Chemically Designed Nanoscale Materials for Controlling Cellular Processes.

Nanoparticles are widely used in various biomedical applications as drug delivery carriers, imaging probes, single-molecule tracking/detection probes, artificial chaperones for inhibiting protein aggregation, and photodynamic therapy materials. One key parameter of these applications is the ability of the nanoparticles to enter into the cell cytoplasm, target different subcellular compartments, and control intracellular processes. This is particularly the case because nanoparticles are designed to interact with subcellular components for the required biomedical performance. However, cells are protected from their surroundings by the cell membrane, which exerts strict control over entry of foreign materials. Thus, nanoparticles need to be designed appropriately so that they can readily cross the cell membrane, target subcellular compartments, and control intracellular processes.In the past few decades there have been great advancements in understanding the principles of cellular uptake of foreign materials. In particular, it has been shown that internalization of foreign materials (small molecules, macromolecules, nanoparticles) is size-dependent: endocytotic uptake of materials requires sizes greater than 10 nm, and materials with sizes of 10-100 nm usually enter into cells by energy-dependent endocytosis via biomembrane-coated vesicles. Direct access to the cytosol is limited to very specific conditions, and endosomal escape of material appears to be the most practical approach for intracellular processing.In this Account, we describe how cellular uptake and intracellular processing of nanoscale materials can be controlled by appropriate design of size and surface chemistry. We first describe the cell membrane structure and principles of cellular uptake of foreign materials followed by their subcellular trafficking. Next, we discuss the designed surface chemistry of a 5-50 nm particle that offers preferential lipid-raft/caveolae-mediated endocytosis over clathrin-mediated endocytosis with minimum endosomal/lysosomal trafficking or energy-independent direct cell membrane translocation (without endocytosis) followed by cytosolic delivery without endosomal/lysosomal trafficking. In particular, we emphasize that the zwitterionic-lipophilic surface property of a nanoparticle offers preferential interaction with the lipid raft region of the cell membrane followed by lipid raft uptake, whereas a lower number of affinity biomolecules (<25) on the nanoparticle surface offers caveolae/lipid-raft uptake, while an arginine/guanidinium-terminated surface along with a size of <10 nm offers direct cell membrane translocation. Finally, we discuss how nanoprobes can be designed by adapting these surface chemistry and size preference principles so that they can readily enter into the cell, label different subcellular compartments, and control intracellular processes such as trafficking kinetics, exocytosis, autophagy, amyloid aggregation, and clearance of toxic amyloid aggregates. The Account ends with a Conclusions and Outlook where we discuss a vision for the development of subcellular targeting nanodrugs and imaging nanoprobes by adapting to these surface chemistry principles.

Spontaneous formation and growth kinetics of lipid nanotubules induced by passive nanoparticles.

Lipid nanotubules (LNTs) are conduits that form on the membranes of cells and organelles, and they are ubiquitous in all forms of life from archaea and bacteria to plants and mammals. The formation, shape and dynamics of these LNTs are critical for cellular functions, supporting the transport of myriad cellular cargoes as well as communication within and between cells, and they are also widely believed to be responsible for exploitation of host cells by pathogens for the spread of infection and diseases. In vitro kinetic control of LNT formation can considerably enhance the scope of utilization of these structures for disease control and therapy. Here we report a new paradigm for spontaneous lipid nanotubulation, capturing the dynamical regimes of growth, stabilization and retraction of the tubes through the binding of synthetic nanoparticles on supported lipid bilayers (SLBs). The tubulation is determined by the spontaneous binding-unbinding of nanoparticles on the LNTs. The presented methodology could be used to rectify malfunctioning cellular tubules or to prevent the pathogenic spread of diseases through inhibition of cell-to-cell nanotubule formation.

Compressibility of Multicomponent, Charged Model Biomembranes Tunes Permeation of Cationic Nanoparticles.

