This work was funded by grants to LKM-K from the National Institutes of Health/National Cancer Institute (R01CA129822 and R01CA140995). Dr. Medina-Kauwe thanks C. Rey, M. M-Kauwe and D. Revetto for continued support.

1. Systemic Delivery In vivo
<ol>
	<li>Mix enough HerDox with sterile saline to equate 0.2 ml of a 0.004 mg/kg dose of HerDox per injection for a 6-8 week old NU/NU mouse bearing subcutaneous bilateral flank xenograft tumors.</li>
	<li>Gently draw the HerDox mixture into a 3/10 cc insulin syringe fitted with a 29G needle, avoiding bubbles.</li>
	<li>Anesthesia is induced by brief isoflurane exposure in an induction chamber equipped with a gas scavenging system (Oxygen flow rates: 0.5-1 L/min, isoflurane concentration: 3-4% (or lower).</li>
	<li>Inject the entire mixture into the tail vein of an anesthetized mouse (0.2 ml per injection). IV injection can also be performed in a restrained, unanesthetized mouse.</li>
	<li>Repeat injections on the same mouse for six more sequential days, once per day.</li>
</ol>
2. Fluorescence Imaging In vivo
The accumulation of HerDox fluorescence in tumors can be detectable by the last day of injection (Day 7) using a multimode imager. The procedures below entail the use of a customized macro-illumination and detection system (Figure 1) 8.
<ol>
	<li>Turn on the Multimode In vivo Optical Imager.</li>
	<li>Select an emission bandpass filter (590 nm &plusmn; 30 nm) suited for doxorubicin fluorescence detection.</li>
	<li>Turn on Argon-Krypton laser and place an excitation bandpass filter (488 nm &plusmn; 10 nm) at the laser optical path.</li>
	<li>Turn on the anesthesia system and then place a mouse into the anesthetizing chamber (Oxygen flow rates: 0.5-1 L/min, isoflurane concentration: 3-4% (or lower)equipped with a gas scavenging system.</li>
	<li>Transfer the mouse from the anesthetizing chamber to the imaging chamber of the Multimode In vivo Optical Imager when the mouse is anesthetized.</li>
	<li>Place a nosecone over the nose of the mouse and open the flow to administer continuous anesthesia during image acquisition (Oxygen flow rates: 0.5-1 L/min, isoflurane concentration: 2-3% (or lower).</li>
	<li>Acquire fluorescence images using an exposure time of 5-15 sec.</li>
	<li>Perform image analysis and processing including background correction or contrast adjustment.</li>
</ol>
3. Fluorescence Imaging Ex vivo
HerDox fluorescence can be imaged in tumors and specific organs (including the liver, kidney, spleen, heart, and skeletal muscle) harvested from euthanized mice at 24 hr after the final (Day 7) injection of HerDox.
<ol>
	<li>Turn on the Multimode In vivo Optical Imager.</li>
	<li>Select an emission bandpass filter (580 nm &plusmn; 20 nm) for Doxorubicin fluorescence detection.</li>
	<li>Turn on Argon-Krypton laser and place an excitation bandpass filter (488 nm &plusmn;10 nm) at the laser optical path.</li>
	<li>Place tumors and specific organs arranged on a Petri-dish into the imaging chamber of the Multimode In vivo Optical Imager.</li>
	<li>Acquire fluorescence images of tissues using an exposure time of 5-15 sec. An example of initial fluorescence image acquisition is shown in Figure 2a. Repeat the same using an empty Petri-dish, which will serve as the Background (Figure 2b).</li>
	<li>Perform image analysis and processing including background correction or contrast adjustment. An example of a corrected image is shown in Figure 2c, resulting from subtraction of Figure 2b from Figure 2a.</li>
</ol>
4. In situ Confocal Imaging of Tumors
In situ confocal imaging allows detection and analysis of HerDox tumor accumulation at the cellular level.
<ol>
	<li>Turn on a Leica SPE confocal microscope.</li>
	<li>Select 488 nm laser light for excitation of doxorubicin and emission wavelengths (560-620 nm) for doxorubicin fluorescence detection.</li>
	<li>Select a 40X or 63X objective and drop immersion oil on the objective lens.</li>
	<li>Extract fresh tumors from euthanized mice that previously received HerDox and mock treatments as described in procedure 2.</li>
	<li>Place tumors on a Petri-dish on ice to avoid tissue degradation, and then transfer the tumors to a Delta T chamber for confocal imaging.</li>
	<li>Acquire confocal images of the tumors at sequential focal depths (step size: 1 &mu;m, thickness: 20 &mu;m). An example of sequentially-acquired images along the z-axis is shown in Figure 3, left panel.</li>
	<li>Perform maximum intensity z-projection of the images. A maximum intensity projection of z-stacked images is shown in Figure 3, right panel.</li>
	<li>Calculate mean fluorescence intensities of the maximum intensity Z-projection images. Mean fluorescence intensities of the images over the overall field of view were calculated using ImageJ.</li>
</ol>
5. Ratiometric Spectral Imaging and Analysis
Ratiometric spectral imaging and analysis allows discrimination between Dox fluorescence and autofluorescence.
<ol>
	<li>Power on a laser scanning fluorescence confocal microscope.</li>
	<li>Acquire 15 images of the HerDox-treated and untreated tumors at a specified depth within the spectral range of 510-650 nm, with a step size of 10 nm, and excitation at 488 nm light using a Leica SPE confocal microscope.</li>
	<li>Prepare a 100 &mu;M solution of doxorubicin.</li>
	<li>Perform spectral imaging of the 100 &mu;M doxorubicin solution to obtain the pure spectral signature of Dox fluorescence (spectral range: 510-650 nm, a step size: 10 nm). Typical results of image acquisition from spectral imaging and resulting fluorescence spectrum plotted as a graph are shown in Figure 4.</li>
	<li>Acquire the autofluorescence spectral signature from an image cube (spectral range: 510-650 nm, a step size: 10 nm) obtained by spectral imaging of untreated tumors. Typical results of image acquisition from spectral imaging and resulting fluorescence spectrum plotted as a graph are shown in Figure 5.</li>
	<li>Generate four reference spectral signatures (pure autofluorescence, 0.1.doxorubin+0.9.autofluorescence, 0.2. doxorubicin+0.8.autofluorescence, 0.3.doxorubin+0.7.autofluorescence) using the program we developed 9. A typical curve showing four reference spectral signatures is shown in Figure 6.</li>
	<li>Perform spectral classification of the images as defined by the reference spectral signatures through Euclidean distance measure using the program we previously developed 9.</li>
	<li>Perform linear spectral unmixing of those images by using a spectral unmixing program (plug-in in ImageJ) we developed 7, 10, for comparison to the ratiometric spectral imaging and analysis. An example of separating HerDox fluorescence from autofluorescence by linear spectral unmixing is shown in Figure 7.</li>
</ol>

