Biocompatible Infinite‐Coordination‐Polymer Nanoparticle–Nucleic‐Acid Conjugates for Antisense Gene Regulation

Herein, we report the synthesis of DNA‐functionalized infinite‐coordination‐polymer (ICP) nanoparticles as biocompatible gene‐regulation agents. ICP nanoparticles were synthesized from ferric nitrate and a ditopic 3‐hydroxy‐4‐pyridinone (HOPO) ligand bearing a pendant azide. Addition of Fe^III to a solution of the ligand produced nanoparticles, which were colloidally unstable in the presence of salts. Conjugation of DNA to the Fe^III–HOPO ICP particles by copper‐free click chemistry afforded colloidally stable nucleic‐acid nanoconstructs. The DNA–ICP particles, when cross‐linked through sequence‐specific hybridization, exhibited narrow, highly cooperative melting transitions consistent with dense DNA surface loading. The ability of the DNA–ICP particles to enter cells and alter protein expression was also evaluated. Our results indicate that these novel particles carry nucleic acids into mammalian cells without the need for transfection agents and are capable of efficient gene knockdown.     

Cell‐Derived Vesicles for Single‐Molecule Imaging of Membrane Proteins

A new approach is presented for the application of single‐molecule imaging to membrane receptors through the use of vesicles derived from cells expressing fluorescently labeled receptors. During the isolation of vesicles, receptors remain embedded in the membrane of the resultant vesicles, thus allowing these vesicles to serve as nanocontainers for single‐molecule measurements. Cell‐derived vesicles maintain the structural integrity of transmembrane receptors by keeping them in their physiological membrane. It was demonstrated that receptors isolated in these vesicles can be studied with solution‐based fluorescence correlation spectroscopy (FCS) and can be isolated on a solid substrate for single‐molecule studies. This technique was applied to determine the stoichiometry of α3β4 nicotinic receptors. The method provides the capability to extend single‐molecule studies to previously inaccessible classes of receptors.     

Poly(N‐methylvinylamine)‐Based Copolymers for Improved Gene Transfection

Poly(N‐methylvinylamines) with secondary amines can form complexes with plasmid DNA (pDNA) and provide transfection efficiency in HeLa cells in the same order as linear polyethyleneimine but with higher cell viability. Chemical modifications of poly(N‐methylvinylamine) backbones are performed to further improve transfection efficiency while maintaining low degree of cytotoxicity. In a first type of polymer, primary amino groups are incorporated via a copolymerization strategy. In a second one, primary amino and imidazole groups are incorporated also via a copolymerization strategy. In a third one, secondary amino groups are substituted with methylguanidine functions through a postpolymerization reaction. Thus, novel polymers of various molecular masses are synthesized, characterized, and their interaction with pDNA studied. Then, their transfection efficiency and cytotoxicity are tested in HeLa cells. Two polymethylvinylamine‐based copolymers, one containing 20% of imidazole moieties and another one composed of 12% of guanidinyl units allow remarkable transfection efficiency of HeLa, pulmonary (16HBE), skeletal muscle (C2C12), and dendritic (DC2.4) cells. Overall, this work thus identifies new promising DNA carriers and chemical modifications that improve the transfection efficiency while maintaining low degree of cytotoxicity.     

Transformation of Cell‐Derived Microparticles into Quantum‐Dot‐Labeled Nanovectors for Antitumor siRNA Delivery

Cell‐derived microparticles (MPs) have been recently recognized as critical intercellular information conveyors. However, further understanding of their biological behavior and potential application has been hampered by the limitations of current labeling techniques. Herein, a universal donor‐cell‐assisted membrane biotinylation strategy was proposed for labeling MPs by skillfully utilizing the natural membrane phospholipid exchange of their donor cells. This innovative strategy conveniently led to specific, efficient, reproducible, and biocompatible quantum dot (QD) labeling of MPs, thereby reliably conferring valuable traceability on MPs. By further loading with small interference RNA, QD‐labeled MPs that had inherent cell‐targeting and biomolecule‐conveying ability were successfully employed for combined bioimaging and tumor‐targeted therapy. This study provides the first reliable and biofriendly strategy for transforming biogenic MPs into functionalized nanovectors.     

Self‐Assembled PAA‐Based Nanoparticles as Potential Gene and Protein Delivery Systems

A series of nanoparticles is prepared via layer‐by‐layer assembly of oppositely charged, synthetic biocompatible polyamidoamine polymers as potential carriers. Particle size, surface charge and internal chain mobility are quantified as a function of the polymer type and number of layers. The effect of addition of surfactant is examined to simulate the effects of nanoparticle dissolution. The cyctotoxicity of these particles (in epithelia and murine cell lines) are orders of magnitude lower than polyethyleneimine controls. Stable nanoparticles may be prepared from mixtures of strongly, oppositely charged polymers, but less successfully from weakly charged polymers, and, given their acceptable toxicity characteristics, such modularly designed constructs show promise for drug and gene delivery.

