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. A cleavable ELP-Intein construct fused with the enzyme chorismate mutase (ELP-I-Cm2) was used to better understand the organic solvent extraction process for ELP and the factors impacting the retention of enzyme activity. Our extraction studies indicated that a cell lysis step was essential to stabilize the ELP-I-Cm2 in the organic phase, prevent intein cleavage, and extract the fusion protein with high efficiency and retained activity. Circular dichroism and infrared spectroscopic characterization of ELP-I-Cm2 in organic solvents and aqueous solutions of the extracted and precipitated material indicated that the ELP secondary structure was retained in both environments. Atomic force microscopy and negative stain transmission electron microscopy imaging of ELP-I-Cm2 in organic solvents revealed highly regular circular features that were ∼50 nm in diameter, in contrast to larger (>100 nm) irregular features found in aqueous solutions. Since reverse micelles have often been used in catalytic processes, we evaluated the enzymatic activity of the ELP-I-Cm2 reversed micelles in different organic solvent mixtures and found that Cm2-mediated reactions in organic media were of comparable rate and efficiency to those in aqueous media. Based on these findings, we report an exciting new opportunity for ELP-enzyme fusion applications by exploiting their ability to form catalytically active reverse micelles in organic media.

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. In addition, like conventional lipofuscins, it appears as an autofluorescent pigment, considered toxic waste, and a biomarker of aging. Ocular lipofuscin accumulates in the retinal pigment epithelium (RPE), whereby it interferes with the support of the neuroretina. RPE cell death is the primary cause of blindness in the most prevalent incurable genetic and age-related human disorders, Stargardt disease and age-related macular degeneration (AMD), respectively. Although retinal lipofuscin is directly linked to the cell death of the RPE in Stargardt, the extent to which it contributes to AMD is a matter of debate. Nonetheless, the number of AMD clinical trials that target lipofuscin formation speaks for the potential relevance for AMD as well. Here, we show that retinal lipofuscin triggers an atypical necroptotic cascade, amenable to pharmacological intervention. This pathway is distinct from canonic necroptosis and is instead dependent on the destabilization of lysosomes. We also provide evidence that necroptosis is activated in aged human retinas with AMD. Overall, this cytotoxicity mechanism may offer therapeutic targets and markers for genetic and age-related diseases associated with lipofuscin buildups.

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. Herein, we present a robust pathway to optically pure PG in fewer steps, utilizing H-phosphonates that features a chromatographically friendly and stable triethyl ammonium H-phosphonate salt. Our route is also amendable to the simultaneous installation of different acyl chains, either saturated or unsaturated, on the glycerol backbone.

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. Four different ELP purification methods were directly compared for their ability to remove host cell protein, nucleic acids, and lipopolysaccharide (LPS) contaminants using a model ELP. On the basis of these findings, an optimized extraction-precipitation method was developed that gave highly pure ELP from bacterial pellets in approximately 2.5 h while removing major host cell contaminants, including LPS to levels below 1 EU/mL, to produce highly pure material that is suitable for in vivo applications. Application of this method to the rapid purification of an ELP-epidermal growth factor fusion gave an isolate that retained its capacity to bind to epidermal growth factor receptor positive cells, thereby demonstrating that this method is capable of producing a functional construct after purification by organic extraction-precipitation.

