Phase Evaporation - Springer Link

Preparation of Drug Liposomes by ReversePhase Evaporation Nian-Qiu Shi and Xian-Rong Qi

Contents 1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Preparation of Liposomes by Reverse-Phase Evaporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 The Determination of Encapsulation Efficiency (%) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Observation of Particle Size and Electron Micrograph . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4 Measurement of Surface Charge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5 Diffusional Exchange Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abstract

Liposomes display increased therapies for a series of biomedical application by stabilizing loaded payloads, overcoming shortcomings to cellular and tissue uptake, and enhancing biodistribution of payloads to target sites in vivo. The Bangham method or thin lipid film hydration technology was the first described method for constructing liposomes. The drawbacks of this method involve sonicator contact with the liposomes, and risk of high-temperature exposure may lead to phospholipid/drug damage and deceased encapsulation. A generally adopted preparative alternative is the reverse-phase evaporation technique that N.-Q. Shi (*) Department of Pharmaceutics, School of Pharmacy, Jilin Medical University, Jilin, Jilin Province, China e-mail: [email protected] X.-R. Qi Department of Pharmaceutics, School of Pharmaceutical Science, Peking University, Beijing, China e-mail: [email protected] # Springer-Verlag GmbH Germany 2018 W.-L. Lu, X.-R. Qi (eds.), Liposome-Based Drug Delivery Systems, Biomaterial Engineering, https://doi.org/10.1007/978-3-662-49231-4_3-1

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N.-Q. Shi and X.-R. Qi

tends to form inverted micelles or water-in-oil emulsions. The water phase carries the drug, and organic phase is made up of the lipids for forming the liposome bilayer. The lipid mixture is dissolved in solvents, and the lipid solvents are evaporated. Formed lipid film after evaporation is redissolved in an organic phase. Under reduced pressure condition, the organic solvent can be slowly evaporated, initially resulting in the conversion of the dispersion into a viscous gel and finally generating an aqueous suspension containing the liposomes. Similar to other preparation methods, the size of liposome generated by reverse-phase evaporation technique needs to be reduced by multiple extrusions through a polycarbonate membrane. The number of extrusion cycles and the size of polycarbonate membrane pore influence the degree of size reduction and the final particle size and distribution. This paper summarized the preparation procedure and formation principle of various payload-loaded liposomes by reversephase evaporation technique. Keywords

Liposomes · Reverse- phase evaporation · Encapsulation efficiency · Sizes · Electron micrograph

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Introduction

Liposomes were described first in the 1960s by Bangham et al. (1965) and have been used to be the most common and well-investigated nanocarriers for targeted drug delivery (Xiang et al. 2013; Zhao et al. 2011; Yang et al. 2014). They display increased therapies for a series of biomedical application by stabilizing loaded payloads, overcoming shortcomings to cellular and tissue uptake, and enhancing biodistribution of payloads to target sites in vivo (Bozzuto and Molinari 2015; Koning and Storm 2003; Metselaar and Storm 2005). Over five decades of researches in the field of liposomes have suggested their promise in the medical and cosmetic as well as the food industry (Mu and Sprando 2010; Mozafari et al. 2008; Madni et al. 2014). Liposomes consist of phospholipids, which self-assemble to form sphere-shaped vesicles of lipid bilayer and an aqueous core within the bilayers (Qi et al. 1995a, b, 1997). Liposomes seem to be an almost ideal carrier, because their structure is similar to that of cellular membrane and they can incorporate many substances. Hydrophilic as well as lipophilic compounds could be encapsulated into an aqueous core and a lipid bilayer of liposomes through hydrogen bonding, van der Waals forces, and other electrostatic interactions (Israelachvili et al. 1980; Lasic 1998). Many advantages of liposomes have been displayed including solubility enhancement of the encapsulated drugs, prevention of chemical and biological degradation, reduction of the nonspecific side effects and toxicity, versatile modification with various functional moiety, and improved biocompatibility in vivo (Madni et al. 2014; Qi et al. 1995a, b). These merits of liposomes have resulted in many

