Quality of liposomal phospholipids at different stages in the ...

Pharmaceutical Chemistry Journal, Vol. 46, No. 3, June, 2012 (Russian Original Vol. 46, No. 3, March, 2012)

QUALITY OF LIPOSOMAL PHOSPHOLIPIDS AT DIFFERENT STAGES IN THE MANUFACTURING PROCESS E. V. Sanarova,1 E. A. Kotova,1 S. G. Kozeev,2 A. V. Lantsova,1 A. P. Polozkova,1 I. I. Krasnyuk,2 and N. A. Oborotova1 Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 46, No. 3, pp. 50 – 53, March, 2012. Original article submitted July 8, 2011.

We report here studies of the effects of liposome preparation methods on measures of the quality of phosphatidylcholine used, this being the main component of the liposomal bilayer and extremely unstable to the temperatures of manufacturing processes containing oxygen and transition metal ions. One process destructive for this phospholipid is peroxidation, the level of which is generally assessed in terms of the malondialdehyde concentration and the Klein oxidation index. Extrusion and homogenization of large multilamellar liposomes were found to be the least damaging methods of preparing liposomes, as compared with sonication. Attempts to introduce an antioxidant (vitamin E) did not lead to any significant improvement in the parameters assessed. Key words: liposomes, lipid peroxidation, extrusion, homogenization, sonication, antioxidant.

Liposomes are artificial phospholipid vesicles used in medicine as universal containers for delivery of drugs directly to target cells [1, 2]. They protect drugs packaged within them from the degrading actions of plasma enzymes, decrease the toxicity of encapsulated substances, and prolong their residence in the body. Methods for preparing liposomal medicinal formulations (LMF) have significant influences on their quality. The manufacturing technology also determines vesicle size, the level of lipid peroxidation (LPO) in the membrane (liposomes with high LPO product contents can be toxic to the body), pH, and other quality indicators [3]. The main destructive process in lipids is oxidation. Unsaturated fatty acids (UFA) are subject to oxidation, this occurring the more rapidly the more unsaturated the fatty acids are. Most affected by oxidation are UFA in position 2 of the phospholipid (PL) molecule [4]. This is associated with the specific characteristics of the distribution of fatty acid residues in the PL molecule. Peroxidation of liposomal PL is a typical free-radical process. Lipid peroxidation products are diene conjugates, hydroperoxides, aldehyde alcohols, oxy1 2

and ketoacids, dibasic carboxylic acids, and epoxides, which have adverse effects on the quality of liposomal preparations [5]. Electron microscopic studies (freeze fracture) have demonstrated morphological changes induced by LPO of phospholipid membranes consisting of egg phosphatidylcholine (PC) in the presence of calcium ions. Oxidation of PC to an oxidation index of 0.6 – 0.9 is known to impair the lamellar packaging of PL into bilayers, producing a distorted hydrophobic surface, while higher levels lead to shredding of PL aggregates [6]. Free-radical oxidation is generally studied by determination of the Klein peroxidation index (the ratio of optical densities at 233 and 215 nm [7, 9], which characterizes the level of PL oxidation (content of conjugated dienes), and the well-known reaction with 2-thiobarbituric acid (TBA) [8, 9], which assays the concentration of the LPO end product malondialdehyde (MDA). Antioxidants (AO) – substances slowing or preventing free-radical oxidation processes – are added to LMF to prevent LPO [10]. Water-soluble AO include ascorbic acid, thiol-containing compounds, phenols, and uric acid and its salts; fat-soluble AO include a-tocopherol acetate (vitamin E) and b-carotene. In terms of mechanisms of action, AO are divided into two classes: preventive, which decrease the rate of initiation of the chain reaction, and quenching (chain-

N. N. Blokhin Russian Oncological Scientific Center, Russian Academy of Medical Sciences, Moscow, Russia. State Higher Professional Educational Institution I. M. Sechenov First Moscow State Medical University, Russian Ministry of Health and Social Development, Moscow, Russia.

