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. Y., Park, 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=Multimodality+Imaging+In+vivo+for+Preclinical+Assessment+of+Tumor-Targeted+Doxorubicin+Nanoparticles.">Multimodality Imaging In vivo for Preclinical Assessment of Tumor-Targeted Doxorubicin Nanoparticles.</a> PLoS ONE. 7 (4), e34463 (2012).</li><li>Hwang, J. Y., Wachsmann-Hogiu, S., 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=A+Multimode+Optical+Imaging+System+for+Preclinical+Applications+In+Vivo:+Technology+Development,+Multiscale+Imaging,+and+Chemotherapy+Assessment.">A Multimode Optical Imaging System for Preclinical Applications In Vivo: Technology Development, Multiscale Imaging, and Chemotherapy Assessment.</a> Mol. Imaging Biol. , (2011).</li><li>Hwang, J. Y., Gross, Z., 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=Ratiometric+spectral+imaging+for+fast+tumor+detection+and+chemotherapy+monitoring+in+vivo.">Ratiometric spectral imaging for fast tumor detection and chemotherapy monitoring in vivo.</a> J. Biomed. Opt. 16 (6), 066007 (2011).</li><li>Fujimoto, J. G., Farkas, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomedical Optical Imaging. , (2009).</li><li>Hwang, J. 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><tbody><tr><td>Fluorescence laser scanning confocal microscope</td><td>Leica</td><td>SPE</td><td></td></tr><tr><td>&lt;em&gt;In Vivo&lt;/em&gt; Optical Imager</td><td>Spectral Molecular Imaging</td><td>Multimode &lt;em&gt;In Vivo&lt;/em&gt; 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&quot; 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

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Bioengineering

11.0K Views.  University of Southern California. 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.

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

Polymalic Acid-based Nano Biopolymers for Targeting of Multiple Tumor Markers: An Opportunity for Personalized Medicine?

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this diversity, personalized medicine is developed for silencing malignancy associated genes. Nano drugs fit these needs by targeting tumor and delivering antisense oligonucleotides for silencing of genes. As drugs for the treatment are often administered repeatedly, absence of toxicity and negligible immune response are desirable. In the example presented here, a nano medicine is synthesized from the biodegradable, non-toxic and non-immunogenic platform polymalic acid by controlled chemical ligation of antisense oligonucleotides and tumor targeting molecules. The synthesis and treatment is exemplified for human Her2-positive breast cancer using an experimental mouse model. The case can be translated towards synthesis and treatment of other tumors.

An example of a nano drug based on polymalic acid is presented towards the rational design of personalized medicine that is applicable to cancer. It describes the synthesis of a nano drug to treat Her2-positive human breast cancer in a nude mouse.

Tumors with similar grade and morphology often respond differently to the same treatment because of variations in molecular profiling. To account for this ...

Chemistry

Synthesis of Immunotargeted Magneto-plasmonic Nanoclusters

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications including contrast enhancement in novel magnetomotive imaging modalities, simultaneous capture&#160;and detection of circulating tumor cells (CTCs),&#160;and multimodal molecular imaging combined with photothermal therapy of cancer cells. These applications have stimulated significant interest in development of protocols for synthesis of magneto-plasmonic nanoparticles with optical absorbance in the near-infrared (NIR) region and a strong magnetic moment. Here, we present a novel protocol for synthesis of such hybrid nanoparticles that is based on an oil-in-water microemulsion method. The unique feature of the protocol described herein is synthesis of magneto-plasmonic nanoparticles of various sizes from primary blocks which also have magneto-plasmonic characteristics. This approach yields nanoparticles with a high density of magnetic and plasmonic functionalities which are uniformly distributed throughout the nanoparticle volume. The hybrid nanoparticles can be easily functionalized by attaching antibodies through the Fc moiety leaving the Fab portion that is responsible for antigen binding available for targeting.

Here, we describe a protocol for synthesis of magneto-plasmonic nanoparticles with a strong magnetic moment and a strong near-infrared (NIR) absorbance. The protocol also includes antibody conjugation to the nanoparticles through the Fc moiety for various biomedical applications which require molecular specific targeting.

Magnetic and plasmonic properties combined in a single nanoparticle provide a synergy that is advantageous in a number of biomedical applications ...