Preparation of Drug Liposomes by Reverse-Phase Evaporation

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successful basic and clinical applications. Currently approved commercial liposomal drug products include Myocet ®, Lipodox ®, DaunoXome ®, AmBisome ®, DepoDur ®, Inflexal V ®, etc. for the therapy of various cancers, fungal infections, pain, and influenza. More and more liposome-based formulations are being tested in phase I, II, or III stages (Pattni et al. 2015; Sercombe et al. 2015). The Bangham method or thin lipid film hydration technology was the first described method for constructing liposomes. The drawbacks of this method involve sonicator contact with the liposomes, and risk of high-temperature exposure may lead to phospholipid/drug damage and deceased encapsulation (Mozafari 2005). A generally adopted preparative alternative is the reverse-phase evaporation technique that tends to form inverted micelles or water-in-oil emulsions. The water phase carries the drug, and organic phase is made up of the lipids for forming the liposome bilayer. Briefly, the lipid mixture is dissolved in solvents, and the lipid solvents are evaporated. Formed lipid film after evaporation is redissolved in an organic phase comprised of diethyl ether or isopropyl ether. A two-phase system may generate after the addition of the aqueous phase through forming a homogeneous dispersion by sonication. Under reduced pressure condition, the organic solvent can be slowly evaporated, initially resulting in the conversion of the dispersion into a viscous gel and finally generating an aqueous suspension containing the liposomes. Compared with the thin film hydration method, this method causes a higher internal aqueous loading. The residual solvent can be removed by centrifugation, dialysis, or passage via a Sepharose gel column. The disadvantage is trace remaining organic solvent that can probably interrupt the chemical or biological stability of lipid or loaded drugs/genes. Similar to other preparation methods, the size of liposome generated by reverse-phase evaporation technique needs to be reduced by multiple extrusions through a polycarbonate membrane. The number of extrusion cycles and the size of polycarbonate membrane pore influence the degree of size reduction and the final particle size and distribution (Szoka and Papahadjopoulos 1978; Elorza et al. 1993; Cortesi et al. 1999; Qi et al. 1995c).

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Materials

2.1

Chemicals

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Cholesterol Palmitic acid Phosphatidylcholine (PtdCho) Phosphatidylglycerol (PtdGro) Phosphatidylserine (PtdSer) Phosphatidic acid Dipalmitoyl phosphatidylcholine (Pal2PtdCho) Sphingomyelin (bovine brain) [3H] poly(A) Ferritin

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11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.


Albumin Rabbit Ig G Porcine insulin Alkaline phosphatase Ribonuclease (RNase) 125 I-labeled sucrose 22 Na-labeled sucrose 14 C-labeled sucrose [3H]Cytosine arabinoside (araC) 25S[32P]RNA 50-ml round-bottom flask Nitrogen Ether Chloroform Methanol Phosphate-buffered saline Isopropyl ether 0.1% Triton X-100 Tirfluorotrichloroethane

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Methods

3.1

Preparation of Liposomes by Reverse-Phase Evaporation

1. Add lipid mixtures (several phospholipids, cholesterol, and long-chain alcohols) to a 50-ml round-bottom flask with a long extension neck. 2. Remove the solvent under reduced pressure by a rotary evaporator. 3. Purges nitrogen into the system for protecting the lipid mixtures from degradation. 4. Redissolve lipids in the organic phase such as diethyl ether, isopropyl ether, halothane, and trifluorotrichloroethane. 5. Increase the solubility of lipids also using ether, chloroform, or methanol. 6. Add the aqueous phase and keep the system continuously under nitrogen. 7. Sonicate the resulting two-phase system briefly (2–5 min) in a bath-type sonicator until the mixture becomes either a clear one-phase dispersion or a homogeneous opalescent dispersion. 8. Place the mixture on the rotary evaporator, and remove the organic solvent under reduced pressure at 20–25 C, rotating at approximately 200 rpm. 9. A viscous gel forms and an aqueous suspension appears. 10. Add excess water or buffer, and evaporate the suspension for an additional 15 min at 20 C to remove traces of solvent. 11. Dialyze the preparation, and pass through a Sepharose 4B column, or centrifuged.


