Stability Of Liposomes Research Articles

Dietary interference on the oxidation and hydrolysis of liposomes during in vitro digestion

SummaryIn this study, the influence of dietary compounds, including antioxidants (ethylenediaminetetraacid, ascorbic acid, catechin), chitosan and lactoferrin, on the stability of liposomes during in vitro adult and infant digestion was investigated in terms of formation of thiobarbituric acid‐reactive substances (TBARS) and free fatty acid release. Liposomes were more sensitive to degradation in adults than in infants, suggesting lower pH (1.5) and higher concentration of pancreatic enzymes (3.2 mg mL−1) and bile salts (5 mg mL−1) that indicated greater damage to the structure of liposomes. Compared to ethylenediaminetetraacid and ascorbic acid, catechin presented the most apparent protective effect against liposomal oxidation by scavenging free radicals. Chitosan promoted the formation of TBARS, while lactoferrin facilitated the hydrolysis rate of liposomes under both adult and infant conditions. This study provided an insight into the development of healthcare products and functional foods related to liposomes for special populations.

Liposome-chitosan hydrogel bead delivery system for the encapsulation of linseed oil and quercetin: Preparation and in vitro characterization studies

This study concentrates on developing linseed oil and quercetin co-loaded liposomes-chitosan hydrogel beads to enhance their stability and solubility. Scanning electron microscope (SEM) analysis revealed that the constructed hydrogel beads had three-dimensional network structure and liposomes were entrapped evenly within polymer network. X-ray diffraction (XRD) study illustrated that quercetin was in an amorphous or dissolved form in hydrogel beads. Thermodynamic properties of chitosan and soybean lecithin with phosphatidyl cholines of 60% (PC60) changed after ion crosslinking. After loaded into hydrogel beads, the release of quercetin and ɑ-linolenic acid became slow and can be explained by Higuchi model which indicated that bioactives were released from the skeleton of the hydrogel beads by the mechanism of Fick diffusion. The in vitro simulated digestion study showed that hydrogel beads could improve the stability of liposomes in gastrointestinal tract and inhibit the rapid release of fatty acids. The accelerated oxidation and photostability studies showed that the chemical stability of functional bioactives could be improved by loading liposomes into hydrogel beads. Hence, chitosan hydrogel bead is an attractive candidate for the encapsulation of functional bioactives for use in food industry.

The stabilization and release performances of curcumin-loaded liposomes coated by high and low molecular weight chitosan

A comprehensive stability evaluation for curcumin-loaded liposomes (Cur-LP) coated by low (LCS) or high (HCS) molecular weight chitosan with three gradient concentrations (L: low; M: medium; H: high) was the main objective of this study. Apart from leading to a higher encapsulation efficiency (>90%), all chitosan-coated Cur-LP displayed an improved stability with respect to resistant to salt, sunlight, heat, accelerated centrifugation and long-term storage at 4 °C. Increasing the molecular weight and concentration of chitosan could effectively improve the stability of Cur-LP, in which HCS-H coatings displayed the best performance. According to the fluorescence probe analysis, the mechanical reinforcement of liposomes and the concomitant reduction in membrane fluidity accounts for the major contribution to vesicle stability. Secondly, a simulated digestion model was used to prove the applicability of sustained curcumin release, achieved by adjusting the molecular weight and concentration of the chitosan stabilizer for Cur-LP. The results of this study show that high molecular weight chitosan used at relatively high concentrations, is a promising coating material for improving the stability and sustained release of Cur-LP in vitro.

Co‐encapsulation of resveratrol and epigallocatechin gallate in low methoxyl pectin‐coated liposomes with great stability in orange juice

SummaryTo improve long‐term stability of the liposomes, resveratrol (RES) liposomes, epigallocatechin gallate (EGCG) liposomes, RES+EGCG liposomes, RES+EGCG liposomes coated with low methoxyl pectin (LMP) were prepared, and their physical properties were evaluated. The prepared liposomes have a particle sizes of about 110–160 nm. The LMP‐coated composite liposomes have the largest particle size and zeta potential, the latter indicating higher system stability. Liposome systems were pasteurised and placed in an orange juice system as to study liposome stability in a real the food environment. LMP bridge with metal ions forming a network‐like gel in the LMP‐coated composite liposomes, thereby enhancing the stability of liposomes. LMP‐decorated liposomes could be developed as a formulation for encapsulating and delivering functional ingredients.