<ol>
	<li>Agadjanian, H., Chu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemotherapy+Targeting+by+DNA+Capture+in+Viral+Protein+Particles.">Chemotherapy Targeting by DNA Capture in Viral Protein Particles.</a> Nanomedicine. 7 (3), 335-352 (2012).</li><li>Agadjanian, H., Ma, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+detection+and+elimination+by+a+targeted+gallium+corrole.">Tumor detection and elimination by a targeted gallium corrole.</a> Proc. Natl. Acad. Sci. U.S.A. 106 (15), 6105-6110 (2009).</li><li>Agadjanian, H., Weaver, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+delivery+of+corroles+to+cells+via+noncovalent+conjugates+with+viral+proteins.">Specific delivery of corroles to cells via noncovalent conjugates with viral proteins.</a> Pharm. Res. 23 (2), 367-377 (2006).</li><li>Medina-Kauwe, L. K., Maguire, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-viral+gene+delivery+to+human+breast+cancer+cells+by+targeted+Ad5+penton+proteins.">Non-viral gene delivery to human breast cancer cells by targeted Ad5 penton proteins.</a> Gene Therapy. , 81753-81761 (2001).</li><li>Medina-Kauwe, L. K., Kasahara, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3PO,+a+novel+non-viral+gene+delivery+system+using+engineered+Ad5+penton+proteins.">3PO, a novel non-viral gene delivery system using engineered Ad5 penton proteins.</a> Gene Therapy. , 8795-8803 (2001).</li><li>Rentsendorj, A., Xie, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Typical+and+atypical+trafficking+pathways+of+Ad5+penton+base+recombinant+protein:+implications+for+gene+transfer.">Typical and atypical trafficking pathways of Ad5 penton base recombinant protein: implications for gene transfer.</a> Gene Ther. 13 (10), 821-836 (2006).</li><li>Hwang, J. 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Y., Moffatt-Blue, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimode+optical+imaging+of+small+animals:+development+and+applications.">Multimode optical imaging of small animals: development and applications.</a> Proc. of SPIE. 6411, (2007).</li><li>Ducros, M., Moreaux, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+unmixing:+analysis+of+performance+in+the+olfactory+bulb+in+vivo.">Spectral unmixing: analysis of performance in the olfactory bulb in vivo.</a> PLoS One. 4 (2), e4418 (2009).</li><li>Zimmermann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+imaging+and+linear+unmixing+in+light+microscopy.">Spectral imaging and linear unmixing in light microscopy.</a> Adv. Biochem. Eng. Biotechnol. , 95245-95265 (2005).</li></ol>

