Structure-Function Relationships in the Modification of Liposomes for Targeted Drug Delivery in Infectious Diseases

Abstract

The introduction of liposomes has caused a paradigm shift in medicine, offering novel solutions to problems that are ancient to the drug discovery and development for HIV, TB, and malaria. These are the three deadliest infectious diseases that are endowed with complex pathophysiological and biological mechanisms that allow them to thrive in their hosts through escaping the immune system and capturing key pathways. Disease heterogeneity and lack of suitable models to replicate the disease states make compounds the poor pharmacokinetic issues associated with these diseases. Liposomes are lipid-based nanocarriers that are employed for drug formulations, preservation, and storage. Importantly, they can be tailored for targeted and controlled release. Structure–function relationships are crucial to consider in liposome design as they affect key interactions between the carrier drug and the target cell, which impact on drug release, cellular uptake, bioavailability, biodistribution, and toxicity. Herein, lipid composition, size, lamellarity, zeta potential/charge as well as surface modification with cholesterol, PEG, peptides, and antibodies are discussed with respect to selectivity in targeting diseased cells. The role of computational tools in expediting the liposome technology is reviewed, highlighting the impact of forces of interaction between biomolecules and the conditions of the environment.

1. Introduction

The phenomenon of “pandemic readiness” cannot be overemphasized, and through innovative management and preventive tools, the global manifestation of infectious diseases can be overcome. It is of paramount importance that these measures, which encompass therapeutics, vaccines, and diagnostics be developed with consideration for sustainability, biocompatibility, and biodegradability—safety for human and animal consumption, and eco-friendliness are imperative. Nanotechnology has provided an invigorating landscape for exploring solutions to ancient problems that still persist in modern-day medicine, hence the coining of the term nanomedicine. The introduction of liposomes has had a paradigm shift in medicine; their derivation from biological molecules has implored their service as valuable nano-vehicles in various bio-applications. Not only were they the first nanomedicine to be applied to human patients but also their success in clinical applications is undoubtedly apparent [1, 2].

Infectious diseases (also communicable diseases) are caused by pathogenic microorganisms, which are transmitted between animals through direct and indirect contact [3]. Pathogenic microorganisms have existed for centuries evolving mechanisms that would facilitate their evasion of the human immune system, including human-borne interventions that were developed to curb their spread such as vaccines, therapeutic drugs, and diagnostic tools. Low-resourced countries are the most vulnerable and burdened with infectious diseases which accounted for the highest mortality rate of about 2.5 million in 2020 from malaria, human immunodeficiency virus (HIV), and tuberculosis (TB) [3, 4, 5]. Malaria, TB, and HIV/AIDS were the most prominent infectious diseases in WHO’s top 10 leading causes of death in low-income countries for the year 2019 and are the three deadliest infectious diseases globally [5, 6]. This has necessitated the development of cost-effective methods that are efficient and accessible. The heterogeneity in the pathophysiology of these diseases, their canny mechanisms of capturing, and exploiting their hosts’ systems allow them to thrive while making it difficult to formulate drugs that are nontoxic for the host. Moreover, models that can replicate the disease states do not exist, which further complicates drug validation processes. Poor pharmacokinetics and drug toxicity are thus prominent issues that require novel approaches.

Liposomes are broadly used in industry, including in the pharmaceutical, nutraceutical as well as other applications in biotechnology [7]. They act as cell membrane models and as carriers for targeted delivery systems, such as drugs, vaccines, and various biomolecules: nucleic acids, proteins, peptides, lipids, carbohydrates, antibiotics, dyes, antioxidants, and enzymes [7].

1.1 The history and biochemistry of liposomes

Liposome is a portmanteau of Greek words “lipos” and “soma” meaning “fat” and “body,” respectively, which in essence describes their amphiphilic phospholipid building blocks [8]. Consequently, they are vesicle-forming, with the phospholipid bilayer enclosing an aqueous core, which dissolves hydrophilic compounds, while the lipid layer entraps lipophilic compounds [7]. When mixed with therapeutic compounds, lipids can transform into NP that entrap and protect their cargo, releasing them upon encounter with their target cells. The structure is depicted in Figure 1.

Figure 1.

Structure of a liposome. (The figure has been reproduced from a review by Rommasi and colleagues [9], with permission from SpringerOpen and open access from the creative commons CC-BY license).

Liposomes were first evidenced using electron microscopy in the 1960s by Alec Bangham while studying phospholipids and blood clotting factors [8]. The history of liposomes is outlined in Figure 2. Major strides were reported between the late 1980s and 1990s when the anticancer drug doxorubicin was successfully encapsulated and later approved in 1995 - Doxil® [10]. These were important discoveries where liposomes demonstrated their ability to reduce doxorubicin-induced cardiotoxicity [11, 12] and increase blood residence [13]. The upscaling of liposome production, introduction of new lipid raw material, and the tailorability of the lipid material were some of the major progressive events in the history of liposomes as drug delivery systems [8]. To date, a variety of lipid formulations are used from natural and/or synthetic lipids and surfactants [14].

Figure 2.

Timeline history of liposomes. (The figure was reproduced from Elsevier [10], with open access from the creative commons CC-BY license).

The size, lamellarity, surface charge, zeta potential, lipid composition, and organization of liposomes are imperative properties in their efficacy, playing key roles in their interaction with the cell, blood residence or half-life, tissue permeability, and final fate in vivo [14]. The morphology and physicochemical properties of liposomes, including the dimension and lamellarity dictates their classification and preferred applications [7, 14]. This will be discussed in detail in the sections to follow.

