Name: protein kinase c-delta inhibitor peptide formulation using gold nanoparticles
Uploaded: 2019-03-09T14:00:00.000+00:00
Duration: 6 min 6 s
Description: Watch this Scientific Journal Video about Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles at JoVE.com

This work is supported by research grants from Canadian Institutes of Health Research (PJT-148847), Ministry of Research and Innovation of Ontario (RE-08-029), and Canada First Research of Excellence Program, Medicine by Design at University of Toronto. Dr. Mingyao Liu is James and Mary Davie Chair in Lung Injury, Repair, and Regeneration.

1. Preparation of Peptide Solutions
<ol>
	<li>Retrieve the peptides (P2: CAAAAE, P4: CAAAAW, PKC&#948;i: CSFNSYELGSL) from the -20 &#176;C freezer and thaw at room temperature (RT). 
	NOTE: Keep the bottle closed to prevent moisture from condensing on the peptides.</li>
	<li>Weigh 0.01 g of each peptide on a microscale. Put each peptide into a separate 50 mL conical tube.</li>
	<li>Add 18.74 mL of deionized (DI) water to the P2 tube.</li>
	<li>Add 16.93 mL of DI water to the P4 tube.</li>
	<li>Add 8.21 mL of 50% acetonitrile diluted in DI water to the PKC&#948;i tube.</li>
	<li>Vortex the peptide solutions briefly. Put the 50 mL conical tubes in a sonicator (40 MHz) for 5 min.</li>
	<li>Bring the peptide solutions to a biosafety cabinet. All peptide solutions prepared should be 1 mM.</li>
	<li>Transfer 1 mL of each peptide solution to a new 50 mL conical tube. Add 19 mL of DI water to the tubes of P2 and P4 and add 19 mL of 50% acetonitrile to the PKC&#948;i tube, such that each solution is diluted to 50 &#181;M and stored in its own tube.</li>
</ol>
2. Formulation of GNP/PKCi
<ol>
	<li>Peptides should be added to the GNP solution while still in the BSC</li>
	<li>Add 475 &#956;L of P2, 25&#956;L of P4 and 500 &#956;L of &#948;PKCi solution into a 15 mL tube. Add 9 mL of 20 nm GNP solution (7.0x1011 particle/mL) to the same 15 mL tube.</li>
	<li>Exit the biosafety cabinet. Wrap the 15 mL tube with aluminum foil. Leave it on a shaker at RT overnight.</li>
	<li>Return the samples to the biosafety cabinet. Aliquot 1 mL of GNP/PKCi into each 1.5 mL microtubes.</li>
	<li>Centrifuge the tubes in a micro-centrifuge for 30 min at 15,294 x g at 4 &#176;C.</li>
	<li>Remove the supernatant from each tube under a biosafety cabinet. 
	NOTE: Be careful to remove the supernatant while ensuring that the GNP pellet remains intact and is not aspirated.</li>
	<li>Re-suspend the pellet in the desired solvent according to the concentration required. Applicable solvents can be DI water, PBS, and 0.9% NaCl. 
	NOTE: Starting from 1 mL of GNP/PKCi, the GNP pellet contains 6.3 x 1011 particles, based on the GNP concentration provided by the manufacturer. To administer 1.3 x 1012 particles in 500 &#181;L of 0.9% NaCl, we add 232 &#181;L of 0.9% NaCl to each of three pellets. After pooling them together, we can then collect 500 &#181;L of GNP/PKCi solution. 
	NOTE: Mix the desired solvent well before diluting the GNP/PKCi pellet, otherwise the GNP/PKCi will aggregate.</li>
</ol>
3. Assessment of GNP/PKCi Hybrid Solubility
<ol>
	<li>Pour 0.5 mL of GNP/PKCi solution into an acryl cuvette. Place the acryl cuvette on a UV-Vis spectrophotometer and test the peak absorption15.</li>
</ol>

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The authors have nothing to disclose on this project.