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breaking), which block development of the chain reaction. The former include such metalloenzymes as catalase and peroxidase, which degrade H2O2, as well as organic peroxides and agents forming chelate complexes with metals (EDTA). Chain-breaking AO often include phenols, aromatic amines, and vitamin E, whose main functions are to prevent the formation of fatty acid hydroperoxides, to degrade them, and to inactivate free radicals [4]. The aim of the present work was to study changes in the quality of liposomes at all stages of their preparation using a variety of methods. The quality of phospholipid vesicles was assessed in terms of the level of oxidation of PL bilayers, changes in mean particle diameter, and the pH of liposomal dispersions.

Sterilizing Filtration and Sublimation Drying of Liposomal Dispersions

EXPERIMENTAL SECTION

During experiments with lyophilized LMF, flask contents were rehydrated with 4.8 ml of deionized water to prepare dilute homogeneous dispersions. At all stages of liposome preparation, vesicle size was measured using a Nicomp 380 Submicron Particle Sizer (USA). The liposomal dispersions (1 ml) was placed in a 100-ml measuring flask and deionized water was added to the mark. pH was measured potentiometrically in liposomal dispersions in lyophilized samples.

Preparation of Large Multilamellar Liposomes Large multilamellar liposomes (LML) were prepared by lipid film hydration. Accurately weighed samples of phosphatidylcholine (PC, LIPOID E PC S, Germany), cholesterol (Chol; Sigma, Germany), and PEG-2000-distearoylphosphatidylethanolamine (PEG-2000-DSPE; LIPOID, Germany) at a molar ratio of 165:8:1 and 13 mg of 10% oil solution of a-tocopherol acetate (for the antioxidant-containing formulation) were dissolved in 15 ml of chloroform (reagent grade, Khimmed, Russia) and transferred to a 2000-ml round-bottomed flask. The chloroform solution was evaporated on a rotary evaporator (BUCHI Rotavapor R-200, Switzerland) at a water bath temperature of 35 ± 2°C to formation of a thin film of lipid, followed by drying for 50 – 60 min in vacuo to complete removal of solvent residues. The lipid film was then hydrated with 150 mM sodium chloride solution to complete removal from the vessel wall. The resulting LML dispersion was initially filtered through membrane filters with pore diameters of 1.2 and 0.45 mm (Pall, Russia). Preparation of unilamellar liposomes The LML emulsion was divided into three parts to prepare unilamellar liposomes by different methods: Extrusion – filtered solutions were extruded 10 times through membrane filters with pore diameter 0.2 mm using a LIPEX™ extruder (northern Lipids, Canada); Sonication (SON) – filtered LML solutions were sonicated using a Transsonic 310 disperser (Elma, Germany) with a frequency of 20 kHz for 25 min; Homogenization – filtered LML solutions were dispersed using a Microfluidizer M-110S (Microfluidics, USA) homogenizer at a pressure of 40 mmHg for 4 min.

Unilamellar liposomes were sterilized by filtration through nylon membrane filters with a pore diameter of 0.22 mm and mixed with 40% filtered sucrose solution at a ratio of 3:1. Prepared samples of empty liposomes and empty liposomes with AO (vitamin E) in 5-ml aliquots in 25-ml flasks were subjected to sublimation drying using an Edwards Minifast apparatus (UK). Lyophilization was performed using a previously developed protocol of cooling to –55°C and heating the shelves to +20°C. Measurement of Mean Size, pH, and Phospholipid Oxidation Products in Liposomes

Presence of PL Oxidation Products in Liposomes PL oxidation products were determined in terms of the: a) MDA Concentration The liposome sample (0.5 ml) was placed in a heat-resistant tube, as was 3 ml of the reference solution (15 g of trichloroacetic acid and 0.67 g of thiobarbituric acid (TBA) were dissolved in 100 ml of purified water and heated in a water bath to complete dissolution), and these were heated in a boiling water bath for 20 min to formation of a pink solution. The optical densities of the resulting mixtures were measured relative to the reference solution on a Cary 100 spectrophotometer (Varian Inc., Australia) at wavelengths of 532 nm and 580 nm. MDA concentrations (nmol/ml) were calculated as: C=

( D 532 - D 580 ) ´ 6 ´ 1000 155

(1)

where D532 and D580 are the optical densities of the analytical sample measured relative to reference solution at wavelengths of 532 and 580 nm respectively, 6 is the coefficient of dilution of the analytical sample, and 155 is the molar extinction coefficient of the MDA-TBA chromogen, ml × mmol – 1 × cm – 1. b) Klein Oxidation Index The analytical liposome sample (0.1 ml) was placed in a measuring flask and 95% ethanol was added to 10 ml. The optical density of the ethanol dilution was measured relative

