Shikonin‐loaded liposomes as a new drug delivery system: Physicochemical characterization and in vitro cytotoxicity

Alkannin and shikonin are naturally occurring hydroxynaphthoquinones with a well‐established spectrum of wound healing, antimicrobial, anti‐inflammatory, and antioxidant activities. Recently, extensive scientific effort has been focused on their effectiveness on several tumors and mechanism(s) of antitumor activity. Liposomes have been proved as adequate drug carriers offering significant advantages over conventional formulations, such as controlled release and targeted drug delivery, leading to the appearance of several liposomal formulations in the market, some of them concerning anticancer drugs. The aim of the present study was to prepare shikonin‐loaded liposomes for the first time in order to enhance shikonin therapeutic index. An optimized technique based on the thin film hydration method was developed and liposomes characterization was performed in terms of their physicochemical characteristics, drug entrapment efficiency, and release profile. Results indicated the successful incorporation of shikonin into liposomes, using both 1,2‐dipalmitoylphosphatidylcholine and egg phosphatidylcholine lipids. Liposomes presented good physicochemical characteristics, high entrapment efficiency and satisfactory in vitro release profile. In vitro cytotoxicity of liposomes was additionally tested against three human cancer cell lines (breast, glioma, and non‐small cell lung cancer) showing a moderate growth inhibitory activity. Practical applications: Shikonin is a naturally occurring hydroxynaphthoquinone and extensive scientific research (in vitro, in vivo, and clinical trials) has been conducted during the last years, focusing on its effectiveness on several tumors and mechanism(s) of antitumor action. The purpose of this work was to prepare and characterize shikonin‐loaded liposomes as a new drug delivery system for shikonin. Liposomal formulations provide significant advantages over conventional dosage forms, such as controlled release and targeted drug delivery for anticancer agents. Thus, liposomes could reduce shikonin's side effects, enhance selectivity to cancer cells and protect shikonin from internal biotransformations and instability matters (oxidization and polymerization). Furthermore, liposomal delivery helps overcome the low aqueous solubility of shikonin, which is the major barrier to its oral and internal administration, since it cannot be dissolved and further absorbed from the receptor.     

Nano–Liposomes Double Loaded with Curcumin and Tetrandrine: Preparation,Characterization, Hepatotoxicity and Anti–Tumor Effects

(1) Background: Curcumin (CUR) and tetrandrine (TET) are natural compounds with various bioactivities, but have problems with low solubility, stability, and absorption rate, resulting in low bioavailability, and limited applications in food, medicine, and other fields. It is very important to improve the solubility while maintaining the high activity of drugs. Liposomes are micro–vesicles synthesized from cholesterol and lecithin. With high biocompatibility and biodegradability, liposomes can significantly improve drug solubility, efficacy, and bioavailability. (2) Methods: In this work, CUR and TET were encapsulated with nano–liposomes and g DSPE–MPEG 2000 (DP)was added as a stabilizer to achieve better physicochemical properties, biosafety, and anti–tumor effects. (3) Results: The nano–liposome (CT–DP–Lip) showed stable particle size (under 100 nm) under different conditions, high solubility, drug encapsulation efficiency (EE), loading capacity (LC), release rate in vitro, and stability. In addition, in vivo studies demonstrated CT–DP–Lip had no significant toxicity on zebrafish. Tumor cytotoxicity test showed that CT–DP–Lip had a strong inhibitory effect on a variety of cancer cells. (4) Conclusions: This work showed that nano–liposomes can significantly improve the physical and chemical properties of CUR and TET and make them safer and more efficient.     

Protective and retentive effects of liposomes on water-degradable hydrocortisone acetate in dermatological applications

Various dermatological samples containing Liposomes as a drug carrier were prepared, and the effects of variations in the dermatological formulations, such as liposornal encapsulation, base materials, and the purity of lipid products, on drug stability and characteristics for effective topical drug delivery were investigated. Hydrocortisone-21-acetate, a hydrophobic and water-degradable, anti-inflammatory agent, was used as the model drug. It was found that the liposomally encapsulated drug was more stable than the free-form drug in an ointment formulation. Also, the hydrogel base was found to be effective in maintaining drug stability in spite of its high water content. Another evidence that Liposomes were surrounding drug particles in the base was obtained from anin vitro test of drug permeation from liposome-hydrogel through the pig ear skin. The permeability of hydrocortisone acetate through the skin membrane was found to be 2.7-fold lower in the case of liposome-hydrogel than in the case of free-drug hydrogel. The results also suggested that Liposomes play a role in localizing drug molecules in the skin membrane.     

