Development of an Elastin-like Polypeptide-Based Nucleic Acid Delivery System Targeted to EGFR+ Bladder Cancer Cells Using a Layer-by-Layer Approach.

Nucleic acid (NA)-based therapies are revolutionizing biomedical research through their ability to control cellular functions at the genetic level. This work demonstrates a versatile elastin-like polypeptide (ELP) carrier system using a layer-by-layer (LbL) formulation approach that delivers NA cargos ranging in size from siRNA to plasmids. The components of the system can be reconfigured to modulate the biochemical and biophysical characteristics of the carrier for engaging the unique features of the biological target.

Methods optimization for the expression and purification of human calcium calmodulin-dependent protein kinase II alpha.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a complex multifunctional kinase that is highly expressed in central nervous tissues and plays a key regulatory role in the calcium signaling pathway. Despite over 30 years of recombinant expression and characterization studies, CaMKII continues to be investigated for its impact on signaling cooperativity and its ability to bind multiple substrates through its multimeric hub domain. Here we compare and optimize protocols for the generation of full-length wild-type human calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα).

Unravelling the Mechanism of Elastin-like Polypeptide-Enzyme Fusion Stabilization in Organic Solvents.

Elastin-like polypeptides (ELP) are a class of materials that are widely used as purification tags and in potential therapeutic applications. We have used the hydrophobic nature of ELP to extract them into organic solvents and precipitate them to obtain highly pure materials. Although many different types of ELP have been rapidly purified in this manner, the underlying mechanism for this process and its ability to retain functional proteins within organic phase-rich media has been unclear.

Synthesis of Phosphatidyl Glycerol Containing Unsymmetric Acyl Chains Using H-Phosphonate Methodology.

Naturally occurring phospholipids, such as phosphatidyl glycerol (PG), are gaining interest due to the roles they play in disease mechanisms. To elucidate the metabolism of PG, an optically pure material is required, but this is unfortunately not commercially available. Our previous PG synthesis route utilized phosphoramidite methodology that addressed issues surrounding fatty acid substrate scope and glycerol backbone modifications prior to headgroup phosphorylation, but faltered in the reproducibility of the overall pathway due to purification challenges.

Proteins Adsorbing onto Surface-Modified Nanoparticles: Effect of Surface Curvature, pH, and the Interplay of Polymers and Proteins Acid-Base Equilibrium.

Protein adsorption onto nanomaterials is a process of vital significance and it is commonly controlled by functionalizing their surface with polymers. The efficiency of this strategy depends on the design parameters of the nanoconstruct. Although significant amount of work has been carried out on planar surfaces modified with different types of polymers, studies investigating the role of surface curvature are not as abundant.

Continuous Flow Synthesis of A2E Guided by Design of Experiments and High-Throughput Studies.

-Retinylidene--retinylethanolamine (A2E) is the most studied lipid bisretinoid. It forms lipofuscin deposits in the retinal pigment epithelium (RPE), causing vision impairment and blindness in eye conditions, such as Stargardt's disease, cone-rod dystrophy, Best's macular dystrophy, and potentially age-related macular degeneration. Synthetic A2E is often used for inducing the accumulation of lipofuscins within the lysosomes of RPE cells in culture as an in vitro surrogate of retinal lipofuscin buildup, providing insights into the mechanisms of these eye conditions.

Immunostimulatory Response of RWFV Peptide-Targeted Lipid Nanoparticles on Bladder Tumor Associated Cells.

Bladder carcinoma is the most expensive tumor type to treat on a cost-per-patient basis from diagnosis to death. Treatment with Bacillus Calmette Guerin (BCG) instillation is the only approved immunotherapy in the clinic for the remission of superficial bladder carcinoma. Unfortunately, frequent relapses, high local morbidity, risk of systemic mycobacterial infection, and occasional supply chain interruptions limit the utility of BCG for bladder cancer treatment.

Lipofuscin causes atypical necroptosis through lysosomal membrane permeabilization.

Lipofuscin granules enclose mixtures of cross-linked proteins and lipids in proportions that depend on the tissue analyzed. Retinal lipofuscin is unique in that it contains mostly lipids with very little proteins. However, retinal lipofuscin also presents biological and physicochemical characteristics indistinguishable from conventional granules, including indigestibility, tendency to cause lysosome swelling that results in rupture or defective functions, and ability to trigger NLRP3 inflammation, a symptom of low-level disruption of lysosomes.

Affinity Capture of p97 with Small-Molecule Ligand Bait Reveals a 3.6 Å Double-Hexamer Cryoelectron Microscopy Structure.