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Table 1 Effect of lipid composition on encapsulation of araC and sucrose in liposomes prepared through reverse-phase evaporation method Lipid composition 1. PtdGro/PtdCho/Chol (1:4:5) 2. PtdGro/PtdCho (1:4) 3. PtdGro/PtdCho (1:4) 4. SA/PtdCho (1:4) 5. PtdGro 6. PtdGro 7. Pal2PtdCho 8. PtdGro/PtdCho/Chol (1:4:5) MLV 9. PtdGro/PtdCho/Chol (1:4:5) SUV

3.2

Captured volumn, μl/mg 13.7 9.2 8.1 15.6 10.5 8.7 11.7 4.1 0.5

% encapsulation ara C Sucrose 55.0 3.9 64.6 24.2 0.5 30.1 42.8 0.6 ND 59.7 2.7 63.0 27.7 2.4 18.7 46.7 2.7 ND 28.9 2.7 35.5 16.5 ND 1.8 ND

The Determination of Encapsulation Efficiency (%)

1. Dialyze the vesicles overnight against 300 vol of phosphate-buffered saline at 4 C to determine the amount of encapsulated small smalls such as sodium, sucrose, or [3H] araC (as shown in Table 1, Figs. 1 and 2). 2. Apply column chromatography on Sepharose 4B (1.5 42 cm) to separate encapsulated iodinated proteins from unencapsulated proteins. 3. Use centrifugation (1000 g for 30 min) to separate encapsulated [3H] poly (A) from unencapsulated materials. 4. Separate encapsulated 25S[32P]RNA from unencapsulated materials by first treating with RNase (5 μg) and alkaline phosphatase (10 μg) and separating the encapsulated materials from the hydrolyzed RNA on a Sepharose column (Table 2). 5. Degrade the unencapsulated RNA totally by above procedure. Establish the latency of alkaline phosphatase by measuring enzyme activity in the presence and absence of 0.1% Triton X-100.

3.3

Observation of Particle Size and Electron Micrograph

1. Observe electron micrographs picture of liposomes through negative stain method (as shown in Fig. 1). 2. After diluting liposomes to an appropriate volume with water, determine the particle size of liposomes using a dynamic light scattering instrument (Table 3).

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Fig. 1 (a) Freeze-fracture electron micrograph of PtdGro/PtdCho/cholesterol (1/4/5) liposomes containing ferritin prepared by reverse-phase evaporation technique. Caliper indicated 100 nm. (b–d) Negative-stain electron micrographs of liposomes prepared by the standard procedure: (b) PtdGro/PtdCho/cholesterol (1/4/5), a typical field of an unfiltered preparation; (c) PtdGro/PtdCho/ cholesterol (1/4/5), a typical field of preparation filtered through a 0.2-μm Unipore filter; (d) PtdGro/PtdCho (1/4), a typical field of an unfiltered preparation. Bar indicates 200 nm

3.4

Measurement of Surface Charge

1. Measure the electrophoretic mobility of phospholipid particles in 130 mM KCl containing Tris-HC1 (15 mM, pH 7.4). 2. Convert the electrophoretic mobility to ξ-potential using the formula: ξ = 12.9 V/ Eapp, where V is the electrophoretic mobility in μ.sec 1 and Eapp is the applied voltage in V-cm 1 and ξ in mV.

3.5

Diffusional Exchange Measurements

1. Separate bulk-phase tracer ions from those contained in the lipid particles either by dialysis or by Sephadex filtration. 2. For direct dialysis, pipette 0.5-ml aliquots of a dispersion, each containing 2–10 μmol of phosphorus into dialysis bags (0. 9 6 cm, previously rinsed with the


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Fig. 2 Diagram of the formation of liposomes prepared by reverse-phase evaporation technique. Dissolved lipids in appropriate solvents, lipids indicated by lollipop structures; addition of aqueous phase containing compound to be encapsulated, indicated by filled square Table 2 Encapsulation of various molecules in PtdGro/PtdCho/cholesterol (1:4:5) liposomes prepared through reverse-phase evaporation method Encapsulation materials Sodium Carboxyfluorescein Poly(A) Poly(A) 25S RNA Insulin Ferritin Alkaline phaosphatase Albumin IgG

Buffer PBS 1/10 PBS PBS 1/10 PBS 1/10 PBS PBS 1/10 PBS PBS PBS 1/10 PBS

% encapsulation 42 57 24 43 40 34 54 34 38 28–40

exchange medium) and dialyze them against 5–30-min changes of 500 ml of the exchange fluid which was the same as the dispersing medium but without tracer. 3. Accomplish more rapid removal of the bulk tracer (5 min) by the passage of the dispersion (1–2 ml) through a column of Sephadex (3 g, G-50, coarse grade), packed in the “cold” exchange medium.

