Primary Human Breast Cancer-Associated Endothelial Cells Favor Interactions with Nanomedicines.

Cancer nanomedicines predominately rely on transport processes controlled by tumor-associated endothelial cells to deliver therapeutic and diagnostic payloads into solid tumors. While the dominant role of this class of endothelial cells for nanoparticle transport and tumor delivery is established in animal models, the translational potential in human cells needs exploration. Using primary human breast cancer as a model, the differential interactions of normal and tumor-associated endothelial cells with clinically relevant nanomedicine formulations are explored and quantified. Primary human breast cancer-associated endothelial cells exhibit up to ≈2 times higher nanoparticle uptake than normal human mammary microvascular endothelial cells. Super-resolution imaging studies reveal a significantly higher intracellular vesicle number for tumor-associated endothelial cells, indicating a substantial increase in cellular transport activities. RNA sequencing and gene expression analysis indicate the upregulation of transport-related genes, especially motor protein genes, in tumor-associated endothelial cells. Collectively, the results demonstrate that primary human breast cancer-associated endothelial cells exhibit enhanced interactions with nanomedicines, suggesting a potentially significant role for these cells in nanoparticle tumor delivery in human patients. Engineering nanoparticles that leverage the translational potential of tumor-associated endothelial cell-mediated transport into human solid tumors may lead to the development of safer and more effective clinical cancer nanomedicines.

Gold Nanoparticles Inhibit Macropinocytosis by Decreasing KRAS Activation.

The RAS-transformed cells utilize macropinocytosis to acquire amino acids to support their uncontrolled growth. However, targeting RAS to inhibit macropinocytosis remains a challenge. Here, we report that gold nanoparticles (GNP) inhibit macropinocytosis by decreasing KRAS activation. Using surface-modified and unmodified GNP, we showed that unmodified GNP specifically sequestered both wild-type and mutant KRAS and inhibited its activation, irrespective of growth factor stimulation, while surface-passivated GNP had no effect. Alteration of KRAS activation is reflected on downstream signaling cascades, macropinocytosis and tumor cell growth in vitro, and two independent preclinical human xenograft models of pancreatic cancer in vivo. The current study demonstrates NP-mediated inhibition of macropinocytosis and KRAS activation and provides translational opportunities to inhibit tumor growth in a number of cancers where activation of KRAS plays a major role.

Rapid Mitochondria Targeting by Arginine-Terminated, Sub-10 nm Nanoprobe via Direct Cell Membrane Penetration.

Although mitochondria have been identified as a potential therapeutic target for the treatment of various diseases, inefficient drug targeting to mitochondria is a major limitation for related therapeutic applications. In the current approach, drug loaded nanoscale carriers are used for mitochondria targeting via endocytic uptake. However, these approaches show poor therapeutic performance due to inefficient drug delivery to mitochondria. Here, we report a designed nanoprobe that can enter the cell via a nonendocytic approach and label mitochondria within 1 h. The designed nanoprobe is <10 nm in size and terminated with arginine/guanidinium that offers direct membrane penetration followed by mitochondria targeting. We found five specific criteria that need to be adjusted in a nanoscale material for mitochondria targeting via the nonendocytic approach. They include <10 nm size, functionalization with arginine/guanidinium, cationic surface charge, colloidal stability, and low cytotoxicity. The proposed design can be adapted for mitochondria delivery of drugs for efficient therapeutic performance.

Small-Molecule-Functionalized Hyperbranched Polyglycerol Dendrimers for Inhibiting Protein Aggregation.

Amyloid protein aggregation is responsible for a variety of neurodegenerative diseases, and antiamyloidogenic small molecules are identified for inhibiting such protein aggregation at extra-/intracellular space. We show that the nanoparticle form of small molecules offers better antiamyloidogenic performance via enhanced bioavailability and multivalent binding with protein. Here, we report hyperbranched polyglycerol dendrimers terminated with antiamyloidogenic small molecules such as gallate, tyrosine, and trehalose and their potential in inhibiting lysozyme/huntingtin protein aggregation under intra-/extracellular space. The synthesized functional dendrimers are ∼5 nm in size having an average molecular weight of ∼2000 Da, and they are highly biocompatible in nature. We found that functional dendrimers are efficient in micromolar doses with respect to molecular forms that are effective at millimolar concentration. It is observed that the trehalose-terminated dendrimer is more effective in inhibiting protein aggregation, whereas the gallate-terminated dendrimer is more effective in disintegrating mature protein fibrils. This approach can be used to design functional dendrimers as potential nanodrugs for the treatment of various neurodegenerative diseases.