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. When delivered by inhalation to a rat model of monocrotaline-induced PAH, cerivastatin significantly reduced PA pressure from 55.13 ± 9.82 to 35.56 ± 6.59 mm Hg (P < 0.001) and diminished PA wall thickening. Echocardiography showed that cerivastatin significantly reduced right ventricle thickening (monocrotaline: 0.34 ± 0.02 cm vs. cerivastatin: 0.26 ± 0.02 cm; P < 0.001) and increased PA acceleration time (monocrotaline: 13.98 ± 1.14 milliseconds vs. cerivastatin: 21.07 ± 2.80 milliseconds; P < 0.001). Nanoliposomal cerivastatin was equally effective or slightly better than cerivastatin in reducing PA pressure (monocrotaline: 67.06 ± 13.64 mm Hg; cerivastatin: 46.31 ± 7.64 mm Hg vs. liposomal cerivastatin: 37.32 ± 9.50 mm Hg) and improving parameters of right ventricular function as measured by increasing PA acceleration time (monocrotaline: 24.68 ± 3.92 milliseconds; cerivastatin: 32.59 ± 6.10 milliseconds vs. liposomal cerivastatin: 34.96 ± 7.51 milliseconds). More importantly, the rate and magnitude of toxic cerivastatin metabolite lactone generation from the intratracheally administered nanoliposomes was significantly lower as compared with intravenously administered free cerivastatin. These studies show that nanoliposome encapsulation improved in vitro and in vivo pharmacologic and safety profile of cerivastatin and may represent a safer approach as a disease-modifying therapy for PAH.

Gd3+:DOTA-Modified 2-Hydroxypropyl-ß-Cyclodextrin/4-Sulfobutyl Ether-ß-Cyclodextrin-Based Polyrotaxanes as Long Circulating High Relaxivity MRI Contrast Agents.

A family of five water-soluble Gd3+:1,4,7,10-tetraazacyclododecane-1,4,7-tetraacetic acid-modified polyrotaxane (PR) magnetic resonance contrast agents bearing mixtures of 2-hydroxypropyl-ß-cyclodextrin and 4-sulfobutylether-ß-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 Gd3+ chelation. The PR products were characterized by 1H NMR, gel permeation chromatography/multiangle light scattering, dynamic light scattering, and analytical ultracentrifugation. Nuclear magnetic relaxation dispersion and molar relaxivity measurements at 23 °C revealed that all the PR contrast agents displayed high spin-spin T1 relaxation and spin-lattice T2 relaxation rates relative to a DOTAREM control. When injected at 0.05 mmol Gd/kg body weight in BALB/c mice, the PR contrast agents increased the T1-weighted MR image intensities with longer circulation times in the blood pool than DOTAREM. Excretion of the agents occurred predominantly via the renal or biliary routes depending on the polyrotaxane structure, with the longest circulating L81 Pluronic-based agent showing the highest liver uptake. Proteomic analysis of PR bearing different ß-cyclodextrin moieties indicated that lipoproteins were the predominant component associated with these materials after serum exposure, comprising as much as 40% of the total protein corona. We infer from these findings that Gd(III)-modified PR contrast agents are promising long-circulating candidates for blood pool analysis by MRI.

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. We report a robotic high throughput technique to execute reactions in multi-well plates that were coupled with fast mass spectrometric analysis using an autosampler to accelerate the reaction screening process. Palladium-catalyzed Suzuki-Miyaura cross-coupling reactions were screened in this system and a heat map was generated to identify the best reaction conditions for downstream scale-up under continuous flow. Here, high throughput experimentation reactions in 96-well plates were performed for 1 h at 50 °C, 100 °C, 150 °C, and 200 °C before diluting them into 384-well plates for mass analysis. With the aid of high throughput tools, 648 unique experiments were conducted in duplicate. The cross-coupling reactions were evaluated as a function of stoichiometry, temperature, concentration, order of addition, and substrate type. The hotspots from high throughput experimentation were examined using a microfluidic Chemtrix system that confirmed the positive reaction leads as true positives. Negative outcomes identified by these experiments were found to be true negatives by microfluidic reaction evaluation. Quantitation of product yields was performed using high-performance liquid chromatography-mass spectrometry (HPLC/MS-MS).

Phospholipase Cß3 Membrane Adsorption and Activation Are Regulated by Its C-Terminal Domains and Phosphatidylinositol 4,5-Bisphosphate.

