Analysis of Targeted Viral Protein Nanoparticles Delivered to HER2+ Tumors

Summary

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.

Abstract

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.

Introduction

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.

Protocol

1. Systemic Delivery In vivo

1. 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.
2. Gently draw the HerDox mixture into a 3/10 cc insulin syringe fitted with a 29G needle, avoiding bubbles.
3. 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).
4. 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.
5. Repeat injections on the same mouse for six more sequential days, once per day.

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.

1. Turn on the Multimode In vivo Optical Imager.
2. Select an emission bandpass filter (590 nm ± 30 nm) suited for doxorubicin fluorescence detection.
3. Turn on Argon-Krypton laser and place an excitation bandpass filter (488 nm ± 10 nm) at the laser optical path.
4. 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.
5. Transfer the mouse from the anesthetizing chamber to the imaging chamber of the Multimode In vivo Optical Imager when the mouse is anesthetized.
6. 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).
7. Acquire fluorescence images using an exposure time of 5-15 sec.
8. Perform image analysis and processing including background correction or contrast adjustment.

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.

1. Turn on the Multimode In vivo Optical Imager.
2. Select an emission bandpass filter (580 nm ± 20 nm) for Doxorubicin fluorescence detection.
3. Turn on Argon-Krypton laser and place an excitation bandpass filter (488 nm ±10 nm) at the laser optical path.
4. Place tumors and specific organs arranged on a Petri-dish into the imaging chamber of the Multimode In vivo Optical Imager.
5. 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).
6. 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.

4. In situ Confocal Imaging of Tumors

In situ confocal imaging allows detection and analysis of HerDox tumor accumulation at the cellular level.

1. Turn on a Leica SPE confocal microscope.
2. Select 488 nm laser light for excitation of doxorubicin and emission wavelengths (560-620 nm) for doxorubicin fluorescence detection.
3. Select a 40X or 63X objective and drop immersion oil on the objective lens.
4. Extract fresh tumors from euthanized mice that previously received HerDox and mock treatments as described in procedure ².
5. 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.
6. Acquire confocal images of the tumors at sequential focal depths (step size: 1 μm, thickness: 20 μm). An example of sequentially-acquired images along the z-axis is shown in Figure 3, left panel.
7. Perform maximum intensity z-projection of the images. A maximum intensity projection of z-stacked images is shown in Figure 3, right panel.
8. 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.

5. Ratiometric Spectral Imaging and Analysis

Ratiometric spectral imaging and analysis allows discrimination between Dox fluorescence and autofluorescence.

1. Power on a laser scanning fluorescence confocal microscope.
2. 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.
3. Prepare a 100 μM solution of doxorubicin.
4. Perform spectral imaging of the 100 μM 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.
5. 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.
6. 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.
7. Perform spectral classification of the images as defined by the reference spectral signatures through Euclidean distance measure using the program we previously developed ⁹.
8. 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.

Results

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 ...

Discussion

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...

Materials

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