Cholesterol is mandatory to the liposome formulation, making up 30-50% of the membrane on average, which is higher than any membrane component [15, 16, 17]. It intercalates readily in the membrane core due to its amphipathic properties resembling the phospholipids, with a predominantly hydrophobic structure. Cholesterol has a polar hydroxyl (OH) group that connects to four fused hydrocarbon rings that are, in turn, linked to a branched chain of eight carbon residues [18]. In essence, the arrangement of cholesterol in the liposomes is not coincidental, and it is positioned perpendicularly to the plane of the bilayer, wherein the polar OH group, that is, the 3β-OH is in proximity with the phosphate and carbonyl of the glycerol ester linkage of the phospholipids, allowing the potential formation of hydrogen bonds (H-bonds), which are considered the most important of interactions [19]. Hydrogen bonds can also form through water bridges between the cholesterol OH group and any acceptor or donor group from the phospholipid, and finally, the oxygen of the OH group and phospholipids CH₃ group [19, 20]. However, the rigidity or increased stability of the phospholipid bilayer is not only endowed by the hydrogen bonding but also the aromatic rings of cholesterol, which contain major and minor grooves, and the rings are proposed to reduce the flexibility of the aliphatic carbon chain of the phospholipids [21]. The reduced membrane permeability induced by cholesterol also owes to the hydrophobic effect between the lipid hydrocarbon chains and cholesterol, which is imperative for conferring rigidity [22]. Through these interactions, cholesterol is capable of inducing morphological changes that modify the bilayer stability, curvature, membrane fluidity, diffusion rates of proteins and lipids, and permeability, which directly impact drug leakage.

Phospholipid derivatives: Fluidity and charge of the membrane are greatly dependent on the phospholipid derivatives. These can be in the form of phosphatidylglycerol, phosphatidylserine, phosphatidylcholine, or phosphatidylethanolamines [23]. Permeability or drug leakage can be controlled by the type of lipid used; whereas saturated acyl chain lipids confer rigidity and impermeability, unsaturated lipids tend to be more permeable [23]. The types of lipid composition and effect on drug delivery systems are discussed in detail in the below sections, with specific references to the disease.

Size and lamellarity: Liposomes assume a spherical structure, which can range in size from nanometers to micrometers; however, in medicine, nanoliposomes have been more acceptable [14], providing high-surface areas that afford the loading of high concentrations of drugs and vaccines. Size is an important effector in drug loading and release, bioavailability, biodistribution, mucoadhesion, and cellular uptake [24]. Particle size as well as the polydispersity index (PDI) are essential for cellular uptake—a process that mainly occurs via an endocytosis-dependent manner. Efficiency in the systemic delivery of drugs and uptake by tissues is dependent on the capillary perfusion of tissues, wherein the efficiency in cargo exchange depends on the size of the cargo delivered and of the membranous fenestration [24].

The aerosol delivery of drugs is favored for diseases of the lung, wherein the harsh systemic conditions are avoided to enable proximal localization of the drug to its target. The aerodynamic volume of the liposome-encapsulated system becomes important in the pulmonary localization of the drug, wherein drugs with a mass median aerodynamic diameter (MMAD) of 3 μm have an estimated 50-60% chance of localizing in the alveoli and 80% chance of residence in the lower airways [25]. In comparison, the oropharyngeal and large conducting airways regions are mostly adsorbed with larger particles with a size range of 5–10 μm [25]. The impermeability of the blood-brain barrier (BBB) has imposed challenges in the development of efficacious drugs targeting the brain tissue requiring innovative methods of enhancing their bioavailability and biodistribution. Liposomes ≤100 nm have been reported to improve the delivery of drugs to the brain and central nervous system for cancer therapy [24]. Insight from such studies can facilitate the design of liposome-based delivery systems for the treatment of TB meningitis.

Malarial drugs targeting the hepatic stages of the disease also have to consider liposome size as it determines the hepatic uptake and accumulation where capillary exchange of particles ≤150 nm is acceptable [24]. In addition to size, the curvature of particles impacts on the internalization in the Kupffer cells. While particles greater than 1 μm with elongated shapes enhance particle to membrane contacts, the high-aspect rations often inhibit membrane spread over the smaller dimensions, limiting the completion of phagocytosis. In contrast, with particles that are less than 1 μm, the shape determines the rate and pathway of internalization [26]. Delivery to the parenchymal cells is achieved by particles that are less than 50 nm in size.

To reiterate that size matters, liposomes are classified according to their size-lamellarity, single unilamellar vesicles (SUV), medium unilamellar vesicles (MUV), large unilamellar vesicles (LUV), and giant unilamellar vesicles (GUV). Further, annotations are given in terms of lamellarity, which denotes the number of phospholipid bilayers, either being unilamellar, oligolamellar, or multilamellar [14]. This makes liposomes differ in their functionality. The LUV liposomes are ideal for encapsulating hydrophilic cargo in higher amounts due to their larger entrapped aqueous volume, making them indifferent to the phospholipid types making them up [27].

