Soft materials such as micelles and liposomes hold promise in the pharmaceutical sector as drug delivery vehicles. As the interest in accurate drug delivery is increasing, these microemulsions need to be examined in a liquid environment such as in a living system.
However, since they have a fragile structure and are vulnerable to damage, using a transmission electron microscope (TEM) to image liposome delivery vehicles can be a challenge.
To address this issue, researchers at the Virginia Tech and Sandia National Laboratory have used Protochips' Poseidon system for TEM to carry out in situ imaging of functional liposome nanoparticles. 1- palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) liposomes were imaged by Khalid Hattar’s group at Sandia National Laboratory in Albuquerque, NM and this shown in Figure 1A.
The POPC liposomes were observed as they passed through the holder tip and examined the impact of surface treatment and lipid additives on liposome structure. Pegylated liposomal control vehicles were successfully imaged under low dose conditions by Deborah Kelly’s group at the Virginia Tech Carilion Research Institute in Roanoke, VA and this is shown in Figure 1B.
Figure 1A. POPC lipids imaged under continuous flow at 200KV acceleration voltage
Figure 1B. Pegylated liposomes imaged in static liquid conditions using a 120KV acceleration voltage
The article describes the experiment of TEM imaging of functional liposomes in liquid with the help of the Poseidon system.
The experimental setup includes a pair of Poseidon E-chips with amorphous silicon nitride windows of 50nm thickness.
The bottom and top E-chips were mounted in the Poseidon holder tip and secured with a lid and three brass screws in order to create a hermetic seal to secure the hydrated sample from the microscope vacuum.
The experiment also involved the use of an external syringe pump to ensure continuous flow of the liposome solution via microfluidic tubing that is attached through the holder shaft to the tip.
Preparation of POPC Liposomes in Liquid
Two 10nm polycarbonate pore filters were used to extrude POPC, obtained from Avanti Polar Lipids to prepare POPC liposome. To make the E-chips hydrophilic, they were immersed in an alcian blue stain solution prior to the assembly of sample chamber.
The sample was made by diluting POPC liposomes such that the solution flows at a rate of 100µL/h into the holder tip. The sample imaging is performed using a JEOL 2100 TEM operating at 200kV. With continuous delivery of the fresh sample at the holder tip, the images were captured in real time at a frame rate of 12fps having an exposure time of 500ms for 2h.
Preparation of Pegylated Liposome Suspensions
The sample was prepared from pegylated liposomes purchased from Avanti Polar Lipids having a nominal diameter of 80 to 90nm. It was initially diluted with Milli-Q water to achieve ~0.3mg/ml solution.
Following this, 0.5µL of sample was fed on to the E-chip at 150nm static spacer thickness. The samples were imaged using an FEI Spirit Biotwin operating at 120kV under low dose conditions.Imaging Structural Effects of Lipid Additives to POPC Liposomes.
The structural effects of lipid additives in POPC liposomes were investigated by spiking the sample with distearylglycero triethylglycyl iminodiacetic acid (DSIDA). The study of the impact of lipid additives on the vesicle structure was carried out by passing 10% DSIDA/POPC liposomes solution via the holder over alcian blue-treated E-chips, at a rate of 100µL/h.
The DSIDA/POPC liposomes were imaged under the same TEM conditions as the POPC liposomes.
Effect of Bovine Serum Albumin on POPC Liposome Stability
The E-chips surfaces were passivated using a Bovine Serum Albumin (BSA) solution in deionized water via the holder for an hour to investigate the effect of BSA on the POPC liposome stability.
This procedure was carried out prior to the feeding of liposomes into the chamber. Further, the passage of POPC liposome solution into the imaging chamber at 100µL/h enabled the capture of TEM images.
The TEM images with altered, non-circular, liposome-like structures are shown in the Figure 2A. It was evident from the results that the average size of DSIDA/POPC liposomes was 183nm, showing lesser stability than that of large-sized POPC liposome.
The entering of the liposomes into the liquid chamber between the E-chips, caused them to be attracted to the positively charged E-chip surface and immobilized upon contact. Hence liposomes introduced early during the flow may not have reached the chamber area containing the transparent E-chip windows. The liposomes average diameter measured in TEM was 497 nm. The large diameter was due to liposomes combining together during the experiment.
By preparing liposomes comprising a mixture of POPC and DSIDA lipids, structural changes resulting due to change in the liposome composition were studied. DSIDA are highly rigid POPC, and may not form curved structures easily. It was observed that at room temperature, the DSIDA lipids separated from the POPC, forming DSIDA enriched domains within the liposome, causing high rigidity localized regions.
Figure 2A shows the altered, non-circular, liposome-like structures visible in the TEM images. The DSIDA/POPC liposome average size was 183nm, showing that the DSIDA/POPC liposomes exhibited lesser stability at larger sizes than the POPC liposomes.
By passivating with BSA, the impact of surface chemistry on the stability and structure of the liposomes was studied. The denatured liposome material shown in the Figure 2B reveals that the structures are too small to produce liposomes with stability.
Highly concentrated samples resulted to the crowding of the liquid chamber containing liposomes as shown in Figure 2C. As a result, the structure of individual liposomes could be identified easily.
The TEM images of pegylated liposomes using Poseidon is shown in Figure 1B. From the figure, a heterogeneous mixture of liposomes having a diameter of 50 to 125nm was identified. The diameter of the liposomes was more or less near to the predicted average diameters of 80 to 90nm.
Multiple sequential images of liposomes present in the solution were recorded by a dose of 0.5 electrons per square Angstrom. All the images were observed with continuous blurring in liposomes' outer boundaries that demonstrate the susceptibility of the liposomes to the electron beam.
In order to obtain high-quality images, optimizing the liposome sample concentration was important. Highly concentrated samples resulted in the crowding of the liquid chamber with liposomes, and consequently individual liposome structures could not resolved appropriately, as shown in Figure 2C.
Figure 2. Effects of the liquid environment on liposomal structure and packing. (A) DSIDA/POPC liposomes imaged under continuous flow with a 200 KV acceleration voltage. DSIDA/POPC liposomes exhibit distorted structural features due to the increased bending rigidity of DSIDA. (B) Denatured POPC liposomes. The liposomes denatured on contact with the BSA treated window surface. (C) Closely packed liposomes. The high concentrations of liposomes in the fluid cell make it difficult to distinguish the structure of individual liposomes.
Micelle and liposome suspensions play a very important role in modern pharmaceutical applications. These nanomaterials are utilized in the clinic for encapsulating and transporting chemotherapeutics to specific targets within the body.
Further to their role in drug delivery, micelles are a model system for the study of cellular interactions and membrane behavior. Hence there is a need for studying these systems in a physiologically relevant, dynamic liquid environment.
The results prove the benefits of TEM imaging of functional liposomes in liquid with the help of the Poseidon system. They also prove the potential of high-resolution, real-time images of dynamic processes including formation, drug delivery, deterioration and interplay of liposomes.
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