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to 95% ethanol on a Cary 100 spectrophotometer at wavelengths over the range 200 – 300 nm. The oxidation index (OI) was calculated as: D 233 - D 300 D 215 - D 300

(2)

where D233, D215, and D300 are the optical densities of the analytical sample measured relative to 95% ethanol at wavelengths of 233, 215, and 300 nm respectively. RESULTS AND DISCUSSION The destructive potential of LMF preparation methods was assessed in terms of quality measures such as the extent of liposomal PL oxidation (Figs. 1, 2), mean particle size, and the pH of liposomal dispersions (Table 1). Comparative studies of the level of LPO of liposomes prepared using different technical methods yielded the following measurements of MDA concentrations and Klein OI values. When homogenization was used to prepare unilamellar liposomes, the OI values of empty liposomes (0.31) and empty liposomes with vitamin E (0.26) were great than those of LML (0.13) by factors of 2 – 2.5. Lyophilization of LMF increased OI for both types of liposomes, to 0.35 in the case of empty liposomes and 0.33 for liposomes with vitamin E. MDA concentrations in homogenized liposomes with AO (3.29 nmol/ml) and without AO (3.02 nmol/ml) were similar and increased significantly after stabilization of liposomal dispersions by lyophilization. The pH of empty liposomes with vitamin E during homogenization and lyophilization increased to 7.3 ± 0.1, compared with an increase to 7.5 ± 0.1 for empty liposomes; vesicle size was not greater than 130 nm. The plots (Figs. 1 and 2) show that MDA concentrations after extrusion of empty liposomes (3.52 nmol/ml) and

Extrusion + Lyo

Extrusion

SON

SON + Lyo

Homogenization + Lyo

Homogenization

LML

Extrusion + Lyo

SON + Lyo

Extrusion

SON

0.1

Homogenization

0.2

Homogenization + Lyo

0.3

Empty liposomes

Fig. 1. Malondialdehyde conditions in empty liposomes (a) and empty liposomes with vitamin Ex (b) prepared by different methods (MDA – malondialdehyde; LML – large multilamellar liposomes; SON – sonication; Lyo – lyophilization).

IO =

b

0.4

0

Empty liposomes with vitamin Ex

a

LML

Extrusion + Lyo

Extrusion

SON

SON + Lyo

Homogenization + Lyo

LML

Empty liposomes

Homogenization

Extrusion

Extrusion + Lyo

0

LML

1

SON

2

SON + Lyo

3

Homogenization + Lyo

4

Homogenization

MDA concentration, nmol/ml

0.5

b

MDA concentration, nmol/ml

a

5

Empty liposomes with vitamin Ex

Fig. 2. Klein oxidation index in empty liposomes (a) and empty liposomes with vitamin Ex (b) prepared by different methods (LMLlarge multilamellar liposomes; SON – sonication; Lyo – lyophilization).

liposomes with vitamin E (3.63 nmol/ml), as compared with LML (0.65 – 0.68 nmol/ml), increased more than five-fold. MDA levels changed insignificantly after lyophilization of extruded liposomes. The OI of liposomal PL for this type of liposomes changed little before and after lyophilization, while the presence of AO produced virtually no decrease in OI. The size of empty liposomes obtained by extrusion was 173 ± 10 nm before lyophilization and 166 ± 10 nm after. The mean diameter of liposomes with vitamin E before (182 ± 10 nm) and after lyophilization (169 ± 10 nm) remained below 200 nm. After extrusion, the pH of empty liposomes and liposomes with AO was 7.0 ± 0.1 and 6.8 ± 0.1 respectively. Lyophilization led to minor increases in pH in both types of liposomes. Preparation of liposomes by sonication produced sharp increases in both OI and the MDA concentrations in liposomes. Assessment of OI in empty liposomes showed that before lyophilization, the value was 0.34, increasing to 0.46 after lyophilization; for empty liposomes with vitamin E, IO values before (0.32) and after lyophilization (0.42) were also different. After assessment of MDA concentrations for empty liposomes produced by sonication, it became apparent that sonication promoted increases in dialdehyde levels both for empty liposomes and for liposomes with vitamin E. For empty liposomes, lyophilization increased the MDA concentration from 4.11 to 4.93 nmol/ml, compared with an increase from 3.83 to 4.64 nmol/ml for empty liposomes with vitamin E. The initial small size (less than 100 nm) of vesicles with and without AO prepared by sonication increased to 197 ± 20 nm for empty liposomes and 237 ± 20 nm for liposomes with vitamin E; after lyophilization, vesicle diameters were 251 ± 20 and 249 ± 20 nm respectively. Potentiometric measurement of the pH of vesicles prepared by this method gave the following results: 7.3 ± 0.2 for empty liposomes, 7.4 ± 0.2 for