Zeta potential and charge: Zeta potential is the potential difference between the layer of fluid that is stationary, remaining attached to the surface of a dispersed medium, and the dispersion medium; it can be used as an indirect measure of surface charge [27]. The any extreme shifts can result in structural changes, stability, and functionality of the liposomes, and temperature and viscosity are also effectors to consider [27, 28]. Surface functionalization of liposomes with (bio)molecules such as antibodies, nucleic acids, proteins, and dyes depends on their zeta potential as well as overall charge and polarity. Liposomes are considered stable at a minimum zeta potential of ±30 mV, with extremely positive or negative values indicating that colloidal molecules repel each other displaying dispersed colloidal particle [28]. A nano-quantitative structure-property relationships (QSPR) model study reports that the hydrophilic-lipophilic balance (HLB) and enthalpy of formation are structural features affecting the zeta potential; wherein a lower HLB value displayed higher zeta values, which means, the more lipophilic the higher the stability, and conversely, high enthalpy of formations was associated with lower zeta potential values and instability [29].

With respect to charge, electrostatic interactions between the cargo and vehicle are key players in the efficiency of encapsulation and delivery [28]. Cationic liposomes are predominantly used over their anionic counterparts due to their ease of binding to bacterial membranes and increased drug delivery. However, issues of toxicity have been evident and successfully circumvented — this is discussed in detail in the latter sections. Peetla and co-workers describe four types of interactions between the vehicle and cargo: hydrophilic and electrostatic interactions with a bilayer, water-soluble and non-bilayer interacting, non-water-soluble and non-bilayer interactions, and lastly, lipophilic and bilayer interacting [30].

The partitioning of drugs between the aqueous core and the lipid bilayer is a common occurrence for amphiphilic drugs, which is influenced by changes in the pH of the aqueous core. The non-bilayer is the aqueous core of the liposome, where the water-soluble drugs are encapsulated. Thus, a total entrapped aqueous volume that is large allows for higher concentrations of drug loading. In contrast, non-water-soluble drugs that are non-bilayer interacting have very poor encapsulation efficiencies, unless molecules with functionally compatible moieties are attached to the drug and/or the liposome is reformulated with a different type of lipid and/or accessory molecules. The length of the acyl chain of the phospholipid together with fluidity impacts the loading capacity or intercalation of lipophilic drugs on the bilayer. Incorporation of cholesterol can then be used to manipulate the fluidity of the lipid membrane [27, 30].

2. Liposomes as drug delivery systems in HIV, TB, and malaria

Drug delivery systems (DDS) are pivotal in managing and treating diseases, acting as vehicles for drug formulations, preservation, storage, and targeted release—usually entailing the encapsulation of the cargo. Administration of local and systemic drugs is usually through oral methods and various other routes, including cutaneous, nasal, ophthalmic, anal, vaginal as well as buccal and sublingual routes [31, 32, 33]. Once introduced into the system, the exposure of drugs to abrasive physicochemical environments and antagonizing physiological conditions pose serious limitations to their pharmacokinetics. Conditions that promote the decomposition or premature degradation of drugs and their excretion are associated with extreme pH and temperature, hydrolyzing enzymes, free radicals, and others. Thus, novel designs in DDS have to counteract poor selectivity, uncontrollable drug release, low solubility, short plasma residence, and premature drug metabolism and excretion, which are effectors of low drug efficacy, overdosage, high levels of toxicity, and patient incompliance. In principle, high efficacy, automation, precision as well as high biocompatibility and biodegradability are defining properties of a successful DDS [33]. These qualities are mitigating factors to the trend in acquired drug resistance of microbes, which is a result of overcompensation of poor pharmacokinetics by misuse and overuse of antimicrobials [34].

Liposomes are lipid-based NP, a category of DDS where the most FDA-approved nanomedicine originates [35] — warranted by their versatility in loading capacity, which enables the encapsulation of amphipathic, hydrophilic, and lipophilic bioactive compounds in their aqueous core and intercalation within the lipid leaflets, respectively. Their favorability also owes to their ability to self-assemble and carry high loads, amenability to functionalization, high biocompatibility, and bioavailability [35]. Structure-function relations are inherent in endowing these properties, which need to be unpacked and comprehended. The liposomes’ manner of interaction with cells differs depending on the respective physicochemical properties; these interactive pathways occur via lipid exchange, liposome fusion, adsorption, and lastly and most vital is by endocytosis [36].

2.1 Human immunodeficiency virus (HIV)

Since the approval of the first antiviral drug in 1963, drug discovery against viruses has evolved simultaneously with genomic sequencing and the structure-function-based drug design. This era also brought up interest and effort in deconvolution of the HIV lifecycle, which shed insight into how the virus exploits the hosts’ resources for propagation—an attribute to the difficulty in developing viral therapy without eliciting toxicity in bacteria [37, 38]. This led to the discovery of other drugs strategically targeting the different stages of the life cycle surpassing antibacterial drugs with better specificity, and with respect to HIV, the discovery of the protease was one of major breakthroughs. However, some improvement is warranted—existing side effects, and continued innovation is important to safeguard against drug resistance as well as achieve a cure. Moreover, comorbidities and polypharmacy are compounding factors of drug-drug interactions that lead to poor pharmacokinetics and pharmacodynamics, especially in adults [39]. To date, the highly active antiretroviral therapy (HAART) is still in effect, although efficient, some challenges are ongoing, including low oral bioavailability due to premature metabolism and degradation in the gut and virus’ ability to evade therapy since it resides in unattainable anatomical and cellular reservoirs – the minimum drug concentration is not affected; toxicity is caused by high dosages as compensation to the low half-life, leading to noncompliance in patients [40]. Africa is still the most burdened, accounting for more than 65% of people living with HIV, but with a remarkable 38 and 51% decline in new incidences and mortality compared to the year 2010, respectively.