Azide-Modified Membrane Lipids: Miscibility with Saturated Phosphatidylcholines.

In this study, we describe the miscibility of four azide-modified membrane phospholipids (azidolipids) with conventional phospholipids. The azidolipids bear an azide group at different positions of the sn-1 or sn-2 alkyl chain and they further differ in the type of linkage (ester vs ether) of the sn-2 alkyl chain. Investigations regarding the miscibility of the azidolipids with bilayer-forming phosphatidylcholines will evaluate lipid mixtures that are suitable for the production of stable azidolipid-doped liposomes. These vesicles then serve as model membranes for the incorporation of model peptides or proteins in the future. The miscibility of both types of phospholipids was studied by calorimetric assays, electron microscopy, small-angle X-ray scattering, infrared spectroscopy, and dynamic light scattering to provide a complete biophysical characterization of the mixed systems.

Topical photodynamic therapy with chloroaluminum phthalocyanine liposomes is as effective as systemic pentavalent antimony in the treatment of experimental cutaneous leishmaniasis.

In the Americas, one of the main causative species of cutaneous leishmaniasis is Leishmania (Leishmania) amazonensis. The systemic antimonials remain the most largely used option for disease control. However, this drug has significant toxicity. The development of new alternative therapies, including the identification of effective drugs for topical treatment of cutaneous leishmaniasis, is of utmost interest. In this sense, photodynamic therapy emerges as a new strategy. The aim of this study was to develop the chloroaluminum phthalocyanine-loaded liposome, characterize it, and evaluate its stability and efficacy in the topical treatment of cutaneous leishmaniasis caused by L. (L.) amazonensis. Liposomes composed of egg phosphatidylcholine were prepared by Bangham's method. Storage stability of phthalocyanine-loaded liposomes was evaluated at 30 and 60 days after preparation. For the in vivo evaluation, the animals were infected with L. (L.) amazonensis and divided into groups: chloroaluminium phthalocyanine-loaded liposome, blank liposome, meglumine antimoniate (200 mgSb+5/Kg/day), and control. The lesion size was determined weekly after the beginning of the treatment. Upon completion, parasites were recovered from the skin lesion and spleen and evaluated by limiting dilution assay. Chloroaluminum phthalocyanine-loaded liposomes were stable and showed adequate characteristics for topical administration. The topical chloroaluminum phthalocyanine-loaded liposome was as effective as systemic pentavalent antimony in reducing the parasitic load in the lesion and spleen in infected animals. The present study showed that photodynamic therapy with chloroaluminum phthalocyanine-loaded liposomes is a promising strategy for the treatment of American cutaneous leishmaniasis caused by L. (L.) amazonensis.

Potential antioxidant and anti-inflammatory action of Hypericum hookerianum extracts in a liposome system evaluated with zebrafish embryos

The purpose of this study was to assess the antioxidant and anti-inflammatory potential of liposomal formulations enriched with Hypericum hookerianum (Hyp) aqueous extracts. Cotyledon segments derived from protocorms of H. hookerianum were cultured on Murashige and Skoog (MS) media supplemented with Kinetin (KN, 1 mgl−1) and Naphthalene acetic acid (NAA, 0.1 mgl−1) to induce hypericin-rich red shoots (HypR, 0.87 mg/G DW). Highly stable liposomes (−29.4 mV) were successfully developed which encapsulated 63 ± 0.8% Hyp extracts, respectively. MTT assay subsequently confirmed the biocompatibility of liposome compositions using fibroblast cell lines. This work also evaluated acute toxicity of L-HypR and L-HypG formulations using Danio rerio (Zebrafish) embryos for 96 hpf. The expression of antioxidant and anti-inflammatory genes were found to be upregulated for L-HypR than L-HypG (green shoots without hypericin) formulations. These properties of L-HypR may be extremely useful for incorporating lipophilic substances into the food or pharmaceutical industry.

