Liposomes, Part F (eBook)

Nejat Duzgunes (Herausgeber)

eBook Download: PDF | EPUB

2009 | 1. Auflage
416 Seiten
Elsevier Science (Verlag)
978-0-08-096102-6 (ISBN)

Infos

Liposomes are cellular structures made up of lipid molecules, which are water insoluable organic molecules and the basis of biological membranes. Important as a cellular model in the study of basic biology, liposomes are also used in clinical applications such as drug delivery and virus studies. Liposomes Part ,F is a continuation of previous MIE Liposome volumes A through E.

* One of the most highly respected publications in the field of biochemistry since 1955 * Frequently consulted, and praised by researchers and reviewers alike * Truly an essential publication for anyone in any field of the life sciences

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Liposomes are cellular structures made up of lipid molecules, which are water insoluble organic molecules and the basis of biological membranes. Important as a cellular model in the study of basic biology, liposomes are also used in clinical applications such as drug delivery and virus studies. Liposomes Part F is a continuation of previous MIE Liposome volumes A through E. One of the most highly respected publications in the field of biochemistry since 1955 Frequently consulted and praised by researchers and reviewers alike Truly an essential publication for anyone in any field of the life sciences

Inhalt

Front Cover 
1 
Methods in Enzymology 
4 
Copyright 
5 
Contents 
6 
Contributors 
12 
Preface 
18 
Section 1: Bioactive Liposomes 
48 
Chapter 1: Tubular Liposomes with Variable Permeability for Reconstitution of FtsZ Rings 
50 
1. Introduction 
51 
2. Reagents 
51 
3. Bacterial Expression of Membrane Targeting FtsZ 
52 
4. Purification of FtsZ-mts and FtsZ-YFP-mts 
52 
5. Renatured Preparation of FtsZ-YFP-mts 
54 
6. Tubular Multilamellar Liposome Preparation 
54 
7. Permeability of the Multilamellar Liposomes 
56 
8. Z-ring Formation in Liposomes 
58 
9. A Crude Flow Chamber to Exchange Buffer Outside Liposomes 
58 
10. Factors Affecting Z-ring Formation in Liposomes 
61 
11. Utility of the Liposomes Beyond FtsZ 
63 
References 63
Chapter 2: Detection and Analysis of Protein Synthesis and RNA Replication in Giant Liposomes 
66 
1. Introduction 
67 
2. Methods 
68 
3. Analysis of the FACS Data 
74 
4. Conclusions 
75 
Acknowledgments 76
References 76
Chapter 3: Construction of Cell-Sized Liposomes Encapsulating Actin and Actin-Cross-linking Proteins 
78 
1. Introduction 
79 
2. Experimental Section 
84 
3. Morphogenesis of Giant Liposomes Encapsulating Actin and Its Cross-linking Proteins 
89 
4. Concluding Remarks 
96 
Acknowledgments 97
References 97
Chapter 4: Reconstitution of Membrane Budding with Unilamellar Vesicles 102
1. Introduction 
103 
2. M Protein Purification 
104 
3. Evaluation of the Membrane Activity of M Protein Through its Interaction with Intermediate-Sized Unilamellar Liposomes 
105 
4. Reconstitution of M-Protein-Driven Membrane Budding on GUVs 
113 
5. Concluding Remarks 
120 
References 120
Section 2: Liposomes and Nanotechnology 
124 
Chapter 5: Detection of Antimycolic Acid Antibodies by Liposomal Biosensors 126
1. Introduction 
127 
2. Experimental 
128 
3. Conclusion 
149 
Acknowledgments 149
References 149
Chapter 6: Solid Lipid Nanoparticle Formulations: Pharmacokinetic and Biopharmaceutical Aspects in Drug Delivery 152
1. Introduction 
153 
2. Production of SLN 
154 
3. Pharmacokinetics and Pharmacodynamics 
154 
4. Modified Release Profile 
159 
5. Biopharmaceutical Aspects of Administration Routes 
161 
6. Clinical Pharmacology 
164 
7. Concluding Remarks 
168 
References 169
Chapter 7: Preparation of Complexes of Liposomes with Gold Nanoparticles 178
1. Introduction 
179 
2. Preparation of Complexes of EYPC Liposomes with Au NPs 
181 
3. Time-Dependent SPR of the Complexes 
181 
4. TEM Analysis of the Complexes 
183 
