Screening for Optimal Liposome Preparation Conditions by Using Dual Centrifugation and Time-Resolved Fluorescence Measurements

Dual centrifugation (DC) is a novel in-vial homogenization technique for the preparation of liposomes in small batch sizes under gentle and sterile conditions which allows encapsulation efficiencies (EE) for water soluble compounds of >50%. Since liposome size, size distribution (PDI), and EE depend on the lipid concentration used in the DC process, a screening method to find optimal lipid concentrations for a defined lipid composition was developed. Four lipid mixtures consisting of cholesterol, hydrogenated or non-hydrogenated egg PC, and/or PEG-DSPE were screened and suitable concentration ranges could be identified for optimal DC homogenization. In addition to the very fast and parallel liposome preparation of up to 40 samples, the screening process was further accelerated by the finding that DC generates homogeneously mixed liposomes from a macroscopic lipid mixture without the need to initially prepare a molecularly mixed lipid film from an organic solution of all components. This much simpler procedure even works for cholesterol containing lipid blends, which could be explained by a nano-milling of the cholesterol crystals during DC homogenization. Furthermore, EE determination was performed by time-resolved fluorescence measurements of calcein-loaded liposomes without removing the non-entrapped calcein. The new strategy allows the rapid characterization of a certain lipid composition for the preparation of liposomes within a working day.

Ocular Application of Oleuropein in Dry Eye Treatment: Formulation Studies and Biological Evaluation

Background. Oleuropein is already known for its numerous pharmacological properties, but its activity in the ocular field has not yet been investigated. The study aims to verify a possible use of oleuropein (OLE)-based eye drops both in terms of efficacy in dry eye syndrome and stability in aqueous solution. Methods. OLE was co-precipitated with HP-β-cyclodextrin, and the obtained complex was encapsulated into liposomes prepared by hydration of a lipid film composed of Lipoid S100 and cholesterol with different pH buffer solutions. The hydrated vesicles were shrunk by ultrasonication or extrusion. The preparations were characterized from the physicochemical point of view by subjecting them to differential scanning calorimetry, ATR-FTIR, dynamic light scattering analysis, and microscopy. Subsequently, OLE protective activity against hyperosmotic and oxidative stress on rabbit corneal epithelial cells (RCE) was evaluated. Results. The liposomal vesicles obtained after extrusion showed a tendency towards greater encapsulation efficiency (up to 80.77%) compared to that obtained by sonication, and the liposomes hydrated in pH 5.5 solution tended to incapsulate more than the neutral ones. Ultrasonication produced two-dimensional populations of liposomes, the largest of which reached 2149 nm. On the contrary, the extruded liposomes showed homogeneous diameters of about 250 nm. Complexation with cyclodextrin and subsequent encapsulation in liposomes greatly increased the OLE stability in aqueous solution, especially at 4 °C and for the extruded formulations. OLE aqueous solution (OLE7.4-sol, reference) and neutral extruded liposomes (F7.4-e) were well tolerated on RCE cells. Moreover, OLE was able to control the effects of hyperosmolarity on ocular surface cells and to prevent oxidative stress-induced loss of cell viability.

Development of Lactoferrin-Loaded Liposomes for the Management of Dry Eye Disease and Ocular Inflammation

Dry eye disease (DED) is a high prevalent multifactorial disease characterized by a lack of homeostasis of the tear film which causes ocular surface inflammation, soreness, and visual disturbance. Conventional ophthalmic treatments present limitations such as low bioavailability and side effects. Lactoferrin (LF) constitutes a promising therapeutic tool, but its poor aqueous stability and high nasolacrimal duct drainage hinder its potential efficacy. In this study, we incorporate lactoferrin into hyaluronic acid coated liposomes by the lipid film method, followed by high pressure homogenization. Pharmacokinetic and pharmacodynamic profiles were evaluated in vitro and ex vivo. Cytotoxicity and ocular tolerance were assayed both in vitro and in vivo using New Zealand rabbits, as well as dry eye and anti-inflammatory treatments. LF loaded liposomes showed an average size of 90 nm, monomodal population, positive surface charge and a high molecular weight protein encapsulation of 53%. Biopharmaceutical behaviour was enhanced by the nanocarrier, and any cytotoxic effect was studied in human corneal epithelial cells. Developed liposomes revealed the ability to reverse dry eye symptoms and possess anti-inflammatory efficacy, without inducing ocular irritation. Hence, lactoferrin loaded liposomes could offer an innovative nanotechnological tool as suitable approach in the treatment of DED.