The author, Daniel Farkas, is Chairman of Spectral Molecular Imaging. The remaining authors have no competing interests.

Dox fluorescence can be detectable in vivo using the multimode imager when tumors are subcutaneous. However, the therapeutically effective dose of HerDox (0.004 mg/kg) is below the detection threshold after a single dose. In contrast, after 7 daily injections (1x/day for 7 days), the tumor accumulation and retention of the particle is sufficient to enable visualization of Dox fluorescence.
It is critical when working with Dox or any other fluorophore for in vivo imaging that...

Tumor-targeting of chemotherapy has the potential to eliminate cancer cells at lower dose compared to untargeted drugs because more of the delivered therapy can accumulate at its intended destination rather than distribute to non-tumor tissue. As the latter situation would dilute out the efficacy of the drug and thus require higher doses to be effective, tumor-targeting has both therapeutic and safety advantages over standard non-targeted treatment.
Targeting chemotherapy by encapsulation in self-assembled nanoparticles allows the drug to remain chemically unmodified in contrast to drugs that are covalently linked to targeting molecules. As such linkage has the potential to alter the activity of both the drug and the targeting molecule; non-covalent assembly allows drug potency to be retained.
We have previously shown that the novel three-component, self-assembled complex, HerDox, targets HER2+ tumors in vivo and elicits tumor-growth ablation while sparing normal tissue, including the heart 1. HerDox is formed through non-covalent interactions between the receptor-binding cell-penetration protein, HerPBK10, and the chemotherapeutic agent, doxorubicin (Dox), via a small nucleic acid linker. HerPBK10 binds the human epidermal growth factor receptor (HER) and triggers receptor-mediated endocytosis 2-4, while endosomal membrane penetration is accomplished through incorporation of the adenovirus-derived penton base capsid protein 4-6. A positively-charged domain on the protein enables nucleic acid binding 4, 5, through which DNA-intercalated Dox can be transported for targeted delivery. Electrophilic, intercalation, and possibly protein oligomerization interactions facilitate self-assembly into round 10-20 nm particles that are stable in blood and under extended storage at different temperatures 1. Preferential targeting to HER2+ tumor cells is facilitated by the enhanced ligand affinity when HER2 is elevated.
Our previous studies have shown that systemic delivery of HerDox yields preferential accumulation in tumors over non-tumor tissue and in comparison to untargeted Dox 1, and penetration into tumor cells in vivo 7. We have observed that HerDox releases Dox after tumor cell entry, allowing Dox accumulation into the nucleus 1. Tumor-accumulation appears to correlate with receptor level, as relatively low HER2-expressing tumors accumulate less HerDox compared to those with comparatively higher HER2 levels 1. Moreover, the effective cell death concentration exhibits an inverse correlation with HER2 display on tumor cell lines expressing differing cell surface HER2 levels 1. HerDox exhibits a therapeutic and safety advantage over untargeted Dox, as tumor killing occurs at over 10-times lower dose compared to the untargeted drug and yields no detectable adverse effect on heart (detected by echocardiography and histological stain) or liver (detected by TUNEL stain) tissue, in contrast to untargeted Dox 1. Despite its derivation from a viral capsid protein, HerPBK10 exhibits no detectable immunogenicity at therapeutic levels 2. Whereas pre-existing antibodies to whole adenovirus can recognize HerPBK10, they are unable to prevent cell binding 2.
Tumor volume measured over time is a standard method of assessing therapeutic efficacy of targeted therapeutics, and has been employed for assessing the therapeutic efficacy of HerDox. Supplementing this approach with in vivo and ex vivo fluorescence intensity imaging has allowed us to better assess targeting efficiency 7. We have specifically integrated in situ confocal imaging of excised tumors with spectral analysis of Dox fluorescence to verify that HerDox not only accumulated at tumors in vivo but penetrated into tumor cells and delivered Dox into the cytoplasm and nucleus 7. Spectral analysis furthermore enabled us to distinguish Dox fluorescence from autofluorescence 7.
Here we demonstrate in greater detail our approach for assessing HerDox in vivo after systemic delivery, and most importantly, for assessing targeting through multimode imaging methods and analyses.

