Targeted mRNA Therapy for Ornithine Transcarbamylase Deficiency.

We describe a novel, two-nanoparticle mRNA delivery system and show that it is highly effective as a means of intracellular enzyme replacement therapy (i-ERT) using a murine model of ornithine transcarbamylase deficiency (OTCD). Our Hybrid mRNA Technology delivery system (HMT) comprises an inert lipid nanoparticle that protects the mRNA from nucleases in the blood as it distributes to the liver and a polymer micelle that targets hepatocytes and triggers endosomal release of mRNA. This results in high-level synthesis of the desired protein specifically in the liver. HMT delivery of human OTC mRNA normalizes plasma ammonia and urinary orotic acid levels, and leads to a prolonged survival benefit in the murine OTCD model. HMT represents a unique, non-viral mRNA delivery method that allows multi-dose, systemic administration for treatment of single-gene inherited metabolic diseases.

Stable and Functionalizable Quantum Dots with a Thin Zwitterionic Carboxybetaine Layer.

A new type of ligand chemistry, with a zwitterionic carboxybetaine headgroup and a bidentate thiol end group (CBSS), is introduced to promote the stability of quantum dots (QDs) with targeting capability. Results show that QDs are stable over a broad range of pH values after surface modification. Surface binding assays and cellular internalization studies show that QDs capped with CBSS exhibit low nonspecific adsorption. The CBSS ligand also allows the conjugation of highly specific targeting ligands while effectively maintaining the nonfouling background. This QD chemistry offers a unique approach to presenting abundant functional groups for ligand immobilization in a thin layer with an ultralow background and holds significant potential for imaging applications.

Design, synthesis, and X-ray structural studies of BACE-1 inhibitors containing substituted 2-oxopiperazines as P1'-P2' ligands.

We report the design and synthesis of a series of BACE1 inhibitors incorporating mono- and bicyclic 6-substituted 2-oxopiperazines as novel P1' and P2' ligands and isophthalamide derivative as P2-P3 ligands. Among mono-substituted 2-oxopiperazines, inhibitor 5a with N-benzyl-2-oxopiperazine and isophthalamide showed potent BACE1 inhibitory activity (Ki=2nM). Inhibitor 5g, with N-benzyl-2-oxopiperazine and substituted indole-derived P2-ligand showed a reduction in potency. The X-ray crystal structure of 5g-bound BACE1 was determined and used to design a set of disubstituted 2-oxopiperazines and bicyclic derivatives that were subsequently investigated. Inhibitor 6j with an oxazolidinone derivative showed a BACE1 inhibitory activity of 23nM and cellular EC50 of 80nM.

Design of novel HIV-1 protease inhibitors incorporating isophthalamide-derived P2-P3 ligands: Synthesis, biological evaluation and X-ray structural studies of inhibitor-HIV-1 protease complex.

Based upon molecular insights from the X-ray structures of inhibitor-bound HIV-1 protease complexes, we have designed a series of isophthalamide-derived inhibitors incorporating substituted pyrrolidines, piperidines and thiazolidines as P2-P3 ligands for specific interactions in the S2-S3 extended site. Compound 4b has shown an enzyme Ki of 0.025nM and antiviral IC50 of 69nM. An X-ray crystal structure of inhibitor 4b-HIV-1 protease complex was determined at 1.33Å resolution. We have also determined X-ray structure of 3b-bound HIV-1 protease at 1.27Å resolution. These structures revealed important molecular insight into the inhibitor-HIV-1 protease interactions in the active site.

Structure-based design, synthesis, X-ray studies, and biological evaluation of novel HIV-1 protease inhibitors containing isophthalamide-derived P2-ligands.

We describe the design, synthesis and biological evaluation of a series of novel HIV-1 protease inhibitors bearing isophthalamide derivatives as the P2-P3 ligands. We have investigated a range of acyclic and heterocyclic amides as the extended P2-P3 ligands. These inhibitors displayed good to excellent HIV-1 protease inhibitory activity. Also, a number of inhibitors showed very good antiviral activity in MT cells. Compound 5n has shown an enzyme Ki of 0.17 nM and antiviral IC50 of 14 nM. An X-ray crystal structure of inhibitor 5o-bound to HIV-1 protease was determined at 1.11Å resolution. This structure revealed important molecular insight into the inhibitor-HIV-1 protease interactions in the active site.

Sensitive and fast detection of fructose in complex media via symmetry breaking and signal amplification using surface-enhanced Raman spectroscopy.