5. DLS Analysis of the Complexes 
184 
6. Calcein Release from the Complexes 
184 
7. Estimation of Numbers of the Au NP and the Liposome in the Complexes 
186 
8. Optimization of Lipid Components of the Complexes 
187 
9. Concluding Remarks 
189 
Acknowledgment 
190 
References 191
Chapter 8: Bio-Nanocapsule-Liposome Conjugates for In Vivo Pinpoint Drug and Gene Delivery 
194 
1. Introduction 
195 
2. First-Generation Bio-Nanocapsules 
196 
3. Second-Generation BNCs 
197 
4. Retargeting of BNC-LP Conjugates 
199 
5. Overexpression of BNCs in S. cerevisiae 
200 
6. Conjugation of BNCs with LPs 
202 
7. Preparation of Antibody-Displaying BNC-LP Conjugates 
208 
8. Preparation of Biotin-Displaying BNC-LP Conjugates 
210 
9. Concluding Remarks 
210 
Acknowledgments 211
References 211
Chapter 9: Nanoliposomal Dry Powder Formulations 214
1. Introduction 
215 
2. Preparation of Nanoliposomal DPFs 
216 
3. Physicochemical Characterization of NLDPFs 
221 
4. Concluding Remarks 
234 
References 235
Chapter 10: Lanthanide-Loaded Paramagnetic Liposomes as Switchable Magnetically Oriented Nanovesicles 240
1. Introduction 
241 
2. Paramagnetic Ln(III)-Based Shift Reagents 
242 
3. Preparation of Osmotically Shrunken Liposomes 
244 
4. NMR Characterization of Magnetically Oriented Nonspherical Liposomes 
245 
5. Sample Experiments 
247 
6. Concluding Remarks 
255 
Acknowledgments 255
References 255
Chapter 11: Reconstitution of Membrane Proteins in Phospholipid Bilayer Nanodiscs 258
1. Introduction 
259 
2. Overview of Nanodisc Technology 
259 
3. Reconstitution Considerations 
265 
4. Optimizing the Reconstitution for P-glycoprotein 
270 
Acknowledgments 275
References 275
Chapter 12: DNA-Controlled Assembly of Liposomes in Diagnostics 280
1. Introduction 
281 
2. Probe Design 
282 
3. General Description of Materials and Techniques 
291 
4. Concluding Remarks 
294 
Acknowledgment 
294 
References 295
Chapter 13: Soft Hybrid Nanostructures Composed of Phospholipid Liposomes Decorated with Oligonucleotides 
296 
1. Introduction 
297 
2. Materials 
298 
3. Liposome Preparation and Determination of Lipid Content 
299 
4. Incorporation of Oligonucleotides 
300 
5. Characterization of the Soft Hybrid Nanostructure 
303 
6. Applications of Oligo-Decorated Liposomes 
309 
7. Challenges and Perspectives 
322 
Acknowledgments 323
References 323
Chapter 14: Synthesis, Characterization, and Optical Response of Gold Nanoshells Used to Trigger Release from Liposomes 326
1. Introduction 
327 
2. Synthesis of HGNs 
330 
3. Optimization of HGN Dimensions for Maximum Absorption in the NIR 
333 
4. HGN Response to Femtosecond NIR Laser Pulses 
337 
5. Coupling HGN to Liposomes 
341 
6. Liposome Disruption and CF Release Due to Pulsed Laser Irradiation 
346 
7. Mechanism of Triggered Liposome Release 
347 
8. Effect of Proximity of HGNs to Liposomes 
350 
9. Conclusions 
351 
Acknowledgments 351
References 351
Chapter 15: Complex Nanotube-Liposome Networks 356
1. Introduction 
356 
2. Network Fabrication Protocols 
357 
3. Complexity and Topology 
361 
4. Internal and Membrane Functionalization 
362 
5. Transport Phenomena and Controlled Mixing Procedures 
365 
6. Enzymatic Reactions in NVN 
367 
7. Concluding Remarks 
370 
Acknowledgments 370
References 371
Chapter 16: Bionanotubules Formed from Liposomes 374
1. Introduction 
375 
2. Bionanotubule Formation by Applying Electric Fields to Surface-Attached Liposomes 
376 
3. Bionanotubule Formation from Liposomes in Solution Using Electric Fields 
381 
4. Other Methods of Bionanotubule Formation from Liposomes 
384 
5. Concluding Remarks 
386 
References 386
Chapter 17: Engineering Cationic Liposome: siRNA Complexes for In Vitro and In Vivo Delivery 
390 
1. Introduction 
391 
2. Cationic Liposome Systems for siRNA Delivery 
392 
3. Experimental Methods 
394 
4. Troubleshooting 
399 
5. Concluding Remarks 
400 
References 401
Author Index 
402 
Subject Index 
410 
Color Plates 
418 

