Jun 2020



Surface Engineering and Multimodal Imaging of Multistage Delivery Vectors in Metastatic Breast Cancer

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The design of effective nanoformulations that target metastatic breast cancers is challenging due to a lack of competent imaging and image analysis protocols that can capture the interactions between the injected nanoparticles and metastatic lesions. Here, we describe the integration of in vivo whole-body PET-CT with high temporal resolution, ex vivo whole-organ optical imaging and high spatial resolution confocal microscopy to deconstruct the trafficking of injectable nanoparticle generators encapsulated with polymeric doxorubicin (iNPG-pDox) in pulmonary metastases of triple-negative breast cancer. We describe the details of image acquisition and analysis in a step-wise manner along with the development of a mouse model for metastatic breast cancer. The methods described herein can be easily adapted to any nanoparticle or disease model, allowing a standardized pipeline for in vivo preclinical studies that focus on delineating nanoparticle kinetics and interactions within metastases.

Keywords: In vivo imaging (体内成像), Positron emission tomography (正电子成象术), Optical imaging (光学成像), Cancer (癌症), Metastasis (新陈代谢), Mouse models (小鼠模型)


Metastatic cancer accounts for >90% of mortality in cancer patients; yet, effective therapeutic strategies are missing due to the small size, heterogeneity, and dispersed nature of metastatic cancers (Bianchini et al., 2016; Elia et al., 2019). A series of biological barriers hinder the accumulation, and hence, the efficacy of conventional small molecule drugs and nanoparticle-based delivery systems in metastatic cancers (Blanco et al., 2015). Our group has previously demonstrated the sequential and successful negotiation of most, if not all, biological barriers using iNPG-pDox composed of porous silicon microparticle-based injectable nanoparticle generators (iNPG) packaged with poly(lactic-co-glycolic acid) polymer-doxorubicin (pDox) conjugates (Xu et al., 2016). pDox molecules self-assemble into nanoparticles after their release from iNPGs, and iNPG-pDox treatment significantly inhibited tumor metastasis, including a functional cure in 40-50% of mice with pulmonary metastatic breast cancer (Xu et al., 2016). Visualization of the spatiotemporal kinetics of such nanomedicines that effectively target metastases can allow the directed development and rational improvement of other advanced therapies in the future (Goel, 2017).

Despite significant progress in the development of drug delivery systems, limited studies have evaluated the distribution and interactions of such systems in in vivo metastatic settings. Molecular imaging modalities can uncover a wealth of information across multiple length- and time-scales in disease settings; however, the application of rationally combined multiscale imaging methods to assess the biodistribution of complex drug delivery systems in metastatic tumors remains to be performed (Goel, 2017). Such knowledge, in conjunction with mathematical modeling approaches, can predict and evaluate therapeutic responses and efficacies of complex drug delivery systems, eventually being employed to guide the reverse engineering of advanced therapies (Michor, 2011). In this protocol, we provide a detailed guide to designing and implementing a multiscale and multimodal imaging toolkit for the systematic deconstruction of the trafficking and interactions of nanotherapeutics in a metastatic model of breast cancer, as described in our earlier work (Goel et al., 2020).

We first designed a modular orthogonal surface engineering approach for the modification of iNPG-pDox to render them amenable to multimodal imaging (both nuclear and fluorescence imaging) and microscopy without affecting their intrinsic biophysical properties. Such an approach is independent of carrier type, cargo, formulation method, or delivery route, and can be adopted for any nano- or bioengineered platform and implemented in any disease model. Next, we designed and implemented a systematic imaging toolkit composed of: (1) positron emission tomography/computed tomography (PET/CT) for longitudinal in vivo whole-body imaging at a high spatiotemporal resolution; (2) multispectral optical imaging (OI) for ex vivo whole-organ imaging of metastatic lungs; and (3) multispectral confocal microscopy for intra-tissue imaging of iNPG-pDox distribution in metastatic lungs with high spatial resolution (Goel et al., 2020). This protocol includes methods for each of the approaches described above.

Materials and Reagents

  1. T-75 cell culture flasks

  2. Serological pipettes (Thermo Fisher Scientific, catalog numbers: 13-676-10F and 13-676-10M)

  3. FalconTM conical centrifuge tubes (Thermo Fisher Scientific, catalog number: 14-959-53A)

  4. 1 cc Exel insulin syringes (28 G, 1 ml, Fisher, catalog number: 14-841-31)

  5. BD 1 ml insulin syringe with slip tip (Fisher, catalog number: 22-253-260)

  6. Sterile filter pipette tips

  7. EppendorfTM tubes (various sizes)

  8. Coverslips

  9. 31 G disposable insulin syringes (BD, catalog number: 324921)

  10. ParafilmTM M PM996 all-purpose laboratory film

  11. MilliporeSigmaTM silica gel 60G TLC plates, glass-backed (MilliporeSigma, Catalog number: 1.00384.0001)

  12. 26 G × ¾” mouse and rat tail vein Monoject IV catheter (Patterson Veterinary, catalog number: 07-836-8403)

  13. BalB/c mice: Female, 4-6 weeks old (Invigo or The Jackson Laboratory) or another strain suitable for the selected cancer cell line

  14. Murine triple-negative breast cancer cell line expressing luciferase: 4T1-GFP-Luc (ATCC) or another cancer cell line

  15. Discoidal porous silicon particles (iNPG) and pDox nanoparticles (prepared in-house) or another amine-modified nanoparticle of choice (Xu et al., 2016)

  16. 2-S-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid (p-SC-Bn-NOTA) (Macrocyclics, Dallas, ID: B-605)

  17. 0.9% sodium chloride injection USP, 100 ml fill in 150 ml PAB® (B. Braun, NDC number: 00264-1800-32)