Phospholipase Cß (PLCß) enzymes hydrolyze phosphatidylinositol 4,5-bisphosphate to produce second messengers that regulate intracellular Ca2+, cell proliferation, and survival. Their activity is dependent upon interfacial activation that occurs upon localization to cell membranes. However, the molecular basis for how these enzymes productively interact with the membrane is poorly understood. Herein, atomic force microscopy demonstrates that the ∼300-residue C-terminal domain promotes adsorption to monolayers and is required for spatial organization of the protein on the monolayer surface. PLCß variants lacking this C-terminal domain display differences in their distribution on the surface. In addition, a previously identified autoinhibitory helix that binds to the PLCß catalytic core negatively impacts membrane binding, providing an additional level of regulation for membrane adsorption. Lastly, defects in phosphatidylinositol 4,5-bisphosphate hydrolysis also alter monolayer adsorption, reflecting a role for the active site in this process. Together, these findings support a model in which multiple elements of PLCß modulate adsorption, distribution, and catalysis at the cell membrane.

Targeting and Internalization of Liposomes by Bladder Tumor Cells Using a Fibronectin Attachment Protein-Derived Peptide-Lipopolymer Conjugate.

A synthetic peptidolipopolymer conjugate, incorporated into liposomes to promote specific binding to the fibronectin (FBN) matrix surrounding bladder tumor cells and promote cellular internalization of FBN-integrin complexes, is reported. The peptide promotes association with MB49 mouse model bladder tumor cells in a sequence-specific and concentration-dependent manner, with the maximum cell association occurring at 2 mol % RWFV-PEG2000-DSPE. Double PEGylation of the liposome membrane (i.e., 4 mol % mPEG1000-DSPE + 2 mol % RWFV-PEG2000-DSPE) enhanced binding by >1.6-fold, by improving ligand presentation on the liposome surface. The sequence specificity of the peptide-lipopolymer construct was confirmed by comparing liposomes containing RWFV-PEG2000-DSPE with scrambled and nonpeptidic lipopolymer liposomal formulations. MB49 tumor-bearing mice showed greater mean radiance values for FAP peptide-targeted liposomes in tumor-associated regions of interest than for nontargeted and scrambled peptide liposome formulations. These findings suggest that peptide-modified liposomes may be an attractive vehicle for targeted delivery to bladder tumors in vivo.

Reaction screening and optimization of continuous-flow atropine synthesis by preparative electrospray mass spectrometry.

Preparative electrospray (ES) exploits the acceleration of reactions in charged microdroplets to perform a small scale chemical synthesis. In combination with on-line mass spectrometric (MS) analysis, it constitutes a rapid screening tool to select reagents to generate specific products. A successful reaction in preparative ES triggers a refined microfluidic reaction screening procedure which includes the optimization for stoichiometry, temperature and residence time. We apply this combined approach for refining a flow synthesis of atropine. A successful preparative ES pathway for the synthesis of the phenylacetyl ester intermediate, using tropine/HCl/phenylacetyl chloride, was optimized for solvent in both the preparative ES and microfluidics flow systems and a base screening was conducted by both methods to increase atropine yield, increase percentage conversion and reduce byproducts. In preparative ES, the first step yielded 55% conversion (judged using MS) to intermediate and the second step yielded 47% conversion to atropine. When combined in two discrete steps in continuous-flow microfluidics, a 44% conversion of the starting material and a 30% actual yield of atropine were achieved. When the reactions were continuously telescoped in a new form of preparative reactive extractive electrospray (EES), atropine was synthesized with a 24% conversion. The corresponding continuous-flow microfluidics experiment gave a 55% conversion with an average of 34% yield in 8 min residence time. This is the first in depth study to utilize telescoped preparative ES and the first use of dual ESI emitters for multistep synthesis.

Mechanistic Insight into Receptor-Mediated Delivery of Cationic-ß-Cyclodextrin:Hyaluronic Acid-Adamantamethamidyl Host:Guest pDNA Nanoparticles to CD44(+) Cells.