Recent progress in the development of affinity grids for cryoelectron microscopy (cryo-EM) typically employs genetic engineering of the protein sample such as histidine or Spy tagging, immobilized antibody capture, or nonselective immobilization via electrostatic interactions or Schiff base formation. We report a powerful and flexible method for the affinity capture of target proteins for cryo-EM analysis that utilizes small-molecule ligands as bait for concentrating human target proteins directly onto the grid surface for single-particle reconstruction. This approach is demonstrated for human p97, captured using two different small-molecule high-affinity ligands of this AAA+ ATPase.

Development of a Fast Organic Extraction-Precipitation Method for Improved Purification of Elastin-Like Polypeptides That Is Independent of Sequence and Molecular Weight.

Elastin-like polypeptides (ELP), an increasingly popular tag for protein purification, commonly rely upon inverse transition cycling (ITC) to exploit their lower critical solution temperature characteristics for purification. While considerably faster than chromatography, ITC is still time consuming and often fails to remove host cell contaminants to an acceptable level for in vivo experiments. Here, we present a rapid purification workflow for ELP of broadly varying molecular weight and sequence using a polar organic solvent extraction and precipitation strategy.

Aldol Reactions of Biorenewable Triacetic Acid Lactone Precursor Evaluated Using Desorption Electrospray Ionization Mass Spectrometry High-Throughput Experimentation and Validated by Continuous Flow Synthesis.

Desorption electrospray ionization-mass spectrometry (DESI-MS) was used as a high-throughput experimentation (HTE) tool to rapidly identify derivatives of the biobased platform molecule triacetic acid lactone (TAL). TAL is a platform molecule capable of conversion to a wide range of useful commodity chemicals, agrochemicals, and advanced pharmaceutical intermediates. In the present study, a diverse family of aldol reaction mixtures were prepared in high-density microtiter plates with a liquid handling robot, then printed with a pin tool onto a PTFE surface for analysis by DESI-MS.

High-throughput screening of organic reactions in microdroplets using desorption electrospray ionization mass spectrometry (DESI-MS): hardware and software implementation.

This study describes an automated system used for high throughput screening of reaction conditions based on accelerated reactions occurring in small volumes of reagents. Reaction mixtures are prepared in array format using a fluid handling robot and spotted on a flat polytetrafluoroethylene plate at densities up to 6144 per plate. The reaction and analysis steps are performed simultaneously using desorption electrospray ionization (DESI) to release microdroplets containing the reaction mixture from the plate for reaction prior to arrival at a mass spectrometer.

Stereospecific synthesis of phosphatidylglycerol using a cyanoethyl phosphoramidite precursor.

Phosphatidylglycerols (PG) are a family of naturally occurring phospholipids that are responsible for critical operations within cells. PG are characterized by an (R) configuration in the diacyl glycerol backbone and an (S) configuration in the phosphoglycerol head group. Herein, we report a synthetic route to provide control over the PG stereocenters as well as control of the acyl chain identity.

High-Throughput Experimentation and Continuous Flow Evaluation of Nucleophilic Aromatic Substitution Reactions.

Nucleophilic aromatic substitution (SAr) reactions were optimized using high-throughput experimentation techniques for execution under flow conditions. A total of 3072 unique reactions were evaluated with an analysis time of ∼3.5 s per reaction using a system that combines a liquid handling robot for reaction mixture preparation with desorption electrospray ionization (DESI) mass spectrometry (MS) for analysis.

Development And In Vitro Characterization Of Bladder Tumor Cell Targeted Lipid-Coated Polyplex For Dual Delivery Of Plasmids And Small Molecules.

Background: Bladder cancer is the fourth most common cancer in men and eleventh most common in women. Combination therapy using a gene and chemotherapeutic drug is a potentially useful strategy for treating bladder cancer in cases where a synergistic benefit can be achieved successfully. This approach relies on developing drug combinations using carrier systems that can load both hydrophilic genes and hydrophobic drugs.

High Throughput Experimentation Using DESI-MS to Guide Continuous-Flow Synthesis.

We demonstrate the use of accelerated reactions with desorption electrospray ionization mass spectrometry (DESI-MS) as a tool for predicting the outcome of microfluidic reactions. DESI-MS was employed as a high throughput experimentation tool to provide qualitative predictions of reaction outcomes, so that vast regions of chemical reactivity space may be more rapidly explored and areas of optimal efficiency identified. This work is part of a larger effort to accelerate reaction optimization to enable the rapid development of continuous-flow syntheses of small molecules in high yield.