Azidothymidine (AZT), also known as zidovudine (ZDV), is a reverse transcriptase inhibitor and the first anti-HIV drug to be approved in 1987, and it is also the first to be encapsulated by liposomes from work that was initiated in 1991 by Phillips and team [41]. Conceptually, the work was built on the knowledge that the phagocytic system of the reticuloendothelial system (REM), and the macrophages, in particular, are highly active in the uptake of liposomes [42, 43]. The liposome-encapsulated AZT was able to reduce hematopoietic toxicity, enhancing antiretroviral activity and prophylaxis in mice infected with HIV [41], and the distearoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DSPC/ DMPG) liposomes showed three-fold higher retention time versus dimyristoylphosphatidylglycerol/dimyristoylphosphatidylglycerol (DPPC/DMPG) liposomes. Given that there are limitations in liposome-encapsulation of ARV, such as poor hydrophilic loading capacity, instability associated with the physical and biological make-up, poor scale-up and high cost, short shelf-life, and toxicity — through modern technology and by varying lipid properties such as compositions, size, and charge, these challenges may be overcome. AZT is amphiphilic, thus, leakage is caused by the split affinity between the liposome aqueous core and the hydrophobic lipid bilayer [42]. Jin and co-workers enhanced liposome retention of AZT by encapsulating the lipophilic prodrug form which intercalates better with the lipid bilayer [43]. Subsequently, the encapsulated prodrug displayed a half-life that was enhanced in the rat model. Modifying liposomes with polyethylene glycol (PEG), so-called PEGylation, is a well-accepted strategy that not only increases hydrophilicity but acts also as a steric barrier—preventing opsonins in the serum from tagging the liposome cargo for clearance [3]. Consequently, PEGylation increases retention time, improving bioavailability of the drug; this has been excellently demonstrated in the PEGylated-liposome encapsulating doxorubicin, commercially Doxil® — the first liposome-encapsulated drug to be approved and used commercially in treating AIDS-related Kaposi’s sarcoma [44]. Saquinavir, a HIV protease inhibitor, showed more stability in protein supplemented media and a more sustainable release when encapsulated in PEGylated liposomes compared to free drug and non-PEG liposomes. The PEGylated liposomes were also less toxic to Jurkat T-cells [40].

The localization of encapsulated drugs on the liposome is crucial to consider as it affects drug leakage and can be exploited for controlled release. Saquinavir and nevirapine were observed to concentrate at different regions of the nanocarrier when they were simultaneously loaded; moreover, their release was in a timely manner, wherein the saquinavir was dominant during the later phase and nevirapine at the early stages [45]. Simultaneous release of the drugs is an approach that was aimed at circumventing the metabolism of saquinavir prior reach of the effective concentration. The acyl group of the bilayer allows association of the more hydrophobic nevirapine, while saquinavir localizes to the aqueous core causing its delay in release. The composition of liposome and surface modification affects entrapment of the drugs—PEGylation increases the hydrophilicity of the nanocarrier, significantly decreasing the encapsulation of nevirapine by 45%, while a slight increase was observed for saquinavir. Hydrophobicity was promoted by the incorporation of cholesterol, hence nevirapine loading was significantly increased but not alter that of saquinavir. An optimized encapsulation of 45% and 30% was achieved for nevirapine and saquinavir, respectively, at a combinatory ratio of 9:1:1 for EPC (1,2 Dioleoyl sn-glycero 3 ethylphosphocholine): Cholesterol: distearoylphosphatidylethanolamine (DSPE)-PEG. The study also employed specific targeting, homing on HIV-infected T-cells and macrophages by surface modification of the liposomes with anti-CD4 antibodies. Overall, the anti-CD4 immuno-liposomes demonstrated enhanced uptake into the CD4-positive Jurkat cells, exhibited preferential delivery to HIV-infected cells, and the proliferation of the virus was markedly reduced by dual activity of the encapsulated drugs. These effects were at a lower concentration versus with the free drugs.

2.2 Tuberculosis

The Mycobacterium tuberculosis (MTB) is a pathogenic bacterium with inherently robust transmission and survival mechanism, causing one of the first and second deadliest infectious diseases —tuberculosis (TB) [46]. It mainly affects the lungs, but its target organs are vast, including the pleura, bones, joints, meninges, genitourinary tract, and the skin, challenging both diagnosis and therapeutic interventions [46]. Like HIV, it exploits various mechanisms to evade the hosts’ immune system as well as exogenous drugs, wherein its escape from the lungs’ first line of defense, the macrophages leads to the progression of the disease from the development of granulomas to cavity formation where unabated replication of MTB occurs. Significant challenges in the treatment of TB exist at different levels of disposition, including at the molecular, physiological, clinical, and geographical levels, which has led scientists to consider disease subgroupings for selecting appropriate disease biomarkers [47]. Hence, to date, TB remains a global dilemma, which was responsible for 1.3 million deaths in 2023 [48]. Although this is a 19% decline from 2015, drug resistance is a continuous concern with the development of multiple drug resistance (MDR) and extensively drug-resistant (XDR) TB —a challenge due to the lack of established clinical management systems in terms of specific/tailored diagnosis and curative drug treatments. This is a call for innovative methods of treatment, such as resistance to first-line and second-line drugs, and importantly, the scarcity in the approval of the new drugs: bedaquiline, delamanid, and pretomanid, which were discovered 40 years after the approval of the latest of the second generation of drugs [49, 50], are indications for improving the drug delivery system as opposed to discovery of new drugs.

