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The advent of nanotechnology has revolutionized drug delivery systems, with liposomes standing out as one of the most promising carriers for targeted and controlled delivery of therapeutic agents. This edited volume, Liposomal Drug Delivery: A Novel Approach for Therapeutics, is a comprehensive compilation of the scientific principles, formulation strategies, analytical methodologies, and regulatory perspectives associated with liposomal drug delivery systems. Liposomes, first described by Alec Bangham in the 1960s, have evolved from a basic model of biological membranes to clinically relevant drug delivery platforms. Today, liposomal formulations have been successfully commercialized for various diseases including cancer, fungal infections, and viral illnesses, marking their significance in modern pharmaceutics. This book aims to serve as an academic and practical guide for researchers, formulators, industry professionals, and regulatory scientists involved in the development of advanced drug delivery systems. The first chapter introduces the fundamental concept of liposomal drug delivery, encompassing its history, structural aspects, and a survey of FDA-approved liposomal products. Following this, the book systematically addresses the critical components used in liposome formulation, highlighting the role of excipients and formulation challenges. The transition from labscale preparation to industrial manufacturing is discussed in detail, outlining key process parameters and scale-up considerations. Drug loading strategies form a cornerstone of liposomal development, and an entire chapter is devoted to exploring passive and active loading techniques along with associated hurdles. The analytical characterization chapter provides insights into the physicochemical evaluation and quality control measures vital for product success. Recognizing the growing emphasis on quality, a dedicated chapter discusses the application of Quality by Design (QbD) approaches in optimizing liposomal formulations. The book further explores targeted and long-acting liposomes, essential for achieving site-specific delivery and prolonged circulation. Sterilization, a critical aspect of parenteral liposomal products, is examined with a focus on technical challenges and potential solutions. Stability studies and shelflife determination are also covered to ensure the long-term viability of formulations.

Introduction Liposomes are bubble-like structures made up of (phospho)lipids that form on their own and surround an area of water in the middle with two or more overlapping layers of lipids. Liposomes are between 30 and micrometers in size and are made up of two phospholipid layers that are 4-5 nm thick. British scientists Alec Bangham and his colleagues at Babraham Cambridge were the first to describe the structure of liposomes in 1964 (Kozubek et al., 2000; Samad et al., 2007). This was in the middle of the 1960s and was a big step forward for the field of liposomology. Following this, liposomes have been studied in great detail because they can carry imaging agents, proteins, nucleic acids, and small chemicals. Many ways of giving medicine have been created to make treatment more effective and help patients stick with it. These include parenteral, pulmonary, oral, transdermal, ophthalmic, and nasal methods. In addition, liposomes are used a lot in the food and beauty industries (Maurer et al., 2001). Liposomes are very good at delivering drugs because they keep the parts inside from breaking down naturally, increase the half-life of the drug, manage the release of drug molecules, and are very safe and compatible with living things. Liposomes can improve therapeutic benefits by passively or actively targeting to deliver their payload to the affected area. This raises the maximum dose that can be tolerated, lowers the risk of systemic side effects, and improves treatment results. When compared to normal tissue, which has strong links between endothelial cells, abnormal tissues like solid tumors or inflammatory sites have more permeable capillaries (Szoka, 2019; Allen and Cullis, 2013; Fielding, 1991). The enhanced permeability and retention (EPR) effect is when liposomes can quietly gather and stay in damaged tissues by passing through the broken neovasculature. When the liposome interacts with certain molecules and receptors on the surface of tumor cells, this is called active targeting (Saraf et al., 2020). Some receptors, like folic acid, integrin, CD44, CD13, prostate-specific membrane antigen, vascular endothelial growth factor, and epidermal growth factor, may be overexpressed in tumor cells. Certain ligands could be used to change the surface of liposomes, as shown by the receptors. Antibodies, nucleic acids, protein peptides, iNGR, small molecules like folic acid, and carbohydrates are some of these ligands. Besides being used for specific medicines, liposomes are also a great way to get drugs to people who need them. However, liposomes have not yet reached their full potential because there are only 14 different types of liposomal goods on the market. We have learned more about commercial liposomal products that have been approved by both the FDA and the EMA through this research. The materials used in commercial goods and the ways they are made are both carefully thought out (Nekkanti and Kalepu, 2015; Olusanya et al., 2018).