  18. Sterile de-ionized water

  19. Phosphate-buffered saline (PBS) solution, pH 7.4 (Thermo Fisher Scientific, catalog number: 10010023)

  20. 100 ml filter-sterilized 50 mM EDTA solution

  21. N,N′-dimethylformamide (DMF; MiliporeSigma, catalog number: 227056-100ML)

  22. Dimethyl sulfoxide (DMSO; MiliporeSigma, catalog number: 317275-100ML)

  23. Diisopropylethylamine (MiliporeSigma, catalog number: 387649-100ML)

  24. Isopropanol

  25. 0.1 M sodium acetate solution

  26. 0.1 M HCl solution

  27. Copper-64 (64Cu; Washington University St. Louis)

  28. AlexaFluorTM 647 NHS ester (Thermo Fisher, catalog number: A20006)

  29. Mouse serum (MiliporeSigma, catalog number: M5905-5ML)

  30. OCT freezing compound (Tissue-Tek® O.C.T. Compound, Sakura® Finetek; VWR, catalog number: 25608-930)

  31. Tissue-Tek® Cryomold® molds/adapters (Sakura® Finetek)

  32. 4′,6-diamidino-2-phenylindole (DAPI; Thermo Scientific, prepared according to vendor instructions; catalog number: 62247)

  33. ProLongTM Gold Antifade Mountant (ProLong, catalog number: P36930)

  34. RPMI-1640 medium (ATCC, catalog number: ATCC® 30-2001TM)

  35. Fetal bovine serum (FBS) (GibcoTM, catalog number: 10-082-147)

  36. Pencillin-streptomycin antibiotics (GibcoTM, catalog number: 15-640-055)

  37. Isoflurane (MiliporeSigma, catalog number: Y0000858)

  38. Aluminium stubs (PELCO® pin mount starter kit; PELCO, catalog number: 76250-10)

  39. Recombinant Anti-CD31 antibody [EPR17260-263] (Abcam, catalog number: ab222783)

  40. Goat anti-rabbit IgG H&L (FITC) (Abcam, catalog number: ab6717)

  41. Complete culture medium (see Recipes)

  42. 50 mM EDTA solution (see Recipes)


  1. InveonTM PET/CT (Siemens Medical Inc., INVEON, catalog number: 138757)

  2. IVIS® Spectrum In vivo Imaging System (PerkinElmer, catalog number: 124262)

  3. Bioscan AR-2000 Radio-TLC Imaging Scanner (Eckert & Ziegler, Valencia, CA)

  4. Scanning electron microscope (Nova NanoSEM 230, Thermo Fisher Scientific, USA)

  5. Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK)

  6. Ultraviolet-visible-NIR spectrophotometer (Synergy H4 Hybrid, BioTek, USA)

  7. Wizard2 2-Detector Gamma Counter (PerkinElmer, catalog number: 2470-0020)

  8. EVOS Auto FL System (Thermo Fisher Scientific, USA)

  9. Nikon Eclipse Ti microscope

  10. Centrifuge (Beckman Coulter, model: Avanti® J-26 XPI, catalog number: 393127)

  11. Tissue culture incubator (Sanyo Scientific, catalog number: 133060)

  12. Biological safety cabinet (The Baker company, SterilGARD, catalog number: 101951)

  13. FisherbrandTM IsotempTM Hot Plate Stirrer, Ambient to 540 °C, Ceramic (Fisherbrand, catalog number: SP88854200)

  14. PCR thermal cycler (Bio-Rad Laboratories, model: T100TM, catalog number: 1861096)

  15. Braintree Scientific Diversity Partner Mouse Tail Illuminator Restrainer (Fisher, catalog number: NC0772753)

  16. SimportTM Scientific StainTrayTM Slide Staining System (Fisher, catalog number: 22-045-035)


  1. Inveon Research Workplace (INVEON, Siemens)

  2. Living Image Software (PerkinElmer)

  3. Nikon Elements software (Nikon)

  4. FIJI (NIH)

  5. Microsoft Office Suite

  6. GraphPad Prism


  1. Animal models for pulmonary metastasis of triple-negative breast cancer

    1. Grow and expand the cells according to the recommended conditions. Typically, 4T1-GFP-Luc cells are cultured in RPMI-1640 medium supplemented with 10% FBS and 1% Pencillin-streptomycin.

    2. Harvest cells while in the exponential growth phase (approximately 80-90% confluence) using trypsin or an appropriate enzyme for the specific cell line. Resuspend cells in medium containing serum.

    3. Count cells.

    4. Centrifuge cells at 225 × g, 4°C for 5 min. Resuspend cells in 1× PBS to a concentration of 1 × 105 cells/200 µl.

    5. Place cells on ice and transport to the vivarium.

    6. Restrain the mouse in a restrainer and position the tail such that the vein is facing upwards.

    7. Draw 200 μl cells into a 28 G disposable insulin syringe and gently inject into the tail vein. The contents of the needle should inject easily without resistance.

    8. Monitor tumor development regularly via bioluminescence imaging. Typically, 4T1-Luc cells take 10 days to develop lung metastases.

  2. Surface modification of iNPG-pDox or other amine-modified nano/microparticles

    1. For details of the structure of iNPG-pDox vectors and chemistry, readers are referred to the following references (Goel et al., 2020; Xu et al., 2016).

    2. Dissolve 3 × 109 amine-modified iNPG (or other nanoparticles of choice) in 0.5 ml DMF in a 1.5-ml EppendorfTM tube. Shake gently.

    3. Dissolve 0.8 mg p-SCN-Bn-NOTA in 0.5 ml DMSO and add to the iNPG solution.

    4. Sonicate the mixture until a homogenous solution is formed.

    5. Add 20 µl diisopropylethylamine and incubate the reaction mixture at 25°C for 4 h under vigorous shaking at 700 rpm. This step can be performed on a hot plate stirrer or in a thermal cycler.

    6. After the 4-h incubation, wash the iNPG-NOTA conjugates by centrifugation at 5,000 rpm for 5 min with DMSO as the solvent. Briefly, spin the conjugates down, carefully discard the supernatant and resuspend the pelleted nanoparticles in 0.5 ml DMSO by sonication. Repeat the wash step with DMSO (for a total of 2 times), followed by 0.5 ml isopropanol (one time) and 0.5 ml DI water (one time). The speed and time of the centrifugation can be adjusted according to the nanoparticles used.

    7. After the final wash step, resuspend the iNPG-NOTA pellet in DI water and freeze at -80°C or freeze-dry (depending on the nanoparticle used) for further use (for up to 1 week).

    8. Use the same process to obtain iNPG-AF647 by conjugating fluorescent dyes (e.g., AlexaFluorTM 647 NHS ester) for optical imaging.