Liposomes create a new dimension in solving the pharmacokinetic and pharmacodynamic associated with TB drugs, such as the long duration of treatment, side effects that are debilitating in physical and mental health with subsequent relapse rates and the development of drug resistance. By encapsulating TB drugs in liposomes, which can be designed to target specific cells, this reduces the chances of off-targeting; while improving concentration efficacies, multi-drugs can then be loaded and simultaneously delivered in a single dose. PEGylation (and other methods) can be used to increase the longevity and bioavailability of the drug, and encapsulation can facilitate the reduction in the premature metabolism of the drug. Consequently, lower doses and shortened periods of treatment will negate issues of patient compliance and decrease relapse rates, lowering the prospective of drug resistance.

Since TB is chiefly a disease of the lungs, the pulmonary and parenteral routes of administration are better qualified as they avoid rapid metabolic degradation and gastrointestinal absorption, which result in the delivery of suboptimal concentrations in the diseased alveoli. Aerosol diagnostics in the form of volatile organic compounds (VOC) and inhaled therapeutics have been in the sphere of TB research since the 1940s, promising an efficient route for theranostics and prevention of drug resistance [51]. These present as easier and less invasive methods of sampling and drug administration. Encapsulated drugs can be delivered in suspension or dry powder form by pulmonary devices in small doses using pressurized metered inhalers (pMDIs), dry powder inhalers (DPIs), and soft mist inhalers (SMIs) versus medical nebulizers, which deliver larger doses of medicine [23]. Liposomes present an ideal mode of transport due to their remarkable uptake by macrophages, biocompatible nature, and surfactant-like properties similar to the lungs [52], which lowers the chances of localized toxicity and adverse reactions. In this regard, a few interesting studies have been dedicated to investigating the purpose of old and new drugs for inhalation therapy [53].

In a study by Bhardwaj and colleagues, liposomes were surface modified with mannan, a macrophage-specific receptor that is associated with MTB binding and activation of the innate immune and loaded with the first-line antituberculosis drugs rifampicin (RIF), isoniazid (INH), and pyrazinamide (PYZ) [54]. The mannan-liposome encapsulating RIF, INH, and PYZ were lyophilized into an inhalable powder, which was cryopreserved using sucrose, an important component for preserving hydration of the phospholipid heads and preventing drug leakage. Due to their differing hydrophobicity indices, their localization was specific. Whereas, the lipophilic RIF intercalated with lipid bilayer, the more hydrophilic INH and PYZ were entrapped proximal the aqueous core. This may also be the cause for their drug release of 67.4 ± 2.43, 65.8 ± 2.56, and 58.1 ± 1.65% for RIF, INH, and PYZ, respectively. This was similar to the neutral liposomes and followed the Korsemeyer-Peppas model for drug release; in contrast, the lung uptake of the mannan-liposomes was higher, 51.36 and 48.66% for the neutral liposomes [54]. As it has been clear throughout the chapter, the lipid composition of liposomes can be varied to perform specific functions and dual functionality. This was demonstrated by Chimote and Banarjee, who successfully prepared DPPC-based liposomes entrapping INH, thus acting as anti-TB and anti-atelectatic surfactant. The authors exploited the well-known fact that DPPC is the most abundant lipid of the lung surfactant as well as an exogenous surfactant [55]. The surfactant plays an essential role in preventing the lung collapse that becomes apparent in the late stages of pulmonary (PTB), enhancing the anti-TB reach of INH; moreover, the drug release was sustained for over 24 hours. This is an insightful finding, which presents a strong case for designing liposomes that can target the different phenotypic stages of the disease such as the granulomatous, necrotic granulomatous, caseous necrotic granulomatous, and cavitary disease, especially where evasion is prominent.

The physicochemical properties of the microenvironment of the diseased tissue must be considered as factors such as pH can alter the structure and function of the liposome and are especially relevant for MTB infection, which is characterized by acidification steps during phagolysosomal fusion. A follow-up study by Bhardwaj and team, who designed a pH-sensitive mannan-anchored encapsulating INH and Ciprofloxacin HCl (CIP HCl), shows a drug release of 58-64% in alkaline pH compared to 81-87% release in macrophage pH [54]. Some of the prominent research into aerosol therapeutics for TB have been reviewed by Hickey et al., highlighting the advantages, progresses, and limitations in targeting infected alveoli by inhalers versus nebulizers, mostly advocating for the drug powder inhalers as being more efficient [51].

Macrophages can likewise be specifically targeted by tufstin-modified liposomes. The treatment of MTB-infected mice with tufstin liposomes loaded with RIF displayed an impressive 2000-fold higher reduction in bacterial load in the lungs compared to the free drug [56]. This was achieved without continuous administration of the drug, thus lowering toxicity that is associated with high-drug dosages —owing to specific targeting of macrophages.