Introduction The term “drug delivery systems” (DDSs) describes sophisticated dose forms that are utilized in the management of illness. The idea behind the design is to deliver the right quantity of medication to the diseased locations at the right time in order to maximize therapeutic efficacy while minimizing negative effects. The “3R” delivery principle—right dose, right place, and right time—is a concise way to describe it. Drug delivery technology can increase a candidate’s draggability while also enhancing the potency and safety of the medication. For example, liposomal drug delivery methods can be used to address issues with poor solubility and significant side toxicity in a variety of medications. The hazardous side effects of conventional chemotherapy medications can be greatly decreased by employing liposomal technology, which increases patient compliance (Andresen et al., 2004). Liposomes are the most widely used and researched nanocarrier for targeted medication delivery. They have improved therapies for a range of biomedical applications by stabilizing medicinal molecules, eliminating obstacles to cellular and tissue absorption, and boosting the biodistribution of compounds to specific locations in vivo. One or more concentric lipid bilayers enclosing discrete aqueous zones are described as the features of phospholipid vesicles, also known as liposomes. Due to the unique ability of liposomal systems to ensnape both lipophilic and hydrophilic molecules, a broad range of pharmaceuticals can be stored within these vesicles. Hydrophilic molecules may become trapped in the aqueous center of the bilayer membrane when hydrophobic molecules are introduced into it (Antohe et al., 2004). There are many potentials uses for liposomes, and some recent reviews have offered useful details. In order to address the ongoing demand for the R&D of novel liposomal medications, significant efforts must be done. There are still some unanswered questions in this field. This article will summarize technological advancements, address issues, and explore the future of pharmaceutical liposomal delivery by focusing on three key areas: effective application, overcoming obstacles, and the move from idea to clinical application (Allen et al., 2013). Additionally, because of the large aqueous center and biocompatible lipid exterior, a variety of macromolecules, including as DNA, proteins, and imaging agents, can be administered. Liposomes are a drug delivery technique that have several advantages, including biocompatibility, the capability to self-assemble, a large drug payload, and a range of physicochemical and biophysical properties that may be modified to control their biological features (Allen, 1994). Among the features that characterize liposomal formulations and ascertain their durability in vitro and in vivo are particle size, charge, lamellar number, lipid content, and surface modification with polymers and ligands. Encapsulated compounds are protected from dilution, degradation, and early inactivation in the circulation by liposomes. Liposomes are generally believed to be low toxicity and pharmacologically inactive because they are frequently composed of phospholipids that occur naturally. But more and more studies are showing that liposomes are not as immunologically inactive as was once thought. Although liposomal formulations have demonstrated efficacy in vivo, their clinical translation has been gradual. The advancements, biological difficulties, biomedical uses, and translational barriers of liposomal technology will all be covered in this review (Dams et al., 2000).