  3. Radiolabeling of iNPG-NOTA

    1. Resuspend iNPG-NOTA in 0.5 ml 0.1 M sodium acetate solution.

    2. Adjust the pH of the solution to 5.5 using 0.1 M HCl.

    3. Add 37 MBq 64CuCl2 to the nanoparticle solution. All handling of radioisotopes and radiolabeled solutions should be performed behind a lead shield in a fume hood, in accordance with institutional radiosafety guidelines.

    4. Carefully wrap parafilm around the cap and incubate the tube at 37°C for 1 h with gentle shaking. This step should be performed in a thermal cycler or on a hot plate stirrer behind a protective lead shield.

    5. Remove the excess unchelated 64Cu by centrifugation at ~1,610 × g for 5 min. Wash twice with DI water to obtain the final radiolabeled constructs ([64Cu]NOTA-iNPG). All radioactive waste should be appropriately discarded.

    6. To calculate radiolabeling yield, radio thin-layer chromatography (radio TLC) is performed (Figure 1). Briefly, drop 2 µl [64Cu]NOTA-iNPG on one end (origin) of a silica gel TLC plate. Dip the strip vertically in 2 ml 50 mM EDTA solution in a 50-ml conical tube.

      Figure 1. Schematic of the radio TLC procedure to calculate radiolabeling yield

    7. The solution will slowly travel upward by capillary action. Once the solution has moved 2–4 cm upward (or 1 cm from the top of the strip; solvent front), remove the strip from the EDTA solution and allow to dry.

    8. Scan the strip on a Bioscan AR-2000 RadiobTLC Imaging Scanner according to the vendor’s instructions.

    9. Typical radiolabeling yields using with this procedure range from 50% to 70%, with >99% radiochemical purity.

  4. Loading pDox into [64Cu]NOTA-iNPG or iNPG-AF647

    1. Suspend 3 mg polymeric pDox monomers in DMSO (70 mg/ml) and add the solution to the [64Cu]NOTA-iNPG or iNPG-AF647 conjugates.

    2. Incubate the mixture for 3 h at room temperature with vigorous shaking. This step can be performed in a thermal cycler.

    3. Remove unloaded pDox polymers by washing twice with DI water using centrifugation, as described in B above, to obtain the final constructs: [64Cu]NOTA-iNPG-pDox for PET/CT imaging and iNPG-AF647-pDox for optical imaging and confocal microscopy. Resuspend the final conjugates in 1 ml PBS.

    4. Draw 200 μl [64Cu]NOTA-iNPG-pDox (~60-80 µCi per mouse) solution into a 1-ml syringe for injections in the next step.

  5. Small animal PET/CT imaging

    1. Anesthetize 4T1 tumor-bearing mice under 2% isoflurane (in 100% O2 gas).

    2. Transfer each mouse to a mouse restrainer, carefully placing the nose cone to maintain anesthesia.

    3. Insert the micro-catheter into the tail vein and flush with 50 μl PBS. Leave the syringe with PBS attached to the catheter.

    4. Secure the mouse onto the microPET/CT scanner bed under 2% isoflurane anesthesia administered via the nose cone manifold. To minimize movement, the limbs can be secured to the side of the bed with tape.

    5. Switch the PBS syringe in the catheter with the syringe containing [64Cu]NOTA-iNPG-pDox.

    6. Acquire a whole-body CT calibration image of the mouse.

    7. The PET/CT acquisition protocol will depend on the particular experiment and the requirements of the researcher.

    8. For dynamic PET scans, a 30-min dynamic PET mode, data are acquired using the 3D-OSEM iterative algorithm with a 256 × 256 × 256 voxel volume.

    9. Immediately after the dynamic PET scan is started, slowly inject the radiolabeled nanoparticle solution as a single bolus dose over 30 s.

    10. Continue the dynamic PET acquisition.

    11. If PET images are required at more time points, data are acquired using the standard 3D PET acquisition protocol. To account for radioactive decay, longer scan times may be necessary.

    12. Datasets are acquired as: standard static 3D PET reconstruction and dynamic 3D PET reconstruction with 6 × 300 s frames. CT attenuation correction is applied to all PET images.

  6. Ex vivo gamma counting

    1. When the PET/CT scans are at the final time point, euthanize the animal according to institutional protocols.

    2. Harvest and weigh the major organs including the tumor-bearing lungs.

    3. Radioactivity in the organs is measured using a gamma counter per the manufacturer’s instructions.

  7. Ex vivo whole-organ optical imaging

    1. In a separate cohort of 4T1-tumor bearing mice, inject 200 μl iNPG-AF647-pDox prepared in section D via the tail vein (described in section A).

    2. At the desired time point post-injection, euthanize the mouse according to institutional protocols.

    3. Harvest the lungs and place on a black sheet in the IVIS Spectrum Scanner.

    4. Acquire multiplexed ex vivo fluorescence images using the vendor’s preset filter sets encompassing the excitation and emission spectra for GFP (tumor), doxorubicin (pDox), and AF647. For this combination of fluorophores, 18 filter sets are automatically selected when using the IVIS Spectrum system.

    5. Set the field of view, acquisition time, and binning number. Data acquisition settings will vary from user to user but should be kept constant throughout the experimental study.

    6. After image acquisition, rinse the tissues in PBS, preserve in OCT compound in cryomolds, and store at -80°C for sectioning.

  8. Immunofluorescence microscopy

    1. Cryo-section the frozen tissues into 6-µm-thick sections using a cryotome.

    2. Incubate the sectioned tissues in cold acetone for 10 min. All steps for staining and incubation in this section should be performed in a staining tray or similarly fashioned light-blocking device.

    3. Prepare the blocking solution as follows: Mix horse and goat serum at a 1:1 v/v ratio and add 0.1% (by volume) Triton X-100.

      Note: The choice (species) of blocking serum depends on the host of the primary and secondary antibodies used for staining.

    4. Add 100 μl blocking solution per tissue section. Incubate at 4°C for 1 h.

    5. Prepare the solution of primary antibody for a total volume of 100 μl per tissue section. The anti-CD31 antibody can be used at a 1:200 dilution. For other primary antibodies, refer to the vendor’s instructions for the suggested dilution.

