We describe protocols for assessing the degree of transduction by cell-penetrating peptides utilizing ex vivo imaging systems followed by paraffin embedding, sectioning, and confocal fluorescent microscopy using cardiac targeting peptide as an example. In our protocol, a single animal can be used to acquire both types of imaging assessment of the same organs, thereby cutting the number of animals needed for studies by half.
Since the initial description of protein transduction domains, also known as cell penetrating peptides, over 25 years ago, there has been intense interest in developing these peptides, especially cell-specific ones, as novel vectors for delivering diagnostic and therapeutic materials. Our past work involving phage display identified a novel, nonnaturally occurring, 12 amino acid-long peptide that we named cardiac targeting peptide (CTP) due to its ability to transduce normal heart tissue in vivo with peak uptake seen in as little as 15 min after an intravenous injection. We have undertaken detailed biodistribution studies by injecting CTP labeled with fluorophore cyanine5.5, allowing it to circulate for various periods of time, and euthanizing, fixing, and sectioning multiple organs followed by fluorescent microscopy imaging. In this publication, we describe these processes as well as ex vivo imaging of harvested organs using an in vivo imaging system in detail. We provide detailed methodologies and practices for undertaking transduction as well as biodistribution studies using CTP as an example.
The cell plasma membrane is a semipermeable barrier that is essential for cell integrity and survival and serves to regulate the interior of the cell by controlling movement of substances into the cell. Although vital for survival, it also presents a barrier to delivery of cargo to the cell. In 1988, the trans-activator of transcription (Tat) protein of the human immunodeficiency virus was shown to enter cultured cells and promote viral gene expression1,2, with the domains responsible for this transduction limited to an arginine and lysine rich 13-amino acid region of the third helix3 These were thus named cell penetrating peptides (CPPs). This was followed by research showing the ability of the Tat peptide to carry functional β-galactosidase into multiple cell types4. Since the initial description, the number of cell-penetrating peptides has increased dramatically. These transduction domains are naturally occurring or synthetic short peptides, typically 6−20 amino acids long, that are able to carry functional cargoes across cell membranes. These cargoes can range from other small peptides, full-length proteins, nucleic acids, nanoparticles, viral particles, fluorescent molecules, and radioisotopes5. The initial CPPs described were not cell specific, with Tat being taken up by multiple cell types and even crossing the blood-brain barrier4, hence limiting its therapeutic potential. In order to identify cell-specific CPPs, investigators have undertaken phage display utilizing large, commercially available phage libraries6. Our own work using a combinatorial in vitro and in vivo phage display methodology led to the identification of a CPP named cardiac targeting peptide (CTP)7, a 12-amino acid, nonnaturally occurring peptide (NH2-APWHLSSQYSRT-COOH) that targets the heart with peak uptake at 15 min after a peripheral intravenous injection8. Using immunofluorescent colocalization with actin, an intracellular marker, and exclusion of laminin, a cell membrane marker, we showed that CTP is internalized into mouse cardiomyocytes after an intravenous injection7. Additionally, we incubated human induced pluripotent stem cell-derived beating cardiomyocytes with dual-labeled CTP, labeled with 6-carboxyfluorescein at its C-terminus and rhodamine at its N-terminus through an ester linkage that could only be cleaved off by intracellular esterases. Rapid accumulation of rhodamine into beating cardiomyocytes was observed within 15 min on confocal microscopy8.
In this article, we present two complimentary methodologies that can be used to track biodistribution and confirm tissue-specific internalization of CPPs using CTP as an example. For these methodologies, CTP was synthesized, fluorescently labeled at the N-terminus with cyanine5.5 (CY5.5), and amide-capped at the C-terminus for greater peptide stability. The two methodologies used are in vivo fluorescent optical imaging systems and fluorescent microscopy of tissue sections. Both methods are very useful in studying biodistribution, uptake, and elimination of fluorescently labeled CPPs. The advantage of assessing biodistribution using these methods over others, such as single-photon emission tomography (SPECT) or positron emission tomography (PET), is that there is no need for the time-intensive radiolabeling of CPPs compared with fluorescent labeling, which is relatively easy and in routine use at all peptide synthesis facilities. The use of in vivo imaging quickly produces biodistribution data in the context of a living system, while sectioning provides greater in-depth information about peptide uptake and localization within cells via the preservation of morphological detail. This protocol can be applied to a wide variety of organs and tissues such as the heart, lung, liver, kidney, brain, spleen, stomach, large intestine, small intestine, skeletal muscle, bone, testes/ovaries, and eyes.