Textauszug

Chapter One

Tubular Liposomes with Variable Permeability for Reconstitution of FtsZ Rings

Masaki Osawa; Harold P. Erickson    Department of Cell Biology, Duke University Medical Center, Durham, North Carolina, USA

Abstract

We have developed a system for producing tubular multilamellar liposomes that incorporate the protein FtsZ on the inside. We start with a mixture of spherical multilamellar liposomes with FtsZ initially on the outside. Shearing forces generated by applying a coverslip most likely distort some of the spherical liposomes into a tubular shape, and causes some to leak and incorporate FtsZ inside. We describe protocols for liposome preparation, and for preparing membrane-targeted FtsZ that can assemble contractile Z rings inside the tubular liposomes. We also describe the characterization of the multilamellar liposomes in terms of the permeability or leakiness for a small fluorescent dye and larger protein molecules. These liposomes may be useful for reconstitution of other biological systems.

1 INTRODUCTION

FtsZ is a bacterial tubulin homologue that forms a ring structure called the “Z ring” at the division plane in bacteria. The Z ring is anchored to the membrane and constricts to divide the bacteria. FtsZ recruits a dozen other essential division proteins, which are mostly involved in remodeling the peptidoglycan cell wall. We recently succeeded in reconstituting Z rings inside tubular liposomes, and found that they generated a constriction force on the liposome wall (Osawa et al., 2008). The assembly of Z rings and the generation of the constriction force were achieved with FtsZ alone, and did not require any other division protein. This was an important discovery itself for understanding the mechanism of bacterial cell division. Now the liposome system we developed should provide a simple in vitro system for studying molecular details of how FtsZ works.

To achieve these results we had to overcome two technical problems. The first problem was to tether FtsZ to the membrane. Pichoff and Lutkenhaus (2005) discovered that the carboxy terminus of FtsZ binds to FtsA, and FtsA has an amphipathic helix at its carboxy terminal that inserts into the membrane. We made an FtsZ that could tether itself to the membrane by fusing an amphipathic helix (membrane targeting sequence: mts) to the carboxy terminus of FtsZ. To visualize the protein, we inserted a yellow fluorescent protein (YFP) before the mts, giving FtsZ-YFP-mts. Here we provide detailed protocols for the purification of FtsZ-YFP-mts.

The second problem was how to get the protein inside liposomes. We have succeeded in getting FtsZ-YFP-mts inside spherical unilamellar liposomes using the emulsion method (Noireaux and Libchaber, 2004; Pautot et al., 2003). However, we have never found Z rings assembled in such spherical unilamellar liposomes.

Eventually, we discovered a procedure that produced tubular multilamellar liposomes, and incorporated FtsZ-YFP-mts inside, where it formed Z rings. Initially this was a fortunate accident, since the cylindrical geometry was not designed, and the FtsZ was initially on the outside. We have since refined the procedures for producing tubular multilamellar liposomes, and we now understand some aspects of the permeability or leakiness that lets FtsZ inside. We describe here our protocols for producing the tubular liposomes and the tests of permeability.

2 REAGENTS

The following reagents are used in our experiments:

• Column buffer: 50 mM Tris/HCl, pH 7.9, 50 mM KCl, 1 mM EDTA, 10% (v/v) glycerol

• HMKCG buffer: 50 mM HEPES/KOH, pH 7.7, 5 mM MgAc, 300 mM KAc, 50 mM KCl 10% (v/v) glycerol

• HMK50-350 buffer: 50 mM HEPES/KOH, pH 7.7, 5 mM MgAc, 50–350 mM KAc

• DOPG:1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (Avanti)

• Egg PC: phosphatidylcholine (Avanti)

• HccA: 7-Hydroxycoumarin-3-carboxylic acid (Invitrogen)

• Teflon disc, 37 mm diameter

3 BACTERIAL EXPRESSION OF MEMBRANE TARGETING FTSZ

The FtsZ-YFP-mts is expressed from a pET-11b expression vector, with FtsZ366-YFP-mts or FtsZ366-mts genes inserted at NdeI/BamHI sites (366 indicates that the FtsZ was truncated there, removing the FtsA-binding C-terminal peptide). The YFP we use is the variety Venus (Nagai et al., 2002), which gave superior results in FtsZ fusions in E. coli (Osawa and Erickson, 2005). The mts used here is the amphipathic helix from E. coli MinD (Szeto et al., 2003). We have not yet tested the amphipathic helix from FtsA, which has three to five additional extra amino acids that extend the amphipathic helix (Pichoff and Lutkenhaus, 2005). The expression vector is transformed into E. coli strain C41 (Miroux and Walker, 1996), which gives better yields of soluble proteins than BL21.