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Medicine

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 ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Cancer Research

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few decades, the field of antibody-drug conjugates, bispecific antibodies, chimeric antigen receptors (CAR) and cancer immunotherapy has emerged as the most promising area of basic and therapeutic investigations. With numerous successful human trials targeting immune checkpoint receptors and CAR-T cells in leukemia and melanoma at a breakthrough pace, it is highly exciting times for oncologic therapeutics derived from variations of antibody engineering. Regrettably, a significantly large numbers of antibody and CAR based therapeutics have also proven disappointing in human trials of solid cancers because of the limited availability of immune effector cells in the tumor bed. Importantly, nonspecific distribution of therapeutic antibodies in tissues other than tumors also contribute to the lack of clinical efficacy, associated toxicity and clinical failure. As faithful translation of preclinical studies into human clinical trails are highly relied on mice tumor xenograft efficacy and safety studies, here we highlight a method to test the tumor and general tissue distribution of therapeutic antibodies. This is achieved by labeling the protein-A purified antibody with near Infrared fluorescent dye followed by live imaging of tumor bearing mice.

Here we present a protocol to study the in vivo localization of antibodies in mice tumor xenograft models.

Monoclonal antibodies are high affinity multifunctional drugs that work by variable independent mechanisms to eliminate cancer cells. Over the last few ...

Isolation of Primary Cancer-Associated Fibroblasts from a Syngeneic Murine Model of Breast Cancer for the Study of Targeted Nanoparticles

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor cells, CAFs regulate tumor progression and provide protection from antitumor immunity. Emerging anticancer strategies aim to remodel the tumor microenvironment through the ablation of pro-tumorigenic CAFs or reprogramming of CAFs functions and their activation status. A promising approach is the development of nanosized delivery agents able to target CAFs, thus allowing the specific delivery of drugs and active molecules. In this context, a cellular model of CAFs may provide a useful tool for in vitro screening and preliminary investigation of such nanoformulations.
This study describes the isolation and culture of primary CAFs from the syngeneic 4T1 murine model of triple-negative breast cancer. Magnetic beads were used in a 2-step separation process to extract CAFs from dissociated tumors. Immunophenotyping control was performed using flow cytometry after each passage to verify the process yield. Isolated CAFs can be employed to study the targeting capability of different nanoformulations designed to tackle the tumor microenvironment. Fluorescently labeled H-ferritin nanocages were used as candidate nanoparticles to set up the method. Nanoparticles, either bare or conjugated with a targeting ligand, were analyzed for their binding to CAFs. The results suggest that ex vivo extraction of breast CAFs may be a useful system to test and validate nanoparticles for the specific targeting of tumorigenic CAFs.

This paper aims to provide a protocol for the isolation and culture of primary cancer-associated fibroblasts from a syngeneic murine model of triple-negative breast cancer and their application for the preclinical study of novel nanoparticles designed to target the tumor microenvironment.&#160;

Cancer-associated fibroblasts (CAFs) are key actors in the context of the tumor microenvironment. Despite being reduced in number as compared to tumor ...

Nanodrug Targeting Oncogenic miRNA-10b for Treating Metastatic Breast Cancer

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or &#34;nanodrug&#34;) administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have ...

Iron Oxide Nanoparticles for Image-Guided Therapy Delivery in Murine Glioblastoma

Nanoparticle Delivery of an Oligonucleotide Payload in a Glioblastoma Multiforme Animal Model

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a significant hurdle in the delivery of therapies to GBM. Reported here is an image-guided, iron oxide-based therapeutic delivery nano platform capable of bypassing this physiological barrier by virtue of size and accumulating in the tumor region, delivering its payload. This 25 nm nano platform consists of crosslinked dextran-coated iron oxide nanoparticles labeled with Cy5.5 fluorescent dye and containing antisense oligonucleotide as a payload. The magnetic iron oxide core enables tracking of the nanoparticles through in vivo magnetic resonance imaging, while Cy5.5 dye allows tracking by optical imaging. This report details the monitoring of the accumulation of this nanoparticle platform (termed MN-anti-miR10b) in orthotopically implanted glioblastoma tumors following intravenous injection. In addition, it provides insight into the in vivo delivery of RNA oligonucleotides, a problem that has hampered the translation of RNA therapeutics into the clinic.

Author Spotlight: Innovative Cancer Therapies with Iron Oxide Nanoparticles for Glioblastoma Treatment

The blood-brain barrier is a significant hurdle in the delivery of therapies for glioblastoma, a disease for which there is no cure. Here, we report an in vivo image-guided iron oxide therapeutic nano platform that can bypass this physiological barrier by virtue of size and accumulate in the tumor.

Glioblastoma multiforme (GBM) is the most common and aggressive form of primary brain malignancy for which there is no cure. The blood-brain barrier is a ...