Liposomes in wool dyeing — the stability of dye-liposome systems and their application to untreated wool fibres

The use of two different types of liposome suspensions (multilamellar vesicles, MLV, and large unilamellar vesicles, LUV) as carriers in the commercial dyeing of untreated wool with a milling acid dye is described. Liposomes prepared with egg phosphatidylcholine and containing the dye CI Acid Blue 90 were used. The physico-chemical stability of liposomes was studied by measuring the mean particle size distribution of phospholipidic vesicles during dyeing. The possible hydrolysis of phospholipid molecules was also determined. Kinetic aspects involving dye adsorption and bonding were investigated. Dye exhaustion on untreated wool fibres was inhibited and dye bonding was improved. The lipid concentration and type of liposomes were important factors in this process.     

Impact of Liposomal Drug Formulations on the RBCs Shape,Transmembrane Potential,and Mechanical Properties

Liposomal technologies are used in order to improve the effectiveness of current therapies or to reduce their negative side effects. However, the liposome–erythrocyte interaction during the intravenous administration of liposomal drug formulations may result in changes within the red blood cells (RBCs). In this study, it was shown that phosphatidylcholine-composed liposomal formulations of Photolon, used as a drug model, significantly influences the transmembrane potential, stiffness, as well as the shape of RBCs. These changes caused decreasing the number of stomatocytes and irregular shapes proportion within the cells exposed to liposomes. Thus, the reduction of anisocytosis was observed. Therefore, some nanodrugs in phosphatidylcholine liposomal formulation may have a beneficial effect on the survival time of erythrocytes.     

A Comparative Approach for the Preparation and Physicochemical Characterization of Lecithin Liposomes Using Chloroform and Non‐Halogenated Solvents

Organic solvents used in various pharmaceutical preparations may be associated with chronic health effects, with special emphasis on halogenated solvents. Liposomes, lipid bilayer membrane carriers, have potential applications in targeted drug delivery systems. The non‐halogenated solvents, acetonitrile and ethanol, were used in comparison to commonly used chloroform. The effect of solvents and dispersion medium was demonstrated using physicochemical properties, stability studies and hemolytic activity. Increased sonication time showed decreased particle size in phosphate buffer saline and water medium. Vesicles prepared from all solvents exhibited better stability in phosphate saline buffer than water when evaluated by particle size and zeta potentials. Liposomes showed a positive zeta potential in buffer solution whereas liposomes in water showed negative zeta potential. In vitro hemolytic activity of liposomes was done with fresh human red blood cells. Results in buffer solution were in the range of 1–4 % which further proved this medium superior to pure water. The findings of this study are helpful in suggesting the formulation of thin films by less hazardous solvents in terms of the environmental integrity and human health.     

Microfluidic production of liposomes through liquid--liquid phase separation in ternary droplets

Liposomes, the self-assembled phospholipid vesicles, have been extensively used in various fields such as artificial cells, drug delivery systems, biosensors and cosmetics. However, current microfluidic routes to liposomes mostly rely on water-in-oil-in-water double emulsion droplets as templates, and require complex fabrication of microfluidic devices, and tedious manipulation of multiphase fluids. Here we present a simple microfluidic approach to preparing monodisperse liposomes from oil-in-water droplets. For demonstration, we used butyl acetate-water-ethanol ternary mixtures as inner phase and an aqueous solution of surfactants as outer phase to make oil-in-water droplets, which can evolve into water-in-oil-in-water double emulsion droplets by liquid–liquid phase separation of ternary mixtures. Subsequently, the resultant water-in-oil-in-water droplets underwent a dewetting transition to form separated monodisperse liposomes and residual oil droplets, with the assistance of surfactants. The method is simple, does not require complex microfluidic devices and tedious manipulation, and provides a new platform for controllable preparation of liposomes.     