After transforming, colonies are selected on an LB (Luria broth) agar plate containing 100 μg/ml ampicillin. A colony is picked and cultured overnight in 50 ml LB media with 100 μg/ml ampicillin at 37 °C.

Five milliliters of the overnight culture is diluted in 500 ml LB and cultured at 37 °C until the optical density at 600 nm reaches 0.8–1.0. Protein expression is induced by addition of 0.5 mM IPTG and at the same time the temperature of the shaker is set to 20 °C (our shaker takes 1–2 h to reach 20 °C).

The cells are cultured overnight and spun down at 3750 rpm for 45 min in a Beckman GPR rotor.

4 PURIFICATION OF FTSZ-MTS AND FTSZ-YFP-MTS

Since FtsZ-mts and FtsZ-YFP-mts are expressed as soluble proteins, we purify them using the same protocol as for wild-type FtsZ.

The packed cells are resuspended in a final volume of 20 ml column buffer, and 1 mM phenylmethanesulphonylfluoride (PMSF) and 0.1–0.2 mg/ml lysozyme are added. The mixture is then incubated on a rotator at 4 °C for 15 min. They are frozen at 80 °C overnight or longer.

Two cycles of freeze–thaw (fresh 1 mM PMSF is added after each thawing) method are performed. The resultant mixture is sonicated on ice until the viscosity is reduced. We usually sonicate it for three cycles of 20 s, with 1 min cooling intervals.

It is then centrifuged at 32,000 rpm for 20 min at 4 °C (Beckman 42.1 Ti rotor). The supernatant is collected and ammonium sulfate is added to 30% saturation (3.52 g dry ammonium sulfate to the 20 ml volume). This mixture is incubated for 20 min on ice and again centrifuged at 32,000 rpm for 20 min at 4 °C (Beckman 42.1 Ti rotor). The supernatant is discarded and the pellet is resuspended in 10 ml column buffer and passed through a 0.22 μm filter.

The protein is purified on an anion exchange column. A 1 × 10 cm Source Q column (Source 15Q, GE Healthcare) is used. The column is eluted with a 100 ml gradient from 50–500 mM KCl in column buffer.

4.1 For FtsZ-mts

FtsZ has very low UV absorbance, so the peak is located by running each fraction on SDS–PAGE.

The peak fractions are pooled and dialyzed into HMK350.

The protein concentration is determined by the BCA method (Pierce). FtsZ produces 75% as much color as BSA (Lu et al., 1998), so it is necessary to correct for this.

Aliquots are frozen and stored at − 80 °C.

4.2 For FtsZ-YFP-mts

After elution from the Source 15Q column, the peak fractions are pooled. There are typically two peaks: a large main peak and a following small peak, and both peaks have an indistinguishable activity. These peaks can be identified by yellow fluorescence and confirmed by SDS–PAGE.

They are concentrated using an Amicon Ultra-15 with centrifugation at 5000×g.

We have noted that incomplete boiling of FtsZ-YFP-MTS with SDS sample buffer generates two bands on the gel. The upper band (68 Kd) results from completely denatured protein and the lower band (60 Kd), which still has yellow fluorescence in the gel, is due to FtsZ-YFP-mts where the YFP is not denatured.

The concentration of FtsZ-YFP-mts can be determined from its absorption at 515 nm, using the extinction coefficient for YFP-Venus 92,200 M− 1 cm− 1.

Our preferred buffer for FtsZ-YFP-mts is now HMKCG because FtsZ-YFP-mts seems to be more stable, as described below; we now use HMKCG for dialysis, dilution, reaction, and storage buffer.

5 RENATURED PREPARATION OF FTSZ-YFP-MTS

In our previous study (Osawa et al., 2008), we used a renaturing technique to prepare FtsZ366-YFP-mts. We developed this protocol because an early preparation...

Produkt-Details

• Erscheint lt. Verlag: 6.11.2009
• Sprache: englisch
• Themenwelt: Medizin / Pharmazie
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
• Themenwelt: Naturwissenschaften ► Biologie ► Zellbiologie
• Themenwelt: Naturwissenschaften ► Physik / Astronomie ► Angewandte Physik
• Themenwelt: Technik
• ISBN-10: 0-08-096102-9 / 0080961029
• ISBN-13: 978-0-08-096102-6 / 9780080961026