    6. After 1 h, gently tap the slide on its side to discard the blocking solution.

    7. Add 100 μl primary antibody cocktail to each tissue section and incubate overnight at 4°C.

    8. After overnight incubation, wash the tissue sections gently in PBS. For each wash, incubate the tissue section in PBS for 3 min, tap the slide on its side to drain the solution, and add fresh PBS. Repeat the wash step 3 times.

    9. Prepare the secondary antibody in blocking solution at the vendor’s recommended dilution. For the goat anti-rabbit-FITC secondary antibody, a 1:50 dilution is recommended.

    10. Incubate the sections in secondary antibody solution for 1 h at 4°C.

    11. Wash the secondary antibody with PBS as described in Step H8.

    12. Add 50 μl DAPI solution to the tissue sections and incubate for 10 min.

    13. Wash twice with PBS as outlined in Step H8.

    14. Clean and dry the portions of the slide adjacent to the tissue, taking care not to disturb or dislodge the tissue.

    15. Add a drop (or 5 μl) Gold Anti-fade Mounting Medium and gently place a coverslip on the tissue sections. Care must be taken to prevent the introduction of bubbles.

    16. Dry the slide by tapping gently with Kimwipes and keep protected from light in a black box or slide cassette covered with aluminum foil. It is recommended to perform confocal microscopy within a day of staining.

    17. Observe and photograph immunofluorescent slides by mounting on the stage of a confocal microscope according to the vendor’s instructions, using the appropriate objectives. Typically, 10× and 40× objectives are sufficient for nanoparticle uptake studies. Take images of 5-10 fields of view per tissue section distributed over the entire section.

    18. Prevent overexposure or stain bleaching of the samples. All images are acquired under identical conditions.

    19. FITC and DAPI can be observed with their dedicated filter sets. To oberve iNPG-AF647, use the cy5 common filter set. To observe fluorescence from pDox nanoparticles, the RFP common filter can be used.

Data analysis

  1. MicroPET/CT analysis

    1. PET/CT analysis varies by the user experimental system and institutional software and hardware set up. In this work, the following steps were followed:

    2. Input the injected radioactivity dose, time of injection, name of isotope, and time of scan into the software.

    3. Analyze the whole-body PET images of the mouse using the Inveon Workstation Software (Siemens Medical Inc.).

    4. Manually draw the region-of-interest (ROI) centered around the organs of interest (e.g., lungs, liver, spleen, kidney, bone, muscle, and heart) on the co-registered PET/CT images (Figure 2). The decay-corrected radioactivity in each ROI is computed automatically by the software as the percentage of the injected dose per gram of organ (%ID/g).

      Figure 2. Example of PET image quantitation using the Inveon Workstation Software (Siemens Medical Inc.). Coronal view of the whole-body distribution of [64Cu]NOTA-iNPG-pDox in a mouse bearing lung 4T1 lung metastases. White arrows depict the different organs, while ROI outlines are shown in green.

    5. Dynamic time-activity curves are plotted for each ROI using the dynamic 3D dataset.

    6. To save images, select a coronal, axial, or sagittal view and export as .TIFF files. 3D maximum intensity projection (MIP) reconstructions can be also obtained using the software.

  2. Optical imaging analysis

    1. Analysis of multiplexed optical imaging will vary from user to user depending on the experimental and hardware/software setups. The following steps were used in this protocol.

    2. Analyze the data using the LivingImageTM software (PerkinElmer).

    3. Perform spectral unmixing using the automatic mode with four-component principal components analysis. Autofluorescence is subtracted from all images during analysis.

    4. Draw volume- and area-matched ROIs on the spectrally unmixed images for each component (GFP for the tumor, AF647 for iNPG, and doxorubicin for pDox) (Figure 3).

      Figure 3. Example of ex vivo multiplexed optical imaging (IVIS® Imaging System) depicting the distribution of AF647-iNPG-pDox in 4T1 lung metastases using the PerkinElmer Living ImageTM software. Fluorescence signal from Dox (excitation: 535 nm, emission 560 nm) is shown.

    5. ROIs are computed automatically and are presented as the average radiant efficiency [units: (p/s/ cm2/ sr)/(μW/cm2)].

    6. Pixelwise line analysis can also be performed using the software.

    7. Export data as .TIFF files.

  3. Confocal microscopy analysis

    1. In the present work, images were analyzed using the Nikon Elements (Figure 4) and FIJI software.

      Figure 4. Example of multiplexed confocal microscopy immunofluorescence imaging analysis using the Nikon NIS-ElementsTM software. Merged (first panel) and single-channel images of DAPI (blue), blood vessels (CD31, green), AF647-iNPG (magenta), and pDox (red) in 4T1 metastases-bearing lungs. Scale bars as shown.

    2. Use constant settings for image analysis throughout the study.

    3. Apply Gaussian blur and automatic thresholding to segment the images for quantitation.

    4. Perform pixelwise quantitation of fluorescence intensity and area fraction coverage by drawing an ROI over the entire organ or individual tumor nodules.

    5. Typically, quantitation is performed over 5 random fields of view per stain.


  1. Complete culture medium for murine 4T1 triple-negative breast cancer cell line:

    500 ml RPMI 1640 Medium

    50 ml 1:10 Gibco fetal bovine serum, Premium Plus

    5.5 ml 1:100 Penicillin-Streptomycin (Pen/Strep) antibiotics


    1. We recommend aliquoting FBS and Pen/Strep into 50 5.5-ml batches (sufficient to make one batch of 500 ml complete cell culture medum) to prevent repeated freezing/thawing of the components.

    2. The complete medium can be pre-prepared in advance and stored in 4°C for an extended period.

    3. We recommend warming the complete cell culture medium in a water bath at 37°C prior to use.

  2. 100 ml filter-sterilized 50 mM EDTA solution:

    1.87 g EDTA (disodium ethylenediaminetetraacetate·2H2O)

    De-ionized water

    1. Add sterile de-ionized water to EDTA powder to a final volume of 100 ml

    2. Pass the solution through a 0.2-µm Nalgene bottle top sterile filter unit

    3. Store in 4°C for an extended period


The protocols described in this work are derived from the original research publication, Sci Adv. 2020 Jun 24;6(26):eaba4498 (Goel et al., 2020). This work was partially supported by the NIH grants U54CA210181, R01CA193880, and R01CA222959, in addition to the U.S. Department of Defense grant W81XWH-17-1-0389.