Cells respond to external stress by altering their membrane lipid composition to maintain fluidity, integrity and net charge. However, in interactions with charged nanoparticles (NPs), altering membrane charge could adversely affect its ability to transport ions across the cell membrane. Hence, it is important to understand possible pathways by which cells could alter zwitterionic lipid composition to respond to NPs without compromising membrane integrity and charge. Here, we report in situ synchrotron X-ray reflectivity (XR) measurements to monitor the interaction of cationic NPs in the form of quantum dots, with phase-separated supported lipid bilayers of different compositions containing an anionic lipid and zwitterionic lipids having variable degrees of stiffness. We observe that the extent of NP penetration into the respective membranes, as estimated from XR data analysis, is inversely related to membrane compression moduli, which was tuned by altering the stiffness of the zwitterionic lipid component. For a particular membrane composition with a discernible height difference between ordered and disordered phases, we were able to observe subtle correlations between the extent of charge on the NPs and the specificity to bind to the charged and ordered phase, contrary to that observed earlier for phase-separated model biomembranes containing no charged lipids. Our results provide microscopic insight into the role of membrane rigidity and electrostatics in determining membrane permeation. This can lead to great potential benefits in rational designing of NPs for bioimaging and drug delivery applications as well as in assessing and alleviating cytotoxicity of NPs.

Penetration and preferential binding of charged nanoparticles to mixed lipid monolayers: interplay of lipid packing and charge density.

Designing of nanoparticles (NPs) for biomedical applications or mitigating their cytotoxic effects requires microscopic understanding of their interactions with cell membranes. Such insight is best obtained by studying model biomembranes which, however, need to replicate actual cell membranes, especially their compositional heterogeneity and charge. In this work we have investigated the role of lipid charge density and packing of phase separated Langmuir monolayers in the penetration and phase specificity of charged quantum dot (QD) binding. Using an ordered and anionic charged lipid in combination with uncharged but variable stiffness lipids we demonstrate how the subtle interplay of zwitterionic lipid packing and anionic lipid charge density can affect cationic nanoparticle penetration and phase specific binding. Under identical subphase pH, the membrane with higher anionic charge density displays higher NP penetration. We also observe coalescence of charged lipid rafts floating amidst a more fluidic zwitterionic lipid matrix due to the phase specificity of QD binding. Our results suggest effective strategies which can be used to design NPs for diverse biomedical applications as well as to devise remedial actions against their harmful cytotoxic effects especially against respiratory diseases.

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.

Trehalose-Conjugated, Catechin-Loaded Polylactide Nanoparticles for Improved Neuroprotection against Intracellular Polyglutamine Aggregates.

Intracellular/extracellular protein aggregation is linked to a variety of neurodegenerative diseases. Current research focuses on identifying antiamyloidogenic small molecules to inhibit such protein aggregation and associated cytotoxicity. We have recently demonstrated that transforming these antiamyloidogenic small molecules into nanoparticle forms can greatly improve their performance, and biocompatible/biodegradable formulation of such nanoparticles is critical for therapeutic applications. Here, we report polylactide (PL)-based biodegradable nanoparticles for improved neuroprotection against polyglutamine (polyQ) aggregation that is responsible for Huntington's disease. PL is terminated with an antiamyloidogenic trehalose molecule or the neurotransmitter dopamine, and the resultant nanoparticle is loaded with the antiamyloidogenic catechin molecule. The self-assembled nanoparticle is ∼200 nm in size and enters into the neuronal cell, inhibits polyQ aggregation, lowers oxidative stress, and enhances cell proliferation against polyQ aggregates. This biodegradable polymer can be used in nanoformulation of other reported antiamyloidogenic molecules for testing various animal models of neurodegenerative diseases.

Nanoscale Heterogeneities Drive Enhanced Binding and Anomalous Diffusion of Nanoparticles in Model Biomembranes.

