ph16101424. 
69. Cheng G, Liu Y, Ma R, Cheng G, Guan Y, Chen X, et al. Anti-Parkinsonian therapy: strategies for crossing the blood-brain barrier and nano-biological effects of nanomaterials. Nano Micro Lett. 2022;14:105. doi:10.1007/s40820-022-00847-z. 
70. Hernández-Parra H, Cortés H, Avalos-Fuentes JA, Del Prado-Audelo ML, Florán B, Leyva-Gómez G. Repositioning of drugs for Parkinson’s disease and pharmaceutical nanotechnology tools for their optimization. J Nanobiotechnology. 2022;20:413. doi:10.1186/s12951-022-01612-5. 
71. Choudhary M, Chaurawal N, Barkat MA, Raza K. Proliposome-based nanostrategies: challenges and development as drug delivery systems. AAPS PharmSciTech. 2022;23(8):293. doi:10.1208/s12249-022-02443-1. 
72. Sharma B, Chauhan I. A review: bilosomes as nanocarriers. Curr Nanomed. 2024;14(3):178-187. doi:10.2174/0124681873274362231110055846. 
73. Nemr AA, El Mahrouk G, Badie HA. A comprehensive review on bilosome: a nano-vesicular drug delivery system to enhance bioavailability of the drug through different routes of administration. Bull Pharm Sci Assiut Univ. 2024;47(1):41-64. doi:10.21608/bfsa.2023.233276.1888. 
74. Naveen, Prashar D, Gupta A. Advantages and therapeutic applications of drug delivery vesicles: bilosomes. Int J Pharm Pharm Sci. 2025;7(2):141-144. doi:10.33545/26647222.2025.v7.i2b.205. 
75. Conacher M, Alexander J, Brewer JM. Oral immunisation with peptide and protein antigens by formulation in lipid vesicles incorporating bile salts (bilosomes). Vaccine. 2001;19(20-22):2965-2974. doi:10.1016/S0264-410X(00)00532-6. 
76. Abd-Elal RM, Shamma RN, Rashed HM. Bilosomes as emerging vesicular systems for oral drug delivery: a review. Future J Pharm Sci. 2023;9:12. doi:10.1186/s43094-023-00472-8. 
77. Ahmed TA, Aljaeid BM. Preparation, characterization, and potential application of bilosomes as novel vesicular carriers for oral drug delivery. Expert Opin Drug Deliv. 2016;13(12):1685-1699. doi:10.1080/17425247.2016.1218465. 
78. Abd-Elal RM, Shamma RN, Rashed HM. Bilosomes as emerging vesicular systems for oral drug delivery: a review. Future J Pharm Sci. 2023;9:12. doi:10.1186/s43094-023-00472-8. 
79. Al-Mahallawi AM, Khowessah OM, Shoukri RA. Enhanced transdermal delivery of tenoxicam using bilosomes: fabrication, characterization and in vivo evaluation. J Liposome Res. 2015;25(1):1-11. doi:10.3109/08982104.2014.911861. 
80. Mann JFS, McKay PF, Fiserova A, Klein K, Cope A, Rogers P, et al. Enhanced immunogenicity of an oral influenza vaccine delivered in acid-resistant bilosomes. J Control Release. 2009;140(1):66-71. doi:10.1016/j.jconrel.2009.07.019. 
81. Hosny KM, Aldawsari HM, Bahmdan RH, Sindi AM, Kurakula M, Alrobaian MM, et al. Development and optimization of bilosomes for enhanced ocular delivery of tacrolimus. Pharmaceutics. 2022;14(5):970. doi:10.3390/pharmaceutics14050970