Targeted delivery is a key element for improving the efficiency and safety of nonviral vectors for gene therapy. We have recently developed a CD44 receptor targeted, hyaluronic acid-adamantamethamidyl based pendant polymer system (HA-Ad), capable of forming complexes with cationic ß-cyclodextrins (CD-PEI(+)) and pDNA. Complexes formed using these compounds (HA-Ad:CD-PEI(+):pDNA) display high water solubility, good transfection efficiency, and low cytotoxicity. Spatial and dynamic tracking of the transfection complexes by confocal microscopy and multicolor flow cytometry techniques was used to evaluate the target specificity, subcellular localization, and endosomal escape process. Our data shows that cells expressing the CD44 receptor undergo enhanced cellular uptake and transfection efficiency with HA-Ad:CD-PEI(+):pDNA complexes. This transfection system, comprised noncovalent assembly of cyclodextrin:adamantamethamidyl-modified hyaluronic acid via host:guest interactions to condense pDNA, is a potentially useful tool for targeted delivery of nucleic acid therapeutics.

Nonfouling NTA-PEG-Based TEM Grid Coatings for Selective Capture of Histidine-Tagged Protein Targets from Cell Lysates.

We report the preparation and performance of TEM grids bearing stabilized nonfouling lipid monolayer coatings. These films contain NTA capture ligands of controllable areal density at the distal end of a flexible poly(ethylene glycol) 2000 (PEG2000) spacer to avoid preferred orientation of surface-bound histidine-tagged (His-tag) protein targets. Langmuir-Schaefer deposition at 30 mN/m of mixed monolayers containing two novel synthetic lipids-1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[(5-amido-1-carboxypentyl)iminodiacetic acid]polyethylene glycolamide 2000) (NTA-PEG2000-DSPE) and 1,2-(tricosa-10',12'-diynoyl)-sn-glycero-3-phosphoethanolamine-N-(methoxypolyethylene glycolamide 350) (mPEG350-DTPE)-in 1:99 and 5:95 molar ratios prior to treatment with a 5 min, 254 nm light exposure was used for grid fabrication. These conditions were designed to limit nonspecific protein adsorption onto the stabilized lipid coating by favoring the formation of a mPEG350 brush layer below a flexible, mushroom conformation of NTA-PEG2000 at low surface density to enable specific immobilization and random orientation of the protein target on the EM grid. These grids were then used to capture His6-T7 bacteriophage and RplL from cell lysates, as well as purified His8-green fluorescent protein (GFP) and nanodisc solubilized maltose transporter, His6-MalFGK2. Our findings indicate that TEM grid supported, polymerized NTA lipid monolayers are capable of capturing His-tag protein targets in a manner that controls their areal densities, while efficiently blocking nonspecific adsorption and limiting film degradation, even upon prolonged detergent exposure.

Decitabine nanoconjugate sensitizes human glioblastoma cells to temozolomide.

In this study, we developed and characterized a delivery system for the epigenetic demethylating drug, decitabine, to sensitize temozolomide-resistant human glioblastoma multiforme (GBM) cells to alkylating chemotherapy. A poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) based nanoconjugate was fabricated to encapsulate decitabine and achieved a better therapeutic response in GBM cells than that with the free drug. After synthesis, the highly efficient uptake process and intracellular dynamics of this nanoconjugate were monitored by single-molecule fluorescence tools. Our experiments demonstrated that, under an acidic pH due to active glycolysis in cancer cells, the PLGA-PEG nanovector could release the conjugated decitabine at a faster rate, after which the hydrolyzed lactic acid and glycolic acid would further acidify the intracellular microenvironment, thus providing positive feedback to increase the effective drug concentration and realize growth inhibition. In temozolomide-resistant GBM cells, decitabine can potentiate the cytotoxic DNA alkylation by counteracting cytosine methylation and reactivating tumor suppressor genes, such as p53 and p21. Owing to the excellent internalization and endolysosomal escape enabled by the PLGA-PEG backbone, the encapsulated decitabine exhibited a better anti-GBM potential than that of free drug molecules. Hence, the synthesized nanoconjugate and temozolomide could act in synergy to deliver a more potent and long-term antiproliferative effect against malignant GBM cells.

Ultrastructural analysis of 3 hyaluronic acid soft-tissue fillers using scanning electron microscopy.

