Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Abstract

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 “nanodrug”) administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

Introduction

Despite many advances in the treatment of breast cancer, clinical options for metastatic disease remain limited. Patients commonly receive therapies targeted against drivers identified in the primary tumor, such as estrogen or HER2, but these drivers are not always conserved in metastases, rendering therapy ineffective¹. Other systemic therapies, such as chemotherapy, are non-specific and known for their side effects. To develop effective options for the treatment of metastatic breast cancer, it is important to consider the biological drivers that allow cancer cells to spread and colonize distant sites. One of these drivers is miR-10b, an oncogenic microRNA, implicated in breast cancer cell viability, invasion, and migration, which has been shown to be sufficient to confer metastatic potential in otherwise-nonmetastatic breast cancer cells^2,3. Importantly, miR-10b is also expressed at higher levels in metastases compared to matched primary tumors⁴, making it a promising target for the treatment of existing metastases.

Although miRNAs such as miR-10b have great potential as therapeutic targets for metastatic disease, the design of therapeutically viable methods for miRNA silencing presents unique challenges. Antisense oligonucleotides (ASOs) that bind their complementary miRNA sequence are commonly transferred to the cells in vitro using lipofection but cannot easily reach tumor cells in vivo due to inherent instability, risk of destruction by nucleases, short blood half-life, and the inability to enter cells due to charge-charge repulsion⁵. To combat these challenges, we developed a clinically applicable carrier for biomolecules using dextran-coated magnetic iron oxide nanoparticles (MNP)⁶. Amine groups on the nanoparticle allow for the conjugation of oligonucleotides, fluorescent dyes (e.g., Cy5.5), and targeting moieties. Additionally, the iron oxide core allows for in vivo monitoring of vehicle delivery using magnetic resonance imaging (MRI). We conjugated anti-miR-10b locked nucleic acid ASO and Cy5.5 to MNP to create a “nanodrug” referred to as MN-anti-miR10b, depicted in Figure 1⁷.

Figure 1: Schematic design of the MN-anti-miR10b “nanodrug.”.

The nanodrug components include the iron oxide core covered with dextran, conjugated to Cy5.5 dye and single stranded anti-miR10b oligonucleotide molecules. Abbreviation: ASO = antisense oligonucleotide. Created with BioRender.com.

In our previous studies, we showed that the nanodrug efficiently causes downregulation of miR-10b and inhibits the migration and invasion of triple-negative breast cancer cells in vitro7. In murine models of metastatic breast cancer, intravenous delivery of the nanodrug prevented the development of lymph node metastases or, if administered after lymph node metastasis formation, halted their growth⁷. Notably, the nanodrug was observed to readily accumulate in cancer tissues. While the nanodrug did not eradicate metastases on its own, in subsequent studies, we showed that combination treatment with adjuvant doxorubicin was curative in both immunocompromised and immunocompetent mouse models^3,8. The effects of miR-10b inhibition by the nanodrug have also been seen in feline mammary carcinoma⁹.

To effectively treat breast cancer, it is imperative to demonstrate that the drug accumulates in tissues of interest. Here, we present a protocol for demonstrating the accumulation of the magnetic nanoparticle carrier used to deliver therapeutic anti-miR-10b ASOs to cancer tissues using multiple modalities in murine models of metastatic breast cancer.

Protocol

The Michigan State University Institutional Animal Care and Use Committee (IACUC) has approved all procedures involving animal subjects. Values for calculations are summarized in Table 1.

Table 1:

Summary of values used for cell concentration and animal dosing calculations. Representative mouse body weight of 20 g is used for all dosing calculations. BW = bodyweight.

Measurement	Value
Cells in PBS	40 × 106 cells/mL
Cell stock (after addition of equal part Matrigel)	20 × 106 cells/mL
Cells per 50 μL of mammary fat pad implantation of stock	1 × 106 cells
Mouse bodyweight (approx.)	20 g
Luciferin stock concentration in PBS	30 mg/mL
Luciferin administration concentration	150 mg luciferin/kg bodyweight
Luciferin dose volume per mouse (approx.)	100 μL
Ketoprofen stock concentration in PBS	1 mg/mL
Ketoprofen administration concentration	5 mg ketoprofen/kg bodyweight
Ketoprofen dose volume per mouse (approx.)	100 μL
Nanodrug concentration in PBS	10 mg Fe/mL
Nanodrug administration concentration	10 mg Fe/kg bodyweight
Nanodrug dose volume per mouse (approx.)	40 μL

1. Key steps of MN-anti-miR10b synthesis

NOTE: Details of the MN-anti-miR10b synthesis have been described previously^9,10,11.

1. Prepare the magnetic nanoparticle (MN) core by co-precipitation method.

2. Crosslink and aminate the prepared nanoparticles using sodium hydroxide, epichlorohydrin, and ammonium hydroxide.

3. Conjugate a Cy5.5-NHS ester to MN through the heterobifunctional cross-linker N-succinimidyl 3-[2-pyridyldithio]-propionate (SPDP) to obtain MN-Cy5.5 to enable fluorescence imaging and microscopy.

