Introduction
Liposomes are phospholipid vesicles composed of concentric phospholipid bilayers that enclose to create an aqueous space [1] (Figure 1). The phospholipid tails are made of two hydrophobic long fatty acid chains that aggregate together to minimize interactions with water molecules, i.e. through hydrophobic interactions. Liposomes are of important interest in the field of drug delivery because they are non-toxic, biocompatible, and biodegradable. Most of all, they can deliver drugs by having them inserted into the hydrophobic part of the bilayer or aqueous interior of the liposomes [2]. In addition, liposomes can protect drugs from being degraded by enzymes and filtered out by the kidneys [2].
Figure 1. Structure of a liposome and possible modifications to deliver drugs to specific cells.
To deliver liposomes to a specific type of cell, liposomes are modified with an antibody, ligand [2], or other proteins or peptides as shown in Figure 1. Once the modified liposomes are recognized by the specific receptors on target cells, they are taken up by the cell in a process called receptor-mediated endocytosis (Figure 2), hence delivering the drug molecules into the cytoplasm [3, 4]. For example, folate (a ligand) and its binding receptor, folate binding protein (FBP), are often used to study drug delivery strategies in cancer research because the FBP is over-expressed in a broad range of human cancers such as ovarian, brain, kidney, and lung [3, 4].
Fig. 2. Receptor-mediated endocytosis of ligand-modified liposomes. The first liposome is enlarged to show the ligands clearly. Receptors for the ligand are seen on a typical cell membrane. Upon ligand binding to the receptor, the cell membrane closes in around the liposome to form a vesicle, trapping the drug-carrying liposome inside it, which would then be further processed inside the cell to release the drug into the cytoplasm.
Poly(ethylene glycol) (PEG) is a well known substance that is commonly used to prevent surface adsorption of proteins [5]. For liposome applications, PEG is often added to extend the circulation time of liposomes in the human body because without it, the unmodified liposomes would be removed within a few hours by phagocytes involved with immune response [2, 3]. This is because the presence of PEG prevents non-specific adsorption by phagocytes, for example [3]. However, the downside to modifying liposomes with PEG is that it may decrease the ability of the ligand on the liposome to bind to its specific receptor due to steric hindrance. Thus, there must be a compromise between extending the liposome circulation time and maximizing the delivery efficacy of the liposomes to target cells [2].
Liposomes have been immobilized on surface plasmon resonance (SPR) sensing surfaces to study drug and liposome interactions [6] and as an amplification strategy to decrease the limits of detection of interferon and bacterial toxins, for instance [7]. The purpose of the following experiment is to demonstrate that the addition of PEG 2000 to folate-modified liposomes would lead to decreased binding to FBP that are immobilized on the SPR gold sensing surface of Affinité's P4SPR™ (Figure 3). SPR is a powerful tool to characterize modified liposomes in real-time and in a label-free fashion. Moreover, having a SPR instrument such as the P4SPR directly in a research laboratory allows one to perform experimental optimizations on the fly instead of making multiple trips to a centralized facility.
Figure 3. The detection scheme of PEGylated and unPEGylated folate-modified liposomes using Affinité's P4SPR™
Experimental Procedures
The SPR measurements were performed using Affinité's P4SPR under static conditions. To immobilize the folate receptor, the 16-MHA-coated gold prism was activated through the injection of 500 uL of EDC:NHS 20:5 mM in MilliQ water and reacted for 20 minutes. The surface was rinsed throughout with the injection of 1 mL of 100 mM phosphate buffer (KH2PO4/K2HPO4, pH 5). Then, 300 uL of 40 nM folate binding protein (FBP) in a buffer composed of 100 mM phosphate buffer (pH 5), 4 mM mercaptoethanol, and 10% v/v glycerol, were injected and reacted for 1 h. Following FBP immobilization, the surface was rinsed with 1 mL of 100 mM phosphate buffer to assess the adsorbed FBP on the biosensor surface. 1 mL of 1 M ethanolamine was injected and reacted for 5 minutes to deactivate unreacted NHS groups. The sensor was rinsed with 1 mL of 1 x PBS (pH 7.4), and 500 uL of liposome samples were injected. All liposome suspensions were at a concentration of 10 uM, and was immobilized for a period of 10 minutes. The samples injected are listed Table 1, and they differ only in the addition of DOPE-PEG 2000-NH2 in the second sample. Between the immobilizations of the liposomes, the surface was rinsed sequentially with 1 mL 1 x PBS (pH 7.4), and 1 mL of 10 mM glycine (pH 1.5) for a period of 5 minutes, and finally rinsed with 1 x PBS (pH 7.4).