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The University of Pittsburgh's Institutional Animal Care and Use Committee approved all animal protocols specified in this publication prior to undertaking any of these animal experiments.
NOTE: This protocol details how to perform ex vivo biodistribution studies utilizing ex vivo optical fluorescent imaging followed by in vivo biodistribution studies by embedding the organs in paraffin, sectioning, and performing fluorescent microscopy. Although CTP is used as an example, this method can be applied to any fluorescently labeled CPP.
1. In vivo imaging
2. Histology
NOTE: Follow steps 1.1−1.8 to obtain different organs. Alternatively, the same organs that were imaged can be placed in formalin immediately and used for sectioning as detailed below.
Using this protocol, we treated three mice with a 10 mg/kg dose of Cy5.5-CTP through a retro-orbital injection (Figure 1A). After allowing the peptide to circulate for 15 min, the mice were euthanized using a CO2 chamber, the chest opened through a median sternotomy incision, the right atrium was nicked, and the mice perfusion fixed using 3 mL of 10% phosphate-buffered formalin (Figure 1B,C). After fixation, the heart, lungs, liver, kidney, spleen, and brain were dissected out and arranged in a 12 well plate for ex vivo optical fluorescent imaging (Figure 1D and Figure 2A). Three control mice with no injections were also perfused, dissected, and imaged (Figure 1B−D and Figure 2B). The fluorescence data acquired for each set of organs can be compared due to the counts being converted to radiant efficiency, the relative fluorescence unit of the imaging software. This data can be quantified to yield both qualitative images and quantitative data for each organ in question (Figure 2C) across different time points for biodistribution studies. The autofluorescence of different organs in response to different excitation wavelengths is demonstrated in Figure 3A−E. Shorter excitation wavelengths, such as enhanced green fluorescent protein (EGFP), are associated with higher autofluorescence, especially in liver and brain, than far-red (Cy5.5) or near infrared (Cy7) excitation wavelengths (Figure 3). The imaged organs were fixed in 10% phosphate buffered formalin at RT for a minimum of 48 h, followed by a transfer to tissue processing and embedding cassettes (Figure 1E). The organs were processed and paraffinized in a tissue processing machine, positioned in molds filled with paraffin, frozen on a -20 °C stage, and stored overnight at -20 °C (Figure 1F). The samples were sectioned (Figure 1G) and treated with solution exchanges to rehydrate the tissue (Figure 1H). The prepared slides were then mounted with DAPI fluorescent mounting medium (Figure 1I). Once dry, usually overnight, the slides were imaged using fluorescent microscopy. Representative images of each organ from a mouse are shown in Figure 4A. The images from different organs were acquired using identical settings to allow for comparison across mice, and to allow for quantification (Figure 4B).
Figure 1: Harvesting mouse organs for ex vivo optical fluorescent imaging and sectioning. (A) Wild type mouse injected retro-orbitally with CTP-Cy5.5. (B) Mouse dissected with chest opened, right atrium snipped, for perfusion fixation. (C) Heart injected with 3 mL of 10% buffered formalin phosphate for perfusion fixation of the animal. (D) Organs of interest harvested and arranged in a 12 well plate for fluorescent optical imaging. (E) Heart loaded into a cassette and processed using a tissue processor. (F) Heart embedded in paraffin. (G) Heart sectioned on a microtome. (H) Slides deparaffinized through a series of solution exchanges. (I) Sections mounted with coverslips using DAPI. Please click here to view a larger version of this figure.
Figure 2: Representative images from treated and control organs. (A) Organs from a mouse treated with 10 mg/kg CTP-Cy5.5. (B) Organs from an untreated control mouse. (C) Quantification of fluorescence for each set of organs. Please click here to view a larger version of this figure.
Figure 3: Untreated mouse organs imaged at different excitation emission wavelengths to demonstrate how longer wavelengths produce less autofluorescence. (A) Cy7: 740−790. (B) Cy5.5: 660−710. (C) Cy5: 620−670. (D) Cy3: 520−570. (E) EGFP: 480−520. Please click here to view a larger version of this figure.
