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; 
&#160; 
GNP-based drug formulation provides several advantages. First, GNPs can be easily synthesized in well controlled sizes, ranging from a few nanometers to ~100 nm. Usually, the smaller sized GNPs can deliver drugs into cells more efficiently than the larger ones as the smaller ones can easily diffuse into their target region24.&#160; Second, GNPs are non-toxic in vitro and in vivo25, making them safe drug carriers.&#160; Third, hydrophobic drugs can be loaded onto the modified GNPs26.&#160; Forth, the surface chemistry of GNPs is readily modified for specific applications. In our studies, two short peptides are used to modify the GNP surface, to stabilize them in physiological conditions and impart new bioactivities. The peptides were thoughtfully designed with three regions including gold binding, spacing, and functional regions.&#160; Specifically, the N terminus of the peptide has cysteine (C) residue containing thiol group that can bind with gold. The middle part has four hydrophobic alanine residues to promote peptide assembly into a densely packed monolayer on the GNPs. The amino acid at the C terminus is a functional amino acid pointing outward, which can be used to manipulate the surface properties of the GNPs. The 95:5 ratio of these two peptides was systemically selected in a previous study14. The ratio between PKCi and P2/P4 peptides was also systemically tested and selected15.&#160;&#160;
On the other hand, the GNP drug delivery system has limitations. GNPs are not cell type or tissue specific. GNPs are mainly accumulated in the lung, liver, and spleen after intravenous administration27,28,29. So far, this formula has only been tested in cell culture and small animal models. To translate it to clinical application, further studies on large animal models and its pharmacokinetics, tissue distribution and potential toxicity need to be determined.

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 absorption will be shifted to the right. As an alternate method of analysis, when aggregates have formed the &#916;Optical Density (&#916;OD = OD at 525 nm - OD at 440 nm) decreases.&#160;
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58741/58741fig1v2.jpg" /> 
Figure 1: Quality of GNP/PKCi. (A) Properly prepared GNP/PKCi is pink in color (left). Aggregated GNP/PKCi appears light purple (right). (B) Good GNP/PKCi preparation is stable in water, PBS, or in 0.9% NaCl solution. Readings on a UV-Vis spectrometer indicated that the peak of absorption was at 525 nm in all solutions. (C) An example of good and bad GNP/PKCi preparations. When the GNP was aggregated, the peak of absorption was shifted to the right. Moreover, &#916;OD decreased. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/58741/58741fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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. 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.

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

Formulation of Diblock Polymeric Nanoparticles through Nanoprecipitation Technique

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale (nanoparticles). Nanoparticles can be engineered in a precise fashion where their size, composition and surface chemistry can be carefully controlled. This enables unprecedented freedom to modify some of the fundamental properties of their cargo, such as solubility, diffusivity, biodistribution, release characteristics and immunogenicity. Since their inception, nanoparticles have been utilized in many areas of science and medicine, including drug delivery, imaging, and cell biology1-4. However, it has not been fully utilized outside of "nanotechnology laboratories" due to perceived technical barrier. In this article, we describe a simple method to synthesize a polymer based nanoparticle platform that has a wide range of potential applications. 
The first step is to synthesize a diblock co-polymer that has both a hydrophobic domain and hydrophilic domain. Using PLGA and PEG as model polymers, we described a conjugation reaction using EDC/NHS chemistry5 (Fig 1). We also discuss the polymer purification process. The synthesized diblock co-polymer can self-assemble into nanoparticles in the nanoprecipitation process through hydrophobic-hydrophilic interactions.
The described polymer nanoparticle is very versatile. The hydrophobic core of the nanoparticle can be utilized to carry poorly soluble drugs for drug delivery experiments6. Furthermore, the nanoparticles can overcome the problem of toxic solvents for poorly soluble molecular biology reagents, such as wortmannin, which requires a solvent like DMSO. However, DMSO can be toxic to cells and interfere with the experiment. These poorly soluble drugs and reagents can be effectively delivered using polymer nanoparticles with minimal toxicity. Polymer nanoparticles can also be loaded with fluorescent dye and utilized for intracellular trafficking studies. Lastly, these polymer nanoparticles can be conjugated to targeting ligands through surface PEG. Such targeted nanoparticles can be utilized to label specific epitopes on or in cells7-10.

This article describes a nanoprecipitation method to synthesize polymer-based nanoparticles using diblock co-polymers. We will discuss the synthesis of diblock co-polymers, the nanoprecipitation technique, and potential applications.

Nanotechnology is a relatively new branch of science that involves harnessing the unique properties of particles that are nanometers in scale ...

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. ...

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold nanoclusters (Au NCs) within the drug-loaded protein, based on the differential fluorescence signal emitted by the Au NCs. Albumin proteins such as human serum albumin (HSA) and bovine serum albumin (BSA) are selected as the model proteins. Four small molecular drugs (e.g., ibuprofen, warfarin, phenytoin, and sulfanilamide) of different binding affinities to the albumin proteins are tested. It was found that the formation rate of fluorescent Au NCs inside the drug loaded albumin protein under denaturing conditions (i.e., 60 &#176;C or in the presence of urea) is slower than that formed in the pristine protein (without drugs). Moreover, the fluorescent intensity of the as-formed NCs is found to be inversely correlated to the binding affinities of these drugs to the albumin proteins. Particularly, the higher the drug-protein binding affinity, the slower the rate of Au NCs formation, and thus a lower fluorescence intensity of the resultant Au NCs is observed. The fluorescence intensity of the resultant Au NCs therefore provides a simple measure of the relative binding strength of different drugs tested. This method is also extendable to measure the specific drug-protein binding constant (KD) by simply varying the drug content preloaded in the protein at a fixed protein concentration. The measured results match well with the values obtained using other prestige but more complicated methods.

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

We demonstrate a new drug screening method for determining the binding affinity of small drug molecules to a target protein by forming fluorescent gold ...

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 ...

Cancer Research

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 ...

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 ...