Introduction At the moment, liposomes, which are tiny bubble-like structures made of phospholipid bilayers, are an important part of all drug transport methods. Their unique ability to hold medicinal chemicals that both like and don’t like water is what makes them stand out. These molecules’ ability to change forms is very important for making different medicines more bioavailable, stable, and effective (Shah et al., 2020; Wagner and Vorauer-Uhl, 2011). Since Alec D. Bangham first found liposomes in the 1960s, they have gone from being simple experimental models to highly advanced carriers that are now used in both clinical and business settings. Liposome production has changed over time from simple lab methods to complex systems used on a commercial scale. At the moment, these methods can make liposomal formulations that are of high quality and consistently good traits (Alavi et al., 2022; Wagner et al., 2006). It is very important to know how to control the physicochemical qualities of liposomes during the manufacturing process. This includes things like size, charge, lamellarity, and how well they encapsulate (Mozafari et al., 2005; Watwe and Bellare, 1995). It is important to know the qualities of liposomes in order to figure out how they act in biological systems, like how fast drugs are released, how long they stay in circulation, and how they spread throughout the body. What method is used to make liposomal pharmaceutical compositions has a big effect on how safe and useful they are. Over the years, many methods have been developed to make liposomes with specific properties that are useful for their purpose (Sorgi and Huang, 1996; Patil and Jadhav, 2014). One of the first and easiest ways to make liposomes was through thin-film hydration. It was mostly used in places where study was being done. “Thin-film hydration” is the process of an organic liquid evaporating after lipids dissolve in it, which makes a thin film (Redziniak et al., 2017). After that, a watery phase is added to the film, which causes multilamellar vesicles (MLVs) to form. This method is easy to use and works well for smallscale applications, but it often creates liposome populations that aren’t all the same size or level of encapsulation. In order to get around these problems, methods like sonication and extrusion are used to make liposomes that are smaller and more uniform (Vemuri et al., 1990). There is a method called “sonication” that can be used to efficiently change larger liposomes into smaller unilamellar vesicles (SUVs). The size of liposomes can be reduced very well with this method, which is known for being one of the first to do so. With this method, liposomes with diameters generally smaller than 100 nm can be made very efficiently. Because of this, these liposomes work well for many cellular purposes. Despite this, sonication could create heat and mechanical forces that could damage the structure of the liposome membrane or make it less stable for molecules that are particularly fragile. Improving the sonication settings is necessary to find the best balance between making the liposomes smaller while still protecting the inside parts (Liu and Meng, 2021). An effective way to make liposomes with a precise and uniform size distribution is through extrusion. A pressure-based method can let liposomes pass through

Introduction Liposomes as Drug Delivery Vehicles Liposomes, first described by Alec Bangham in 1961, are self-assembled vesicular structures composed of one or more lipid bilayers surrounding an aqueous core(Nakhaei et al., 2021). These lipid bilayers typically consist of natural or synthetic phospholipids, which can spontaneously form closed vesicles in an aqueous environment due to their amphiphilic nature. The resulting liposomal structure resembles a miniature cell membrane, with hydrophilic heads facing outward towards the aqueous environment and hydrophobic tails facing inward, forming the bilayer structure(Nogueira, Gomes, Preto, & Cavaco-Paulo, 2015). Liposomes are important in the field of drug delivery due to their unique structural properties, which enable them to encapsulate a wide range of therapeutic agents, including hydrophilic, hydrophobic, and amphiphilic compounds. (Guimarães, Cavaco-Paulo, & Nogueira, 2021) The size of liposomes can vary from tens to hundreds of nanometers, making them suitable for both systemic and targeted drug delivery applications. Liposomes can be formulated to exhibit specific properties such as prolonged circulation time, enhanced stability and targeted delivery.(Simões, Moreira, Fonseca, Düzgünes, & De Lima, 2004) The versatility of liposomes is due to their ability to encapsulate drugs within the aqueous core, embed drugs within the lipid bilayers, or attach drugs onto the liposomal surface through chemical conjugation or targeting ligands. This flexibility allows for the development of multifunctional liposomal formulations capable of encapsulating multiple drugs which facilitate combination therapy and theranostic applications.(Cheng et al., 2021; Eloy et al., 2014) Liposomes also offer several advantages over conventional drug delivery systems, including biocompatibility, biodegradability, and low immunogenicity. These characteristics minimize the risk of adverse reactions and systemic toxicity which makes the liposomes suitable for repeated dosing and long-term therapeutic applications.(Al-Jamal & Kostarelos, 2011; Alavi, Karimi, & Safaei, 2017; Goyal et al., 2005) However, liposomal drug delivery systems are also associated with several challenges which include issues related to stability, scalability, and manufacturing consistency.(Kraft, Freeling, Wang, & Ho, 2014)