Contribution of headgroup and chain length of glycerophospholipids to thermal stability and permeability of liposomes loaded with calcein

Biological membranes are complex systems that are composed of lipids, proteins and carbohydrates. They are difficult to study, so it is established practice to use lipid vesicles that consist of closed ‘shells’ of phospholipid bilayers as model systems to study various functional and structural aspects of lipid organisation. To define the effects of the structural properties of lipid vesicles on their phase behaviour, we investigated their headgroup and chain length, and the chemical bonds by which their acyl chains are attached to the glycerol moiety of glycerophospholipid species, in terms of phase transition temperature, enthalpy change and calcein permeability. We used differential scanning calorimetry to measure the temperature and enthalpy changes of phase transition, and fluorescence to follow calcein release through the bilayer structure. Our data show that longer acyl chains increase the stability of the lipid bilayers, whereas higher salt concentrations decrease the thermal stability and widen the phase transitions of these lipid bilayers. We discuss the possible reasons for the observed phase transition behaviour.

Physicochemical characterization of dual action liposomal formulations: anticancer and antimicrobial

BackgroundCancer till date remains one of the world's most life threatening disease accompanied by risk of secondary infections. Therefore formulations carrying anticancer drugs which can also decrease the risk of secondary infection are inevitable. Chemotherapeutic drug doxorubicin along with flavonoids quercetin and epigallocatechin gallate (EGCG) is simultaneously loaded on liposomal formulation exploiting the amphiphilic property of the liposomes. ResultsAtomic force microscope imaging reveal the size of liposomal formulation loaded with doxorubicin, quercetin and EGCG to be greater than void liposome confirming the presence of drugs. Liposomal stability is improved by PEGylation; adding to the drug release time in vitro. The charge of phosphatidylcholine is rendered positive by coating the formulation with histone. The average size of the formulation is 342 nm. The encapsulation efficiency of doxorubicin, quercetin and EGCG is found to be 65.8%, 96.8% and 98% respectively. The above formulation demonstrated both anticancer and antimicrobial activity. ConclusionThe formulation will provide dual anticancer and antimicrobial therapy thereby evading secondary infection in cancer patients along with chemotherapy.

Steric stabilization of phycobiliprotein loaded liposome through polyethylene glycol adsorbed cellulose nanocrystals and their impact on the gastrointestinal tract

The goal of the study was to encapsulate phycobiliprotein (PBP), a seaweed bioactive compound with the help of liposome and stabilized through polyethylene glycol adsorbed cellulose nanocrystals (CNC). The study investigated the stability of CNC coating and the release of PBP compounds using a simulated human gastrointestinal tract (GIT) model. Adsorption of polyethylene glycol (PEG) with molecular weights of 400–4000 gmol-1 on desulfated CNCs (DCs) did not show any significant stability of liposomes during in-vitro GIT experiments. The effect of GIT phase on the quality characteristics of the sample revealed that the simulated gastric juice phase of GIT helps on desorption of polyethylene glycol from desulfated CNCs surface followed by precipitation of DCs. This phenomena confirmed the effective agglomeration of liposomes in the gastric phase of the GIT. The liposomes stabilized through the higher molecular weight of PEG adsorbed with DCs (i.e., DPCs-4000, DPCs-1500, and DPCs-1000) displayed a higher degree of anti-agglomeration behavior as compared to the lower molecular weight of PEG with desulfated CNCs (DPCs-600 and DPCs-400). However, liposomes stabilized through pure DCs displayed insignificant (p > 0.05) effect on the stability of PBP loaded liposomes. We concluded that the adsorption of PEG onto DCs surface was enough to stabilize PBP loaded liposomes in a mouth and saliva environment. This finding is relevant to understand the stability of CNC as coating material and PBP as core bioactive compounds in the human digestive system for potential nutraceutical and food applications.

Bioinspired Liposomes for Oral Delivery of Colistin to Combat Intracellular Infections by Salmonella enterica.