A new strategy is proposed to sensitively and rapidly detect analytes with weak Raman signals in complex media using surface-enhanced Raman spectroscopy (SERS) via detecting the SERS signal changes of the immobilized probe molecules on SERS-active substrates upon binding of the analytes. In this work, 4-mercaptophenylboronic acid (4-MPBA) was selected as the probe molecule which was immobilized on the gold surface of a quasi-three-dimensional plasmonic nanostructure array (Q3D-PNA) SERS substrate to detect fructose. The molecule of 4-MPBA possesses three key functions: molecule recognition and reversible binding of the analyte via the boronic acid group, amplification of SERS signals by the phenyl group and thus shielding of the background noise of complex media, and immobilization on the surface of SERS-active substrates via the thiol group. Most importantly, the symmetry breaking of the 4-MPBA molecule upon fructose binding leads to the change of area ratio between totally symmetric 8a ring mode and nontotally symmetric 8b ring mode, which enables the detection. The detection curves were obtained in phosphate-buffered saline (PBS) and in undiluted artificial urine at clinically relevant concentrations, and the limit of detection of 0.05 mM was achieved.

Cross-linked carboxybetaine SAMs enable nanoparticles with remarkable stability in complex media.

A photo-cross-linkable carboxybetaine (CB)-terminated thiol with only one CB headgroup was introduced to modify gold nanoparticles (GNPs) via self-assembled monolayers (SAMs). This CB-terminated thiol consists of three moieties: (a) an anchoring thiol group, which binds directly to the GNP surface, (b) a CB terminal group, which is highly resistant to protein adsorption, and (c) a diacetylene group in the middle, which is converted to a poly(enyne) structure during UV irradiation via 1,4-topochemical polymerization. Results show that, after cross-linking, CB-modified GNPs are highly resistant to protein adsorption from undiluted human blood serum and cell uptake, and are stable at low pH and high temperature. This cross-linkable CB thiol holds tremendous potentials for biomedical applications where stable and thin coatings are needed.

Restraint of the differentiation of mesenchymal stem cells by a nonfouling zwitterionic hydrogel.

The success of human mesenchymal stem cell (hMSC) therapies is largely dependent on the ability to maintain the multipotency of cells and control their differentiation. External biochemical and biophysical cues can readily trigger hMSCs to spontaneously differentiate, thus resulting in a rapid decrease in the multipotent cell population and compromising their regenerative capacity. Herein, we demonstrate that nonfouling hydrogels composed of pure poly(carboxybetaine) (PCB) enable hMSCs to retain their stem-cell phenotype and multipotency, independent of differentiation-promoting media, cytoskeletal-manipulation agents, and the stiffness of the hydrogel matrix. Moreover, encapsulated hMSCs can be specifically induced to differentiate down osteogenic or adipogenic pathways by controlling the content of fouling moieties in the PCB hydrogel. This study examines the critical role of nonspecific interactions in stem-cell differentiation and highlights the importance of materials chemistry in maintaining stem-cell multipotency and controlling differentiation.

Hydrolytic cationic ester microparticles for highly efficient DNA vaccine delivery.

DNA vaccination holds great potential to be a safer and more efficient alternative to traditional vaccination strategies, but the current lack of nontoxic and effective delivery systems is the greatest impediment to its clinical implementation. In this work, a convenient one-step method is used to prepare a degradable "microgel" delivery platform, featuring hydrolytic esters. Prior to hydrolysis, these micrometer-sized gel particles can effectively condense DNA due to their positive surface charge. Upon entering antigen-presenting cells (APCs), the microgels can be hydrolyzed to nontoxic zwitterionic polymers, consequently releasing the DNA and inducing phagosomal escape. Surface charge, DNA loading, cytotoxicity, and gene transfection efficiency of the hydrolysable microparticles with different tertiary to quaternary amine ratios are systematically studied. Nonhydrolysable counterparts and commercially developed PLGA-CTAB particles are used as the control. The passive targeting effect is further evaluated by blocking the phagocytosis pathway of the cells. The hydrolytic microgels prepared in this study possess great potential to become a platform for DNA vaccine delivery.

Engineering buffering and hydrolytic or photolabile charge shifting in a polycarboxybetaine ester gene delivery platform.

Polycarboxybetaine esters (PCB-esters) can condense plasmid DNA into nanosized polyplexes for highly effective gene delivery with low toxicity. The design and characterization of tertiary CB-ester monomers and PCB-ester polymers are presented here to study the effects of molecular variation on functions important to nonviral gene transfer. Both buffering capacity and charge-shifting behavior can be tuned by modifying the distance between the charged groups and the ester size or type. A carbon spacer length (CSL) of one was found to bring the pKa of the tertiary amine into the optimal range for proton buffering. Ester hydrolytic degradation switches this polymer from cationic (DNA binding) to zwitterionic (DNA releasing) form while conferring nontoxicity. To allow rapid and externally controlled degradation, the effect of this charge-switching behavior on DNA release from polyplexes was directly studied with a novel photolabile PCB-nitrobenzyl ester (PCB-NBE). Photoinitiated ester degradation precipitated the rapid release of 72±5% of complexed DNA from PCB-NBE polyplexes. These insights reveal the key parameters important for the PCB-ester platform and the significance of charge switching to an effective and nontoxic nonviral gene delivery platform.