The adaptability of liposomes is once again demonstrated by the thermosensitive liposome-in-hydrogel, which targets the bones [57]. Bone TB is a common EPTB form, accounting for 10% of the cases; as this side is inaccessible to drugs owing to poor blood supply, treatment includes surgical removal of the diseased tissue followed by localized injection of a multi-anti-TB drugs —a painful and invasive method of treatment. INH is the foremost drug of treatment for TB infecting the bones, but its hydrophilic properties predispose it to burst drug release and shortened periods of release, which were greatly overcome by using its hydrophobic derivative N′-Dodecanoylisonicotinohydrazide (DINH). Additionally, the DINH encapsulated in liposomes, followed by the hydrogel adds another network of protective layer, which sustained drug release over 24 hours compared to the 4 hours when only the hydrogel was used [57]. The thermosensitive peptide PLGA-PEG-PLGA with self-healing properties was an excellent incorporation into the liposomes, which meant that upon contact and sensing of bodily temperature the hydrogel transforms into a sol-gel, which is less painful than other implantations while inducing self-healing at the diseased site. Liposome-in-hydrogel seems an effective method for targeting the bone tissue, and adhesion to the bone tissue can be enhanced by appending functional groups, such as octadecylamine, which is an amphiphilic cationic film-forming material, and sulfhydryl groups [57]. These modified liposomes showed better bone regeneration and remodeling. EPTB can be effectively treated using liposome-encapsulated TB drugs, such as rifabutin (RFB), which is used to treat patients who are intolerant to rifampicin including HIV-coinfected individuals. Gaspar and colleagues show that DPPC: DPPG formulated liposomes act as more efficient delivery systems for RFB displaying high concentrations of RFB in the liver, spleen, and lung within a 24-hour period and decreased bacterial loads in these organs of a disseminated-TB rat model [58].

The introduction of asymmetric liposomal systems has broadened the design opportunities for drug and vaccine delivery, with some important breakthroughs such as the commercialized and FDA-approved liposome-based amikacin suspension Arikace® —the first and only approved inhalable drug for treating the nontuberculous Mycobacterium avium complex (MAC), previously difficult to treat [23, 59]. Asymmetry in membranes is a common phenomenon in eukaryotes, which was first described by Bretscher in 1972, where the distribution in the type of lipids within the inner and outer leaflets is deliberately organized differently. It is also noteworthy that the protein composition and cholesterol distribution also differs [23]. Phosphatidylcholine (PC) and sphingomyelin (SM) preferentially makeup the outer leaflet, while the negatively-charged lipids that include phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol are embedded in the inner leaflet [60]. This asymmetry is maintained via the flippases, floppases, and scramblases, performing intricate functions, such as the translocation of the lipids across the membranes [23, 60]. Moreover, other effectors of asymmetry exist, such as the size of the vesicle, which observable augments asymmetry with decreasing diameter size [23].

2.3 Malaria

Malaria is a vector-borne disease that is caused by the protozoan parasite, Plasmodium, transmitted by the female Anopheles species mosquitoes [3, 61]. The incidences of malaria span 85 countries, infecting 249 million people and culminating with 608,000 deaths, with 94-95% of these occurrences based in African regions [62]. There are five different species of Plasmodium that can cause pathogenesis, which is characterized by the well-established cycle between the mosquito and the human, presenting with similar events across the different species. Briefly, the sporozoites from the mosquito’s salivary glands are injected into the human host via a bite, traveling via the bloodstream to the liver where asexual replication occurs. Following that, the thousands of daughter merozoites flood the bloodstream invading the red blood cells, and this is a crucial stage of adverse symptom manifestations and also a great target for drugs. A full circle is achieved upon another event of a mosquito bite, wherein the merozoites turned gametophytes in the erythrocytes are ingested into the salivary glands of the mosquito [3, 61].

The enormous effort toward developing antimalarial drugs has been greatly compromised by the persistent counter evolution of drug resistance in parasites, and this is resistance against almost every known drug for treating humans [63, 64]. Quite concerning is the spontaneity of the mutations occurring without any drug pressure [65]. This is within the existing shortcomings in the drug discovery pipeline of malaria drugs, including lack of specificity in the intracellular parasite-associated biomarker targets, poor drug solubility, low permeability, and poor bioavailability; consequently, high toxicity as a result of overcompensation by giving high doses becomes apparent, which also compounds issues of noncompliance in patients due to accompanying side effects [63, 64]. It is interesting that despite the dramatic reduction in malaria deaths owing to administration of quinoline-based drugs, their mode of action has not been fully deciphered albeit the 400 years following their discovery [66]. Emerging drug resistance has posed a serious bottleneck in the treatment of malaria, this is true for quinolone-based drugs chloroquine, mefloquine, and halofantrine as well as artemisinin, which is not in this group. Precautionary use of primaquine to avoid resistance and relapse has been a continuous discussion. Liposomes have proved distinct adeptness in mitigating these shortfalls among other interventions that exploit nanotechnology, with several studies reporting improved compound pharmacokinetics and pharmacodynamics from in vivo and in vitro studies of treatment with the liposome-encapsulated drug versus the drug on its own [64].

To elaborate on the role of electrostatic interactions in the structure-function relationship in optimizing encapsulation, the drug loading capacity of liposomes with platinum-chloroquine (PtCQ) diphosphate dichloride, a potent combination against chloroquine-resistant cells was investigated [67]. PtCQ was loaded into PEGylated liposomes that were either cationic or neutral. Since the inner membrane of erythrocytes is anionic, the exposure that occurs following eryptosis makes it more likely for cationic liposomes to bind, increasing the chances of drug delivery. Although this warrants using cationic liposomes, the challenge remains with the low half-life and high chance of macrophage recognition, hence a common mitigation was PEGylation of the liposomes [67]. Both PEGylated cationic and neutral liposomes were able to prevent leakage of the drug; moreover, the encapsulation efficiency was as high as 76.1% for neutral liposomes and 96.9% for the cationic liposomes. This outcome is not surprising, given the physicochemical relationship of the interacting liposomes and the erythrocytes.