To ensure proper formulation, it is crucial that the &#948;PKCi solution undergoes the sonication step outlined in 1.6. &#948;PKCi peptide sequence contains hydrophobic moieties, so a sonicator assists in dissolving PKCi in the 50% acetonitrile solution. In addition, it is very important to mix the solvent meticulously, as outlined in step 2.7.&#160; The GNP/PKCi hybrid will not be well formulated if these steps are not done properly, due to the aggregation of the &#948;PKCi peptide23.&#160; ...

Lung transplantation saves patients with end-stage lung disease1. However, serious complications after lung transplantation remain an obstacle. In the early stages following lung transplantation, primary graft dysfunction is the most harmful complication1, and its primary cause is ischemia-reperfusion (IR)-induced acute lung injury2.
Under cold preservation, metabolism in a donor lung is restricted to a very low level. However, reactive oxygen species and nitric oxide synthesis are activated due to the cessation of blood flow3. After transplantation, blood circulation is restored, and reactive oxygen species and nitric oxide generated during cold ischemia enhance inflammation and cell death, resulting in tissue injury.
To prevent IR injury, a protein kinase C&#948; inhibitor (PKC&#948;i) has been used in the heart, brain and lung4,5,6,7,8. These studies showed that PKC&#948;i decreased inflammation and apoptosis during reperfusion. It has also prevented pulmonary IR injury in rats and in a lung transplant model6. PKC&#948;i is usually conjugated with a cell-penetrating peptide, TAT, for intracellular delivery. However, it has been shown that the TAT peptide alone has non-specific biological effects, including promotion of angiogenesis, apoptosis, and inhibition of multiple cytokines9,10,11. Nanoparticles, small particles ranging from 1 to 100 nm in diameter12, have been explored as candidates in facilitating drug delivery13. In particular, gold nanoparticles (GNPs) are regarded as noninvasive and nontoxic. Therefore, we have developed GNPs as drug delivery carriers for peptide-based drugs14,15.
The surface of GNPs can be manipulated for specific applications such as molecular recognition16,17, chemical sensing18, imaging19, and drug delivery. A GNP/peptide hybrid system has been developed, containing 20 nm GNPs and two short peptides (P2: CAAAAE and P4: CAAAAW) at a 95:5 ratio, to modify the surface properties of GNPs. The P2 peptide, with the negatively charged glutamic acid (E) at the end, stabilizes GNPs in an aqueous solution, and the P4 peptide, with the hydrophobic tryptophan (W) at the end, helps GNPs entrance into cells14. The cysteine (C) residue at the N terminus of these peptides contains a thiol group that can conjugate to the gold surfaces14. This peptide/GNP hybrid was further used to deliver PKC&#948;i (CSFNSYELGSL). The optimized molar ratio of P2:P4 to PKC&#948;i is 47.5:2.5:50. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and PBS&#160;containing bovine albumin or fetal bovine serum14. Intravenous injection of GNP/PKCi has been shown to prevent ischemia-reperfusion injury of the lung15. This article outlines a method to formulate GNP/PKCi and describes how to evaluate the physicochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs conjugated to GNP20,21,22. We hope this article will draw more attention to this novel formulation for intracellular drug delivery.