Interaction of functional nanoparticles with cells and model biomembranes has been widely studied to evaluate the effectiveness of the particles as potential drug delivery vehicles and bioimaging labels as well as in understanding nanoparticle cytotoxicity effects. Charged nanoparticles, in particular, with tunable surface charge have been found to be effective in targeting cellular membranes as well as the subcellular matrix. However, a microscopic understanding of the underlying physical principles that govern nanoparticle binding, uptake, or diffusion on cells is lacking. Here, we report the first experimental studies of nanoparticle diffusion on model biomembranes and correlate this to the existence of nanoscale dynamics and structural heterogeneities using super-resolution stimulated emission depletion (STED) microscopy. Using confocal and STED microscopy coupled with fluorescence correlation spectroscopy (FCS), we provide novel insight on why these nanoparticles show enhanced binding on two-component lipid bilayers as compared to single-component membranes and how binding and diffusion is correlated to subdiffraction nanoscale dynamics and structure. The enhanced binding is also dictated, in part, by the presence of structural and dynamic heterogeneity, as revealed by STED-FCS studies, which could potentially be used to understand enhanced nanoparticle binding in raft-like domains in cell membranes. In addition, we also observe a clear correlation between the enhanced nanoparticle diffusion on membranes and the extent of membrane penetration by the nanoparticles. Our results not only have a significant impact on our understanding of nanoparticle binding and uptake as well as diffusion in cell and biomembranes, but have very strong implications for uptake mechanisms and diffusion of other biomolecules, like proteins on cell membranes and their connections to functional membrane nanoscale platform.

Antiamyloidogenic Chemical/Biochemical-Based Designed Nanoparticle as Artificial Chaperone for Efficient Inhibition of Protein Aggregation.

Protein aggregation is linked to variety of neurodegenerative disorders and other diseases. Current research involves understanding the mechanism of protein aggregation, inhibiting protein aggregation under intra/extracellular space, lowering toxicity arising due to soluble oligomers, and augmenting the clearance of protein aggregates from the brain. Toward this direction, different types of antiamyloidogenic small molecules, macromolecules, and nanomaterials are identified that can inhibit protein aggregation, and extensive progress has been made for their effective utilization. Here, we summarize our effort in designing a nanoparticle form of antiamyloidogenic molecules with enhanced performance under in vitro and in vivo conditions. We found that the nanoparticle form of antiamyloidogenic molecules can perform up to 100,000-times better than the respective molecular form due to the combined effect of enhanced bioavailability at intra/extracellular space and multivalent binding property with aggregating protein. This work demonstrates that further research should be directed toward designing nanoparticle forms of antiamyloidogenic molecules for their effective performance.

Trehalose-Functionalized Gold Nanoparticle for Inhibiting Intracellular Protein Aggregation.

Trehalose is a well-known antiamyloidogenic molecule that inhibits protein aggregation under the intracellular/extracellular condition, and recent work shows that the nanoparticle form of trehalose can further enhance this performance. Here we have designed a trehalose-functionalized Au nanoparticle that can inhibit the aggregation of a polyglutamine-containing mutant protein inside the neuronal cell. Designed nanoparticles have a 20-30 nm Au core with about 350 ± 50 trehalose molecules per particle on the surface on average. They enter the cell, inhibit mutant protein aggregation, and enhance the cell survival against toxic protein aggregates. This work extends the application potential of trehalose for the understanding and treatment of different diseases involving protein aggregation.

Phase Transfer and Surface Functionalization of Hydrophobic Nanoparticle using Amphiphilic Poly(amino acid).