Preparation,Characterization, and Gastrointestinal Digestion of Triterpenol-Loaded Liposomes

Triterpenol is an important phytochemical that is beneficial to humans. However, there are limitations of application due to its poor solubility. Liposomes are increasingly utilized as a delivery system for phytochemicals with poor solubility which is suitable for triterpenol. In the present study, the stability of triterpenol-loaded liposomes (Tri-LP) is demonstrated. Tri-LP stability is observed when the mass ratio of phospholipids to triterpenol is 4:1. Sodium alginate (ALG) and chitosan showed different effects on Tri-LP. Tri-LP coated by ALG (Tri-LP-ALG) is stabilized by H-bonding, while electrostatic interactions can stabilize Tri-LP coated by chitosan (Tri-LP-CH). Tri-LP-ALG is more stable than Tri-LP-CH during storage. The bioaccessibility of Tri-LP is 11%. ALG and chitosan show no influence on bioaccessibility of Tri-LP, but reduce the release rate of triterpenol during digestion. Overall, the findings suggest that Tri-LP showed good promise for future application in functional beverages. Practical Applications: Triterpenol has poor solubility both in oil and water, which greatly limits the functional utilization of triterpene in organisms. Liposomes offer a novel approach to triternenol oral delivery system. It meets the current demands for food health. Liposomes can improve the absorption and utilization of triterpenol in vivo digestion. Many studies and applications of liposomes has carried out in medical field while nutritional supplements with liposomes technology are not common in the market. Triterpenol-loaded liposomes can not only increase applications in phytochemicals with poor absorption but also offer an interesting attempt of liposomes in food development.     

Optimization of Phospholipid Nanoparticle Formulations Using Response Surface Methodology

The present study deals with the optimization of phospholipid liposome formulations to mimic red blood cells. Optimization of different concentrations of distearylphosphatidylcholine, dipalmitoylphosphatidylcholine, and phosphatidylserine at a fixed concentration of lecithin and Tween^® 80 was done using response surface methodology. The optimized formulation produced liposomes with a particle size in the range of 112–196 nm. The optimized formulation shows low encapsulation efficiency at low levels of insulin but increases at higher loading levels. Formulated vesicles fulfill the size requirement for intravenous drug delivery. The present system is environmentally friendly with respect to biodegradability and biocompatibility.     

Analysis of process parameters on the characteristics of liposomes prepared by ethanol injection with a view to process scale-up: Effect of temperature and batch volume

Liposomes have been extensively used as carriers of several compounds, due to their structural versatility in terms of size, composition, surface charge, lamellarity, and facility to incorporate hydrophilic and hydrophobic substances. Among the methodologies for liposomes production, the ethanol injection technique is probably the most suitable for implementation at industrial scale, mainly due to its simplicity. The effects of the aqueous phase temperature and preparation volume on the characteristics of liposomes produced by this technique were evaluated, seeking to attain criteria for its implementation in large scale. The aqueous phase temperature significantly affects the final characteristics of liposomes obtained by this methodology. By varying this parameter, it is possible to control size distribution and polydispersity of the lipid vesicles. Subsequently, process scale-up was carried out using a 10-fold scale ratio. The adapted power per unit of volume (P/V) scale-up criterion showed to be appropriate to reproduce in larger scale the final characteristics of liposomes produced in smaller scale. The results demonstrate that using the ethanol injection technique, it is possible to obtain a relatively narrow distribution of small unilamellar vesicles, appropriately modulating the experimental conditions. Therefore, the preparation of vesicles with the desired diameter and size distribution was essentially fulfilled.     