BACKGROUND: Although hyaluronic acid (HA) specifications such as molecular weight and particle size are fairly well characterized, little information about HA ultrastructural and morphologic characteristics has been reported in clinical literature. OBJECTIVE: To examine uniformity of HA structure, the effects of extrusion, and lidocaine dilution of 3 commercially available HA soft-tissue fillers. MATERIALS AND METHODS: Using scanning electron microscopy and energy-dispersive x-ray analysis, investigators examined the soft-tissue fillers at various magnifications for ultrastructural detail and elemental distributions. RESULTS: All HAs contained oxygen, carbon, and sodium, but with uneven distributions. Irregular particulate matter was present in RES but BEL and JUV were largely particle free. Spacing was more uniform in BEL than JUV and JUV was more uniform than RES. Lidocaine had no apparent effect on morphology; extrusion through a 30-G needle had no effect on ultrastructure. CONCLUSION: Descriptions of the ultrastructural compositions and nature of BEL, JUV, and RES are helpful for matching the areas to be treated with the HA soft-tissue filler architecture. Lidocaine and extrusion through a 30-G needle exerted no influence on HA structure. Belotero Balance shows consistency throughout the syringe and across manufactured lots.

Single-step antibody-based affinity cryo-electron microscopy for imaging and structural analysis of macromolecular assemblies.

Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies (i.e., protein complexes and viruses). Although single particle cryo-EM requires less concentrated and smaller amounts of samples than X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques can potentially further extend single particle cryo-EM to these challenging samples by combining sample purification and cryo-EM grid preparation into a single step. Here we report a new design of affinity cryo-EM approach, cryo-SPIEM, that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method. This approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. In the present work, we extensively tested the feasibility of cryo-SPIEM with multiple samples including those of high or low molecular weight, macromolecules with low or high symmetry, His-tagged or native particles, and high- or low-yield macromolecules. Results for all these samples (non-purified His-tagged bacteriophage T7, His-tagged Escherichiacoli ribosomes, native Sindbis virus, and purified but low-concentration native Tulane virus) demonstrated the capability of cryo-SPIEM approach in specifically trapping and concentrating target particles on TEM grids with minimal view constraints for cryo-EM imaging and determination of 3D structures.

Photoassisted one-step aerosol fabrication of zwitterionic chitosan nanoparticles.

Zwitterionic chitosan nanoparticles (ZCNPs) were conveniently obtained by a one-step aerosol method, and their potential for the production of biocompatible materials was investigated. A low-molecular-weight chitosan was conjugated with succinic anhydride to produce zwitterionic chitosan (ZC). Collison-atomized ZC droplets were simultaneously UV-irradiated and dried in a tube furnace in a one-step aerosol process to produce particles. The observed cytotoxicities of ZCNPs (85 ± 3.9% cell viability) were similar to unmodified chitosan nanoparticles (CNPs, 88 ± 6.6%) and UV-irradiated ZCNPs (83 ± 3.3%). The aerosol process described in this work allowed facile production and modification of CNPs, which could then be employed for biomedical purposes.

Effect of pendant group on pDNA delivery by cationic-ß-cyclodextrin:alkyl-PVA-PEG pendant polymer complexes.

We have previously shown that cationic-ß-cyclodextrin:R-poly(vinyl alcohol)-poly(ethylene glycol) (CD+:R-PVA-PEG) pendant polymer host:guest complexes are safe and efficient vehicles for nucleic acid delivery, where R = benzylidene-linked adamantyl or cholesteryl esters. Herein, we report the synthesis and biological performance of a family of PVA-PEG pendant polymers whose pendant groups have a wide range of different affinities for the ß-CD cavity. Cytotoxicity studies revealed that all of the cationic-ß-CD:pendant polymer host:guest complexes have 100-1000-fold lower toxicity than branched polyethylenimine (bPEI), with pDNA transfection efficiencies that are comparable to bPEI and Lipofectamine 2000. Complexes formed with pDNA at N/P ratios greater than 5 produced particles with diameters in the 100-170 nm range and ζ-potentials of 15-35 mV. Gel shift and heparin challenge experiments showed that the complexes are most stable at N/P ≥ 10, with adamantyl- and noradamantyl-modified complexes displaying the best resistance toward heparin-induced decomplexation. Disassembly rates of fluoresceinated-pDNA:CD(+):R-PVA-PEG-rhodamine complexes within HeLa cells showed a modest dependence on host:guest binding constant, with adamantyl-, noradamantyl-, and dodecyl-based complexes showing the highest loss in FRET efficiency 9 h after cellular exposure. These findings suggest that the host:guest binding constant has a significant impact on the colloidal stability in the presence of serum and cellular uptake efficiency, whereas endosomal disassembly and transfection performance of cationic-ß-CD:R-poly(vinyl alcohol)-poly(ethylene glycol) pendant polymer complexes appears to be controlled by the hydrolysis rates of the acetal grafts onto the PVA main chain.