<table><tbody><tr><td>negatively charged glutamic acid peptide (P2)</td><td>CanPeptide</td><td></td><td>Sequence: CAAAAE-NH2&lt;br/&gt; Length: 6aa&lt;br/&gt; Modification: C-terminal amidation&lt;br/&gt; Quantity: 50mg&lt;br/&gt; Purity: &gt;95%</td></tr><tr><td>hydrophobic tryptophan peptide (P4)</td><td>CanPeptide</td><td></td><td>Sequence: CAAAAW-NH2&lt;br/&gt; Length: 6aa&lt;br/&gt; Modification: C-terminal amidation&lt;br/&gt; Quantity: 50mg&lt;br/&gt; Purity: &gt;95%</td></tr><tr><td>&amp;delta;PKCi peptide</td><td>CanPeptide</td><td></td><td>Seqeuence: CSFNSYELGSL-NH2&lt;br/&gt; Length: 11aa&lt;br/&gt; Modification: C-terminal amidation&lt;br/&gt; Quantity: 50mg&lt;br/&gt; Purity: &gt;95%</td></tr><tr><td>Conical tube(50ml)</td><td>Corning Life Sciences</td><td>3582070</td><td></td></tr><tr><td>Conical tube(15ml)</td><td>Corning Life Sciences</td><td>3582096</td><td></td></tr><tr><td>Acetonitrile</td><td>Sigma-Aldrich</td><td>271004-100ML</td><td></td></tr><tr><td>Sonicator</td><td>Branson Ultrasonics Corp.</td><td>Branson 2510MTH</td><td></td></tr><tr><td>Microtube</td><td>Diamed.ca</td><td>AD 150-N</td><td></td></tr><tr><td>Gold nanoparticle solution</td><td>Ted Pella</td><td>15705-5</td><td>A particle size is 20nm</td></tr><tr><td>Rocking Platform shaker</td><td>VWR international</td><td>40000-304&amp;nbsp;</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>5417R</td><td></td></tr><tr><td>Acryl cuvette</td><td>SARSREDT</td><td>67.758</td><td></td></tr><tr><td>UV-Vis spectrophotometer</td><td>Agilent</td><td>Caty 60 UV-Vis</td><td></td></tr></tbody></table>

protein kinase c-delta inhibitor peptide formulation using gold nanoparticles

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a cell-penetrating peptide, TAT, for intracellular delivery. However, TAT has shown non-specific biological activities. Gold nanoparticles (GNPs) can be used as drug delivery carriers without recognized toxicity. Therefore, we have used a GNP/peptide hybrid to deliver PKC&#948;i. Two short peptides (P2: CAAAAE and P4: CAAAAW), at a 95:5 ratio, were used to modify the surface properties of GNP. GNPs conjugated with PKC&#948;i (GNP/PKCi) are stable in distilled water, 0.9% NaCl, and phosphate-buffered saline (PBS) containing bovine serum albumin or fetal bovine serum. Intravenous injection of GNP-PKCi was previously shown to prevent ischemia-reperfusion injury of the lung. This article outlines a protocol to formulate GNP/PKCi and assess the physiochemical properties of GNP/PKCi. We have used similar methods to formulate other peptide-based drugs with GNP. This article will hopefully draw more attention to this novel intracellular drug delivery technology and its applications in vivo.

Care should be taken to evaluate the biophysical properties of the GNP/PKCi hybrid, as GNP tends to aggregate in solvent. When GNP is aggregated, the color of the solution changes from pink to purple (Figure&#160;1a). UV-Vis spectrophotometer is able to detect the changes more sensitively. If the GNP/PKCi is not aggregated the peak of absorption should be at 525 nm (Figure&#160;1b). If the GNP is aggregated, the peak of absorptio...

Watch this Scientific Journal Video about Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles at JoVE.com

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

We have previously used a gold nanoparticle peptide hybrid to intravenously deliver a synthetic peptide, protein kinase C-delta inhibitor, which reduced ischemia-reperfusion-induced acute lung injury. Here we show the detailed protocol of the drug formulation. Other intracellular peptides can be formulated similarly.

Protein kinase C-delta inhibitor (PKC&#948;i) is a promising drug to prevent ischemia-reperfusion-induced organ injury. It is usually conjugated to a ...

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5.5K Views.  University Health Network. Ons unieke drug leveringssysteem, dat 20 nanometer gouddeeltjes in twee specifieke korte peptiden gebruikt, kan gouden nanodeeltjes in fysiologische oplossing stabiliseren en zijn cellulaire opname verbeteren, en zo, effectief peptide-gebaseerde of andere drugs in cellen leveren. Onze GNP peptide hybride is eenvoudig te synthetiseren en stabiel. Door het gebruik van gouden nanodeeltjes peptide hybride als drug dragers, kunnen we de overdracht peptide drugs in de cellen effectief en in overvlo...