Table 1
Results
A set of preliminary results are shown in Figure 4, where the sensorgrams of each sample are displayed. First of all, the data show that the P4SPR was capable of detecting liposomes via the folate and FBP binding affinity as shown by the two association curves from ~60 s to ~750 s (green) and ~60 s to ~800 s (black). In addition, one can see that the PEGylated, folate-modified liposome sample (green) had a lower relative change in resonance units (~40 RU) compared to the unPEGylated folate-modified liposome sample (~110 RU, black). This demonstrates that the presence of PEG on the liposome surface inhibited some binding of the folate-modified liposomes to the FBP-modified sensor surface due to steric hindrance, i.e. PEG blocked folate on the liposomes from binding to FBP immobilized on the sensor surface.
Figure 4. A section of the sensorgram displaying the resonance units obtained by each liposome sample.
This experiment took about 3 hours to complete, from surface activation and folate modification of the sensor surface to the final injections of the liposome samples. The total SPR run time could have been shorter because there were some sample concentration optimizations in between the initial and final injections. The fact that a researcher can change experimental conditions at any moment is an advantage of having a SPR instrument such as the P4SPR in one’s laboratory. Finally, even though injection spikes were observed and other conditions such as surface chemistry and liposome concentrations can be optimized, the experiment can be repeated and further developed on the P4SPR at any time.
Conclusions
Affinité's P4SPR was able to detect folate-modified liposomes and more importantly, distinguishing the differences between PEG and unPEGylated folate-modified liposomes due to the steric hindrance caused by PEG, thus reducing the affinity of folate to FBP. Affinité's P4SPR can be used as a standard platform for researchers to characterize liposomes in a quick and simple manner prior to being further tested in bioassays or even in animals to obtain pharmacokinetic profiles [8], hence preventing the loss of precious research time, resources, and animals. Most importantly, having the P4SPR within reach would accelerate the research development of liposomes because there would not be a need to access a centralized SPR instrument (please see blog on Advantages of a Portable SPR Instrument).
Acknowledgements
We would like to thank Dr. Félix Lussier for providing the sensorgram data. He is from the Department of Cellular Biophysics at the Max Planck Institute for Medical Research.
Affinité Instruments P4SPR™
Affinité Instruments’ P4SPR is an excellent SPR instrument that can provide high quality, real-time data to suit your research needs. It requires no labels or secondary reactions and reduces a significant amount of precious research time compared to performing an ELISA assay. It can also detect low affinity interactions with a higher sensitivity than ELISA due to real-time monitoring.
We help life science labs and biotech companies to do rapid assay development and characterization. Feel free to reach out to us about the expertise we offer at info@affiniteinstruments.com
Thank you for reading and watch out for additional blogs discussing SPR related topics such as wavelength vs. angle interrogation, planar SPR vs. nanoparticle localized SPR, and multiple channel SPR.
References
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[3] Alberto Gabizon, Aviva T. Horowitz, Dorit Goren, Dina Tzemach, Hilary Shmeeda, and Samuel Zalipsky, “In Vivo Fate of Folate-Targeted Polyethylene-Glycol Liposomes in Tumor-Bearing Mice”, Clin. Cancer Res., 9, 6551–6559 (2003).
[4] Mary Jo Turk, David J. Waters, Philip S. Low, “Folate-conjugated liposomes preferentially target macrophages associated with ovarian carcinoma”, Cancer Lett., 213, 165-172 (2004).
[5] Roger Michel, Stephanie Pasche, Marcus Textor, and David G. Castner, ”The Influence of PEG Architecture on Protein Adsorption and Conformation”, Langmuir, 21, 12327-12332 (2005).
[6] Cheryl L. Baird, Elizabeth S. Courtenay, and David G. Myszka, “Surface plasmon resonance characterization of drug/liposome interactions”, Anal. Biochem., 310, 93-99 (2002).
[7] Agustina Gomez-Hens, Juan Manuel Fernandez-Romero, “The role of liposomes in analytical processes”, Trends Analyt. Chem., 24, 9-19 (2005).
[8] B.J. Crielaard, A. Yousefi, J.P. Schillemans, C. Vermehren, K. Buyens, K. Braeckmans, T. Lammers, G. Storm, “An in vitro assay based on surface plasmon resonance to predict the in vivo circulation kinetics of liposomes”, J. Control. Release, 156, 307-314 (2011).
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