Encapsulation of Living Cells within Giant Phospholipid Liposomes Formed by the Inverse‐Emulsion Technique

Liposomes form spontaneously by the assimilation of phospholipids, the primary component of cell membranes. Due to their unique ability to form selectively permeable bilayers in situ, they are widely used as nanocarriers for drug and small‐molecule delivery. However, there is a lack of straightforward methodologies to encapsulate living microorganisms. Here we demonstrate the successful encapsulation of whole cells in phospholipid vesicles by using the inverse‐emulsion technique of generating unilamellar vesicles. This method of liposome preparation allows for a facile encapsulation of large biomaterials that previously was not easily attainable. Using Escherichia coli as a model organism, we found that liposomes can protect the bacterium against external protease degradation and from harsh biological environments. Liposomes prepared by the inverse‐emulsion method were also capable of encapsulating yeast and were found to be naturally susceptible to hydrolysis by enzymes such as phospholipases, thus highlighting their potential role as cell delivery carriers.     

Overview of preparation methods of polymeric and lipid-based (niosome,solid lipid,liposome) nanoparticles: A comprehensive review

A wide number of drug nanocarriers have emerged to improve medical therapies, and in particular to achieve controlled delivery of drugs, genes or gene expression-modifying compounds, or vaccine antigens to a specific target site. Of the nanocarriers, lipid-based and polymeric nanoparticles are the most widely used. Lipid-based systems like niosomes and liposomes are non-toxic self-assembly vesicles with an unilamellar or multilamellar structure, which can encapsulate hydrophobic/hydrophilic therapeutic agents. Polymeric nanoparticles, usually applied as micelles, are colloidal carriers composed of biodegradable polymers. Characteristics such as loading capacity, drug release rate, physical and chemical stability, and vesicle size are highly dependent on experimental conditions, and material and method choices at the time of preparation. To be able to develop effective methods for large scale production and to meet the regulatory requirements for eventual clinical implementation of nanocarriers, one needs to have in-depth knowledge of the principles of nanoparticle preparation. This review paper presents an overview of different preparation methods of polymeric and novel lipid-based (niosome and solid lipid) nanoparticles.     

Liposome Formulation and In Vitro Testing in Non-Physiological Conditions Addressed to Ex Vivo Kidney Perfusion

This work focuses on formulating liposomes to be used in isolated kidney dynamic machine perfusion in hypothermic conditions as drug delivery systems to improve preservation of transplantable organs. The need mainly arises from use of kidneys from marginal donors for transplantation that are more exposed to ischemic/reperfusion injury compared to those from standard donors. Two liposome preparation techniques, thin film hydration and microfluidic techniques, are explored for formulating liposomes loaded with two model proteins, myoglobin and bovine serum albumin. The protein-loaded liposomes are characterized for their size by DLS and morphology by TEM. Protein releases from the liposomes are tested in PERF-GEN perfusion fluid, 4 °C, and compared to the in vitro protein release in PBS, 37 °C. Fluorescent liposome uptake is analyzed by fluorescent microscope in vitro on epithelial tubular renal cell cultures and ex vivo on isolated pig kidney in hypothermic perfusion conditions. The results show that microfluidics are a superior technique for obtaining reproducible spherical liposomes with suitable size below 200 nm. Protein encapsulation efficiency is affected by its molecular weight and isoelectric point. Lowering incubation temperature slows down the proteins release; the perfusion fluid significantly affects the release of proteins sensitive to ionic media (such as BSA). Liposomes are taken up by epithelial tubular renal cells in two hours’ incubation time.     

Elastic and Ultradeformable Liposomes for Transdermal Delivery of Active Pharmaceutical Ingredients (APIs)

Administration of active pharmaceutical ingredients (APIs) through the skin, by means of topical drug delivery systems, is an advanced therapeutic approach. As the skin is the largest organ of the human body, primarily acting as a natural protective barrier against permeation of xenobiotics, specific strategies to overcome this barrier are needed. Liposomes are nanometric-sized delivery systems composed of phospholipids, which are key components of cell membranes, making liposomes well tolerated and devoid of toxicity. As their lipid compositions are similar to those of the skin, liposomes are used as topical, dermal, and transdermal delivery systems. However, permeation of the first generation of liposomes through the skin posed some limitations; thus, a second generation of liposomes has emerged, overcoming permeability problems. Various mechanisms of permeation/penetration of elastic/ultra-deformable liposomes into the skin have been proposed; however, debate continues on their extent/mechanisms of permeation/penetration. In vivo bioavailability of an API administered in the form of ultra-deformable liposomes is similar to the bioavailability achieved when the same API is administered in the form of a solution by subcutaneous or epi-cutaneous injection, which demonstrates their applicability in transdermal drug delivery.     