Video: Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Synthesis and Characterization of Placental Chondroitin Sulfate A (plCSA)-Targeting Lipid-Polymer Nanoparticles

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising approach to cancer therapy. The glycosaminoglycan placental chondroitin sulfate A (plCSA) is expressed on a wide range of cancer cells and placental trophoblasts, and malarial protein VAR2CSA can specifically bind to plCSA. A reported placental chondroitin sulfate A binding peptide (plCSA-BP), derived from malarial protein VAR2CSA, can also specifically bind to plCSA on cancer cells and placental trophoblasts. Hence, plCSA-BP-conjugated nanoparticles could be used as a tool for targeted drug delivery to human cancers and placental trophoblasts. In this protocol, we describe a method to synthesize plCSA-BP-conjugated lipid-polymer nanoparticles loaded with doxorubicin (plCSA-DNPs); the method consists of a single sonication step and bioconjugate techniques. In addition, several methods for characterizing plCSA-DNPs, including determining their physicochemical properties and cellular uptake by placental choriocarcinoma (JEG3) cells, are described.

Synthesis and Characterization of Placental Chondroitin Sulfate A plCSA-Targeting Lipid-Polymer Nanoparticles

Here, we present a protocol for the synthesis of placental chondroitin sulfate A binding peptide (plCSA-BP)-conjugated lipid-polymer nanoparticles via single-step sonication and bioconjugate techniques. These particles constitute a novel tool for the targeted delivery of therapeutics to most human tumors and placental trophoblasts to treat cancers and placental disorders.

An effective cancer therapeutic method reduces and eliminates tumors with minimal systemic toxicity. Actively targeting nanoparticles offer a promising ...

Cancer Research

Preparation and Photoacoustic Analysis of Cellular Vehicles Containing Gold Nanorods

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to their intense optical absorbance in the near-infrared window, low cytotoxicity and potential to home into tumors. However, their delivery to tumors still remains an issue. An innovative approach consists of the exploitation of the tropism of tumor-associated macrophages that may be loaded with gold nanorods in vitro. Here, we describe the preparation and the photoacoustic inspection of cellular vehicles containing gold nanorods. PEGylated gold nanorods are modified with quaternary ammonium compounds, in order to achieve a cationic profile. On contact with murine macrophages in ordinary Petri dishes, these particles are found to undergo massive uptake into endocytic vesicles. Then these cells are embedded in biopolymeric hydrogels, which are used to verify that the stability of photoacoustic conversion of the particles is retained in their inclusion into cellular vehicles. We are confident that these results may provide new inspiration for the development of novel strategies to deliver plasmonic particles to tumors.

We show the preparation and address the feasibility of cellular vehicles containing gold nanorods for the photoacoustic imaging of cancer.

Gold nanorods are attractive for a range of biomedical applications, such as the photothermal ablation and the photoacoustic imaging of cancer, thanks to ...

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

The development of immune checkpoint blockade therapy based on programmed cell death-protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) has revolutionized cancer therapies in recent years. However, only a fraction of patients responds to PD-1/PD-L1 inhibitors, owing to the heterogeneous expression of PD-L1 in tumor cells. This heterogeneity presents a challenge in the precise detection of tumor cells by the commonly used immunohistochemistry (IHC) approach. This situation calls for better methods to stratify patients who will benefit from immune checkpoint blockade therapy, to improve treatment efficacy. Positron emission tomography (PET) enables real-time visualization of the whole-body PD-L1 expression in a noninvasive way. Therefore, there is a need for the development of radiolabeled tracers to detect PD-L1 distribution in tumors through PET imaging.
Compared to their L-counterparts, dextrorotary (D)-peptides have properties such as proteolytic resistance and remarkably prolonged metabolic half-lives. This study designed a new method to detect PD-L1 expression based on PET imaging of 68Ga-labeled PD-L1-targeted D-peptide, a D-dodecapeptide antagonist (DPA), in tumor-bearing mice. The results showed that the [68Ga]DPA can specifically bind to PD-L1-overexpressing tumors in vivo, and showed favorable stability as well as excellent imaging ability, suggesting that [68Ga]DPA-PET is a promising approach for the assessment of PD-L1 status in tumors.