Introduction Liposomal formulations constitute a distinctive category of nanomedicine platforms, showcasing potential applications in pharmaceuticals, nutraceuticals, and cosmetics (Salave et al., 2024). Since their discovery, liposomes have attracted considerable interest because of their biocompatibility, ability to encapsulate both hydrophilic and hydrophobic drugs, and potential to improve drug bioavailability while minimizing toxicity (Bangham, 1992; Dhayalan et al., 2024). The pharmaceutical industry is rapidly recognizing liposomes as effective drug delivery vehicles capable of changing pharmacokinetics, targeting particular tissues, and managing drug release. The unique properties of these vesicles, mainly made up of phospholipids and cholesterol, have created new opportunities for developing drugs that present difficult therapeutic indices (Wang et al., 2023). Nonetheless, the intricate architecture and functional diversity require comprehensive and advanced analytical evaluation, essential for maintaining quality, efficacy, and stability during the formulation’s lifecycle (Fan et al., 2021 a). The swift progress in liposomal drug development has resulted in a heightened need for strong analytical techniques. Establishing reliable, reproducible, and precise analytical methods is a crucial step in the formulation and commercialization of liposomal drugs (Khan et al., 2024). These methods must consider not only the physical and chemical stability but also the entrapment efficiency, particle size distribution, surface charge, and drug release kinetics, along with other essential characteristics. Considering that liposomal formulations consist of multiple components that interact dynamically, a thorough analytical approach is crucial. The complexity of ensuring quality and performance in liposomal products arises from the diverse physicochemical characteristics of liposomes, which can be affected by formulation composition, the encapsulated drug, and external environmental factors (Giordani et al., 2023). The primary focus of liposomal characterization is the assessment of particle size and size distribution, as these factors have a direct impact on pharmacokinetics, biodistribution, and cellular uptake. Dynamic Light Scattering (DLS) and nanoparticle tracking analysis (NTA) are widely used methods for assessing liposome size and distribution, offering important information regarding batch-to-batch consistency and formulation stability (Nadkarni et al., 2024). Furthermore, the size of the particles influences their circulation duration in living organisms, as smaller vesicles typically attain extended circulation and enhanced tissue infiltration. A key parameter, zeta potential, or surface charge, offers insights into the electrostatic stability of liposomal suspensions, which is essential for predicting aggregation behavior and shelf-life (Biscaia-Caleiras et al., 2024; Paramshetti et al., 2024). Encapsulation efficiency is a crucial metric for therapeutic effectiveness, indicating the ratio of the drug contained within the liposome to the overall quantity utilized in the formulation. This aspect is essential, particularly for powerful medications where precise dosing is required. Measuring encapsulation efficiency typically requires the use of separation methods such as ultracentrifugation,

Introduction Nanomedicine, utilizing nanoparticles for therapeutic purposes, has captured significant interest in both research and industry. Numerous FDA-approved products demonstrate its clinical potential. Liposomes, spherical vesicles with phospholipid bilayers, have emerged as a prominent platform for drug delivery. They offer a versatile carrier system for a wide range of therapeutics, including the successful encapsulation of doxorubicin, an anticancer agent, leading to improved efficacy and reduced side effects. Additionally, liposomes have been explored extensively for nucleic acid delivery, such as siRNA and DNA, enhancing cellular penetration and protecting drugs from degradation. Liposomes were among the first nanotechnologies to reach the market in 1995 and remain a major platform today. Notably, the first FDA-approved mRNA vaccine for COVID-19 (2020) utilizes lipidic/liposomal nanocarriers for delivery. However, despite their advantages, formulating, developing, and manufacturing liposomes presents significant challenges. The complexity of nano formulations and nanomanufacturing stems from the unique physicochemical properties at the nanoscale. A greater number of variables need to be understood and optimized compared to traditional drug delivery systems. This lack of comprehensive understanding often leads to sensitivity and poor reproducibility in nano-preparations and manufacturing. For such complex systems, experimental approaches that identify critical parameters and their influence on the final product are crucial. Quality by Design (QbD) offers a solution, advocated for by various industries and regulatory bodies. QbD begins with defining the Quality Target Product Profile (QTPP), outlining the essential quality attributes (QA) of the final product for safety and efficacy. These QAs are influenced by critical material attributes (CMA) and process parameters (CPP). QbD then focuses on optimizing CMA and CPP by setting target specifications that ensure the desired QAs and ultimately achieve the QTPP (Alshaer et al., 2022). A proper experimental design is employed to link CMA and CPP to QA. This facilitates establishing targeted specifications for materials, processes, and the final product. Furthermore, QbD enables the evaluation of multiple factors simultaneously and prioritizes QA through risk assessments. Given the success of liposomal-based drugs in clinical use and their diverse pre-clinical applications, a strategic and systematic approach to liposome development is necessary. QbD offers a framework to create better drug delivery systems for enhanced therapeutic efficacy. While previous research has explored QbD for liposomal drug delivery, there is a growing need to understand and describe the latest advancements in using QbD for liposomal formulations. This will pave the way for liposomalbased drug delivery systems with superior therapeutic outcomes and potential for industrial development (Alshweiat et al., 2019).