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</th
 ></tr>
 <tr>
 <td >Fluorescence laser scanning confocal microscope</td>
 <td >Leica</td>
 <td >SPE</td>
 <td></td>
 </tr>
 <tr>
 <td>In Vivo Optical Imager</td>
 <td>Spectral Molecular Imaging</td>
 <td>Multimode In Vivo Optical Imager</td>
 <td></td>
 </tr>
 <tr>
 <td >Doxorubicin-HCl</td>
 <td >Sigma-Aldrich</td>
 <td >D4035</td>
 <td></td>
 </tr>
 <tr>
 <td >Nude (NU/NU) mouse, female, 6-8 week</td>
 <td >Charles River</td>
 <td >Strain code 088</td>
 <td></td>
 </tr>
 <tr>
 <td >MDA-MB-435 human HER2+ tumor cells</td>
 <td >NCI-Frederick Cancer DCTD Tumor/Cell Line Repository</td>
 <td >0507292</td>
 <td></td>
 </tr>
 <tr>
 <td >3/10 cc insulin syringe U-100 with 29G x 1/2" Ultra-FineIV permanently attached needle</td>
 <td >BD</td>
 <td >309301</td>
 <td></td>
 </tr>
 <tr>
 <td >Delta T chamber </td>
 <td >Bioptechs </td>
 <td >04200417B</td>
 <td></td>
 </tr>
 </tbody>
</table>

analysis of targeted viral protein nanoparticles delivered to her2+ tumors

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. HerDox is formed by non-covalent self-assembly of a tumor targeted cell penetration protein with the chemotherapy agent, doxorubicin, via a small nucleic acid linker. A combination of electrophilic, intercalation, and oligomerization interactions facilitate self-assembly into round 10-20 nm particles. HerDox exhibits stability in blood as well as in extended storage at different temperatures. Systemic delivery of HerDox in tumor-bearing mice results in tumor-cell death with no detectable adverse effects to non-tumor tissue, including the heart and liver (which undergo marked damage by untargeted doxorubicin). HER2 elevation facilitates targeting to cells expressing the human epidermal growth factor receptor, hence tumors displaying elevated HER2 levels exhibit greater accumulation of HerDox compared to cells expressing lower levels, both in vitro and in vivo. Fluorescence intensity imaging combined with in situ confocal and spectral analysis has allowed us to verify in vivo tumor targeting and tumor cell penetration of HerDox after systemic delivery. Here we detail our methods for assessing tumor targeting via multimode imaging after systemic delivery.