Development of a 68Gallium-Labeled D-Peptide PET Tracer for Imaging Programmed Death-Ligand 1 Expression

This study developed a noninvasive and real-time method to evaluate the distribution of programmed death-ligand 1 in the whole body, based on positron emission tomographic imaging of [68Ga] D-dodecapeptide antagonist. This technique has advantages over conventional immunohistochemistry and improves the efficiency of identifying appropriate patients who will benefit from immune checkpoint blockade therapy.

The development of immune checkpoint blockade therapy based on programmed cell death-protein 1 (PD-1)/programmed death-ligand 1 (PD-L1) has revolutionized ...

Synthesis of Aptamer-PEI-g-PEG Modified Gold Nanoparticles Loaded with Doxorubicin for Targeted Drug Delivery

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the designing of poly(ethylenimine) grafted with polyethylene glycol (PEI-g-PEG) copolymer functionalized gold nanoparticles (AuNPs) with loaded aptamer (AS1411) and DOX through amide reactions. AS1411 is specifically bonded with targeted nucleolin receptors on cancer cells so that DOX targets cancer cells instead of healthy cells. First, PEG is carboxylated, then grafted to branched PEI to obtain a PEI-g-PEG copolymer, which is confirmed by 1H NMR analysis. Next, PEI-g-PEG copolymer coated gold nanoparticles (PEI-g-PEG@AuNPs) are synthesized, and DOX and AS1411 are covalently bonded to AuNPs gradually via amide reactions. The diameter of the prepared AS1411-g-DOX-g-PEI-g-PEG@AuNPs is ~39.9 nm, with a zeta potential of -29.3 mV, indicating that the nanoparticles are stable in water and cell medium. Cell cytotoxicity assays show that the newly designed DOX loaded AuNPs are able to kill cancer cells (A549). This synthesis demonstrates the delicate arrangement of PEI-g-PEG copolymers, aptamers, and DOX on AuNPs that are achieved by sequential amide reactions. Such aptamer-PEI-g-PEG functionalized AuNPs provide a promising platform for targeted drug delivery in cancer therapy.

In this protocol, doxorubicin-loaded AS1411-g-PEI-g-PEG modified gold nanoparticles are synthesized via three-step amide reactions. Then, doxorubicin is loaded and delivered to target cancer cells for cancer therapy.

Due to drug resistance and toxicity in healthy cells, use of doxorubicin (DOX) has been limited in clinical cancer therapy. This protocol describes the ...

Chemistry

Formulation and Characterization of Bioactive Agent Containing Nanodisks

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. Nanodisks are organized as a disk-shaped lipid bilayer whose perimeter is circumscribed by the scaffold protein, usually a member of the exchangeable apolipoprotein family. Numerous hydrophobic bioactive agents have been efficiently solubilized in nanodisks by their integration into the hydrophobic milieu of the particle&#39;s lipid bilayer, yielding a largely homogenous population of particles in the range of 10-20 nm in diameter. The formulation of nanodisks requires a precise ratio of individual components, an appropriate sequential addition of each component, followed by bath sonication of the formulation mixture. The amphipathic scaffold protein spontaneously contacts and reorganizes the dispersed bilayer forming lipid/bioactive agent mixture to form a discrete, homogeneous population of nanodisk&#160;particles. During this process, the reaction mixture transitions from an opaque, turbid appearance to a clarified sample that, when fully optimized, yields no precipitate upon centrifugation. Characterization studies involve the determination of bioactive agent solubilization efficiency, electron microscopy, gel filtration chromatography, ultraviolet visible (UV/Vis) absorbance spectroscopy, and/or fluorescence spectroscopy. This is normally followed by an investigation of biological activity using cultured cells or mice. In the case of nanodisks harboring an antibiotic (i.e., the macrolide polyene antibiotic amphotericin B), their ability to inhibit the growth of yeast or fungi as a function of concentration or time can be measured. The relative ease of formulation, versatility with respect to component parts, nanoscale particle size, inherent stability, and aqueous solubility permits myriad in vitro and in vivo applications of nanodisk technology. In the present article, we describe a general methodology to formulate and characterize nanodisks containing amphotericin&#160;B as the hydrophobic bioactive agent.