Functionalization of nanoparticles with chemical and biochemical is essential for their biomedical and other application. However, most of the high quality nanoparticles are hydrophobic in nature due to surfactant capping and their conversion into water-soluble functional nanoparticle via appropriate coating and conjugation chemistry is extremely critical issue. Here we report amphiphilic poly(amino acid)-based one-pot coating and conjugation approach that can transform hydrophobic nanoparticle into water-soluble nanoparticle functionalized with primary amine, thiol, and biomolecule. We have designed amphiphilic polyaspartimide that can anchor hydrophobic nanoparticle through octadecyl groups, leaving the polar polyethylene glycol and aspartimide groups exposed outwards. The aspartimide group is then reacted with primary amine containing chemical/biomolecule with the formation of water-soluble functional nanoparticle. This approach has been extended to different hydrophobic nanoparticles and biomolecules. The present approach has advantages over existing approaches as coating and functionalization can be performed in one pot and functional nanoparticles have <12 nm hydrodynamic size, high colloidal stability, and biocompartibility. This developed approach can be used to derive biocompatible nanobioconjugates for various biomedical applications.

Spontaneous unbinding transition of nanoparticles adsorbing onto biomembranes: interplay of electrostatics and crowding.

Cellular membranes are constantly bombarded with biomolecules and nanoscale particles, and cell functionality depends on the fraction of the bound/internalized entities. Understanding the biophysical parameters underlying this complex process is very difficult in live cells. Model membranes provide an ideal platform to obtain insight into the minimal and essential parameters involved in determining cell membrane-nanoparticle (NP) interaction. Here we report spontaneous binding and unbinding of semiconductor NPs, carrying different net charges and interacting with model biomembranes, using in situ neutron reflectivity (NR) and fluorescence microscopy studies. We observe a critical concentration of NPs above which they spontaneously unbind along with lipids from lipid monolayer membranes, leaving behind fewer bound NPs. This critical concentration varies depending on whether the NPs carry a net charge or are neutral, and is also governed by the extent of NP crowding for a fixed NP charge. The observations suggest a subtle interplay between electrostatics, membrane fluidity, and NP crowding effects, which eventually determines the adsorbed concentration for unbinding transition. Our study provides valuable microscopic insight into the parameters that could determine the biophysical process underlying NP uptake and ejection by cells which, in turn, can be utilized for their potential applications in bioimaging and drug delivery.

Harnessing extracellular vesicles-mediated signaling for enhanced bone regeneration: novel insights into scaffold design.

The increasing prevalence of bone replacements and complications associated with bone replacement procedures underscores the need for innovative tissue restoration approaches. Existing synthetic grafts cannot fully replicate bone vascularization and mechanical characteristics. This study introduces a novel strategy utilizing pectin, chitosan, and polyvinyl alcohol to create interpenetrating polymeric network (IPN) scaffolds incorporated with extracellular vesicles (EVs) isolated from human mesenchymal stem cells (hMSCs). We assess the osteointegration and osteoconduction abilities of these modelsin vitrousing hMSCs and MG-63 osteosarcoma cells. Additionally, we confirm exosome properties through Transmission Electron Microscopy (TEM), immunoblotting, and Dynamic Light Scattering (DLS).In vivo, chick allantoic membrane assay investigates vascularization characteristics. The study did not includein vivoanimal experiments. Our results demonstrate that the IPN scaffold is highly porous and interconnected, potentially suitable for bone implants. EVs, approximately 100 nm in size, enhance cell survival, proliferation, alkaline phosphatase activity, and the expression of osteogenic genes. EVs-mediated IPN scaffolds demonstrate promise as precise drug carriers, enabling customized treatments for bone-related conditions and regeneration efforts. Therefore, the EVs-mediated IPN scaffolds demonstrate promise as precise carriers for the transport of drugs, allowing for customized treatments for conditions connected to bone and efforts in regeneration.

Matrimeres are systemic nanoscale mediators of tissue integrity and function.