Fabrication and Release Kinetics of Liposomes Containing Leuprolide Acetate

The present work is focused on the preparation and in vitro release kinetics of liposomal formulation of Leuprolide Acetate. In this work, “Thin Lipid Film Hydration Method” was used for preparation of Leuprolide Acetate loaded liposomes. Prepared liposomal formulations of Leuprolide acetate was evaluated by drug entrapment study, in-vitro drug release kinetics and stability studies. The percentage drug entrapment of Leuprolide acetate for F1 and F2 formulations were found to be 78.14 ± 0.67 and 66.70 ± 0.81% respectively. In-vitro drug release study of liposomal formulations had shown zero order release pattern. Regression co-efficient (R2) value of Zero order kinetics for F1 and F2 formulations were 0.9912 and 0.9676 respectively. After storing formulations for 1 month, stability testing was done at 40C.It was found that all batches were stable. These liposomal formulations of Leuprolide acetate can be formulated for parenteral application to treat prostate cancer and in women, to treat symptoms of endometriosis (overgrowth of uterine lining outside of the uterus) or uterine fibroids.

UV Absorption Spectroscopy for the Diffusion of Plasma-Generated Reactive Species through a Skin Model

Skin applications of non-thermal atmospheric pressure plasma (NTAPP) have been at-tracting attention from medical and cosmetic aspects. The reactive species generated from plasma sources have been known to play important roles in the skin. For proper applications, it is essential to know how they diffuse into the skin. In this study, the penetration of active species from NTAPP through a skin model was analyzed by UV absorption spectroscopy. The diffusions of hydrogen peroxide, nitrite, and nitrate were quantified through curve fitting. We utilized an agarose gel to mimic epidermis and dermis layers, and we used a lipid film or a pig skin sample to mimic the stratum corneum (SC). The diffusion characteristics of reactive species through this skin model and the limitations of this method were discussed

Formulation, Design and Optimization of Glycerosomes for Topical Delivery of Minoxidil

Background: Present work reports the formulation design and optimization of minoxidil loaded glycerosomes for topical application. The delivery system enhances the vesicular properties of vesicles by modifying the fluidity of lipid bilayer. The major component of formulation consists of phospholipid, glycerol, and cholesterol. Methodology: Glycerosomes were prepared by using lipid film hydration method. Prepared formulations were optimized using Box behnken 32 full factorial experimental designs. Two independent variables were selected which were Sonication time (X1), and Glycerol Concentration (X2) and with respect to these two dependent variables were selected which were % cumulative drug release after eight hours (Y1), and Entrapment efficiency (Y2). Nine formulations of (G1-G9) were prepared based on factorial design for optimization. Result and Discussion: Prepared formulations were evaluated in terms of surface analysis, charge distribution, encapsulation efficiency, in-vitro diffusion studies, stability testing and release kinetics. The fabricated glycerosomes found to possess entrapment efficiency in the range of 70.29±0.75 to 87.91±0.23%, cumulative drug release: 73.12 to 89.39%; a shelf life of 356 days at 4± 1°C and show higuchi release kinetics, fickian diffusion. Conclusion: As glycerol present in formulation in high quantity, this is itself used as humectant, emollient and penetration enhancer. So this formulation is best suitable for topical delivery of drugs.

Formulation Development and Evaluation of Transfero-somes Nano Gel Loaded with Levocetirizine Transfero-somes