Here, we describe the production and characterization of bioactive agents containing nanodisks. Amphotericin B nanodisks are taken as an example to describe the protocol in a stepwise manner.

The term nanodisk refers to a discrete type of nanoparticle comprised of a bilayer forming lipid, a scaffold protein, and an integrated bioactive agent. ...

Biochemistry

Synthesis of Functionalized 10-nm Polymer-coated Gold Particles for Endothelium Targeting and Drug Delivery

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting and drug delivery are some of the applications of these AuNPs, but endothelial extracellular matrices&#39; defensive properties hamper particle uptake. To address this issue, we describe a synthesis method for ultrasmall gold nanoparticles to improve vascular delivery, with customizable functional groups and polymer lengths for further adjustments. The protocol yields 2.5 nm AuNPs that are capped with tetrakis(hydroxymethyl)phosphonium chloride (THPC). The replacement of THPC with hetero-functional polyethylene glycol (PEG) on the surface of the AuNP increases the hydrodynamic radius to 10.5 nm while providing various functional groups on the surface. The last part of the protocol includes an optional addition of a fluorophore to allow the AuNPs to be visualized under fluorescence to track nanoparticle uptake. Dialysis and lyophilization were used to purify and isolate the AuNPs. These fluorescent nanoparticles can be visualized in both in vitro and in vivo experiments due to the biocompatible PEG coating and fluorescent probes. Additionally, the size range of these nanoparticles render them an ideal candidate for probing the glycocalyx without disrupting normal vasculature function, which may lead to improved delivery and therapeutics.

We describe a method of synthesizing biocompatible 10-nm gold nanoparticles, functionalized by coating poly-ethylene glycol onto the surface. These particles can be used in vitro and in vivo for delivering therapeutics to nanoscale cellular and extracellular spaces that are difficult to access with conventional nanoparticle sizes.

Gold nanoparticles (AuNPs) have been used extensively in medical research due to their size, biocompatibility, and modifiable surface. Specific targeting ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

Biosensor-based High Throughput Biopanning and Bioinformatics Analysis Strategy for the Global Validation of Drug-protein Interactions

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation of the drug-protein interactions is highly desirable for not only understanding the molecular mechanisms underlying therapeutic efficacy but also for assessing drug characteristics, such as adsorption, distribution, metabolism, excretion, and toxicity (ADMET) for clinical use. Here, we present a biosensor-based high throughput strategy for the biopanning of T7 phage-displayed short peptides that can be easily displayed on the phage capsid. Subsequent analysis of the amino acid sequences of peptides containing short segments, as &#34;broken relics&#34;, of the drug-binding sites using bioinformatics programs in receptor ligand contact (RELIC) suite, is also shown. By applying this method to two clinically approved drugs, an anti-tumor irinotecan, and an anti-flu oseltamivir, the detailed process for collecting the drug-recognizing peptide sequences and highlighting the drug-binding sites of the target proteins are explained in this paper. The strategy described herein can be applied for any small molecules of interest.

This study aimed to present a strategy for identifying drug-peptide interactions. The strategy involves the biopanning of drug-recognizing short peptides based on a quartz-crystal microbalance (QCM) biosensor, followed by bioinformatics analysis for quantitatively assessing the information obtained for the drug recognition and annotation of the drug-binding sites on proteins.

Receptors and enzyme proteins are important biomolecules that act as binding targets for bioactive small molecules. Thus, the rapid and global validation ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Protein Kinase C-delta Inhibitor Peptide Formulation using Gold Nanoparticles

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