Tissue barriers must be rapidly restored after injury to promote regeneration. However, the mechanism behind this process is unclear, particularly in cases where the underlying extracellular matrix is still compromised. Here, we report the discovery of matrimeres as constitutive nanoscale mediators of tissue integrity and function. We define matrimeres as non-vesicular nanoparticles secreted by cells, distinguished by a primary composition comprising at least one matrix protein and DNA molecules serving as scaffolds. Mesenchymal stromal cells assemble matrimeres from fibronectin and DNA within acidic intracellular compartments. Drawing inspiration from this biological process, we have achieved the successful reconstitution of matrimeres without cells. This was accomplished by using purified matrix proteins, including fibronectin and vitronectin, and DNA molecules under optimal acidic pH conditions, guided by the heparin-binding domain and phosphate backbone, respectively. Plasma fibronectin matrimeres circulate in the blood at homeostasis but exhibit a 10-fold decrease during systemic inflammatory injury in vivo . Exogenous matrimeres rapidly restore vascular integrity by actively reannealing endothelial cells post-injury and remain persistent in the host tissue matrix. The scalable production of matrimeres holds promise as a biologically inspired platform for regenerative nanomedicine.

Extracellular vesicle-matrix interactions.

The extracellular matrix in microenvironments harbors a variety of signals to control cellular functions and the materiality of tissues. Most efforts to synthetically reconstitute the matrix by biomaterial design have focused on decoupling cell-secreted and polymer-based cues. Cells package molecules into nanoscale lipid membrane-bound extracellular vesicles and secrete them. Thus, extracellular vesicles inherently interact with the meshwork of the extracellular matrix. In this Review, we discuss various aspects of extracellular vesicle-matrix interactions. Cells receive feedback from the extracellular matrix and leverage intracellular processes to control the biogenesis of extracellular vesicles. Once secreted, various biomolecular and biophysical factors determine whether extracellular vesicles are locally incorporated into the matrix or transported out of the matrix to be taken up by other cells or deposited into tissues at a distal location. These insights can be utilized to develop engineered biomaterials where EV release and retention can be precisely controlled in host tissue to elicit various biological and therapeutic outcomes.

Direct Cellular Delivery of Exogenous Genetic Material and Protein via Colloidal Nano-Assemblies with Biopolymer.

Direct cytosolic delivery of large biomolecules that bypass the endocytic pathways is a promising strategy for therapeutic applications. Recent works have shown that small-molecule, nanoparticle, and polymer-based carriers can be designed for direct cytosolic delivery. It has been shown that the specific surface chemistry of the carrier, nanoscale assembly between the carrier and cargo molecule, good colloidal stability, and low surface charge of the nano-assembly are critical for non-endocytic uptake processes. Here we report a guanidinium-terminated polyaspartic acid micelle for direct cytosolic delivery of protein and DNA. The polymer delivers the protein/DNA directly to the cytosol by forming a nano-assembly, and it is observed that <200 nm size of colloidal assembly with near-zero surface charge is critical for efficient cytosolic delivery. This work shows the importance of size and colloidal property of the nano-assembly for carrier-based cytosolic delivery of large biomolecules.

Cell-Matrix Interactions Regulate Functional Extracellular Vesicle Secretion from Mesenchymal Stromal Cells.

Extracellular vesicles (EVs) are cell-secreted particles with broad potential to treat tissue injuries by delivering cargo to program target cells. However, improving the yield of functional EVs on a per cell basis remains challenging due to an incomplete understanding of how microenvironmental cues regulate EV secretion at the nanoscale. We show that mesenchymal stromal cells (MSCs) seeded on engineered hydrogels that mimic the elasticity of soft tissues with a lower integrin ligand density secrete ∼10-fold more EVs per cell than MSCs seeded on a rigid plastic substrate, without compromising their therapeutic activity or cargo to resolve acute lung injury in mice. Mechanistically, intracellular CD63+ multivesicular bodies (MVBs) transport faster within MSCs on softer hydrogels, leading to an increased frequency of MVB fusion with the plasma membrane to secrete more EVs. Actin-related protein 2/3 complex but not myosin-II limits MVB transport and EV secretion from MSCs on hydrogels. The results provide a rational basis for biomaterial design to improve EV secretion while maintaining their functionality.