4. Activate anti-miR-10b locked nucleic acid with 3% tris(2-carboxyethyl)phosphine (TCEP) and conjugate to MN-Cy5.5 to yield the MN-anti-miR10b.

5. Perform characterization of the conjugate to determine iron content (by iron assay), the number of Cy5.5 molecules per nanoparticle (by spectrophotometry) and the amount of conjugated LNA (by agarose gel electrophoresis).

2. Acquire study animals

1. Outline the study in advance to plan the experimental groups and the number of animals per group. House up to 5 mice per cage. If multiple cages of 5 mice are used during treatment, be sure to have control and experimental mice within each cage to remove the cage as a confounding variable.

2. Obtain mice at 6–7 weeks of age. Allow for at least 1 week of acclimation to the housing conditions prior to the induction of orthotopic tumors.

   NOTE: Procedures using the MDA-MB-231 (human-derived cell line) model of spontaneous breast cancer metastasis are described below. For this model, athymic nude mice (Foxn1^nu/Foxn1^nu) are commonly used. Other compatible immunocompromised mouse strains may be used, and the procedures are also applicable to allograft models in immunocompetent mice (e.g., 4T1 breast cancer cells in BALB/c mice).

3. Culture cells

1. Supplement Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (complete growth medium) to grow MDA-MB-231 cells expressing luciferase (e.g., MDA-MB-231-luc-D3H2LN). Grow the cells under aseptic conditions at 37 °C with 5% CO₂ and 95% humidity, generally in a T75 flask. If frozen, thaw cells and passage at least once prior to tumor implantation. Record the passage number on the flask.

   NOTE: In this study, a previously frozen vial of 1 × 10⁶ cells was thawed and passaged twice before use.

   1. To passage, apply 0.25% trypsin for 3 min at 37 °C to trypsinize cells at a confluency of <80%. Add 4x volume of complete growth media to neutralize the trypsin.
   2. Centrifuge the suspension at 200 × g for 5 min in a conical tube. Resuspend the cell pellet in 5 mL of complete growth medium after decanting the supernatant. Aliquot a portion of the cells to a new flask and add additional complete growth medium based on the new flask volume. Update the passage number on the new flask.

2. Thaw new cells after 10 passages to minimize genetic drift across studies.

4. Preparation of cells for induction of orthotopic tumors

1. Place frozen Matrigel (basement membrane matrix extract) at 4 °C for 24 h prior to preparation to allow the matrix extract to liquefy.

2. Determine the concentration and volume of cells needed for the study. Implant the mice with 1 × 106 cells per mouse in a 50 μL volume, composed of 1 part of chilled PBS and 1 part of the matrix extract.

3. Pellet the trypsinized cells as described in step 3.1.2. Resuspend the cell pellet in at least 10 mL of PBS to wash the cells, then centrifuge once more at 200 × g for 5 min. Resuspend the pellet in 500 μL of chilled PBS (cell stock).

4. Count the cells using a hemocytometer. As the cells will be at a high concentration, dilute a small aliquot (e.g., 10 μL) as needed, keeping track of the dilution factor until accurate measurements can be taken.

5. Dilute the required total number of cells to 40 × 10⁶ cells/mL, then add an equal volume of the chilled matrix extract to achieve a final concentration of 1 × 10⁶ cells per 50 μL. Keep on ice to prevent the extract from solidifying prior to implantation.

   NOTE: Cell concentrations are summarized in Table 1.

5. Induction of orthotopic tumors

1. Anesthetize a mouse using 2% isoflurane and then transfer it to a nosecone, maintaining the surgical plane of anesthesia using 0–3% isoflurane on a heating pad. Confirm the surgical plane of anesthesia by lack of corneal reflex and/or toe pinch response. Protect against corneal drying by applying ophthalmic ointment to the eyes.

2. Clean the skin near the injection site with a 70% alcohol wipe and allow a few seconds for the alcohol to dry. Induce at mammary gland #4 to reduce the risk of overlap of bioluminescence imaging signals between the primary tumor and most common sites of metastasis (lung and axillary lymph node).

   NOTE: Mammary gland numbering has been described previously¹². Briefly, a mouse in supine position with head oriented upwards will have glands 1 to 5 on the injector’s right side (mouse’s left) beginning from the closest to the head (cervical - gland 1) and descending to the most caudal (inguinal - gland 5). Glands 6 to 10 are oriented similarly on the opposite side of the animal.

3. Pipet the cell stock up and down to resuspend the cells. Draw 50 μL of the ice-cold cell suspension into an insulin syringe with a 29 G needle and inject the cells immediately. Keep the syringe on ice if not able to inject immediately.

   NOTE: Pipet up and down between each draw of cells to prevent the cells from settling.

4. Insert the bevel directly below the nipple of the desired mammary gland parallel to the body of the mouse at that location and inject the cells at a steady, slow rate. Leave the needle within the skin for at least 5 s after completion of the injection to allow the Matrigel to solidify and prevent leakage.

