Role of Curvature-Sensing Proteins in the Uptake of Nanoparticles with Different Mechanical Properties.

Nanoparticles of different properties, such as size, charge, and rigidity, are used for drug delivery. Upon interaction with the cell membrane, because of their curvature, nanoparticles can bend the lipid bilayer. Recent results show that cellular proteins capable of sensing membrane curvature are involved in nanoparticle uptake; however, no information is yet available on whether nanoparticle mechanical properties also affect their activity. Here liposomes and liposome-coated silica are used as a model system to compare uptake and cell behavior of two nanoparticles of similar size and charge, but different mechanical properties. High-sensitivity flow cytometry, cryo-TEM, and fluorescence correlation spectroscopy confirm lipid deposition on the silica. Atomic force microscopy is used to quantify the deformation of individual nanoparticles at increasing imaging forces, confirming that the two nanoparticles display distinct mechanical properties. Uptake studies in HeLa and A549 cells indicate that liposome uptake is higher than for the liposome-coated silica. RNA interference studies to silence their expression show that different curvature-sensing proteins are involved in the uptake of both nanoparticles in both cell types. These results confirm that curvature-sensing proteins have a role in nanoparticle uptake, which is not restricted to harder nanoparticles, but includes softer nanomaterials commonly used for nanomedicine applications.

Comparison of the uptake mechanisms of zwitterionic and negatively charged liposomes by HeLa cells.

Zwitterionic molecules are used as an alternative to PEGylation to reduce protein adsorption on nanocarriers. Nonetheless, little is known on the effect of zwitterionic modifications on the mechanisms cells use for nanocarrier uptake. In this study, the uptake mechanism of liposomes containing zwitterionic or negatively charged lipids was characterized using pharmacological inhibitors and RNA interference on HeLa cells to block endocytosis. As expected, introducing zwitterionic lipids reduced protein adsorption in serum, as well as uptake efficiency. Blocking clathrin-mediated endocytosis strongly decreased the uptake of the negatively charged liposomes, but not the zwitterionic ones. Additionally, inhibition of macropinocytosis reduced uptake of both liposomes, but blocking actin polymerization had effects only on the negatively charged ones. Overall, the results clearly indicated that the two liposomes were internalized by HeLa cells using different pathways. Thus, introducing zwitterionic lipids affects not only protein adsorption and uptake efficiency, but also the mechanisms of liposome uptake by cells.

Targeting tumor microenvironment with PEG-based amphiphilic nanoparticles to overcome chemoresistance.

Multidrug resistance is one of the biggest obstacles in the treatment of cancer. Recent research studies highlight that tumor microenvironment plays a predominant role in tumor cell proliferation, metastasis, and drug resistance. Hence, targeting the tumor microenvironment provides a novel strategy for the evolution of cancer nanomedicine. The blooming knowledge about the tumor microenvironment merging with the design of PEG-based amphiphilic nanoparticles can provide an effective and promising platform to address the multidrug resistant tumor cells. This review describes the characteristic features of tumor microenvironment and their targeting mechanisms with the aid of PEG-based amphiphilic nanoparticles for the development of newer drug delivery systems to overcome multidrug resistance in cancer cells. FROM THE CLINICAL EDITOR: Cancer is a leading cause of death worldwide. Many cancers develop multidrug resistance towards chemotherapeutic agents with time and strategies are urgently needed to combat against this. In this review article, the authors discuss the current capabilities of using nanomedicine to target the tumor microenvironments, which would provide new insight to the development of novel delivery systems for the future.

Tuning Liposome Stability in Biological Environments and Intracellular Drug Release Kinetics.

Ideal drug carriers should be stable in biological environments but eventually release their drug load once inside the targeted cells. These two aspects can be in contrast with each other, thus they need to be carefully tuned in order to achieve the desired properties for specific applications. Quantifying drug release profiles in biological environments or inside cells can be highly challenging, and standard methods to determine drug release kinetics in many cases cannot be applied to complex biological environments or cells. Within this context, the present work combined kinetic studies by flow cytometry with aging experiments in biological fluids and size-exclusion chromatography to determine drug release profiles in biological environments and inside cells. To this purpose, anionic and zwitterionic liposomes were used as model nanomedicines. By changing lipid composition, liposome stability in serum and intracellular release kinetics could be tuned and formulations with very different properties could be obtained. The methods presented can be used to characterize liposome release profiles in complex biological media, as well as inside cells. In this way, liposome composition can be tuned in order to achieve formulations with optimal balance between stability and release kinetics for specific applications.

Effects of Protein Source on Liposome Uptake by Cells: Corona Composition and Impact of the Excess Free Proteins.