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.

Quercetin Encapsulated Polymer Nanoparticle for Inhibiting Intracellular Polyglutamine Aggregation.

Quercetin is a dietary flavonoid that shows effective neuroprotective action in cellular and animal models of Alzheimer's disease and Huntington's disease. However, its therapeutic application is limited due to low water solubility and cytotoxicity at the working concentration in the 20-100 µM range. Here we report a nanoparticle form of quercetin (nanoquercetin) that shows antiamyloidogenic performance at lower quercetin concentration (one micromolar) and inhibits polyglutamine (mutant huntingtin) aggregation in Huntington's disease cell model. Nanoquercetin is composed of a polyaspartic acid-based polymer micelle encapsulated with quercetin (3-5 wt %) and colloidal in nature with <100 nm hydrodynamic size. It enters into the cell via endocytosis and slowly releases molecular quercetin in a >3 day time scale that offers better antiamyloidogenic performance via up-regulated autophagy processes. This work shows that nanoformulation of antiamyloidogenic molecule can have better performance as compared to respective molecule.

Designed Polymer Micelle for Clearing Amyloid Protein Aggregates via Up-Regulated Autophagy.

Inhibiting protein aggregation under intra-/extracellular space and clearing protein aggregates from the brain are two critical issues for the treatment of various neurodegenerative diseases. Although a variety of anti-amyloidogenic chemicals/biochemicals have been identified for inhibiting such protein aggregation, clearing protein aggregates is a challenging issue. Here we report a designed biopolymer micelle of 15-30 nm hydrodynamic size that can clear protein aggregates from cells via an up-regulated autophagy process. The polymer has a polyaspartic acid backbone and is functionalized with fatty amine, arginine, and primary amine for inducing self-assembly, enhancing cell uptake, and up-regulating autophagy processes, respectively. The polymer micelle (PM) enters into the cell via lipid raft endocytosis, is transported to the perinuclear region where the protein oligomer/aggregate predominantly localizes, clears aggregated protein from the cell, and enhances the cell's survival against toxic protein aggregates. The designed PM may be used as a drug delivery carrier for anti-amyloidogenic drugs for enhanced efficacy in the treatment of neurodegenerative diseases.

Poly(trehalose) Nanoparticles Prevent Amyloid Aggregation and Suppress Polyglutamine Aggregation in a Huntington's Disease Model Mouse.

Prevention and therapeutic strategies for various neurodegenerative diseases focus on inhibiting protein fibrillation, clearing aggregated protein plaques from the brain, and lowering protein-aggregate-induced toxicity. We have designed poly(trehalose) nanoparticles that can inhibit amyloid/polyglutamine aggregation under extra-/intracellular conditions, reduce such aggregation-derived cytotoxicity, and prevent polyglutamine aggregation in a Huntington's disease (HD) model mouse brain. The nanoparticles have a hydrodynamic size of 20-30 nm and are composed of a 6 nm iron oxide core and a zwitterionic polymer shell containing ∼5-12 wt % covalently linked trehalose. The designed poly(trehalose) nanoparticles are 1000-10000 times more efficient than molecular trehalose in inhibiting protein fibrillation in extra-cellular space, in blocking aggregation of polyglutamine-containing mutant huntingtin protein in model neuronal cells, and in suppressing mutant huntingtin aggregates in HD mouse brain. We show that the nanoparticle form of trehalose with zwitterionic surface charge and a trehalose multivalency (i.e., number of trehalose molecules per nanoparticle) of ∼80-200 are crucial for efficient brain targeting, entry into neuronal cells, and suppression of mutant huntingtin aggregation. The present work shows that nanoscale trehalose can offer highly efficient antiamyloidogenic performance at micromolar concentration, compared with millimollar to molar concentrations for molecular trehalose. This approach can be extended to in vivo application to combat protein-aggregation-derived neurodegenerative diseases.