Figure 1 shows the in vivo optical imager prototype, which was built for the purpose of image acquisition under multiple modalities, including fluorescence intensity, spectral, lifetime, 2-photon, intra-vital confocal, and bioluminescence imaging. In addition, the cooled high sensitive camera and high power laser lines incorporated in this system yields higher contrast fluorescence images compared to commercial optical imaging systems 11, especially for the in vivo detection ...

Watch this Scientific Journal Video about Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors at JoVE.com

Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

This article details the procedures for optical imaging analysis of the tumor-targeted nanoparticle, HerDox. In particular, detailed use of the multimode imaging device for detecting tumor targeting and assessing tumor penetration is described here.

The HER2+ tumor-targeted nanoparticle, HerDox, exhibits tumor-preferential accumulation and tumor-growth ablation in an animal model of HER2+ cancer. ...

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Research

JoVE Journal

Bioengineering

11.0K ビュー.  University of Southern California. この記事の詳細腫瘍標的ナノ粒子、HerDoxの光学イメージング解析のための手続き。特に、ターゲティングおよび腫瘍浸透性を評価する腫瘍を検出するためのマルチモードの撮像装置の詳細な使用がここで記載されている。

ビデオ: Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

Introduction to the Ultrasound Targeted Microbubble Destruction Technique

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid microbubble contrast agents7-9. In mice these vector-carrying microbubbles can be administered intravenously or directly to the left ventricle of the heart. In larger animals they can also be infused through an intracoronary catheter. The subsequent delivery from the circulation to a target organ occurs by acoustic cavitation at a resonant frequency of the microbubbles. It seems likely that the mechanical energy generated by the microbubble destruction results in transient pore formation in or between the endothelial cells of the microvasculature of the targeted region10. As a result of this sonoporation effect, the transfection efficiency into and across the endothelial cells is enhanced, and transgene-encoding vectors are deposited into the surrounding tissue. Plasmid DNA remaining in the circulation is rapidly degraded by nucleases in the blood, which further reduces the likelihood of delivery to non-sonicated tissues and leads to highly specific target-organ transfection.

Ultrasound Targeted Microbubble Destruction (UTMD) can be used to direct site-specific delivery of bioactive molecules, including therapeutic genes, to target organs accessible to ultrasound, such as the heart and liver1-6.

In UTMD, bioactive molecules, such as negatively charged plasmid DNA vectors encoding a gene of interest, are added to the cationic shells of lipid ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

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. ...

Viral Nanoparticles for In vivo Tumor Imaging

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being investigated for applications in imaging and therapy. Viral nanoparticles (VNPs) derived from plants can be regarded as self-assembled bionanomaterials with defined sizes and shapes. Plant viruses under investigation in the Steinmetz lab include icosahedral particles formed by Cowpea mosaic virus (CPMV) and Brome mosaic virus (BMV), both of which are 30 nm in diameter. We are also developing rod-shaped and filamentous structures derived from the following plant viruses: Tobacco mosaic virus (TMV), which forms rigid rods with dimensions of 300 nm by 18 nm, and Potato virus X (PVX), which form filamentous particles 515 nm in length and 13 nm in width (the reader is referred to refs. 1 and 2 for further information on VNPs).
From a materials scientist's point of view, VNPs are attractive building blocks for several reasons: the particles are monodisperse, can be produced with ease on large scale in planta, are exceptionally stable, and biocompatible. Also, VNPs are "programmable" units, which can be specifically engineered using genetic modification or chemical bioconjugation methods 3. The structure of VNPs is known to atomic resolution, and modifications can be carried out with spatial precision at the atomic level4, a level of control that cannot be achieved using synthetic nanomaterials with current state-of-the-art technologies.
In this paper, we describe the propagation of CPMV, PVX, TMV, and BMV in Vigna ungiuculata and Nicotiana benthamiana plants. Extraction and purification protocols for each VNP are given. Methods for characterization of purified and chemically-labeled VNPs are described. In this study, we focus on chemical labeling of VNPs with fluorophores (e.g. Alexa Fluor 647) and polyethylene glycol (PEG). The dyes facilitate tracking and detection of the VNPs 5-10, and PEG reduces immunogenicity of the proteinaceous nanoparticles while enhancing their pharmacokinetics 8,11. We demonstrate tumor homing of PEGylated VNPs using a mouse xenograft tumor model. A combination of fluorescence imaging of tissues ex vivo using Maestro Imaging System, fluorescence quantification in homogenized tissues, and confocal microscopy is used to study biodistribution. VNPs are cleared via the reticuloendothelial system (RES); tumor homing is achieved passively via the enhanced permeability and retention (EPR) effect12. The VNP nanotechnology is a powerful plug-and-play technology to image and treat sites of disease in vivo. We are further developing VNPs to carry drug cargos and clinically-relevant imaging moieties, as well as tissue-specific ligands to target molecular receptors overexpressed in cancer and cardiovascular disease.