A new drug delivery technology called transferosomes were came into existence which is an artificial vesicle designed to show the characteristics of a cell vesicle suitable for controlled and potentially directed drug delivery. Transferosome is a highly flexible and stress- responsive compound, complex compound and highly deformed vesicle with an aqueous core surrounded by the complex lipid bilayer thus enabling it to deliver both hydrophilic and hydrophobic drug. Urticaria is a general condition distinguished as brief erythematous and oedematous plaques or papules with defined erythematous borders and central clearing, identified as hives/wheals. Levocetirizine which is used for the treatment is a H1 anti histamine that is actively used in treatment of urticaria. The aim of the present research work was to investigate the potential of transferosome formulations for transdermal delivery of levocetirizine. The transferosomes were formulated by lipid film hydration technique using Rotary vacuum Evaporator. In the present work levocetirizine vesicle was efficaciously formulated using an appropriate ratio of tween 80 and soya lecithin and was incorporated into gel since gel formulations are easy to administer and patient compliance. Levocetirizine transferosomes was evaluated for vesicle characteristicslike zeta potential, poly-dispersibility index, TEM, and stability study. Theaverage sizes of transferosome were found to be 566.6nm and poly dispersity index (PI) was found to be 0.532, zeta potential of the transferosome was found to be -2.3 mV which indicates that transferosome formulation is stable. Levocetirizine gel was prepared by using various concentrations of Carbopol 934 and is evaluated for their gel characteristics like pH, viscosity, Spreadability, extrudability, homogeneity, drug content, diffusion etc. Gel containing 2% Carbopol show best and promising results. Levocetirizine transferosomal gel were efficaciously formulated by utilizing levocetirizine transferosome which was prepared by thin film hydration method by using soya lecithin and tween 80 in the ratio 85:15. Other Oral formulation used in treatment for urticaria have disadvantage like poor bioavailability, 1st pass metabolism, patient non-compliance etc. Transferosome nano gel loaded with levocetirizine transferosome was found to be more effective than other oral formulations used in treatment for urticaria since transferosome are capable of passing through lipid layer and delivering drug into systemic circulation with maximum bioavailability.

Formulation of Liposomes Containing Royal Jelly and Their Quality Assessment

Royal jelly, a gelatinuous consistency bee product produced and secreted by the hypopharyngeal and mandibular glands of worker honeybees, is beneficial in the treatment of dermatological conditions, likely through its content of the fatty acid 10-hydroxy-2-decenoic acid (10-HDA). However, 10-HAD poorly penetrates into skin. Thus, in this work, we produced royal jelly incorporated liposomes with the aim of increasing skin penetration of 10-HDA. Lipid nanocarriers were prepared by the thin lipid-film hydration method. Size and polydispersity index of the nanocarrier particles, and their stability over 30 days were measured. The effects of royal jelly and 10-HDA liposomal formulations on the viability of immortalized human keratinocyte cells were tested with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The skin penetration of 10-HDA from liposomal formulations and royal jelly solution was studied in vitro with Franz type vertical diffusion cells using porcine skin as limiting membrane. As result, small liposomes were achieved, and the efficacy of the obtained nanoformulations was examined by means of in vitro cell assays with a HaCaT immortalized human keratinocyte cell culture line. Finally, the skin penetration experiments showed that liposomal incorporation greatly increased 10-HDA penetration into skin layers.

Lipidated Peptidomimetic Ligand-Functionalized HER2 Targeted Liposome as Nano-Carrier Designed for Doxorubicin Delivery in Cancer Therapy

The therapeutic index of chemotherapeutic agents can be improved by the use of nano-carrier-mediated chemotherapeutic delivery. Ligand-targeted drug delivery can be used to achieve selective and specific delivery of chemotherapeutic agents to cancer cells. In this study, we prepared a peptidomimetic conjugate (SA-5)-tagged doxorubicin (Dox) incorporated liposome (LP) formulation (SA-5-Dox-LP) to evaluate the targeted delivery potential of SA-5 in human epidermal growth factor receptor-2 (HER2) overexpressed non-small-cell lung cancer (NSCLC) and breast cancer cell lines. The liposome was prepared using thin lipid film hydration and was characterized for particle size, encapsulation efficiency, cell viability, and targeted cellular uptake. In vivo evaluation of the liposomal formulation was performed in a mice model of NSCLC. The cell viability studies revealed that targeted SA-5-Dox-LP showed better antiproliferative activity than non-targeted Dox liposomes (Dox-LP). HER2-targeted liposome delivery showed selective cellular uptake compared to non-targeted liposomes on cancer cells. In vitro drug release studies indicated that Dox was released slowly from the formulations over 24 h, and there was no difference in Dox release between Dox-LP formulation and SA-5-Dox-LP formulation. In vivo studies in an NSCLC model of mice indicated that SA-5-Dox-LP could reduce the lung tumors significantly compared to vehicle control and Dox. In conclusion, this study demonstrated that the SA-5-Dox-LP liposome has the potential to increase therapeutic efficiency and targeted delivery of Dox in HER2 overexpressing cancer.