Competing interests

Mauro Ferrari is the inventor of a U.S. patent from The University of Texas-Houston (patent no. US 8,920,625 B2, granted 30th December, 2014). Mauro Ferrari and Haifa Shen are inventors of a non-provisional patent application from the Houston Methodist Hospital (PCT/US 2014/0010879 A1). Both inventions are related to the scientific research described herein. More recently, Mauro Ferrari has formed a company (BrYet LLC) and has secured certain commercial rights on said patents. Though not an executive officer, he remains a majority shareholder, director, and scientific advisor of the company. All other authors declare that they have no competing interests.


All animal studies were performed in accordance with the guidelines from the Institutional Animal Care and Use Committee (IACUC ID: IS00004452; Validity: 01/02/2018-01/01/2021) at the Houston Methodist Research Institute.


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  2. Blanco, E., Shen, H. and Ferrari, M. (2015). Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat Biotechnol 33(9): 941-951.
  3. Goel, S., England, C. G., Chen, F. and Cai, W. (2017). Positron emission tomography and nanotechnology: A dynamic duo for cancer theranostics. Advanced Drug Delivery Reviews 113: 157-176.
  4. Goel, S., Zhang, G., Dogra, P., Nizzero, S., Cristini, V., Wang, Z., Hu, Z., Li, Z., Liu, X., Shen, H. and Ferrari, M. (2020). Sequential deconstruction of composite drug transport in metastatic breast cancer. Science Advances 6(26): eaba4498.
  5. Michor, F., Liphardt, J., Ferrari, M. and Widom, J. (2011). What does physics have to do with cancer? Nat Rev Cancer 11(9): 657-670.
  6. Elia, I., Rossi, M., Stegen, S., Broekaert, D., Doglioni, G., van Gorsel, M., Boon, R., Escalona-Noguero, C., Torrekens, S., Verfaillie, C., Verbeken, E., Carmeliet, G. and Fendt, S. M. (2019). Breast cancer cells rely on environmental pyruvate to shape the metastatic niche. Nature 568(7750): 117-121.
  7. Xu, R., Zhang, G., Mai, J., Deng, X., Segura-Ibarra, V., Wu, S., Shen, J., Liu, H., Hu, Z., Chen, L., Huang, Y., Koay, E., Huang, Y., Liu, J., Ensor, J. E., Blanco, E., Liu, X., Ferrari, M. and Shen, H. (2016). An injectable nanoparticle generator enhances delivery of cancer therapeutics. Nat Biotechnol 34(4): 414-418.


[摘要]的d ESIGN的有效纳米制剂靶向转移性乳癌是具有挑战性由于一个缺少的主管成像和图像分析协议,可以捕获注入的纳米颗粒和转移病灶之间的相互作用。在这里,我们描述了体内全身PET-CT与高时间分辨率,离体全身器官光学成像和高空间分辨率共聚焦显微镜的整合,以解构封装有聚合物阿霉素(iNPG-pDox )在三重的肺转移瘤-阴性的乳腺癌。我们描述的图像采集和分析的细节在逐步地沿的开发一个用于转移性乳腺癌的小鼠模型。这些方法在此描述中可以很容易地适应于任何纳米颗粒或疾病模型,允许一个标准化管道体内临床前研究,侧重于划定转移内的纳米粒子动力学和相互作用。

[背景技术] Metasta抽动癌症占> 90%的癌症患者中的死亡率; 然而,有效的治疗策略缺少由于小尺寸,异质性,和转移性癌症的分散性质(比安基尼等人,2016;埃利亚。等人,2019)。一系列的生物屏障阻碍积累,并且因此,传统的小分子药物和纳米颗粒的功效- (Blanco的基于递送系统在转移性癌症。等人,2015)。我们小组先前已经证明了的最顺序和协商成功,如果不是全部,生物屏障使用INPG-PDOX由多孔硅微粒基可注射的纳米颗粒发生器(的INPG与聚(乳酸-共-乙醇酸)封装)聚合物-多柔比星(pDox )共轭物(Xu et al。,2016)。PDOX分子自组装成纳米颗粒后其从释放iNPGs ,和INPG-PDOX治疗显著抑制肿瘤转移,包括一个在40功能治愈-肺转移性乳腺癌的小鼠的50%(徐等人,2016)。有效地针对转移的此类纳米药物的时空动力学的可视化可以在未来进行其他先进疗法的定向开发和合理改进(Goel,2017)。

尽管在药物传递系统的发展进步显著,有限的研究评估这些系统的分布和相互作用在体内转移的设置。分子成像模态可以发现丰富的跨多个长度的信息-和时间-在疾病设置秤; ħ H但是,所述的合理组合的多尺度成像方法应用到评估的在转移性肿瘤仍然复杂的药物递送系统的生物分布将被执行(戈埃尔,2017)。这样的知识,与数学建模方法结合,可以预测和评估治疗反应和efficac IES复杂的药物递送系统的,最终被荷兰国际集团用来引导所述的先进的疗法(逆向工程Michor ,2011)。在该p rotocol,邮局我们提供了一个详细的指南来设计和实现一个多尺度和多模成像工具包的系统解构的离子在贩卖和纳米治疗剂的相互作用一个乳腺癌的转移模型,如在我们早期的工作(描述戈埃尔等人。,2020)。

我们首先设计了一个模块化的垂直表面工程方法的修饰的的ication INPG-PDOX以使它们适合于多模式成像(核和荧光成像)和显微镜检查,而不影响其固有的生物物理特性。这种方法与载体类型,货物,配制方法或运输路线无关,并且可以用于任何纳米或生物工程平台,并可以在任何疾病模型中实施。接下来,我们设计并实现的构成的系统的成像工具包:(1)正电子发射断层成像/计算机断层扫描(PET / CT)为纵向体内整体-在体温成像一个高时空分辨率; (2)多光谱光学成像(OI)为体外全-转移性肺器官成像; (3)多光谱共聚焦显微镜以高空间分辨率对转移性肺中iNPG-pDox分布进行组织内成像(Goel等,2020)。此p rotocol包括每个上述方法中描述的方法。