Viral Nanoparticles for In vivo Tumor Imaging

Plant viral nanoparticles (VNPs) are promising platforms for applications in biomedicine. Here, we describe the procedures for plant VNP propagation, purification, characterization, and bioconjugation. Finally, we show the application of VNPs for tumor homing and imaging using a mouse xenograft model and fluorescence imaging.

The use of nanomaterials has the potential to revolutionize materials science and medicine. Currently, a number of different nanoparticles are being ...

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

Production of Metal Nanoparticles by Pulsed Laser-ablation in Liquids: A Tool for Studying the Antibacterial Properties of Nanoparticles

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of medicine. In addition to efforts to identify novel small-molecule antimicrobial drugs, there has been great interest in the use of metal nanoparticles as coatings for medical devices, wound dressings, and consumer packaging, due to their antimicrobial properties. The wide variety of methods available for nanoparticle synthesis results in a broad spectrum of chemical and physical properties which can affect antibacterial efficacy. This manuscript describes the pulsed laser-ablation in liquids (PLAL) method to create nanoparticles. This approach allows for the fine tuning of nanoparticle size, composition, and stability using post-irradiation methods as well as the addition of surfactants or volume excluders. By controlling particle size and composition, a large range of physical and chemical properties of metal nanoparticles can be explored which may contribute to their antimicrobial efficacy thereby opening new avenues for antibacterial development.

The antimicrobial properties of metals such as copper and silver have been recognized for centuries. This protocol describes pulsed laser-ablation in liquids, a method of synthesizing metal nanoparticles that provides the ability to fine tune the properties of these nanoparticles to optimize their antimicrobial effects.

The emergence of multidrug-resistant bacteria is a global clinical concern leading some to speculate about our return to a "pre-antibiotics" era of ...

Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing virus-infected or transfected cells via EM are the low efficiencies of infection or transfection, hindering the examination of these cells. In order to overcome this difficulty, light microscopy (LM) can be performed first to allocate the subpopulation of infected or transfected cells. Thus, taking advantage of the use of fluorescent proteins (FPs) fused to viral proteins, LM is used here to record the positions of the "positive-transfected" cells, expressing a FP and growing on a support with an alphanumeric pattern. Subsequently, cells are further processed for EM via high pressure freezing (HPF), freeze substitution (FS) and resin embedding. The ultra-rapid freezing step ensures excellent membrane preservation of the selected cells that can then be analyzed at the ultrastructural level by transmission electron microscopy (TEM). Here, a step-by-step correlative light electron microscopy (CLEM) workflow is provided, describing sample preparation, imaging and correlation in detail. The experimental design can be also applied to address many cell biology questions.

Correlative Light Electron Microscopy CLEM for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells

A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.

Due to its high resolution, electron microscopy (EM) is an indispensable tool for virologists. However, one of the main difficulties when analyzing ...