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Table 3 Summary of size, encapsulation efficiency, and internal volume of different liposome preparations

Liposomes Liposomes prepared by reverse evaporation method (PtdGro/PtdCho/Chol, 1:4:5) Liposomes prepared by reverse evaporation method (PtdGro/PtdCho, 1:4) Multilamellar liposomes (PtdGro/PtdCho/ Chol, 1:4:5) Sonicated unilamellar liposomes (PtdGro/ PtdCho/Chol, 1:4:5) Large unilamellar liposomes (PtdSer)

Encapsulation efficiency 35–65

Captured volumn, μl/ mg 13.7

Diameter, nm (range) 200–1000

30–45

8.1

100–300

5–15

4.1

400–3500

0.5

20–50

9.1

200–1000

0.5–1 5–15

4. Wash the column with the exchange medium and collect the main lipid peak (usually 5 ml, between 13 and 17 ml of eluted volume). 5. Pipette aliquots (0.5 ml) into rinsed dialysis bags. 6. Transfer dialysis bags into duplicate stoppered tubes each containing 10 ml of the exchange medium after removal of bulk-phase tracer. Rotate the tubes gently at a speed of a rev./min. 7. Determine the tracer ions in each 10-ml dialysate, and those remaining inside the bags, were d with the appropriate counting equipment. 8. Take the amount of tracer appearing in the dialysate during the first hour after the removal of unincorporated ions to represent the diffusion rate constant, for a concentration difference of 145 mM at zero time. 9. Self-diffusion rate is expressed both as mequiv of ions/mole of lipid per h and as a percentage of the ions present inside the particles at zero time. The total amount of ions present at zero time, referred to as capture, is expressed as equiv/mole of lipid. 10. Estimate the amount of lipid present in each dialysis tube by phosphorus analysis. 11. Calculate activation energies from the Arrhenius equation by plotting the natural log of self-diffusion rates vs. the reciprocal of absolute temperature.

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Notes

1. All above lipids need to be purified on silicic acid column to ensure the quality of lipids. The purity may be verified by thin-layer chromatography. 2. To avoid the degradation or damage of lipids, all mentioned lipids should be stored in chloroform in sealed ampules under nitrogen at 50 C until use. 3. Redistill diethyl ether led from sodium bisulfite immediately before use to eliminate any peroxides for some preparations.


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4. Keep the stabilization of the lipids using the nitrogen protection in the preparation of Rev. 5. Bath-type sonicator, instead of probe-type sonicator, is suitable to prepare reverse evaporation liposomes because metal contamination probably appears through sonicating by metal probe. 6. The sonication process will result in the increase of temperature. Thus, control the temperature at 0~5 C. 7. Change the determination methods of encapsulation efficiency (%) for various encapsulated substances. It is necessary to ensure the removal or degradation of unencapsulated substances. 8. Reverse-phase evaporation method results in the formation of “inverted micelles” that are collapsed into a viscous gel-like state when the organic phase is removed by evaporation. The diagram of the formation of liposomes by reverse-phase evaporation is shown in Fig. 2. 9. The introduction of cholesterol in the liposome preparations will cause the higher encapsulation efficiency generally. 10. Using reverse-phase evaporation method, a substantial fraction of the aqueous phase is up to 65% in liposomes. This preparative method of liposomes has unique advantage for encapsulating valuable water-soluble materials such as drugs, proteins, nucleic acids, and other biochemical reagents.

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Conclusion

Among the methods for preparing liposomes, reverse-phase evaporation is a typical and representative approach. The lipid mixtures are dispersed and dissolved in solvents, and the lipid solvents are evaporated. Yield lipid film after evaporation is redissolved in an organic phase. By reduced pressure method, the organic solvent can be slowly removed, initially leading to the conversion of the dispersion into a viscous gel and finally forming an aqueous suspension containing the liposomes. A substantial fraction of the aqueous phase (up to 65% at low salt concentrations) is entrapped within the vesicles, encapsulating even large macromolecular assemblies with high efficiency. Thus, this relatively simple technique has unique advantages for encapsulating valuable water-soluble materials such as drugs, proteins, nucleic acids, and other biochemical reagents. The preparation and properties of the liposomes are described.

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