Figure 4: Transduction of heart, lung, liver, and kidney after intravenous injection in mice. Wild type mice were injected with 10 mg/kg of Cy5.5-CTP, or a random peptide (RAN-Cy5.5), and euthanized at the indicated time points. (A) Peak transduction of heart tissue was seen at 15 min with steady decrease in fluorescence over time. Some capillary uptake was noted in the lungs with robust transduction in the liver as well at the kidney glomerular capillaries, the latter implying a renal mechanism of excretion. (B) Quantification of fluorescent intensity shows significantly increased heart uptake of CTP-Cy5.5 over RAN-Cy5.5. Scale bar = 500 µm. This figure has been modified from Zahid et al.8. Please click here to view a larger version of this figure.
Animal models are essential for preclinical drug development at every stage of the process from identification of novel CPPs, biodistribution studies, mechanism of transduction, to ultimately testing for efficacy of the delivered cargo using these novel CPPs as vectors. There are many commonly used methods available to assess biodistribution such as histology, nuclear medicine imaging (SPECT and PET), and in vivo optical imaging12. Nuclear imaging methods can be cumbersome due to the limited availability of small animal SPECT and PET systems, as well as the ability to produce radiolabel-drug conjugates, which require the expertise of a radiochemist. In contrast, fluorescent labels are much simpler and can be cost effective to work with. The protocol described in this paper allows for rapid analysis of biodistribution using multiple methods. Ex vivo whole organ fluorescent optical imaging allows for the immediate comparison of fluorescence across different tissues and treatment groups and can identify organ uptake and time to peak uptake in organ of interest in a semiquantitative manner. Quantitative tissue histology requires more extensive processing to treat, section, image, and analyze tissues, but it provides more data on the microscopic level and is a current standard technique. It is worthwhile to note that direct, quantitative comparison across the two techniques is not possible, because the autofluorescence seen with each technique for different organs and excitation wavelengths varies significantly.
An important part of this protocol is selecting the right fluorophore to label candidate CPPs for experiments. One potential issue is spectra overlap, which can be problematic when multiple fluorophores are needed. The DAPI fluorescent mounting media and Cy5.5 do not have overlapping spectra. However, for certain applications where multiple fluorophores are needed, the risk of spectral overlap needs to be carefully considered. Depending on the system being used, fluorophore selection may be limited. Therefore, knowledge of the system's capabilities is key. Fluorescent optical systems are best utilized with far-red or near-infrared fluorophores due to the high tissue absorption of shorter wavelengths13. Fluorophores in the range of enhanced green fluorescent protein have a major limitation, because there is significant organ autofluorescence seen at its excitation wavelength, specifically in brain and liver tissue. Depending on the conditions of an experiment, some fluorophores are best avoided. Some water-soluble organic fluorophores have a strong interaction with lipid bilayers, which can cause false positives. Hence, taking steps to determine if a fluorophore has strong affinity to the tissue of interest is advisable14. Another factor to consider is selecting the appropriate method of fluorophore conjugation, which can be an important parameter affecting the results. CPPs can be labeled fluorescently at the N- or C-terminus through a covalent bond between the N-terminus of the peptide and the carboxyl group of the dye such as Cy5.5-NHS. Care should be taken, because the mechanism of transduction of most CPPs is not understood in detail and conjugation at one end may affect the uptake mechanism more so than at the other end. Another possibility for labeling CPPs is through biotinylating the N-terminus for conjugation to fluorescently labeled streptavidin. Using this strategy has the convenience of allowing different fluorescent streptavidin conjugates to be utilized. However, a possible limitation of this strategy is that a biotin-streptavidin complex is a large construct, which could potentially interfere with transduction.
Fluorescent optical imaging systems are an effective strategy for generating comparison of fluorescence across different organs and treatments efficiently but are incapable of producing a quantitative measure of absolute concentrations in tissue. This is due to light scattering effects within the tissue, which is further compounded by the naturally occurring variety in tissue sizes and densities, and differences in vascularity, with variable fluorescence scattering. Tissue autofluorescence can be a factor as well, due to naturally occurring biochemical sources such as collagen, or dietary sources like chlorophyll in food13.