Polymer vesicles: Mechanism,preparation, application,and responsive behavior

Polymer vesicles, also known as polymersomes, are finding increasing applications in biomedical field, including drug delivery, gene therapy, magnetic resonance imaging, theranostics, etc. This is due to their intrinsic hollow nanostructure and compartmentalized domains with diverse functionalities. This review describes recent advances in the design and synthesis of polymer vesicles, including the formation mechanisms, preparation methods, applications and responsive behaviors toward external stimuli. We first present the rational design and synthesis of polymer vesicles based on different polymeric building blocks, followed by an insight into the structure and formation mechanism of polymer vesicles, as well as the recently developed means to determine the exact thickness of the vesicle membrane. Except for responding to traditional stimuli such as pH, temperature and oxidation/reduction, polymer vesicles are becoming ‘smarter’ owing to the newly developed stimuli including electrical field, magnetic field, sugar molecules, gas, ultrasound, etc. Finally, the potential applications of polymer vesicles beyond biomedical field are highlighted as novel nanoreactors, water remediation materials, etc.     

Delivery of the Radionuclide 131I Using Cationic Fusogenic Liposomes as Nanocarriers

Liposomes are highly biocompatible and versatile drug carriers with an increasing number of applications in the field of nuclear medicine and diagnostics. So far, only negatively charged liposomes with intercalated radiometals, e.g., ⁶⁴Cu, ^99mTc, have been reported. However, the process of cellular uptake of liposomes by endocytosis is rather slow. Cellular uptake can be accelerated by recently developed cationic liposomes, which exhibit extraordinarily high membrane fusion ability. The aim of the present study was the development of the formulation and the characterization of such cationic fusogenic liposomes with intercalated radioactive [¹³¹I]I⁻ for potential use in therapeutic applications. The epithelial human breast cancer cell line MDA-MB-231 was used as a model for invasive cancer cells and cellular uptake of [¹³¹I]I⁻ was monitored in vitro. Delivery efficiencies of cationic and neutral liposomes were compared with uptake of free iodide. The best cargo delivery efficiency (~10%) was achieved using cationic fusogenic liposomes due to their special delivery pathway of membrane fusion. Additionally, human blood cells were also incubated with cationic control liposomes and free [¹³¹I]I⁻. In these cases, iodide delivery efficiencies remained below 3%.     

Proliposomes of lisinopril dihydrate for transdermal delivery: Formulation aspects and evaluation

We formulated and evaluated proliposomal gel of relatively low bioavailable drug lisinopril dihydrate (LDH) for transdermal delivery. Several proliposomal gel formulations of lisinopril dihydrate were prepared by modified coacervation phase separation method and examined for formation of liposomes by optical microscope and characterized by transmission electron microscopy. The formulations were evaluated for size, zeta potential, entrapment efficiency, rheological behavior, ex vivo drug permeation, skin irritation and stability. Differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) studies were performed to understand the phase transition behavior and mechanism for skin permeation, respectively. The microscopic examination revealed the formation of liposomes from proliposomal gel, and the size of the vesicles was found to be in the range of 385 nm to 635 nm. Entrapment efficiency was high for the formulation containing greater amounts of phosphatidylcholine. The DSC studies indicated the amorphous form of LDH in proliposomal gel formulation. Ex vivo permeation studies revealed sustained permeation of drug from proliposomal gels studied. The stability studies reveal that the proliposomal formulations are more stable when stored at refrigeration temperature (4 °C). In conclusion, proliposomal gels offer potential and prove to be efficient carriers for improved and sustained transdermal delivery of lisinopril dihydrate.     