     

Construction of Versatile and Functional Nanostructures Derived from CO2‐based Polycarbonates

The construction of amphiphilic polycarbonates through epoxides/CO₂ coupling is a challenging aim to provide more diverse CO₂‐based functional materials. In this report, we demonstrate the facile preparation of diverse and functional nanoparticles derived from a CO₂‐based triblock polycarbonate system. By the judicious use of water as chain‐transfer reagent in the propylene oxide/CO₂ polymerization, poly(propylene carbonate (PPC) diols are successfully produced and serve as macroinitiators in the subsequent allyl glycidyl ether/CO₂ coupling reaction. The resulting ABA triblock polycarbonate can be further functionalized with various thiols by radical mediated thiol–ene click chemistry, followed by self‐assembly in deionized water to construct a versatile and functional nanostructure system. This class of amphiphilic polycarbonates could embody a powerful platform for biomedical applications.     

Theranostic Layer‐by‐Layer Nanoparticles for Simultaneous Tumor Detection and Gene Silencing

Layer‐by‐layer nanoparticles (NPs) are modular drug delivery vehicles that incorporate multiple functional materials through sequential deposition of polyelectrolytes onto charged nanoparticle cores. Herein, we combined the multicomponent features and tumor targeting capabilities of layer‐by‐layer assembly with functional biosensing peptides to create a new class of nanotheranostics. These NPs encapsulate a high weight percentage of siRNA while also carrying a synthetic biosensing peptide on the surface that is cleaved into a urinary reporter upon exposure to specific proteases overexpressed in the tumor microenvironment. Importantly, this biosensor reports back on a molecular signature characteristic to metastatic tumors and associated with poor prognosis, MMP9 protease overexpression. This nanotheranostic mediates noninvasive urinary‐based diagnostics in mouse models of three different cancers with simultaneous gene silencing in flank and metastatic mouse models of ovarian cancer.     

Monodisperse Poly(lactide‐co‐glycolic acid)‐Based Nanocarriers for Gene Transfection

This contribution describes a simple, aerosol‐based method for fabricating monodisperse particles containing mixtures of poly(lactide‐co‐glycolic acid) [PLGA], protamine sulfate (Prot), and poly(l‐ lysine) [PLL] as nanocarriers for gene transfection. Aqueous solutions of PLGA, Prot, and PLL were collison‐atomized, and the resulting aerosolized droplets were dried “on the fly” to form solid particles, which then were electrostatically size‐classified into 50, 100, and 200 nm mobility diameter samples. Measurements of cell viability and transfection reveal that the fabricated nanocarriers have a lower cytotoxicity (>85% in cell viability) and a higher transfection efficiency [>8.7 × 10⁵ in relative light units (RLU) mg⁻¹] than does 25 kDa polyethyleneimine (≈50% and 6.8 × 10⁵ RLU mg⁻¹).     

Porous Protein‐Based Nanoparticle Hydrogel for Protein Chips with Improved Sensitivity

We have discovered a novel method to prepare a protein‐based hydrogel, that is, a ‘three‐dimensional nanostructured protein hydrogel’ (3D NPH), which is composed of loosely inter‐connected protein–polymer hybrid nanoparticles. The 3D NPH can be easily prepared by spotting a protein/polymer mixture on a substrate. Surprisingly, gold nanoparticles carrying protein molecules easily diffuse into the 3D NPH through pores and spaces. We have shown that the protein chip made by our 3D NPH method has tremendously improved sensitivity in detecting protein–protein interactions compared with that by direct protein immobilization methods.

     

Remote Loading of Small‐Molecule Therapeutics into Cholesterol‐Enriched Cell‐Membrane‐Derived Vesicles

The increasing popularity of biomimetic design principles in nanomedicine has led to therapeutic platforms with enhanced performance and biocompatibility. This includes the use of naturally derived cell membranes, which can bestow nanocarriers with cell‐specific functionalities. Herein, we report on a strategy enabling efficient encapsulation of drugs via remote loading into membrane vesicles derived from red blood cells. This is accomplished by supplementing the membrane with additional cholesterol, stabilizing the nanostructure and facilitating the retention of a pH gradient. We demonstrate the loading of two model drugs: the chemotherapeutic doxorubicin and the antibiotic vancomycin. The therapeutic implications of these natural, remote‐loaded nanoformulations are studied both in vitro and in vivo using animal disease models. Ultimately, this approach could be used to design new biomimetic nanoformulations with higher efficacy and improved safety profiles.     

Cell‐ and Tissue‐Based Proteome Profiling and Bioimaging with Probes Derived from a Potent AXL Kinase Inhibitor

AXL has been defined as a novel target for cancer therapeutics. However, only a few potent and selective inhibitors targeting AXL are available to date. Recently, our group has developed a lead compound, 9im, capable of displaying potent and specific inhibition of AXL. To further identify the cellular on/off targets, in this study, competitive affinity‐based proteome profiling was carried out, leading to the discovery of several unknown cellular targets such as BCAP31, LPCAT3, POR, TM9SF3, SCCPDH and CANX. In addition, trans‐cyclooctene (TCO) and acedan‐containing probes were developed to image the binding between 9im and its target proteins inside live cells and tumor tissues. These probes would be useful tools in the detection of AXL in various biosystems.     

