Have cell membrane-encapsulated NPs been misunderstood?

Negative staining is often used to study the characteristic structure of the cell membrane core (CM) shell of coated polymeric nanoparticles (NPs) via transmission electron microscopy (TEM). However, negative staining can lead to the development of artifacts that present difficulties in detecting the actual NP structure.

Stady: Correct determination of the basic shell structure of cell membrane-coated polymeric nanoparticles. Image Credit: Vink Fan / Shutterstock.com

Recently, scientists analyzed several polymeric core nanoparticles using the fluorescence quenching method and showed that some of the observed crust core structures were in fact artifacts created by the staining process. This study is available at chemistry european journal.

The role of biomimetic nanoparticles

Biomimetic nanoparticles containing functional CM coatings are effectively used in treatments as they provide desirable biological properties. Although many NPs, such as liposomes, iron oxide, gold, porous silica, and metal-organic frameworks (MOFs), have been screened and treated with CM, poly(lactic-co-glycolic acid) (PLGA) has received approval from the US Administration of US Food and Drug Administration (FDA) for the production of NPs coated with CM.

Some of the distinguishing features of PLGA are superior biodegradability, excellent drug loading capacity, and great biocompatibility.

PLGA NPs (polymer nanoparticles) have been applied in many therapies, such as anisotropic red blood-coated PLGA NPs coated with CM for detoxification, platelet membrane-coated PLGA NPs designed to improve cancer immunotherapy, and CM-camouflaged PLGA NPs to target inflammation.

Negative staining and development of basic nanostructures of the structure

When CM and NPs core materials are exposed to ultrasonic energy or other immobilizing forces, they instantly transform into an integrated core-shell nanostructure. This concept emerged from TEM images of negatively stained CM-coated PLGA (CM-PLGA) NPs.

Negative staining is associated with the use of an aqueous solution of electron-disperse heavy metal salts, such as phosphotungstic acid and uranyl acetate. These salts are deposited on the dried sample, which enhances its visualization. The negative stress method helps to preserve the sample by forming the sample mold. However, some negative staining methods (eg negative air drying staining) tend to develop artifacts that are often misinterpreted during TEM image analysis.

Accurate detection of CM-PLGA NPs

Typically, techniques such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), dynamic light scattering (DLS), and fluorescent co-localization are used to detect the presence of CMs in PLGA NPs. The scientists recently sought to analyze whether the polymeric NPs were truly coated with CM, as indicated by previous TEM images with negative staining.

In-depth research revealed that the core nanostructure of the shell, visualized in TEM analysis of negatively stained polymeric NPs, commonly used as the basis for CM coating, for example, poly(caprolactone) (PCL), PLGA, and block-methyl-ether-PLGA (PEG) -PLGA), is an artifact created by the colorization technique.

Co-fusion of CM and PLGA NPs was demonstrated using mouse colon cancer cells. Confocal laser scanning microscopy images confirmed the formation of CM-PLGA NPs. To determine the structure of CM-PLGA NPs, a fluorescent quenching technique was used along with TEM analysis combined with Triton X-100 (TX-100) treatment.

The scientists compared TEM images of PLGA NPs with and without negative strain to accurately determine the CM coating. Interestingly, the uranyl acetate spot revealed that most of the PLGA NPs possessed a core shell structure. To ascertain the original state of PLGA NPs, which is usually misinterpreted by the TEM imaging technique, cryogenic TEM (cryo-TEM) was used, as this method is generally considered to be free of artifacts.

Interestingly, cryo-TEM images revealed a solid structure of PLGA NPs, indicating that the putative core coat structure of the TEM image of negatively stained PLGA NPs could be an artifact. This artifact can be created during the coloring and drying process. Atomic force microscopy (AFM) images of PLGA NPs showed a smooth surface and spherical shape, which was consistent with field emission scanning electron microscopy (FE-SEM) images.

Interestingly, the core-shell artifact was observed to use both uranyl acetate and a phosphotung acid negative stain, indicating that core-shell artifacts were formed regardless of the type of negative stain used. Changes in staining timing also did not affect the thickness of the artifacts.


Taken together, most polymeric NPs, including PLGA, tend to erroneously appear as a core envelope structure in TEM analysis of negatively stained samples. This study confirmed that the core-shell structure of CM-PLGA NPs identified in several TEM images of negatively stained samples are in fact artifacts generated by native PLGA NPs staining and not due to CM coating.


Liu, L.; and others. (2022) Correct determination of the core structure of the cortex of polymeric nanoparticles coated with a cell membrane. chemistry european journal. https://doi.org/10.1002/chem.202200947

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