Bacterial invasion into eukaryotic cells and the establishment of intracellular infection has proven to be an effective means of resisting antibiotic action, as anti-infective agents commonly exhibit a poor permeability across the host cell membrane. Encapsulation of anti-infectives into nanoscaled delivery systems, such as liposomes, is shown to result in an enhancement of intracellular delivery. The aim of the current work is, therefore, to formulate colistin, a poorly permeable anti-infective, into liposomes suitable for oral delivery, and to functionalize these carriers with a bacteria-derived invasive moiety to enhance their intracellular delivery. Different combinations of phospholipids and cholesterol are explored to optimize liposomal drug encapsulation and stability in biorelevant media. These liposomes are then surface-functionalized with extracellular adherence protein (Eap), derived from Staphylococcus aureus. Treatment of HEp-2 and Caco-2 cells infected with Salmonella enterica using colistin-containing, Eap-functionalized liposomes resulted in a significant reduction of intracellular bacteria, in comparison to treatment with nonfunctionalized liposomes as well as colistin alone. This indicates that such bio-invasive carriers are able to facilitate intracellular delivery of colistin, as necessary for intracellular anti-infective activity. The developed Eap-functionalized liposomes, therefore, present a promising strategy for improving the therapy of intracellular infections.

Physicochemical mechanisms of different biopolymers' (lysozyme, gum arabic, whey protein, chitosan) adsorption on green tea extract loaded liposomes

Having various domains of applicability, liposomes have been the issue of many studies since 1960s. Kinetically stable nature of liposomes required incorporation of other substituents to gain storage stability and interaction of liposomes with polymers, electrolytes, proteins or lipids still requires further investigation to explain the underlying mechanism. In this study, polyphenol-rich green tea extract was encapsulated into liposomes by means of microfluidization in two different aqueous media (pH = 3.8 acetate buffer and pH = 6.5 distilled water). Antioxidant loaded vesicles were further mixed with anionic biopolymers (gum arabic, whey protein) and cationic biopolymers (lysozyme, chitosan) separately. The physical and chemical interactions between liposomes and biopolymers were rationalized by particle size, zeta potential, transmission electron microscopy, total phenolic content and antioxidant activity measurements during 28-days storage at 4 °C. Experimental results indicated that the biopolymer incorporated liposomes showed better stability compared to control liposomes during storage, developing resistance against changes in particle size and zeta potential. On the other hand, biopolymer interaction mechanisms were shown to be different for different biopolymers. As was also proved by transmission electron microscopy, lysozyme was absorbed into the liposomes while gum arabic, whey protein and chitosan were adsorbed on the vesicle surface to shield green tea extract loaded liposomes.

Graphene Oxide Supported Liposomes as Red Emissive Theranostics for Phototriggered Tissue Visualization and Tumor Regression.

Selective tissue visualization and localized tumor regression without affecting the surrounding healthy tissues are critical concerns in cancer nanomedicine. Importantly, the complete wrapping of a flimsy matrix like liposome by multifunctional graphene oxide is an interesting engineering idea for nanomedicine design. Moreover, designing a safe and biodegradable nanohybrid with significant theranostic ability is a current need for targeted combined therapies. Here, we report a comprehensive result of in vivo tumor diagnosis and phototriggered tumor regression using a biodegradable red emissive nanotheranostic system, viz., graphene oxide flakes fortified liposome (GOF-Lipo), functionalized with folic acid (FA): GOF-Lipo-FA. Graphene oxide support enhances the stability of drug-loaded liposomes in an extracellular environment that prevents the premature release of loaded anticancer drug from the liposomal cavity. Promising outcomes of tumor regression (∼300 to 25 mm3) from organized cellular and animal studies are demonstrated in this work. These studies reveal superior biocompatibility, deep intracellular localization, 4T1 breast tumor diagnosis, and long time tumor binding ability of an injected emissive nanohybrid. Overall, a single dose of designed multifunctional systems demonstrates the best tumor regression.

The Delivery of a Wnt Pathway Inhibitor Toward CSCs Requires Stable Liposome Encapsulation and Delayed Drug Release in Tumor Tissues.