Lipid-Raft-Mediated Direct Cytosolic Delivery of Polymer-Coated Soft Nanoparticles.

Polymer shelling around a nanoparticle is commonly employed for stabilization, surface chemistry, and bioconjugation. However, this shelling increases the overall size of the nanoparticle which limits many biomedical applications. Here we show that soft and nonionic polymer shelling can induce direct cytosolic delivery of a nanoparticle, as compared to clathrin-mediated uptake and lysozomal trafficking by a similarly sized nanoparticle with molecular shelling. Specifically, we have studied cellular internalization of two classes of colloidal nanoparticles of 10-50 nm hydrodynamic size. In one class, a 4-5 nm quantum dot is coated with a soft polyacrylate shell of varied thickness between 2 and 20 nm and in the other class a Au nanoparticle of varied size between 5 and 45 nm is coated with a molecular shell. We found that polymer shelling has two roles in controlling cellular internalization of nanoparticles. First, it increases the hydrodynamic size and controls the surface charge that influences the binding to the cell membrane, and 10 nm appears to be the minimum size requirement for such binding. Second, it increases softness that induces membrane penetration and directs cytosolic delivery of the nanoparticle. In particular, a soft and nonionic polymer shell induces lipid-raft-mediated direct cytosolic delivery, but a soft and cationic polymer shell induces clathrin-mediated endocytosis with lysozomal trafficking, like that of a nonionic molecular shell. The observed results can be used to design more effective nanoprobes for controlling intracellular processing.

Arginine-Terminated Nanoparticles of <10 nm Size for Direct Membrane Penetration and Protein Delivery for Straight Access to Cytosol and Nucleus.

Although colloidal nanoparticles are known to enter into cells via endocytosis, the direct membrane permeation of nanoparticles is rarely reported, and the underlying mechanism of direct membrane permeation is largely unsolved. However, a direct membrane-penetrating nanoparticle has great advantage as a delivery carrier that offers high delivery efficiency, faster delivery kinetics, and minimal lysosomal degradation. Here we show that arginine-terminated Au nanoparticles of <10 nm size enter via energy-independent direct membrane penetration, but as the size increases, the nanoparticles switch to energy-dependent endocytotic uptake. As a delivery carrier, <10 nm Au nanoparticles directly transport an electrostatically bound protein into the cytosol within a minute and allow direct access of the protein to subcellular compartments. This direct delivery approach has been used for efficient nuclear targeting of proteins and can be adapted for direct cytosolic delivery or subcellular targeting applications with high efficiency.

Surface Chemistry- and Intracellular Trafficking-Dependent Autophagy Induction by Iron Oxide Nanoparticles.

Autophagy is a cellular self-clearance process for maintaining regular cytoplasmic function, and modulation of autophagy can influence cytotoxicity, apoptosis, and clearance of toxic amyloid fibril. In a recent work, functional nanoparticles are used to modulate autophagy. However, the role of nanoparticle uptake mechanisms and their intracellular processing on autophagy is vaguely understood. Here, we show that autophagy is influenced by nanoparticle surface chemistry-directed intracellular trafficking and localization. In particular, we have designed iron oxide nanoparticles functionalized with arginine/arginine methyl ester/octyl/oleyl/cholesterol with a high cell uptake property. We found that autophagy is induced by octyl/oleyl functionalization without appreciable cell death. Further study shows that enhanced cytosolic delivery over membrane localization and increased intracellular aggregation over homogeneous cytosolic distribution lead to autophagy induction via intracellular reactive oxygen species generation. The observed result can be used to design functional nanoparticles/nanodrugs for modulating cellular autophagy that can be used in various biomedical applications.

Arginine-Terminated, Chemically Designed Nanoparticle for Direct Cell Translocation.

Direct cell translocation of nanomaterials is preferred over the endocytotic uptake for various subcellular targeting applications that can bypass the lysosomal trafficking/degradation. Although arginine-rich cell-penetrating peptides are routinely used for cell transfection of wider range materials from molecule to nanoparticle, the direct cell translocation of nanoparticle is not a routine approach, particularly because of the predominate endocytotic uptake. Here we report arginine-terminated, designed nanoparticle of 15-30 nm hydrodynamic size that enters into cell via direct translocation. We found that direct cell translocation of nanoparticle is very efficient without localization at any specific subcellular compartment for 12-24 h. This study shows that nanomaterial can be chemically designed for direct cell translocation and for cytosolic delivery without any biomembrane-coated endosome that can be employed for subcellular targeting applications.