Corona formation in biological fluids strongly affects nanomedicine interactions with cells. However, relatively less is known on additional effects from the free proteins in solution. Within this context, this study aims to gain a better understanding of nanomaterial-cell interactions in different biological fluids and, more specifically, to disentangle effects due to corona composition and those from the free proteins in solution. To this aim, the uptake of liposomes in medium with bovine and human serum are compared. Uptake efficiency in the two media differs strongly, as also corona composition. However, in contrast with similar studies on other nanomaterials, despite the very different corona, when the two corona-coated liposomes are exposed to cells in serum free medium, their uptake is comparable. Thus, in this case, the observed differences in uptake depend primarily on the presence and source of the free proteins. Similar results are obtained when testing the liposomes on different human cells, as well as in murine cells and in the presence of murine serum. Overall, these results show that the protein source affects nanomedicine uptake not only due to effects on corona composition, but also due to the presence and composition of the free proteins in solution.

Tuning liposome composition to modulate corona formation in human serum and cellular uptake.

Nano-sized objects such as liposomes are modified by adsorption of biomolecules in biological fluids. The resulting corona critically changes nanoparticle behavior at cellular level. A better control of corona composition could allow to modulate uptake by cells. Within this context, in this work, liposomes of different charge were prepared by mixing negatively charged and zwitterionic lipids to different ratios. The series obtained was used as a model system with tailored surface properties to modulate corona composition and determine the effects on liposome interactions with cells. Uptake efficiency and uptake kinetics of the different liposomes were determined by flow cytometry and fluorescence imaging. Particular care was taken in optimizing the methods to isolate the corona forming in human serum to prevent liposome agglomeration and to exclude residual free proteins, which could confuse the results. Thanks to the optimized methods, mass spectrometry of replicate corona isolations showed excellent reproducibility and this allowed semi-quantitative analysis to determine for each formulation the most abundant proteins in the corona. The results showed that by changing the fraction of zwitterionic and charged lipids in the bilayer, the amount and identity of the most abundant proteins adsorbed from serum differed. Interestingly, the formulations also showed very different uptake kinetics. Similar approaches can be used to tune lipid composition in a systematic way in order to obtain formulations with the desired corona and cell uptake behavior. STATEMENT OF SIGNIFICANCE: Liposomes and other nano-sized objects when introduced in biological fluids are known to adsorb biomolecules forming the so-called nanoparticle corona. This layer strongly affects the subsequent interactions of liposomes with cells. Here, by tuning lipid composition in a systematic way, a series of liposomes with tailored surface properties has been prepared to modulate the corona forming in human serum. Liposomes with very different cellular uptake kinetics have been obtained and their corona was identified in order to determine the most enriched proteins on the different formulations. By combining corona composition and uptake kinetics candidate corona proteins associated with reduced or increased uptake by cells can be identified and the liposome formulation can be tuned to obtain the desired uptake behavior.

Corona Composition Can Affect the Mechanisms Cells Use to Internalize Nanoparticles.

Nanosized objects, such as nanoparticles and other drug carriers used in nanomedicine, once in contact with biological environments are modified by adsorption of biomolecules on their surface. The presence of this corona strongly affects the following interactions at cell and organism levels. It has been shown that corona proteins can be recognized by cell receptors. However, it is not known whether the composition of this acquired layer can also affect the mechanisms nanoparticles use to enter cells. This is of particular importance when considering that the same nanoparticles can form different coronas for instance in vitro when exposed to cells in different serum amounts or in vivo depending on the exposure or administration route. Thus, in this work, different coronas were formed on 50 nm silica by exposing them to different serum concentrations. The uptake efficiency in HeLa cells was compared, and the uptake mechanisms were characterized using transport inhibitors and RNA interference. The results showed that the nanoparticles were internalized by cells via different mechanisms when different coronas were formed, and only for one corona condition was uptake mediated by the LDL receptor. This suggested that coronas of different composition can be recognized differently by cell receptors, and this in turn leads to internalization via different mechanisms. Similar studies were performed using other cells, including A549 cells and primary HUVEC, and different nanoparticles, namely 100 nm liposomes and 200 nm silica. Overall, the results confirmed that the corona composition can affect the mechanisms of nanoparticle uptake by cells.

Cell membrane tracker based on restriction of intramolecular rotation.

The fluorescence of tetraphenylethylene (TPE), an archetypal luminogen, is induced by restriction of intramolecular rotation (RIR). TPE was grafted with palmitic acid (PA) onto a hydrophilic peptide to yield a cell membrane tracker named TR4. TR4 was incorporated into liposomes, where it showed significant RIR characteristics. When cells were incubated with TR4, cytoplasmic membranes were specifically labeled. TR4 shows excellent photostability and low cytotoxicity.