Introduction The area of drug delivery has progressed remarkably in recent decades propelled by the demand for more efficient therapeutic approaches that can reduce side effects and improve the therapeutic index of medications. Conventional approaches to drug delivery frequently lead to widespread distribution, putting healthy tissues at risk of exposure to harmful levels of therapeutic compounds, while not achieving sufficient concentrations at targeted disease locations (Yu et al., 2023). This issue is especially apparent in addressing intricate conditions like cancer, cardiovascular diseases, and neurological disorders, where the therapeutic window is limited, and the likelihood of adverse effects increases. In this context, the advancement of targeted drug delivery systems has become an essential approach for enhancing the effectiveness and safety of therapeutic agents, with liposomal drug delivery systems receiving significant focus (Mitchell et al., 2021). The concept of targeted drug delivery revolves around the selective direction of therapeutic agents towards specific tissues or cells, aiming to reduce exposure to non-target areas. This method is crucial for enhancing treatment efficacy, especially in conditions marked by variability in drug response, like cancer. For example, traditional chemotherapy frequently results in considerable toxicity and negative side effects because of the non-specific distribution of drugs (Gao et al., 2023). Utilizing specific delivery methods allows healthcare professionals to increase the concentration of therapeutic agents in tumor tissues, leading to better treatment results and minimizing harm to healthy tissues (Bae et al., 2011). Beyond oncology, targeted drug delivery shows potential for various diseases, such as infectious diseases, autoimmune disorders, and neurological conditions. For example, in the management of viral infections, targeted delivery can increase the concentration of antiviral agents within infected cells, thereby enhancing efficacy and minimizing systemic toxicity. Targeted delivery systems can enhance the transport of therapeutics across the blood-brain barrier (BBB) in the context of neurodegenerative diseases, addressing a major challenge in drug development for central nervous system (CNS) disorders. Delivering drugs selectively to their sites of action is essential for achieving optimal therapeutic outcomes and enhancing patient quality of life (Wu et al., 2023 a; Rana et al., 2024). Among various drug delivery systems, liposomes have surfaced as a flexible and efficient platform for targeted drug delivery. Liposomes consist of lipidbased vesicles formed by phospholipid bilayers, capable of encapsulating both hydrophilic and lipophilic drugs. Their distinctive structure and characteristics render them excellent options for various applications in targeted therapy. The encapsulation of a wide variety of therapeutic agents, such as chemotherapeutics, anti-inflammatory drugs, peptides, and nucleic acids, stands out as a key benefit of liposomes. This adaptability facilitates the simultaneous delivery of various drugs or therapeutic approaches within one liposomal formulation, promoting combination therapies that can improve effectiveness and address resistance mechanisms (Wang et al., 2023).

