Cell entry is one of the common prerequisites for nanomaterial applications. Despite extensive studies on a homogeneous group of nanoparticles (NPs), fewer studies have been performed when two or more types of NPs were coadministrated. We previously described a synergistic cell entry process for two heterogeneous groups of NPs, where NPs functionalized with TAT (transactivator of transcription) peptide (T-NPs) stimulate the cellular uptake of coadministered unfunctionalized NPs (bystander NPs, B-NPs). Here, we show that the synergistic cell entry of NPs is driven by free energy decline and depends on B-NP sizes. Simulations showed that when separately placed initially, two NPs first move toward each other instead of initiating cell entry individually. Only T-NP invokes an inward bending of membrane mimicking endocytosis, which attracts the nearby NPs into the same “vesicle”. A two-phase free energy decline of the entire system occurred as two NPs get closer until contact, which is likely the thermodynamic driver for synergistic NP coentry. Experimentally, we found that T-NPs increase the apparent affinity of B-NPs to plasma membrane, suggesting that T-NPs help B-NPs “trapped” in the endocytic vesicles. Next, we varied the sizes of B-NPs and found that bystander activity peaks around 50 nm. Simulations also showed that the size of B-NPs influences the free energy decline, and thus the tendency and dynamics of NP coentry. These efforts provide a system to further understand the synergistic cell entry among individual NPs or multiple NP types on a biophysical basis and shed light on the future design of nanostructures for intracellular delivery.

Liposomes have been widely used as a drug delivery vector. One way to further improve its therapeutic efficacy is to increase the cell entry efficiency. Covalent conjugation with cell-penetrating peptides (CPPs) and other types of ligands has been the mainstream strategy to tackle this issue. Although efficient, it requires additional chemical modifications on liposomes, which is undesirable for clinical translation. Our previous study showed that the transportan (TP) peptide, an amphiphilic CPP, was able to increase the cellular uptake of co-administered, but not covalently coupled, metallic nanoparticles (NPs). Termed bystander uptake, this process represents a simpler method to increase the cell entry of NPs without chemical modifications. Here, we extended our efforts to liposomes. Our results showed that co-administration with the TP peptide improved the internalization of liposome into a variety of cell lines in vitro. This effect was also observed in primary cells, ex vivo tumor slices, and in vivo tumor tissues. On the other hand, this peptide-assisted liposome internalization did not apply to cationic CPPs, which were the main inducers for bystander uptake in previous studies. We also found that TP-assisted bystander uptake of liposome is receptor dependent, and its activity is more sensitive to the inhibitors of the macropinocytosis pathway, underlining the potential cell entry mechanism. Overall, our study provides a simple strategy based on TP co-administration to increase the cell entry of liposomes, which may open up new avenues to apply TP peptides in nanotherapeutics.

While nanoparticles (NPs) can be useful to improve the diagnosis and treatment of various diseases including cancer, a major limiting factor for their applications is the poor efficiency of cell entry. A common solution to this problem is to covalently link NPs with cell-penetrating peptides (CPPs), such as the transactivator of transcription (TAT) peptide. A few cationic CPPs were previously shown to increase the cellular uptake of co-administered, but not covalently coupled, NP cargo through an endocytic pathway, macropinocytosis. This so-called bystander uptake process is of unique advantage for NP intracellular delivery as it bypasses the requirements of additional chemical modifications. Here, we set out to determine whether other classes of CPPs (e.g. hydrophobic and amphiphilic) exhibit similar bystander activities, and what physico-chemical properties of NPs affect the bystander uptake process. First, we discovered that transportan (TP) peptide, an amphiphilic CPP, can initiate similar bystander uptake process for a variety of NPs and solute tracers. TP-induced bystander uptake relies on macropinocytosis as well, and occurs in a variety of cell types and in physiological tissues. Second, using TAT-functionalized NPs to stimulate the bystander effect, we showed that the bystander activity depends on the size of bystander NPs, with an optimal range around 50 nm in diameter. Compared to rod-like and triangular NPs, spherical NPs showed higher uptake in the bystander manner. Additionally, we investigated how CPP-NPs, once inside cells, get out of one cell and enter another, and found again that NP sizes affect the efficiency of CPP-NP intercellular transport as well. Overall, these efforts lay the foundation to further understand the bystander activity of CPP-assisted cell entry and improve the efficacy of nanomedicine in cancer and other human diseases. Citation Format: Yue-Xuan Li, Yushuang Wei, Hong-Bo Pang. Improving the nanomaterial delivery by using cell-penetrating peptides in the bystander manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 369.

Nanomaterials often need to interact with proteins on the plasma membrane to get cross and access their intracellular targets. Therefore, to fully understand the cell entry mechanism, it is of vital importance to gain a comprehensive insight into the proteome at the interface when nanomaterials encounter the cells. However, only very few studies have focused on this aspect. Here, we reported a peroxidase-based proximity labeling method to survey the proteome at the nanoparticle (NP)-cell interface. Horseradish peroxidase (HRP) was conjugated to a variety of NPs and other ligand types while still being able to biotinylate the proteins surrounding NP (or ligand)-receptor complexes. Using two NP-based tracers for macropinocytosis (MP), which is highly relevant to NP internalization, we performed a proteomic survey and revealed the interface proteome difference between traditional and receptor-dependent MP. Moreover, our survey found that E-cadherin (CDH1), while not serving as the primary receptor, is present at the NP-cell interface and is functionally important for the cellular uptake of a wide variety of NPs. Overall, by integrating nanotechnology with proximity labeling, our study provides an approach to map the proteome of NP-cell interface for investigating the molecular mechanism of NP and macromolecule internalization into cells.