关键字:体内成像, 正电子成象术, 光学成像, 癌症, 新陈代谢, 小鼠模型


1. T-75细胞培养瓶     

2.血清移液器(Thermo Fisher Scientific,目录号:13-676-10F和13-676-10M)     


4. 1立方厘米Exel的我nsulin小号yringes(28克,1毫升,费舍尔,目录号:14-841-31)     

5. BD 1米升我nsulin小号yringe与小号唇吨IP(费舍尔,目录号:22-253-260)     


7. Eppendorf TM管(各种尺寸)     


9. 31 G一次性胰岛素注射器(BD ,目录号:324921)     

10. Parafilm TM M PM996通用实验室胶片 

11. MilliporeSigma TM小号利卡克EL 60G TLC p酸酯,克小姑娘-b被ACK (MilliporeSigma ,目录号:1.00384.0001) 

12. 26 G × ¾”鼠尾静脉Mon oject IV导管(帕特森兽医,目录号:07-836-8403) 

13. BALB / c小鼠:女性,4 - 6周龄(Invigo或Ť他杰克逊Laborator Ý )或另一应变适合于所选癌细胞系 


15.盘状多孔硅颗粒(iNPG )和pDox纳米颗粒(内部制备)或其他选择的胺改性纳米颗粒(Xu等人,2016) 

16. 2-S-(4-异硫氰酸根合苄基)-1,4,7-三氮杂环壬烷-1,4,7-三乙酸(p-SC-Bn-NOTA)(Macrocyclics ,Dallas,ID:B-605) 

17. 0.9%小号裂果Ç hloride我njection USP,将100ml ˚F生病在150ml PAB ® (贝朗,NDC号码:00264-1800-32) 


19.磷酸盐-b uffered小号艾琳(PBS)溶液,pH 7.4(赛默飞世尔科技,产品目录号:10010023) 

20. 100毫升˚F ILTER -灭菌的50mM EDTA溶液 

21. N,N'-二甲基甲酰胺(DMF; MiliporeSigma,目录号:227056-100ML) 

22.二甲基亚砜(DMSO; MiliporeSigma ,目录号:317275-100ML) 



25. 0.1 M醋酸钠溶液 

26. 0.1 M HCl溶液 

27. Copper-64(64铜;圣路易斯华盛顿大学) 

28. AlexaFluor TM 647 NHS酯(Thermo Fisher ,目录号:A20006) 

29.小鼠血清(MiliporeSigma ,目录号:M5905-5ML) 

30. OCT ˚F reezing Ç ompound(组织-Tek的® OCT化合物,樱® Finetek ; VWR ,目录号:25608-930) 

31.组织-Tek公司® Cryomold ®米孩子/一个dapters(樱® Finetek ) 

32. 4',6-diamidino-2-phenylindole(DAPI; Thermo Scientific,根据供应商说明制备;目录号:62247) 

33.的ProLong TM Gold抗淬灭封固(的ProLong ,目录号:P36930) 

34. RPMI-1640米edium(ATCC ,目录号:ATCC ® 30-2001 TM ) 

35.胎儿b绵羊小号erum (FBS) (Gibco公司TM ,目录号:10-082-147) 

36. Pencillin -s链霉素抗生素(Gibco TM ,目录号:15-640-055) 


38.铝存根(PELCO ®销安装启动套件; PELCO ,目录号:76250-10) 

39.重组抗CD31抗体[EPR17260-263 ](Abcam ,目录号:ab222783) 

40.山羊一个nti- ř abbit的IgG H&L(FITC)(Abcam公司,目录号:ab6717) 


42. 50 mM EDTA解决方案(请参阅食谱) 


Inveon TM PET / CT(西门子医疗公司,INVEON,目录号:138757)
IVIS ®谱在体内成像系统(珀金埃尔默,目录号:124262)
Bioscan AR-2000 Radio-TLC成像扫描仪(埃克特和齐格勒,巴伦西亚,加利福尼亚)
扫描电子显微镜(Nova NanoSEM 230,美国赛默飞世尔科技公司)
Zetasizer Nano ZS(英国伍斯特郡马尔文仪器有限公司)
紫外可见近红外分光光度计(Synergy H4 Hybrid ,美国BioTek )
向导2 2检测器伽玛计数器(PerkinElmer ,目录号2470-0020)
EVOS Auto FL System(Thermo Fisher Scientific,美国)
尼康Eclipse Ti显微镜
离心机(Beckman Coulter公司,型号:阿凡提® J-26 XPI,目录号:393127)
组织培养培养箱(Sanyo Scientific,目录号:133060)
生物安全柜(The Baker公司,SterilGARD ,目录号:101951)
Fisherbrand TM等温TM热板搅拌器,环境温度540 °C,陶瓷(Fisherbrand ,目录号:SP88854200)
PCR热循环仪(Bio-Rad Laboratories,型号:T100 TM ,目录号:1861096)
Braintree科学多样性合作伙伴鼠标尾部照明约束器(Fisher ,目录号:NC0772753)
Simport TM Scientific StainTray TM幻灯片染色系统(Fisher ,目录号:22-045-035)


Nikon Elements软件(Nikon)
Microsoft Office套件


成长并根据扩增细胞的推荐条件。通常,在补充有10%FBS和1%Pencillin - s链霉素的RPMI-1640培养基中培养4T1-GFP-Luc细胞。
收获细胞,而在该指数生长期(约80 -使用胰蛋白酶或90%汇合)的针对特定细胞系合适的酶。RES uspend介质中的细胞含有血清。
在225 × g ,4°C下将细胞离心5分钟。将细胞重悬于1 × PBS中,浓度为1 × 10 5细胞/ 200 µl。
第200期 将μl细胞放入28 G一次性胰岛素注射器中,然后轻轻注入尾静脉。针头中的内含物应易于注射而不会产生阻力。