Introduction Liposomal drug delivery methods have received a lot of interest in the pharmaceutical industry because of their capacity to improve therapeutic efficacy while also reducing toxicity of encapsulated pharmaceuticals. Liposomes, which are spherical vesicles made up of lipid bilayers, provide targeted delivery of drugs, increased bioavailability, and controlled release of therapeutics (Gupta et al., 2023; Rana et al., 2023; Salave et al., 2023, 2024). However, guaranteeing the sterility of liposomal formulations is critical for their safety and efficacy, particularly in parenteral applications. Sterilization is a validated process that achieves a microorganism-free product without altering its properties. In the pharmaceutical industry, a sterilization process is deemed valid when it successfully attains the necessary Sterility Assurance Level (SAL) of 10-6. The SAL represents the likelihood of contamination occurring after the sterilization process for a product with an initial biological load of 106 colonyforming units (Woedtke et al., 2008). The biological indicator used in this case is one of three bacterial species: Geobacillus stearothermophilus, Bacillus pumilus, or B. atrophaeus (Mcevoy et al., 2023). Accordingly, a product may be deemed “sterile” if the likelihood of a viable microorganism being present is equal to or less than 106. Liposomal formulations provide particular issues for sterilization due to their fragile nature and the physicochemical features of the encapsulated drugs. Sterilization plays a vital role in the production of liposomal formulations for parenteral administration. It is crucial to remove all viable microorganisms to prevent infections, maintain the effectiveness of therapeutic agents, and ensure patient safety. The delicate nature of liposomal carriers presents unique challenges in the sterilization process (Lombardo et al., 2022 a). Traditional sterilization methods, such as autoclaving, dry heat, and chemical sterilization, are frequently not suitable for liposomal structures (Figure 1). The application of these techniques can lead to the breakdown of lipids, changes in phase, or destabilization of the drug contained within the formulation, resulting in a decrease in its therapeutic effectiveness (Zuidam et al., 1993 a). In addition, the physicochemical properties of liposomes, such as their size, lamellarity, and lipid composition, play a crucial role in determining their stability and how they react to sterilization methods. Considering these limitations, the pharmaceutical industry has investigated alternative methods of sterilization that guarantee the elimination of microorganisms while preserving the integrity of liposomes. A commonly used method for sterilizing liposomal formulations is filtration through sterile membrane filters with pore sizes of 0.2 micrometers (Goldbach et al., 1995 a). This

Introduction Phospholipids naturally form liposomes when placed in water. These liposomes consist of one or more phospholipid bilayers that enclose an aqueous interior. The liposomes offer multiple benefits due to their enhanced permeability and retention mechanism [1]. Liposomes have the potential to enhance the pharmacokinetic properties of encapsulated drugs [2], increase their circulation times, and allow passive targeting and accumulation in areas of inflammation and tumors. Liposomes help diminish the overall toxicity caused by unencapsulated drugs [3]. They surge a drug’s solubility and offer a sustained [4], controlled release of the encapsulated drug. Stability studies and shelf-life determination are indispensable for maintaining the integrity and efficacy of liposome-based products. The key factors influencing liposome stability include temperature, pH, lipid composition, and the use of stabilizing agents. These elements must be carefully managed to ensure that liposomal formulations remain effective throughout their intended shelf life [5]. Research indicates that adding tetraether lipids to liposomes can significantly improve their stability in challenging conditions such as low pH, elevated temperatures, and autoclaving, thereby extending their shelf life [6]. The addition of antioxidants such as vitamin C has proven effective in surging the stability and antioxidant properties of liposomes, thereby extending their shelf life across various environmental conditions [7]. Studies have exhibited those liposomal formulations showed superior chemical, physical, and microbiological stability compared to traditional formulations such as ointments, emulsions, and gels. This superior stability makes liposomes a highly auspicious alternative for maintaining product integrity over extended periods

Introduction Liposomal formulations have emerged as a revolutionary platform in drug delivery systems, significantly enhancing the therapeutic efficacy and safety profile of numerous pharmaceutical agents (Crommelin et al., 2015). Liposomes are spherical vesicles composed of lipid bilayers, providing a versatile and biocompatible method for encapsulating both hydrophilic and hydrophobic drugs (Nsairat et al., 2022) (Liu et al., 2022). This encapsulation protects drugs from degradation, reduces systemic toxicity, and enhances their bioavailability (Gubernator, 2011). Beyond their protective capabilities, liposomes serve as an effective platform capable of delivering a diverse array of therapeutic agents, including conventional drugs, proteins, genes, and oligonucleotides (Gupta et al., 2023) (Salave et al., 2022 a) (Juliano et al., 2009). Their attractive biological properties, such as biocompatibility, enhanced solubility of hydrophobic