The Wnt signaling pathway is involved in tumorigenesis and various stages of tumor progression, including the epithelial-mesenchymal transition, metastasis, and drug resistance. Many efforts have been made to develop drugs targeting this pathway. CGX1321 is a porcupine inhibitor that can effectively block Wnt ligand synthesis and is currently undergoing clinical trials. However, drugs targeting the Wnt pathway may frequently cause adverse events in normal tissues, such as the intestine and skin. Formulation of the drug inside liposomes could enable preferential drug delivery to solid tumor tissues and limit drug exposure in normal organs. We developed a strategy to stably encapsulate CGX1321 inside liposomes with minimal drug releases in circulation. The liposomal drugs were shown to interfere with the aberrant Wnt signaling specifically in tumor tissues, resulting in focused effects on LGR5+ CSCs (cancer stem cells), while sparing other cells from significant cytotoxicity. We showed it is feasible to use such a CSC elimination approach to treat malignant cancers prone to rapid progression using a LoVo tumor model as well as a GA007 patient derived xenograft (PDX) model. Nano drug delivery systems may be required for precision medicine in cancer therapy.

Liposomal Vitamin D3 as an Anti-aging Agent for the Skin.

Vitamin D3 is an effective skin protective substance to prevent photoaging. Liposomes were used as a carrier to deliver vitamin D3 to improve the stability and to enhance the treatment effect of vitamin D3. The stability of vitamin D3 liposomes, average cumulative penetration, and retention of vitamin D3 in the skin were then evaluated and compared with free vitamin D3. Finally, the treatment effect of vitamin D3 liposomes in a rat photoaging model was appraised and Haematoxylin-Eosin (H&E) staining was used to assess the histology changes of the skin after vitamin D3 liposome treatment. The results indicated that liposomes could significantly improve the stability of vitamin D3. The average skin retention of vitamin D3 liposomes was 1.65 times that of the vitamin D3 solution. Vitamin D3 liposomes could repair the surface morphology of skin in the photoaging model and promote the production of new collagen fibers. Vitamin D3 liposomes as a potential skin care agent could significantly improve skin appearance and repair damage in the histology of photoaging.

Modulation effect of core-wall ratio on the stability and antibacterial activity of cinnamaldehyde liposomes.

Cinnamaldehyde is an active component of some plant essential oils, which has broad antibacterial activity. However, the strong volatility and instability of cinnamaldehyde limits its application. Cinnamaldehyde was encapsulated by liposomes, and the effects of core-wall ratio on the stability and antibacterial activity during storage were investigated. The particle size during storage showed that cinnamaldehyde liposomes with high core-wall ratios aggregated more easily, and the retention ratio of it can maintained at around 60%. The increase of cinnamaldehyde loading could reduce the fluidity of the liposome membrane. The antibacterial activity of cinnamaldehyde liposomes against Staphylococcus aureus during storage was investigated by fluorescence labeling and the killing log value. It was found that liposome-encapsulated cinnamaldehyde might still inhibit bacteria by destroying cell membrane integrity after storage and the persistence was more efficient than that of pure cinnamaldehyde. Therefore, liposomes could improve the stability and long-term antibacterial activity of cinnamaldehyde.

Liposome-based combination therapy for acne treatment

Acne vulgaris is one of the most common chronic diseases worldwide with the high prevalence ratio of about 80–85% in patients who are in puberty period. For the treatment options, many conventional dosage forms are available; however, existing limitations of systemic administration of drugs (oral antibiotics), such as adverse events and resistance, led for seek of new formulation options. In this study, liposomes containing tetracycline HCl and tretinoin were prepared by the film formation method. In vitro characterization studies revealed that liposomes (111.10 ± 8.02 nm; P.D.I.=0.198 ± 0.03; Z.P.=25.83 ± 0.40 mV) with an encapsulation efficiency more than 80% for both APIs were formulated. In order to maintain a suitable viscosity for topical application, optimized liposomal formulations were dispersed in carbopol-based gel. In vitro release of APIs was sustained for 24 hours with released amounts of 56.44% and 58.44% for tetracycline HCl and tretinoin, respectively. Stability evaluation of both liposomes and liposomes in hydrogels was investigated for 6 months at 4 °C and 25 °C; and no statistically significant change was observed in terms of particle size, zeta potential, encapsulation efficiency, appearance, pH, and viscosity. Cytotoxicity tests confirmed the nontoxic structure of liposomal gel formulations on mice fibroblast cells. In addition, antibacterial efficacy has been proven with Staphylococcus aureus and Streptococcus epidermidis strains as well as the effect on biofilm formation and eradication. As a result, we hereby presented a new combination drug product, which consists of dual active ingredients having comedolytic and bacteriostatic effects in a single, safe, and stable liposome formulation.