关于iNPG-pDox载体的结构和化学的详细信息,读者可参考以下参考文献(Goel等,2020; Xu等,2016)。
将3 ×10 9胺改性INPG (或选择的其它纳米颗粒)在0.5ml DMF中,在1.5 -米升的Eppendorf TM管。轻轻摇动。
将0.8 mg p-SCN-Bn-NOTA溶解在0.5 ml DMSO中,然后添加到iNPG溶液中。
加入20 µl二异丙基乙胺,在700 rpm剧烈振摇下,将反应混合物在25°C孵育4小时。该步骤可以在热板上搅拌器或进行在热循环仪。
后的4小时温育后,冲洗INPG以5,000rpm离心-NOTA缀合物5分钟用DMSO作为所述溶剂。简要地说,将缀合物旋转下来,小心弃去上清液,然后通过超声将沉淀的纳米颗粒重悬于0.5 ml DMSO中。用DMSO重复洗涤步骤(共2次),然后用0.5 ml异丙醇(一次)和0.5 ml去离子水(一次)重复洗涤步骤。离心的速度和时间可以根据所使用的纳米颗粒进行调节。
后的最终洗涤步骤中,重新悬浮在INPG在DI水中和FR -NOTA粒料EEZE在- 80℃或冷冻-DR ý (取决于所使用的纳米颗粒)以供进一步使用(用于最多至1周)。
使用S火焰处理以获得INPG-AF647通过缀合的荧光染料(例如,的AlexaFluor TM 647用于光学成像NHS酯)。

iNPG -NOTA的无线电标记
RES uspend INPG -NOTA于0.5ml的0.1M乙酸钠溶液。
甲djust使用0.1M HCl中的溶液的pH至5.5。
将37 MBq 64 CuCl 2添加到纳米颗粒溶液中。放射性同位素和放射性标记溶液的所有处理均应根据机构的放射安全指南,在通风橱中的铅屏蔽罩后面进行。
小心地将封口膜缠绕在瓶盖上,并在37 °C下轻轻摇动将试管孵育1小时。该步骤应在热循环仪中或在铅防护罩后面的热板搅拌器中进行。
除去的过量的未螯合64通过以铜〜1 ,610 ×克5分钟。用去离子水洗涤两次,以获得最终的放射性标记的构建体([ 64 Cu] NOTA - iNPG )。所有放射性废物均应适当丢弃。
为了计算放射性标记收率,无线电薄-层色谱法(TLC无线电)被执行(图1)。简短地说,在硅胶TLC板的一端(起点)滴2 µl [ 64 Cu] NOTA - iNPG 。垂直于2M浸条升的50mM EDTA溶液,在50 -毫升锥形管中。


溶液将通过毛细管作用缓慢向上传播。溶液向上移动2 – 4厘米(或距离试纸顶部1厘米;溶剂前面)后,将其从EDTA溶液中取出并使其干燥。
扫描上的Bioscan的AR-2000带无线电b TLC根据成像扫描仪的供应商的指令。
使用该程序的典型放射标记收率范围为50 %至70%,放射化学纯度> 99%。

大号oading PDOX到[ 64的Cu] NOTA - INPG或INPG-AF647
暂停3个毫克聚合物PDOX单体在DMSO(70毫克/毫升),并将该溶液添加到该[ 64的Cu] NOTA - INPG或INPG-AF647缀合物。
删除卸载PDOX通过洗涤聚合物利用离心DI水两次,如上述B中描述的,以获得所述最终构建体:[ 64的Cu] NOTA - INPG - PDOX用于PET / CT成像和INPG-AF647-PDOX邻ptical成像和共聚焦显微镜。RES uspend于1ml PBS中的最终缀合物。
绘制200 μ升[ 64的Cu] NOTA - INPG - PDOX (〜60 - 80 μ次每只小鼠)溶液中,以1 -毫升注射器用于在下一步骤中注射。

小一个nimal PET / CT成像
在2%异氟烷(在100%O 2气体中)下麻醉4T1荷瘤小鼠。
插入微导管到尾静脉中,并用冲洗50 μ升PBS。保留注射器与PBS附着在导管上的状态。
在通过鼻锥歧管进行2%的异氟烷麻醉下,将鼠标固定到microPET / CT扫描仪床上。为了最大程度地减少运动,可以用胶带将四肢固定到床的侧面。
切换PBS注射器在cathete随r的注射器含有[ 64的Cu] NOTA - INPG - PDOX 。
的PET / CT采集协议将取决于在特定的实验和所述研究者的要求。
对于动态PET扫描,30 -分钟动态PET模式中,数据被使用采集的3D-OSEM迭代算法使用256 × 256 × 256体素体积。
在开始动态PET扫描后,立即在30 s内以单次大剂量缓慢注入放射性标记的纳米颗粒溶液。
如果在更多的时间,PET图像点,数据被使用获取的所述标准3D PET获取协议。为了说明放射性衰变,可能需要更长的扫描时间。
数据集的获取方式为:具有6 × 300 s帧的标准静态3D PET重建和动态3D PET重建。CT衰减校正适用于所有PET图像。

当PET / CT扫描是在所述最后的时间点,根据机构协议安乐死的动物。

在4T1的一个单独的队列-荷瘤小鼠,注射200 μ升INPG-AF647-PDOX在部分d经由制备的尾静脉(在A节中所述)。
收获肺部并将其放在IVIS Spectrum Scanner中的黑色薄板上。
获取复用离体荧光图像使用供应商的预设过滤套包围的为GFP(肿瘤)的激发和发射光谱,d oxorubicin(PDOX )和AF647。对于荧光团的这种组合,当使用过滤器18个集自动选择的IVIS光谱系统。
图像获取后,冲洗在PBS组织,保存在在cryomolds OCT化合物,并储存在-80℃用于切片。

低温部分的冷冻组织中,以6 -μ米-使用低温切片机厚的切片。
准备的封闭溶液如下:混合马和山羊血清以1:1的体积/体积比率,并添加0.1%(按体积)的Triton X-100。

添加100 μ升阻断每组织部分的解决方案。在4°C孵育1小时。
添加50 μ升DAPI溶液到组织切片并孵育10分钟。
添加一滴(或5 μ升)金甲NTI衰落中号ounting中号edium并轻轻放置在组织切片盖玻片。必须注意防止气泡的产生。
ð RY滑动通过轻敲与轻轻的Kimwipes和保持在一个黑箱或载玻片盒用铝箔覆盖避光。建议在染色的一天内进行共聚焦显微镜检查。
通过在阶段安装观察和照片免疫滑动一个根据共焦显微镜的供应商的说明,使用的合适的目标。通常,10倍和40倍物镜足以进行纳米颗粒吸收研究。拍摄图像的5 - 10个字段的每组织切片视图分布在整个截面。
FITC和DAPI可以通过其专用过滤器集进行观察。为了oberve INPG-AF647,使用的Cy5的普通过滤器设置。从观察荧光PDOX纳米颗粒,所述可用于RFP公共过滤器。