Reversible Re‐programing of Cell–Cell Interactions

The ability to engineer and re‐program the surfaces of cells would provide an enabling synthetic biological method for the design of cell‐ and tissue‐based therapies. A new cell surface‐engineering strategy is described that uses lipid‐chemically self‐assembled nanorings (lipid‐CSANs) that can be used for the stable and reversible modification of any cell surface with a molecular reporter or targeting ligand. In the presence of a non‐toxic FDA‐approved drug, the nanorings were quickly disassembled and the cell–cell interactions reversed. Similar to T‐cells genetically engineered to express chimeric antigen receptors (CARS), when activated peripheral blood mononuclear cells (PBMCs) were functionalized with the anti‐EpCAM‐lipid‐CSANs, they were shown to selectively kill antigen‐positive cancer cells. Taken together, these results demonstrate that lipid‐CSANs have the potential to be a rapid, stable, and general method for the reversible engineering of cell surfaces and cell–cell interactions.     

Cell‐Permeant and Photocleavable Chemical Inducer of Dimerization

Chemical inducers of dimerization (CIDs) have been developed to orchestrate protein dimerization and translocation. Here we present a novel photocleavable HaloTag‐ and SNAP‐tag‐reactive CID (MeNV‐HaXS) with excellent selectivity and intracellular reactivity. Excitation at 360 nm cleaves the methyl‐6‐nitroveratryl core of MeNV‐HaXS. MeNV‐HaXS covalently links HaloTag‐ and SNAP‐tag fusion proteins, and enables targeting of selected membranes and intracellular organelles. MeNV‐HaXS‐mediated translocation has been validated for plasma membrane, late endosomes, lysosomes, Golgi, mitochondria, and the actin cytoskeleton. Photocleavage of MeNV‐HaXS liberates target proteins and provides access to optical manipulation of protein relocation with high spatiotemporal and subcellular precision. MeNV‐HaXS supports kinetic studies of protein dynamics and the manipulation of subcellular enzyme activities, which is exemplified for Golgi‐targeted cargo and the assessment of nuclear import kinetics.     

Cell‐ and Tissue‐Based Proteome Profiling and Dual Imaging of Apoptosis Markers with Probes Derived from Venetoclax and Idasanutlin

Venetoclax (ABT‐199) and idasanutlin (RG7388) are efficient anticancer drugs targeting two essential apoptosis markers, Bcl‐2 and MDM2, respectively. Recent studies have shown that the combination of these two drugs leads to remarkable enhancement of anticancer efficacy, both in vitro and in vivo. In an attempt to disclose the relationships of their protein targets, competitive affinity‐based proteome profiling coupled with bioimaging was employed to characterize their protein targets in the same cancer cell line and tumor tissue. A series of protein hits, including ITPR1, GSR, RER1, PDIA3, Apoa1, and Tnfrsf17 were simultaneously identified by pull‐down/LC–MS/MS with the two sets of affinity‐based probes. Dual imaging was successfully carried out, with the simultaneous detection of Bcl‐2 and MDM2 expression in various cancer cells. This could facilitate the novel diagnostic and therapeutic strategies of dual targeting of Bcl‐2/MDM2.     

Design of Aromatic‐Containing Cell‐Penetrating Peptide Mimics with Structurally Modified π Electronics

Cell‐penetrating peptides (CPPs) and their synthetic mimics (CPPMs) represent a class of molecules that facilitate the intracellular delivery of various cargo. Previous studies indicated that the presence of aromatic functionalities improved CPPM activity. Given that aromatic functionalities play prominent roles in membrane biology and participate in various π interactions, we explored whether these interactions could be optimized for improved CPPM activity. CPPMs were synthesized by ring‐opening metathesis polymerization by using monomers that contained aromatic rings substituted with electron‐donating and electron‐withdrawing groups and covered an electrostatic potential range from ?29.69 to +15.57 kcal mol^?1. These groups altered the quadrupole moments of the aromatic systems and were used to test if such structural modifications changed CPPM activity. CPPMs were added to dye‐loaded vesicles and the release of carboxyfluorescein was monitored as a function of polymer concentration. Changes in the effective polymer concentration to release 50 % of the dye (effective concentration, EC₅₀) were monitored. Results from this assay showed that the strength of the electron‐donating and electron‐withdrawing groups incorporated in the CPPMs did not alter polymer EC₅₀ values or activity. This suggests that other design parameters may have a stronger impact on CPPM activity. In addition, these results indicate that a wide range of aromatic groups can be incorporated without negatively impacting polymer activity.