Application of Statistical Design to Assess the Critical Process Parameters of Ethanol Injection Method for the Preparation of Liposomes

In the present study custom screening design was employed to observe the effect of four critical process parameters on particle size and polydispersity index of the liposomal formulation made by ethanol injection method. The four process parameters selected were lipid ratio, rate of injection, phase volume ratio and rotational speed of magnetic stirring. Eight different liposomal formulations were prepared using the design. The formulations were subjected to particle size analysis. The analysis was done at a significance level p<0.05 and found that the process parameters had significant effect on the particle size and polydispersity index of the formulations. The design was optimized for the individual responses with an overall desirability of more than 50%. Three batches of liposomes were formulated at optimized process parameters which matched the target as predicted by the design. Therefore, it can be concluded that the design was effective in production of nano sized stable monodisperse liposomes by ethanol injection method.
 Dhaka Univ. J. Pharm. Sci. 18(1): 103-111, 2019 (June)

Effects of environmental pH and ionic strength on the physical stability of cinnamaldehyde-loaded liposomes

The physical stability of cinnamaldehyde-loaded liposomes was investigated under various conditions including different storage time, storage temperature, pH and ionic strength of external environment, as well as variations in hydration media. Results showed that the average diameter of liposomes increased first and remained constant, the retention rate (RR) decreased while the liposomes stored at higher temperature exhibited a lower RR during the storage period of 9 days. Furthermore, liposomes under acidic conditions (pH ≤ 6) exhibited a decreased particle size with an increased RR. As for the ionic strength of the external environment and hydration media, the particle size of liposomes increased, whereas the RR and encapsulation efficiency (EE) decreased as ionic strength increased. Atomic force microscopy (AFM) further proved that high ionic strength could result in instability of liposomes. However, the particle size of liposomes in the external environment composed of ion solution were far more than those in the hydration media, which indicated that the liposomes prepared in ion solution could resist instability caused by changes in external ionic strength. These results also provided a guidance for the application of liposomes in different environments and selectively preparing desired liposomes by modulating the pH and ionic strength of environment.

Physicochemical study of the protein–liposome interactions: influence of liposome composition and concentration on protein binding

The aim of the present study is to investigate the interactions between liposomes and proteins and to evaluate the role of liposomal lipid composition and concentration in the formation of protein corona. Liposomes composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or hydrogenated soybean phosphatidylcholine (HSPC) with 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (DPPE-PEG 3000), cholesterol (CH) or mixtures of these lipids, were prepared at different concentrations by the thin-film hydration method. After liposomes were dispersed in HPLC-grade water and foetal bovine serum (FBS), their physicochemical characteristics, such as size, size distribution, and ζ-potential, were determined using dynamic and electrophoretic light scattering. Aggregation of DPPC, HSPC, DPPC:CH (9:1 molar ratio), and HSPC:CH (9:1 molar ratio) in FBS was observed. On the contrary, liposomes incorporating DPPG lipids and CH both in a molar ratio of 11% were found to be stable over time, while their size did not alter dramatically in biological medium. Liposomes containing CH and PEGylated lipids retain their size in the presence of serum as well as their physical stability. In addition, our results indicate that the protein binding depends on the presence of polyethylene glycol (PEG), CH, concentration and surface charge. In this paper, we introduce a new parameter, fraction of stealthiness (Fs), for investigating the extent of protein binding to liposomes. This parameter depends on the changes in size of liposomes after serum incubation, while liposomes have stealth properties when Fs is close to 1. Thus, we conclude that lipid composition and concentration affect the adsorption of proteins and the liposomal stabilization.