PEGylation is also an alternative to using pH gradient methods, which maximize the loading capacity of small-sized liposomes that are characterized by low drug loading [68, 69, 70]. The pH gradient methods conveniently depend on the intrinsic structure of the transmembrane where a progressive drop in pH occurs toward the core of the liposome, and the efficiency of drug upload is dependent on its overall counter charge as well as other physicochemical properties [68, 69]. Knowledge of the ionization states of the drugs can shorten the time spent on optimizing the compatible conditions for encapsulation when predictive models are exploited; the modeling of the liposomal distribution of diprotic antimalarial compounds has been analyzed by Moles and co-workers [71]. The distribution of quinine, primaquine, tafenoquine, quinacrine, and chloroquine in phosphatidylcholine-based liposomes, as well as interest in the application of pH gradients as a method of efficient drug uploading and their responsiveness, were studied [71]. This gradient method is particularly suitable for addressing the non-endocytic uptake of drugs by cells, such as the erythrocytes, wherein their membrane skeleton is a meshwork of spectrin-actin molecules that exploit steric hindrance to inhibit endocytosis, mainly relying on the diffusion pathway for exchange of molecules [72]. With respect to NP, the inhibitory effect is due to membrane-NP interaction, size of NP relative to the meshwork, and skeleton tension as was found through molecular simulation models using dissipative particle dynamics (DPD). This further reiterates the structure-function relationship as a useful comprehension and building tool for expediting the development of predictive computational models that would facilitate the surface modification of liposomes, making them a more specific and efficacious system for drug delivery.

Ligands are convenient in that they can be used as structural moieties to modify biological macromolecules to serve specific functions or applications. Active pharmaceutical compounds that display target promiscuity, with non-validated biomarkers or non-elucidated mechanisms of action, can benefit from liganded liposomes. A wide spectra of ligands have been used to modify liposomes for targeting malaria-infected cells, ranging from derivatives of glycolipids and peptides to antibodies; these studies have been summarized in a review by Memvanga and Nkanga [64]. Varying lipid compositions and ratios have been explored for optimizing the efficiency of the liganded moieties.

Peptide-functionalization of nanomaterials, such as liposomes, is an ingenious practice that has been adopted for enhancing the bioavailability and specificity of small molecule delivery, in principle creating an active targeting system [73]. A 19-amino acid (19-aa)-long peptide sequence from the circumsporozoite protein (CSP) has been used for targeting the hepatic stage of the malaria virus by incorporating it into liposomes. CSP and the thrombospondin-related anonymous protein are highly specific to the hepatocytes, wherein their localization to the liver is observed within minutes of sporozoites being introduced to the bloodstream [74]. Subsequently, liposomes decorated with a 19-aa CSP sequence accumulated with more than several hundred-fold higher in the liver compared to other organs, except for the 10-fold higher accumulation than in the spleen; and the binding is suggested to be via the heparin-associated proteoglycans [74]. Since the macrophages in the spleen are known to clear particles, then this accumulation is warranted. This system indeed presents an efficient solution for preerythrocytic specific targeting of malaria; however, the antigenicity needs to be investigated prior to therapeutic use. Another peptide-based strategy was developed for targeting the intraerythrocytic stage, wherein a macrophage activating tetrapeptide, tuftstin, and mimicking a sequence in the Fc-portion of the heavy chain of the IgG [75] was used to surface modify liposomes. This concept stems from the ability of tuftstin to activate cells of the immune, such as macrophages, to kill intraerythrocytic malarial parasites. Pretreatment of mice with hydrophobic derivatives of tufstin incorporated into liposomes resulted in reduced mortality of Plasmodium berghei infected mice as well as decreased parasitemia [76]. These studies not only open novel avenues for developing prophylactic treatment for malaria but also for the development of vaccines.

Attaching antibodies to nanomaterials is a widely used preparation method that endows specificity to therapeutic and diagnostic products that can be used for liposome DDS. This is evident in the covalent conjugation of an anti-erythrocyte F(ab’)2 and liposomes encapsulating chloroquine used in treating mice infected with Plasmodium berghei as well as chloroquine-resistant mice [77]. Studies showed that the F(ab’) two-bearing antibodies enhanced binding to the erythrocytes, reducing parasitemia in susceptible and resistant mice, and prolonging survival [77]. This strategy presents a more straightforward way of increasing the sensitivity and specificity of liposomes.

3. Computational tools in the design of liposome-based drug delivery systems

To expedite the liposome technology, predictive computational models are proficient tools that can be employed by adopting high-throughput screening methods, which further reduce time spent and cost required in the drug discovery pipeline, and for developing optimal conditions in vitro. Leveraging existing empirical data and structure-function relationships, it is possible to create such models. Layers of complex algorithms and machine learning systems have afforded computational modeling with the ability to handle multiple variables simultaneously, better accuracy of predictions, data output that is simpler to analyze, and improved spatiotemporal landscapes — referred to as coarse grained analysis.

With the capabilities of various simulation methods, different analyses can be extracted, including from molecular dynamics (MD), machine learning (ML), Monte Carlo (MC), finite element analysis (FEA), computational fluid dynamics (CFD), density functional theory (DFT), and dissipative particle dynamics (DPD) [78]. At the molecular level, MD can be used to analyze the behavior of atoms with respect to space, time, and the forces acting on them, giving an overview of molecular interactions. In this case, a motion-based equation that is derived from Newton’s theory of force enables the prediction of behavior of liposomes with the drug and the environment. With MC, statistical analysis and probability theory are exploited in simulation of complex systems, wherein various potential scenarios or outputs are analyzed. MC allows the prediction of drug release and uptake, bioavailability, and toxicity can be predicted [78].