An Implantable Ultrasonically-Powered Micro-Light-Source (µLight) for Photodynamic Therapy.

Photodynamic therapy (PDT) is a promising cancer treatment modality that can selectively target unresectable tumors through optical activation of cytotoxic agents, thus reducing many side effects associated with systemic administration of chemotherapeutic drugs. However, limited light penetration into most biological tissues have so far prevented its widespread adoption beyond dermatology and a few other oncological applications in which a fiber optic can be threaded to the desired locations via an endoscopic approach (e.g.

Gd:DOTA-Modified 2-Hydroxypropyl-β-Cyclodextrin/4-Sulfobutyl Ether-β-Cyclodextrin-Based Polyrotaxanes as Long Circulating High Relaxivity MRI Contrast Agents.

A family of five water-soluble Gd:1,4,7,10-tetraazacyclododecane-1,4,7-tetraacetic acid-modified polyrotaxane (PR) magnetic resonance contrast agents bearing mixtures of 2-hydroxypropyl-β-cyclodextrin and 4-sulfobutylether-β-cyclodextrin macrocycles threaded onto Pluronic cores were developed as long circulating magnetic resonance contrast agents. Short diethylene glycol diamine spacers were utilized for linking the macrocyclic chelator to the PR scaffold prior to Gd chelation. The PR products were characterized by H NMR, gel permeation chromatography/multiangle light scattering, dynamic light scattering, and analytical ultracentrifugation.

High Throughput Experimentation and Continuous Flow Validation of Suzuki-Miyaura Cross-Coupling Reactions.

Traditional methods to discover optimal reaction conditions for small molecule synthesis is a time-consuming effort that requires large quantities of material and a significant expenditure of labor. High-throughput techniques are a potentially transformative approach for reaction condition screening, however, rapid validation of the reaction hotspots under continuous flow conditions remains necessary to build confidence in high throughput screening hits. Continuous flow technology offers the opportunity to upscale the screening hotspots and optimize their output of the target compounds due to the exceptional heat and mass transfer ability of flow reactions that are conducted in a smaller and safer reaction volume.

Cerivastatin Nanoliposome as a Potential Disease Modifying Approach for the Treatment of Pulmonary Arterial Hypertension.

In this study we investigated nanoliposome as an approach to tailoring the pharmacology of cerivastatin as a disease-modifying drug for pulmonary arterial hypertension (PAH). Cerivastatin encapsulated liposomes with an average diameter of 98 ± 27 nm were generated by a thin film and freeze-thaw process. The nanoliposomes demonstrated sustained drug-release kinetics in vitro and inhibited proliferation of pulmonary artery (PA) smooth muscle cells with significantly less cellular cytotoxicity as compared with free cerivastatin.

High throughput reaction screening using desorption electrospray ionization mass spectrometry.

We report the high throughput analysis of reaction mixture arrays using methods and data handling routines that were originally developed for biological tissue imaging. Desorption electrospray ionization (DESI) mass spectrometry (MS) is applied in a continuous on-line process at rates that approach 10 reactions per h at area densities of up to 1 spot per mm (6144 spots per standard microtiter plate) with the sprayer moving at 10 microns per s. Data are analyzed automatically by MS using in-house software to create ion images of selected reagents and products as intensity plots in standard array format.

Development of dihydrochalcone-functionalized gold nanoparticles for augmented antineoplastic activity.

Background: Phloridzin, an antidiabetic and antineoplastic agent usually found in fruit trees, is a dihydrochalcone constituent that has a clinical/pharmaceutical significance as a sodium-glucose linked transport 2 (SGLT2) inhibitor. While the aglycone metabolite of phloridzin, phloretin, displays a reduced capacity of SGLT2 inhibition, this nutraceutical displays enhanced antineoplastic activity in comparison to phloridzin.

Purpose: The objective of this study was to develop gold nanoparticle (AuNP) mediated delivery of phloridzin and phloretin and explore their anticancer mechanism through conjugation of the dihydrochalcones and the AuNP cores.

Rapid and simple purification of elastin-like polypeptides directly from whole cells and cell lysates by organic solvent extraction.

Elastin-like polypeptides (ELP) are a well-known class of proteins that are being increasingly utilized in a variety of biomedical applications, due to their beneficial physicochemical properties. A unifying feature of ELP is their demonstration of a sequence tunable inverse transition temperature (Tt) that enables purification using a simple, straightforward process called inverse transition cycling (ITC). Despite the utility of ITC, the process is inherently limited to ELP with an experimentally accessible Tt.