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). Side-by-side comparison of expression and purification in both insect and bacterial systems shows that the insect expression method provides superior yields of the desired autoinhibited CaMKIIα holoenzymes. Utilizing baculovirus insect expression system tools, our results demonstrate a high yield method to produce homogenous, monodisperse CaMKII in its autoinhibited state suitable for biophysical analysis. Advantages and disadvantages of these two expression systems (baculovirus insect cell versus Escherichia coli expression) are discussed, as well as purification optimizations to maximize the enrichment of full-length CaMKII.

Synthesis, characterization, and evaluation of pluronic-based ß-cyclodextrin polyrotaxanes for mobilization of accumulated cholesterol from Niemann-Pick type C fibroblasts.

Several lines of evidence suggest that ß-cyclodextrin (ß-CD) derivatives initiate the efflux of accumulated, unesterified cholesterol from the late endosomal/lysosomal compartment in Niemann Pick C (NPC) disease models. Unfortunately, repeated injections or continuous infusions of current ß-CD therapies are required to sustain suppression of symptoms and prolong life. In an effort to make CD treatment a more viable option by boosting efficacy and improving pharmacokinetics, a library of Pluronic surfactant-based ß-CD polyrotaxanes has been developed using biocompatible poly(ethylene glycol) (PEG)-polypropylene glycol (PPG)-PEG triblock copolymers. These compounds carry multiple copies of ß-CD as shown by (1)H NMR, 2D nuclear Overhouser effect spectroscopy, gel permeation chromatography/multiangle light scattering, analytical ultracentrifugation analysis, matrix assisted laser desorption/ionization mass spectrometry, and diffusion-ordered spectroscopy. Analyses of free ß-cyclodextrin contamination in the compounds were made by reverse phase high pressure liquid chromatography and hydrophilic interaction liquid chromatography. Dethreading kinetics were studied by reverse phase high pressure liquid chromatography, UV/vis, and (1)H NMR analysis. Filipin staining studies using npc2(-/-) fibroblasts show significant reversal of cholesterol accumulation after treatment with polyrotaxane compounds. The rate and efficacy of reversal is similar to that achieved by equivalent amounts of monomeric ß-CD alone.

Cationic α-cyclodextrin:poly(ethylene glycol) polyrotaxanes for siRNA delivery.

RNA interference has broad therapeutic potential due to its high specificity and ability to potentially evade drug resistance. Three cationic α-cyclodextrin:poly(ethylene glycol) polyrotaxanes derived from polymer axles of different sizes (MW 2,000, 3,400, and 10,000) have been synthesized for delivering siRNA. These polyrotaxanes are able to condense siRNA into positively charged particles that are <200 nm in diameter, enabling their facile internalization into mammalian cells. The cationic polyrotaxanes display cytotoxicity profiles that are >10(2)-fold lower than the commercial standard bPEI and gene silencing efficiencies that are comparable to those of both Lipofectamine 2000 and bPEI. Our findings suggest that the cationic polyrotaxanes display a size-activity relationship, wherein the higher molecular weight polyrotaxanes (PEG3,400 and 10,000) are able to condense and deliver siRNA better than the lower molecular weight material (PEG2,000).