Liposome Characterization Services

Seattle Genova has spent years developing liposome technologies, and our extensive experience qualifies us as experts in liposome preparation and manufacturing. Our liposome platform's goal is to provide you with high-quality liposome services ranging from custom liposome production, analysis, and characterization to application.

Different analytical techniques are used to regulate the liposomal formulations' physico-chemical and biological properties, as well as their stability and drug entrapment effectiveness. Size distribution, lamellarity, surface charge, drug entrapment effectiveness, phospholipid concentration, choleserol concentration, osmolarity, sterility, and pyrogenicity are some of the most crucial liposome properties.

1.     Physical Parameters

The measurement of vesicle form, surface morphology, mean vesicle size and size distribution, surface charge, electrical surface potential, surface pH, lamellarity, phase behaviour, percentage of free drug/percent capture, and drug release is the basis for controlling physical parameters.

        I.            Drug Entrapment Efficiency

When determining whether the introduction of liposomes was justified, the effectiveness of drug entrapment must be established. An organic solvent such as chloroform, chloroform-methanol (2:1 v/v), methanol, ethanol, or phosphate buffer saline and phosphate buffer is used to extract a liposome-encapsulated pellet, and the drug content is then determined. Using the proper analytical technique, such as a spectrophotometer or chromatographic techniques, the free drug can be examined in the supernatant. Additionally, gel permeation chromatography is employed. To distinguish the drug that is entrapped from the drug that is free, ultra-centrifugation is used.

      II.            High Performance Liquid Chromatography (HPLC)

The determination of medications in liposome formulation has seen extensive use of the HPLC method. Some of these techniques analyse medications that have been liposomally encapsulated or that have been liposomally encapsulated in biological materials like liver and plasma. The separation of liposomal and non-liposomal drug forms is a major focus of solid-phase extraction (SPE). The ability of liposomes to cross reversed-phase C18 silica gel cartridges without being retained, in contrast to a non-liposomal drug that is retained on the stationary phase63, is the basis for separation. This is done through the use of high-pressure liquid chromatography (HPLC) or electrophoresis to determine the difference. UV, fluorimetric, and mass spectrometry are the detection methods for drug delivery liposomes that are frequently employed in HPLC (MS). The HPLC techniques for liposomal formulation control are based on modifying parameters.

Depending on the composition and particle size, suspension can range from transparent to milky. While the lamellarity of liposomes is typically assessed using spectroscopic or electron microscopy, the measurement of liposomal size distribution is typically conducted using dynamic light scattering. Liposomal compositions that are physically stable maintain the liposome size distribution. Drug leaking from the vesicles and/or the aggregation or fusing of vesicles to generate bigger particles can both lead to physical instability.

    III.            Size Exclusion Chromatography (SEC)

The size exclusion chromatography has been utilized for fractionation of liposomes. This method is applied to the determination of cisplatin (Hwang et al., 2007), ceftazidime and cefepime encapsulated in liposomes. The unentrapped cisplatin is removed on a Sephadex G-15 column using distilled water, while for the unentrapped quantity of ceftazidime and cefepime a Sephadex G-50 column is used.

   IV.            High-Performance Thin-Layer Chromatography (HPTLC)

A HPTLC technique for the determination of ketorolac tromethamine is introduced by Nava et al.The content of the drug is defined by spotting samples on TLC plates, Alugram® Sil G/UV254 and formulating in ethyl acetate:chloroform:acetic acid.

     V.            Capillary Electrophoresis (CE)

In order to explore the characteristics of liposomes, such as membrane fluidity and stiffness, phospholipid distribution in the membrane, membrane disruption, size distribution, and surface charge density, capillary electrophoresis is used as an efficient instrument. A number of factors, including the liposome radius, the thickness of the electric double layer, and the surface charge, play a significant role in the complex process of liposome electromigration. The drug material free oxaliplatin is separated from liposome-entrapped oxaliplatin using capillary electrophoresis-inductively coupled plasma mass spectrometry (CE-ICP-MS). To separate the free, unencapsulated oxaliplatin, the 31P+, 195Pt+, 127I+, and 115In+ signals are monitored by the ICP-MS. The impact of calcium on the phospholipid coats in fused silica capillaries was investigated by Hautala et al.

   VI.            In Vitro Drug Release Study

Studies on in vitro release are frequently based on the dialysis technique. These experiments are created in a certified diffusion cell using either a dialysis membrane or a cellophane membrane. At a temperature of 37 oC, an aliquot of the produced formulation is deposited on a dialysis membrane, and the sample is tested by UV spectrophotometer for drug realisation. The donor medium is made up of liposomal formulation, and the receptor medium is made up of acetate buffer at pH 5, citrate-phosphate buffer, and phosphate buffer7 at pH 7.4. Drug release when utilising a dialysis membrane is watched over for 30 minutes or eight hours. Franz diffusion cells are also used for the diffusion studies.