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TABLE 1. Effects of Antioxidant and Preparation Method on the Size and pH of Empty Liposomes. Preparation method

Mean liposome diameter, nm PL

PL + E

pH PL

PL + E

LML

602 ± 20

229 ± 20

7.0 ± 0.1

6.8 ± 0.1

Ex

173 ± 10

182 ± 10

7.0 ± 0.1

6.8 ± 0.1

Ex + L

166 ± 10

169 ± 10

7.2 ± 0.1

7.3 ± 0.1

H

123 ± 10

179 ± 10

7.2 ± 0.1

7.2 ± 0.1

H+L

125 ± 10

115 ± 10

7.5 ± 0.1

7.3 ± 0.1

SON

197 ± 20

237 ± 20

7.3 ± 0.2

6.5 ± 0.2

SON + L

251 ± 20

249 ± 20

7.4 ± 0.2

7.2 ± 0.2

Notes. PL – empty liposomes; PL + E – empty liposomes with vitamin E; LML – large multilamellar liposomes; Ex – preparation of liposomes by extrusion; Ex + L preparation of liposomes by extrusion followed by lyophilization; H – preparation of liposomes by homogenization; H + L – preparation of liposomes by homogenization followed by lyophilization; SON – preparation of liposomes by sonication; SON + L – preparation of liposomes by sonication followed by lyophilization.

empty liposomes after lyophilization, 6.5 ± 0.2 for empty liposomes with vitamin E, and 7.2 ± 0.2 for empty liposomes with vitamin E after lyophilization. Assessment of these three methods of preparing liposomes (homogenization, extrusion, sonication) in terms of MDA concentrations, OI levels, pH, and mean diameters of liposomes identify a significant decrease in the quality of

liposomal membrane PL regardless of the presence of AO in sonication (increases in MDA, OI, pH). Lyophilization of vesicles preparing by sonication significantly increased LPO. The best results in terms of these indicators were obtained with extruded vesicles, though the use of extrusion for the industrial-scale manufacture is not economically viable, so homogenization is recommended for the preparation of large volumes, as this maintains adequate LMF quality. REFERENCES 1. Liposome Technology, G. Gregoriadis (ed.) Informa Healthcare USA, New York (2007), Vol. 1, pp. 285 – 295. 2. B. Mishra, B. B. Patel, and S. Tiwari, Nanomedicine: Nanotechnol. Biol. Med., 6, 9 – 24 (2010). 3. Yu. M. Krasnopol’skii, A. E. Stepanov, and V. I. Shvets, Khim.-Farm. Zh., 33(10), 20 – 23 (1999). 4. V. V. Chudinova, S. M. Alekseev, E. I. Zakharova, et al., Bioorgan. Khim., 10(20), 1029 – 1046 (1994). 5. M. C. Dobarganes and J. Velasco, Eur. J. Lipid Sci. Technol., 104, 420 – 428 (2002). 6. I. A. Vasilenko, A. V. Viktorov, R. P. Evstigneeva, et al., Bioorgan. Khim., 9(8), 1281 – 1284 (1982). 7. M. Antolovich, P. D. Prenzler, E. Patsalides, et al., Analyst, 127, 183 – 198 (2002). 8. D. Jardine, M. Antolovich, P. D. Prenzler, and K. Robards, J. Agric. Food Chem., 50, 1720 – 1724 (2002). 9. I. A. Vasilenko, Yu. Krasnopol’skii, A. E. Stepanova, et al., Khim.-Farm. Zh., 32(5), 9 – 15 (1998). 10. M. Laguerre, P. Lecomte, and P. Villeneuve, Progress Lipid Res., 46, 244 – 282 (2007).