In CFD modeling, the flow of fluids in cells and tissues are described; thus, the movement of DDS can be better comprehended using this technique, while the FEA model also exploits mathematical algorithms to facilitate the analysis of DDS under different conditions, thus testing the mechanical properties of the liposome [78].

The CFD model has been greatly used in the design of inhalers and nebulizers, by simulating air flow into the lungs and drug absorption — the design of metered-dose inhaler (MDI), also known as the AeroCup for COVID-19 being exemplary [79]. Another computational model that has been successfully used for DDS is DFT, wherein the thermodynamic nature of interacting systems can be studied, giving insight into the energetic changes that occur at the molecular level; this method is also capable of defining the geometric structure and electrical characteristics of nanocarriers upon drug docking [80].

Machine learning and other AI tools bring another complex element to the computational predictive modeling. By employing training data from known information based on the respective structure-function relationships of DDS or in unknown cases, the data is adopted from quantum models; these tools can facilitate the prediction and modeling of DDS in conditions that are controlled virtually, which would otherwise be impossible to replicate using empirical models [78, 80]. For instance, the stability/dispersity and size of the liposome were predicted using the support-vector machine models and feed-forward artificial neural networks, respectively —initially trained using over 200 liposomes that were varied in their composition, including flow rates, lipid concentrations, and organic: water ratios [81]. This work aimed to optimize the pharmacokinetics of curcumin-loaded liposomes based on the influence of their physicochemical properties and their interactions, which ultimately have an effect on biocompatibility, toxicity, and interaction with biological material [81]. These computational models are adaptable to other DDSs such as dendrimers, polymer-based nanoparticles, solid-lipid nanoparticles, and implantable DDS [78].

Simulations with the lipid bilayers have been carried out, describing the intra- and intermolecular interactions between components of the bilayer itself, and with the cargo as well as distribution of cargo within the liposome [15, 19, 71]. The length of acyl groups in phospholipids influences their packing geometry and conformation of the phospholipid [19], this is due to the type of dominating forces, either stabilizing or de-stabilizing affecting the liposomes’ fluidity. Macromolecules are held together by various forces, including hydrophobic, hydrogen bonds, van der Waals, ionic, and/or electrostatic interactions. These forces can be manipulated to efficiently load specific cargo while controlling their rate of release, wherein the localization of the drug is dependent on its hydrophobicity index, either intercalating in the lipid bilayer or proximal to the aqueous core for a slower release. The ionization state of the cargo is equivocally integral to its retention and release from the liposome, making both the liposome and cellular microenvironments relevant to their efficiency. An example of bilayer simulations is illustrated in Figure 3.

Figure 3.

Simulation of a single-walled nanotube loaded with doxorubicin docking into the bilayer membrane. (This figure is reproduced from a review by Salahshoori and colleagues [78, 82] with open access from the creative commons CC-BY license).

Hydrogen bonds are agreeably one of the vastest macromolecular “glues” in existence, with their directionality highly sought as recognition elements in biomolecules, spanning enzymes, proteins, nucleic acids, lipids, and others. In liposomal formulations, the molecular simulations have been dedicated to studying hydrogen bonds between the phospholipids, cholesterol, and water molecules within the lipid bilayer. Although the existence of H-bonds between the OH group of cholesterol and the polar head group of phospholipids has been supported, Pandit and team went a step further, suggesting that the CH···O H-bonding occurs between the methyl group from phospholipid and the oxygen atom of the cholesterol group, providing the rationale for formations that are larger than ratios of 1:1 [19]. CH···O H-bonding has been debatable; however, quantum studies have characterized them as weaker and with minute directionality than the conventional H-bonds [83]. In structural biology, these bonds were only appreciated about 40 years ago, with immense contributions to the structural integrity of biomolecules, recognition and binding, and catalysis [84, 85]. In conclusion, Pandit and colleagues found that two types of complexes exist in each mixture of dilauroylphosphatidylcholine (DLPC): cholesterol and (DPPC): cholesterol, exhibiting stoichiometries of 1:1 and 2:1; however, the DLPC: cholesterol was more populated with species of a 1:1 ratio and vice versa for the DDPC: cholesterol. This difference was attributed to the different acyl lengths of the phosphatidylcholine lipids. CH···O H-bonds were defined as crucial for the formation of the complexes and in their aggregation, especially in the DPPC: cholesterol [19]. These types of computational models are relevant for multidrug loading and their timeous release; hence, the composition of the liposome can be designed and refined in silico with simultaneous docking of drugs, such that the correct partitioning is achieved to obtain optimized pharmacokinetic and pharmacodynamic properties.

4. Conclusions

The role of liposomes in nanomedicine is futuristic, with the successful approval and commercialization of DaunoXome^®, Doxil^®, AmBisome^®, Arikace^®, RUTI^®, and Mosquirix^®, which are used in either treating or preventing HIV, TB, and malaria and/or associated diseases. The ongoing pipeline developments and clinical trials depict endless possibilities. Computational tools can facilitate the fast tracking of this technology, wherein optimization processes can be minimized in terms of time and cost as well as define the limitations.