MicroPET / CT分析
PET / CT分析因用户实验系统以及机构软件和硬件设置而异。在这项工作中,将遵循以下步骤:
手动绘制该区域的利益(ROI)为中心的周围的兴趣(器官例如,肺,肝,脾,肾,骨骼,肌肉,上和心脏)的共同配准的PET / CT图像(图2)。所述d ecay -在每个ROI校正放射性由软件自动计算的百分比的的每克器官(%ID / g)与注射剂量。

图2. PET图像的实施例孔定量吨通货膨胀使用的Inveon工作站软件(西门子医疗公司)。的冠状视图的全-体[分布64的Cu] NOTA - INPG - PDOX在一个鼠标轴承肺4T1肺转移。白色箭头表示不同的器官,而ROI轮廓以绿色表示。


复用光学成像的分析会有所不同从用户到用户的实验和硬件/软件设置小号。在F ollowing步骤,使用在此协议。
使用LivingImage TM软件(PerkinElmer)分析数据。
绘制体积-和区域匹配的ROI上的每个分量,其频谱的未混合图像(GFP用于所述肿瘤,AF647为INPG ,和d oxorubicin为PDOX )(图3)。

图3的实施例体外复用光学成像(IVIS ®描绘成像系统)的分布的AF647 -iNPG-PDOX在使用珀金埃尔默生活4T1肺转移图片TM软件。显示了来自Dox的荧光信号(激发:535 nm,发射560 nm)。

的ROI自动计算和被表示为所述平均辐射效率[单位:(P / S /厘米2 / SR )/(μW /厘米2 )。

在本工作中,使用Nikon Elements(图4)和FIJI软件对图像进行了分析。

图4.使用尼康NIS- Elements TM软件进行的多重共聚焦显微镜免疫荧光成像分析的例子。合并(第一面板)和单- DAPI(蓝色)的信道的图像,血管(CD31,绿色),AF647 -iNPG(品红色),和PDOX在(红色)4T1转移轴承肺部。比例尺如图所示。



500毫升RPMI 1640培养基

50毫升1:10 Gibco公司˚F等人b绵羊小号erum,特优



我们建议等分˚F BS和Pen / STR ë峰值到50 5.5 -毫升批(足以使一个一批500毫升完全细胞培养medum )反复防止冻结的组分/解冻。
100 ml过滤器-灭菌的50 mM EDTA溶液:
1.87克EDTA(乙二胺四乙酸二钠· 2H 2 O)


通过经过该溶液一个0.2 -微米Nalgene瓶中顶部无菌过滤器单元


该协议描述的这项工作是从衍生的原始研究的出版物,科学进阶。2020 Jun 24; 6(26 ):eaba 4498 (Goel et al。,2020)。这项工作是由部分支持了美国国立卫生研究院授予U54CA210181,R01CA193880,并R01CA222959,除了与美国国防部的拨款W81XWH-17-1-0389。


毛罗·法拉利是从得克萨斯州休斯敦大学的美国专利(专利号US 8920625 B2,授予30的发明者个月,2014)。莫罗法拉利和海法沉是没有的发明者Ñ从休斯敦公会医院(PCT / US 2014/0010879 A1)-provisional专利申请。两项发明都与本文所述的科学研究有关。最近,毛罗·法拉利(Mauro Ferrari)成立了一家公司(BrYet LLC),并获得了上述专利的某些商业权利。尽管不是执行官,但他仍然是公司的大股东,董事和科学顾问。所有其他作者声明他们没有竞争利益。


所有动物研究均根据休斯顿卫理公会科学研究所的机构动物护理和使用委员会(IACUC ID:IS00004452;有效期:01 / 02/2018-01/01 / 2021 )的指导进行。


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              Blanco,E.,Shen,H.和Ferrari,M.(2015年)。纳米颗粒设计的原理克服了药物传递的生物学障碍。Nat Biotechnol 33(9):941-951。              
Goel,S.,England,CG,Chen F.和Cai,W.(2017)。正电子发射断层扫描和纳米技术:癌症治疗学的动态二重奏。先进药物递送评论113:157-176。
Goel,S.,Zhang,G.,Dogra,P.,Nizzero ,S.,Cristini ,V.,Wang,Z.,Hu,Z.,Li,Z.,Liu,X.,Shen,H.和Ferrari,M.(2020年)。转移性乳腺癌中复合药物转运的顺序解构。科学进展6(26):eaba4498。
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埃利亚,I.,罗西,M.,Stegen ,S.,Broekaert ,D.,Doglioni ,G.,面包车Gorsel ,M.,皇家,R.,埃斯卡洛纳-Noguero ,C.,Torrekens ,S.,Verfaillie , C.,Verbeken ,E.,Carmeliet ,G.和Fendt,SM(2019)。乳腺癌细胞依靠环境丙酮酸来塑造转移性利基。自然568(7750):117-121。
徐荣,张庚,麦洁,邓小霞,塞古拉-伊巴拉,V.,吴,S.,沉J.,刘海,胡,Z.,陈L. 。,Huang,Y.,Koay ,E.,Huang,Y.,Liu,J.,Ensor,JE,Blanco,E.,Liu,X.,Ferrari,M.和Shen,H.(2016)。可注射的纳米颗粒产生剂增强了癌症治疗剂的递送。Nat Biotechnol 34(4):414-418。
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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Goel, S., Ferrari, M. and Shen, H. (2021). Surface Engineering and Multimodal Imaging of Multistage Delivery Vectors in Metastatic Breast Cancer. Bio-protocol 11(10): e4030. DOI: 10.21769/BioProtoc.4030.
  2. Goel, S., Zhang, G., Dogra, P., Nizzero, S., Cristini, V., Wang, Z., Hu, Z., Li, Z., Liu, X., Shen, H. and Ferrari, M. (2020). Sequential deconstruction of composite drug transport in metastatic breast cancer.Science Advances 6(26): eaba4498.

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