High viability of cells encapsulated in degradable poly(carboxybetaine) hydrogels.

In this study, we report a degradable poly(carboxybetaine) (pCB) hydrogel, produced via a thiol-disulfide exchange reaction for cell encapsulation. A pCB dithiol was synthesized as a cross-linker and reacted with a pyridyl dithiol-containing CB copolymer to form a hydrogel. We evaluated the biocompatibility of the pCB-based hydrogel via encapsulation of three cell types, including NIH3T3 fibroblasts, MG63 osteoblast-like cells, and HepG2 hepatocarcinoma cells. Up to 90% of cells retained their viability in the pCB hydrogel even at low cell-seeding densities under serum-free conditions after a 9-day culture. Results are compared with a degradable poly(ethylene glycol) methacrylate (PEGMA) hydrogel, which showed very low cell viability under serum-free condition after a 3-day culture. We incorporated an RGD peptide into the CB hydrogel using a cysteine-terminated cross-linker, which was shown to promote cell proliferation.

Reversibly switching the function of a surface between attacking and defending against bacteria.

Attack or defend! A smart polymer surface has two reversibly switchable equilibrium states, a cationic N,N-dimethyl-2-morpholinone (CB-Ring) and a zwitterionic carboxy betaine (CB-OH). CB-Ring will kill bacteria upon contact under dry conditions, whereas CB-OH will release the previously attached and dead bacteria and further resist adhesion of bacteria under wet conditions.

Synthesis of octahydropyrano[3,2-b]pyrrole-2-carboxylic acid derivatives from D-mannose.

Bicyclic amino acids are useful building blocks in synthesizing biologically active molecules and peptidomimetics. 2-Carboxy-6-hydroxyloctahydroindole (Choi) is a novel bicyclic amino acid found in the marine natural products aeruginosins. Many compounds in the aeruginosin family exhibit inhibition activities toward serine proteases including thrombin and trypsin. The unique Choi structure is the common feature of this family of oligopeptides and this motif is important for their observed biological activities. To better understand the influence of the stereochemistry of the Choi core structure on the inhibition activities, we have previously synthesized ring-oxygenated variants from glucose. The preparation of octahydro-pyrano[3,2-b]pyrrole 2-carboxylic acids from d-mannose is reported here. These novel bicyclic amino acids can be used in the preparation of aeruginosin analogs, as well as conformationally constrained peptidomimetics or other biologically active molecules.

Harnessing isomerization-mediated manipulation of nonspecific cell/matrix interactions to reversibly trigger and suspend stem cell differentiation.

Specific protein-cell and drug-cell interactions have been widely used to manipulate stem cell fate. Despite extensive studies, most current platforms cannot realize reversible manipulation of stem cell differentiation. In this work, we report a photodynamic zwitterionic hydrogel capable of reversibly triggering and suspending the differentiation process via manipulating nonspecific interactions between cultured stem cells and the hydrogel. The differentiation state of stem cells can be altered by exposing the hydrogel to a selected light program, while differentiation can be immediately suspended when near-infrared exposure converts the hydrogel into a purely zwitterionic form. While many other studies apply specific interactions to control stem cell fate, this work provides a completely different approach-allowing reversible, real-time and localized manipulation of stem cell fate choice via nonspecific interactions.

Stealth surface modification of surface-enhanced Raman scattering substrates for sensitive and accurate detection in protein solutions.

Reliable surface-enhanced Raman scattering (SERS) based biosensing in complex media is impeded by nonspecific protein adsorptions. Because of the near-field effect of SERS, it is challenging to modify SERS-active substrates using conventional nonfouling materials without introducing interference from their SERS signals. Herein, we report a stealth surface modification strategy for sensitive, specific and accurate detection of fructose in protein solutions using SERS by forming a mixed self-assembled monolayer (SAM). The SAM consists of a short zwitterionic thiol, N,N-dimethyl-cysteamine-carboxybetaine (CBT), and a fructose probe 4-mercaptophenylboronic acid (4-MPBA). The specifically designed and synthesized CBT not only resists protein fouling effectively, but also has very weak Raman activity compared to 4-MPBA. Thus, the CBT SAM provides a stealth surface modification to SERS-active substrates. The surface compositions of mixed SAMs were investigated using X-ray photoelectron spectroscopy (XPS) and SERS, and their nonfouling properties were studied with a surface plasmon resonance (SPR) biosensor. The mixed SAM with a surface composition of 94% CBT demonstrated a very low bovine serum albumin (BSA) adsorption (∼3 ng/cm(2)), and moreover, only the 4-MPBA signal appeared in the SERS spectrum. With the use of this surface-modified SERS-active substrate, quantification of fructose over clinically relevant concentrations (0.01-1 mM) was achieved. Partial least-squares regression (PLS) analysis showed that the detection sensitivity and accuracy were maintained for the measurements in 1 mg/mL BSA solutions. This stealth surface modification strategy provides a novel route to introduce nonfouling property to SERS-active substrates for SERS biosensing in complex media.