compounds, stability of large molecules, and improved efficacy, position them as ideal candidates for advanced drug delivery systems. These properties enable precise targeting and controlled release strategies, thereby optimizing therapeutic outcomes across various medical applications (Polaka et al., 2021). The pharmacokinetic (PK) and pharmacodynamic (PD) properties of liposomal formulations are crucial determinants of their clinical success. Pharmacokinetics involves the study of the absorption, distribution, metabolism, and excretion (ADME) of drugs, while pharmacodynamics focuses on the biochemical and physiological effects of drugs and their mechanisms of action (Li et al., 2019) (Negus et al., 2018). Understanding these parameters in the context of liposomal delivery is essential to optimize therapeutic outcomes and minimize adverse effects. Liposomal encapsulation can significantly alter the PK profile of a drug. By modifying drug release kinetics, liposomes can extend the half-life of encapsulated agents, ensuring sustained therapeutic effects and reducing dosing frequency (Rana et al., 2022). Moreover, the ability of liposomes to target specific tissues or cells through passive and active targeting mechanisms enhances drug distribution to the site of action, increasing efficacy and minimizing off-target effects (Salave et al., 2021)(Salave et al., 2022 b). For instance, the liposome surface can be covalently decorated with targeting ligands, enhancing binding and internalization by cancer cells that express receptors for these ligands (Riaz et al., 2018). These ligands can include immunoglobulins or their subunits, such as immunoglobulin fragment antigen binding (Fab’) and single-chain variable fragments (scFv), or nutrient molecules with affinity for cell surface receptors, such as transferrin and folate (Sapra et al., 2003) (Kang et al., 2011) (Cheng et al., 2010). Liposomes’ unique structural properties facilitate the incorporation of targeting ligands, pHsensitive components, and other modifications to refine the drug’s therapeutic profile (Salave et al., 2022 c)(Salave et al., 2023)(Zangabad et al., 2018). They can also be engineered to release encapsulated drugs at specific sites by sensitizing the bilayer membrane to stimuli such as light exposure, oxidation, enzymatic degradation, heat, or radiation (Lee et al., 2017) (Antoniou et al., 2021). The

Introduction The use of liposomes as carriers for drug delivery has had a profound impact on the pharmaceutical industry. In 1961, Alec Bangham made a significant contribution to the field by introducing liposomes, which sparked a wave of research and development in this area (Bangham, 1992). Over time, liposomes have become incredibly versatile in their ability to transport drugs, biomolecules, and gene therapies. This groundbreaking approach has not only enhanced the effectiveness of treatments but also minimized side effects by precisely targeting specific sites within the body (Lai et al., 2024). Liposomes are vesicular structures composed of one or more concentric lipid bilayers that enclose an aqueous core. Hydrophilic drugs are commonly found within the aqueous compartments, whereas hydrophobic drugs are incorporated into the lipid bilayers. The innovative design of this architecture enables flexible drug encapsulation and precise delivery to specific targets. Liposomes have a wide range of applications in medical science, going beyond drug delivery to encompass diagnostics and vaccines. This versatility greatly enhances their value in the field (Gupta et al., 2023). An important milestone in liposomal delivery technology occurred in 1995 with the approval of Doxil, which was the first-ever liposomal drug delivery system. Since then, there have been notable advancements in improving the physicochemical properties of liposomal formulations. Although there have been advancements in this field, it is important to remember that the core principles established during Doxil’s approval are still relevant for all liposomal delivery systems. These principles encompass the importance of stability, achieving optimal drug release kinetics, and ensuring biocompatibility and safety (Barenholz, 2012; Wang et al., 2022). First and foremost, stability is of utmost importance. It is crucial to ensure that liposomal formulations remain intact throughout storage and administration in order to maintain the integrity of the drug and avoid problems such as leakage or degradation. The stability of liposomes can be affected by various factors, including the lipid composition, preparation method, and storage conditions. Ensuring stability necessitates the implementation of advanced manufacturing processes and rigorous quality control measures (Liu et al., 2022). In addition, it is crucial to achieve the ideal drug release kinetics. Striking the perfect balance between sustained release and efficient delivery to the target site is crucial for optimizing therapeutic benefits and minimizing any potential adverse effects. Designing liposomes to release their payload in a controlled manner, customized to the pharmacokinetics of the encapsulated drug, is a crucial aspect of this process. Optimizing factors like liposome size, surface charge, and the inclusion of targeting ligands can greatly enhance the release profile (Pande, 2024). Furthermore, ensuring biocompatibility and safety is of utmost importance. It is crucial to conduct comprehensive safety evaluations to ensure that liposomal systems do not trigger immune responses or lead to any adverse reactions. The lipid composition and surface characteristics of liposomes play a crucial