Liposomal Formulation of a PLA2-Sensitive Phospholipid–Allocolchicinoid Conjugate: Stability and Activity Studies In Vitro

To assess the stability and efficiency of liposomes carrying a phospholipase A2-sensitive phospholipid-allocolchicinoid conjugate (aC-PC) in the bilayer, egg phosphatidylcholine and 1-palmitoyl-2-oleoylphosphatidylglycerol-based formulations were tested in plasma protein binding, tubulin polymerization inhibition, and cytotoxicity assays. Liposomes L-aC-PC10 containing 10 mol. % aC-PC in the bilayer bound less plasma proteins and were more stable in 50% plasma within 4 h incubation, according to calcein release and FRET-based assays. Liposomes with 25 mol. % of the prodrug (L-aC-PC25) were characterized by higher storage stability judged by their hydrodynamic radius evolution yet enhanced deposition of blood plasma opsonins on their surface according to SDS-PAGE and immunoblotting. Notably, inhibition of tubulin polymerization was found to require that the prodrug should be hydrolyzed to the parent allocolchicinoid. The L-aC-PC10 and L-aC-PC25 formulations demonstrated similar tubulin polymerization inhibition and cytotoxic activities. The L-aC-PC10 formulation should be beneficial for applications requiring liposome accumulation at tumor or inflammation sites.     

Retention of Eucalyptol,a Natural Volatile Insecticide,in Delivery Systems Based on Hydroxypropyl‐β‐Cyclodextrin and Liposomes

Eucalyptol (Euc) is a natural monoterpene with insecticide effects. Being highly volatile and sensitive to ambient conditions, its encapsulation would enlarge its application. Euc‐loaded conventional liposomes (CL), cyclodextrin/drug inclusion complex, and drug‐in‐cyclodextrin‐in‐liposomes (DCL) are prepared to protect Euc from degradation, reduce its evaporation, and provide its controlled release. The liposomal suspension is freeze‐dried using hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as cryoprotectant. The liposomes are characterized before and after freeze‐drying. The effect of Euc on the fluidity of liposomal membrane is also examined. A release study of Euc from delivery systems, in powder and reconstituted forms, is performed by multiple head extraction at 60 °C after 6 months of storage at 4 °C. CL and DCL suspensions are homogeneous, show nanometric vesicles size, spherical shape, and negative surface charge before and after freeze‐drying. Moreover, HP‐β‐CD does not affect the fluidity of liposomes. CL formulations present a weak encapsulation for Euc. The loading capacity of eucalyptol in DCL is 38 times higher than that in CL formulation. In addition, freeze‐dried DCL and HP‐β‐CD/Euc inclusion complex show a higher retention of eucalyptol than CL delivery system. Both carrier systems HP‐β‐CD/Euc and Euc‐loaded DCL decrease Euc evaporation and improve its retention. Practical Applications: Eucalyptol is a natural insecticide. It is highly volatile and poorly soluble in water. To enlarge its application, its encapsulation in three delivery systems (conventional liposomes, cyclodextrin/drug inclusion complex, combined system composed of cyclodextrin inclusion complex and liposome) is studied. In this paper it is proved that cyclodextrin/eucalyptol inclusion complex and eucalyptol‐in‐cyclodextrin‐in‐liposome are effective delivery systems for encalyptol encapsulation, retention, and release.     

Effect of anionic-lipid-molecule geometry on the structure and properties of liposome-polycation complexes

The influence of anionic amphiphilic compounds that have different geometric shapes and become incorporated into bilayers on the polycation-mixed liposome interaction and the structure and properties of the resulting complexes is analyzed. Phosphatidylserine, cardiolipin, and sodium dodecyl phosphate are used as anionic lipids, and poly(N-ethyl-4-vinylpyridinium bromide) and polylysine are used as polycations. Polycation adsorption on the surfaces of all examined types of liposomes is accompanied by the neutralization of their charge, an increase in the size of particles of the systems, and quenching of fluorescence labels. Liposomes whose membranes contain incorporated cylindrical phosphatidylserine molecules retain their integrity during contact with polycations. The resulting complexes quantitatively dissociate into initial components during an increase in the salt concentration in the surrounding solution. In the case of liposomes with asymmetric anionic lipids, that is, cardiolipin and sodium dodecyl phosphate, the conditions of retaining the membrane integrity and reversible complexation are fulfilled only at relatively low molar fractions of both lipids. The obtained data witness the decisive effect of the geometry of anionic lipid molecules on the stability of complexes formed from mixed liposomes and polycations.