Histology is the most commonly used method of measuring biodistribution and can potentially be used to accurately measure and compare uptake across different tissues over time. Light scattering issues are avoided using this method because all tissues are sectioned to the same thickness15. A major advantage of this method is the ability to include additional fluorescent labels postsectioning for immunohistochemistry. Although the addition of another fluorophore could make imaging more challenging, the use of fluorescent labels can be useful for localization of a CPP to particular intracellular compartments, like lysosomes or mitochondria. An antibody could be used in a confocal microscopy experiment to determine if a transduced candidate CPP colocalizes with a structure of interest, which can show the potential of a CPP as a delivery agent. One limitation of this method is that preparation of slides from organ samples can be time consuming, labor intensive, and prone to human error12,15. When imaging slides, care should be taken to not image the same location for too long to avoid photobleaching. Some photobleaching will be inevitable, depending on the sensitivity of the fluorophore. Care should be taken at every step of this protocol to protect the samples from ambient light and store them properly16. We recommend that slides be stored at 4 °C, light-protected, for future imaging.
There are a variety of methods available for measuring the biodistribution of a candidate CPP that require specialized equipment and can produce comparable results, though they may require more complex CPP labeling. The protocol described in this paper uses two compatible methods to efficiently produce biodistribution data in the context of a living system while allowing for the acquisition of greater in-depth information about peptide internalization within cells from the same sample, thus cutting the number of animals needed for a study by half. These methods were used to generate the above data, which demonstrate that both methods can be utilized sequentially in the same animal, and the quality of the data generated by each. Our results also highlight the inability to directly correlate results between the two techniques in a quantitative manner.
M.Z. and K.S.F. are supported by American Heart Association Scientist Development Award 17SDG33411180, and by a grant awarded under the Pitt Innovation Challenge (PInCh®), through the Clinical and Translational Science Institute at the University of Pittsburgh.
Name | Company | Catalog Number | Comments |
10% Buffered Formalin Phosphate | ThermoFisher | SF100-4 | |
10x Tris Buffered Saline (TBS) | ThermoFisher | BP2471-1 | |
12-well Cell Culture Plate | ThermoFisher | 353043 | |
1x TBS Solution | 10x TBS is diluted 1:10 with deionized water. This solution can be stored at room temperature. | ||
20 mL Scintillation Vials | Wheaton | 334173 | |
26G Needles | Becton Dickinson | 305110 | |
28G 0.5ml Insulin syringes | Becton Dickinson | 329461 | |
3mL Syringe | Becton Dickinson | 309657 | |
CD1 Mice | Charles River | 022 | 6 to 12 week old albino, female mice |
Cover Glass | ThermoFisher | 12-544-14 | |
Cy5.5-CTP-amide | Prepared by peptide synthesis, conjugated with Cy5.5 fluorophore, and purified using HPLC. Lyophilized powder is reconstituted in DMSO at 10mM concentration. After reconstituting, store at -80 °C. | ||
Dapi Fluoromount G | SouthernBiotech | 0100-20 | |
Dumont #5 Forceps | Fine Science Tools | 99150-20 | |
Ethanol | |||
Extra Fine Bonn Scissors | Fine Science Tools | 14084-08 | |
Ketamine HCl 100mg/mL | KetaVed | NDC 50989-161-06 | |
Ketamine/Xylazine Solution | Ketamine HCl is mixed with Xylazine 1:1 to produce a stock solution containing 50mg/mL ketamine and 10mg/mL Xylazine. This solution is made fresh before each use. | ||
Leica RM2235 Rotary Microtome | Leica | RM2235 | |
Microscope Slides | ThermoFisher | B9992000TN | |
Paraplast X-TRA | Sigma | P3808-1KG | |
Perkin Elmer Lumina S5 IVIS | Perkin Elmer | CLS148588 | |
Sparkle Optical Lens Cleaner | ThermoFisher | NC0090079 | |
Tissue-Tek Processing/Embedding Cassettes | ThermoFisher | NC9499605 | |
Tissue-Tek VIP Processing machine | Tissue-Tek | VIP 5A-F1 | |
Xylazine 20 mg/mL | AnaSed | NDC 59399-110-20 | |
Xylenes | ThermoFisher | X5-4 |
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