 VII.            Zeta Potential (ζ-Potential)

Zeta potential measurements are used to characterise the surface charges of microspheres. The aggregation of liposomal formulation is inhibited by a larger zeta potential value, which also suggests improved colloidal stability. Microspheres are said to stabilise in suspension when their zeta potential is above (+/-) 30 mV. By delivering voltage across a pair of electrodes at opposite end of a cell holding the particle dispersion, the zetasizer is used to measure the zeta potential of the liposomes.

Particles with charges are drawn to an electrode with an opposing charge. In the interference pattern created by two laser beams, the particles move in an electrical field of known strength and produce scattered light that is influenced by the particle's speed.

VIII.            Differential Scanning Calorimetry (DSC) Study

In this technique drug loaded multilamellar liposomes are introduced to DSC analyzer. This method is utilized to determine phase transition temperature of phospholipids sample. The temperature of maximal excess heat capacity is characterized as the phase transition temperature.Thermograms are received at a scanning rate of 10 ºC/min or 20 ºC/min. Each specimen is scanned between 20 ºC to 200 ºC or 25 ºC to 500 ºC.

2.     Chemical Parameters

It's crucial to conduct a chemical analysis of liposomes in order to assess the potency and purity of different liposomal components. Phospholipid concentration, cholesterol concentration, phospholipid peroxidation, phospholipid hydrolysis, cholesterol auto-oxidation, and osmolarity are the chemical parameters that are most frequently examined. Phospholipid measurement is crucial for monitoring the effectiveness of the preparation process. Using Stewart's approach, the amount of phospholipids in liposomes was assessed based on the development of a colourful complex between the phospholipids and the ammonium ferrothiocyanate reagent.

By measuring the colour of a colourful complex that results from the reaction of cholesterol with newly made acetic anhydride-concentric sulfuric acid mixture (20:1) at 680 nm, or by dissolving cholesterol in glacial acetic acid, it is possible to measure the content of cholesterol. In the literature, an isocratic high-performance liquid chromatographic method is used to simultaneously measure cholesterol, cardiolipin, and 1,2-dioleoyl-sn-glycero-3-phosphocholine in various pharmaceutical formulations. The mobile phase is chloroform-methanol-aqueous ammonium acetate (71:26:3 v/v).

3.     Biological Parameters

The significance of determining biological parameters is beneficial in determining the safety of formulation for therapeutic application. Sterility, pyrogenicity and animal toxicity are specified during the biological characterization of the liposomes.

REFERENCES

1.   Hwang TL, Lee WR, et al “Cisplatin encapsulated in phosphatidylethanolamine liposomes enhances the in Nitro cytotoxicity and in vivo intratumor drug accumulation against melanomas” J Dermatol Sci,2007, 46: 11-20.

2.      Nava G, Piñón E, et al “Formulation and in vitro, ex vivo and in vivo evaluation of elastic liposomes for transdermal delivery of ketorolac tromethamine” Pharmaceutics, 2011, 3: 954-970.

3.      Ghanbarzadeh S, Valizadeh H, et al “Application of response surface methodology in development of sirolimus liposomes prepared by thin film hydration technique” BioImpacts, 2013, 3 (2): 75-81.

4.      Jadhav MP, Nagarsenker MS, et al “Formulation and evaluation of long circulating liposomal amphotericin B: a scinti-kinetic study using 99mTc in BALB/C mice” Indian J Pharm Sci, 2011, 73 (1): 57-64.

5.      Mulye C, Mishra R, et al “Formulation and evaluation of liposome mediated drug delivery” UJP, 2013, 2 (2): 156-160.

6.      Niu M, Lu Y, et al “Liposomes containing glycocholate as potential oral insulin delivery systems: preparation, in Nitro characterization, and improved protection against enzymatic degradation” Int J Nanomedicine, 2011, 6: 1115-1166.

7.      Song J, Shi F, et al “Formulation and evaluation of celastrol-loaded liposomes” Molecules, 2011, 16: 7880-7892.

8.      Divakar P, Kumar DP, et al“Formulation and in vitroevaluation of liposomes containing metformin hydrochloride” IJRPBS, 2013, 4 (2): 479-485.

9.      Makhmalzadeh BS, Azh Z, et al “Preparation and evaluation of mafenide acetate liposomal formulation as eschar delivery system” Int J Drug Dev & Res, 2011, 3 (4): 129-140.

10.  Mirzaee M, Owlia P, et al “Comparison of the bactericidal activity of amikacin in free and liposomal formulation against gram-negative and gram-positive bacteria” JJNPP, 2009, 4 (1): 1-7.

11.  Hathout RM, Mansour S, et al “Liposomes as an ocular delivery system for acetazolamide: in vitro and in vivo studies” AAPS PharmSciTech, 2007, 8 (1): E1-E12.