One-step dip coating of zwitterionic sulfobetaine polymers on hydrophobic and hydrophilic surfaces.

Zwitterionic sulfobetaine polymers with a catechol chain end (DOPA-PSB) were applied to a variety of hydrophobic polymer sheets and fibers. In addition, a silica surface was tested as a representative hydrophilic substrate. The polymer-coated surfaces showed significantly lower fouling levels than uncoated controls. Because of the anti-polyelectrolyte nature of sulfobetaine zwitterionic polymers, the effect of salt concentration on the coating solutions and the quality of the polymer coating against fouling are studied. The coating method involves only water-based solutions, which is compatible with most surfaces and is environmentally friendly. To demonstrate the versatility of the reported method, we evaluated the fouling levels of the polymer coating on commonly used polymeric surfaces such as polypropylene (PP), polydimethylsiloxane (PDMS), polystyrene (PS), nylon, polyvinyl chloride (PVC), and poly(methyl methacrylate) (PMMA).

Achieving One-step Surface Coating of Highly Hydrophilic Poly(Carboxybetaine Methacrylate) Polymers on Hydrophobic and Hydrophilic Surfaces.

It is highly desirable to develop a universal nonfouling coating via a simple one-step dip-coating method. Developing such a universal coating method for a hydrophilic polymer onto a variety of surfaces with hydrophobic and hydrophilic properties is very challenging. This work demonstrates a versatile and simple method to attach zwitterionic poly(carboxybetaine methacrylate) (PCB), one of the most hydrophilic polymers, onto both hydrophobic and hydrophilic surfaces to render them nonfouling. This is achieved by the coating of a catechol chain end carboxybetaine methacrylate polymer (DOPA-PCB) assisted by dopamine. The coating process was carried out in water. Water miscible solvents such as methanol and tetrahydrofuran (THF) are added to the coatings if surface wettability is an issue, as for certain hydrophobic surfaces. This versatile coating method was applied to several types of surfaces such as polypropylene (PP), polydimethyl siloxane (PDMS), Teflon, polystyrene (PS), polymethylmethacrylate (PMMA), polyvinyl chloride (PVC) and also on metal oxides such as silicon dioxide.

Free energy of solvated salt bridges: a simulation and experimental study.

Charged amino acids are the most common on surfaces of proteins and understanding the interactions between these charged amino acids, salt bridging, is crucial for understanding protein-protein interactions. Previous simulations have been limited to implicit solvent or fixed binding geometry due to the sampling required for converged free energies. Using well-tempered metadynamics, we have calculated salt bridge free energy surfaces in water and confirmed the results with NMR experiments. The simulations give binding free energies, quantitative ranking of salt bridging strength, and insights into the hydration of the salt bridges. The arginine-aspartate salt bridge was found to be the weakest and arginine-glutamate the strongest, showing that arginine can discriminate between aspartate and glutamate, whereas the salt bridges with lysine are indistinguishable in their free energy. The salt bridging hydration is found to be complementary to salt bridge orientation with arginine having specific orientations.

Zwitterionic hydrogels implanted in mice resist the foreign-body reaction.

The performance of implantable biomedical devices is impeded by the foreign-body reaction, which results in formation of a dense collagenous capsule that blocks mass transport and/or electric communication between the implant and the body. No known materials or coatings can completely prevent capsule formation. Here we demonstrate that ultra-low-fouling zwitterionic hydrogels can resist the formation of a capsule for at least 3 months after subcutaneous implantation in mice. Zwitterionic hydrogels also promote angiogenesis in surrounding tissue, perhaps owing to the presence of macrophages exhibiting phenotypes associated with anti-inflammatory, pro-healing functions. Thus, zwitterionic hydrogels may be useful in a broad range of applications, including generation of biocompatible implantable medical devices and tissue scaffolds.

Softer zwitterionic nanogels for longer circulation and lower splenic accumulation.

Zwitterionic nanogels of varying stiffness were prepared by tuning their cross-linking densities and reactant contents. In vivo studies of these nanogels show that softer nanogels pass through physiological barriers, especially the splenic filtration, more easily than their stiffer counterparts, consequently leading to longer circulation half-life and lower splenic accumulation. Results from this work emphasize the role of stiffness in designing long-circulating nanoparticles.