5. Move the mouse to a clean cage on a warming pad for recovery and supervise until fully ambulatory and able to maintain sternal recumbency. Do not leave the mouse unattended. Return the mouse to its cage with other mice only after it has recovered.

6. Monitoring tumor growth and metastasis development with bioluminescence imaging (BLI)

NOTE: As the MDA-MB-231 cells utilized here express luciferase, injection of luciferin substrate into mice will produce an optical signal detected by the imaging system scanner. In this model, metastases can be expected at 5–7 weeks post tumor induction. It is recommended to image mice 1–3x per week, depending on the importance of identifying the exact moment when metastases are visualized.

1. Anesthetize the mice using 2% isoflurane to minimize the risk of injury to the mouse when administering luciferin. Protect against corneal drying by applying ophthalmic ointment to the eyes.

2. Inject 150 mg/kg body weight of luciferin intraperitoneally into each mouse and return the mice to their cage on a warming pad to allow the mice to awaken and metabolize luciferin. Supervise and do not leave mice unattended until fully ambulatory and able to maintain sternal recumbency.

   NOTE: Values used for dosing calculations are summarized in Table 1.

3. Image the mice using the imaging system scanner beginning at approximately 10 min post injection with luciferin, re-anesthetizing the mice when needed to allow for time for transfer to the IVIS.

   1. Image up to 5 mice together in supine position, taking care to ensure their entire bodies are included in the field of view guide markings and oriented as straight as possible. Use clear tape to secure their arms for better visualization of the axillary lymph nodes. If imaging several mice at once, use manifold dividers to separate the mice to prevent signals from radiating onto other mice. If dividers are not available, use strips of light-absorbing paper in their place (e.g., thick, black cardstock).
      NOTE: Rates of luciferin metabolism vary across mouse and cell line models, and image acquisition beginning at 10 min post injection may not yield the strongest signal. It is recommended that, at the start of a new study, acquisitions at different time points be performed to determine the timing for peak signal intensity.
   2. Prepare the imaging system software for image acquisition with the following settings for BLI: Exposure = Auto, Binning = Medium, FStop = 1, Excitation = Block, Emission = Open, FOV = D, Height = 1.50.
   3. Generally, primary tumor signals will produce a relatively strong signal due to their superficial location using the setting Exposure = Auto. If monitoring for metastases, use black electrical tape to carefully cover the primary tumor and manually set Exposure = 300 s (or more) to acquire faint signals if present.
      NOTE: In this model, a signal separate from the primary tumor that is visible over multiple imaging sessions when the lower threshold is set as 5 × 10³ radiance is considered indicative of metastasis.

7. Resection of primary tumors

NOTE: Resection of primary tumors is important for longitudinal (e.g., therapeutic) studies in metastases; otherwise, mice may succumb to morbidity related to unrestricted primary tumor growth. Consider primary tumor size (risk of blood loss on resection) and ulceration (risk of infection) when determining time of resection.

1. If considering the absence of BLI signal to test for successful primary tumor resection, perform pre-operation imaging as described in section 6 and confirm that there is no signal post-surgery. Confirm successful resection within the next 1–2 days using freshly administered luciferin.

   NOTE: Immediate re-administration of luciferin is not recommended to avoid unnecessary stress to the animal. Luciferin injected prior to primary tumor resection should be enough to detect the signal if the resection was not complete.

2. Anesthetize the mouse using 2% isoflurane and then transfer it to a nosecone, maintaining the surgical plane of anesthesia using 0–3% isoflurane on a heating pad. Confirm surgical plane of anesthesia by the lack of corneal reflex and/or toe pinch response.

3. Protect against corneal drying by applying ophthalmic ointment to the eyes

4. Inject 5 mg/kg ketoprofen subcutaneously as analgesia for the procedure.

   NOTE: Values used for dosing calculations are summarized in Table 1.

5. Prepare the surgical area by alternating scrubs of 70% alcohol and betadine 3x. Allow the final betadine scrub to dry before proceeding.

6. Use sterile surgical scissors to open the skin above the primary tumor vertically (rostral-caudal). In the case of ulcerated skin, begin opening to the side of the ulceration to avoid leaving the primary tumor behind and to completely remove the ulcerated skin.

7. Continue using scissors and forceps to carefully dissect the connective tissue around the encapsulated tumor to completely remove the mass from the skin, as well as underlying body tissues as any remaining tumor may regrow.

8. If present, control bleeding by applying pressure with a cotton-tipped applicator.

9. Close the surgical opening with 5–0 Vicryl suture.

   NOTE: Interrupted suturing may result in better wound patency as mice are prone to bothering the surgical site.

10. Allow the animal to recover in a clean cage on a warming pad until fully ambulatory. Place moistened food on the bottom of the home cage when returning the animal to the cage.

11. Inject 5 mg/kg ketoprofen subcutaneously once per day for at least 2 days post surgery. Check wound health at these times.

8. Delivery of nanodrug

1. Weigh the mice as nanodrug dosage is based on bodyweight.

2. Anesthetize the mouse using 2% isoflurane and then transfer it to a nosecone, maintaining the surgical plane of anesthesia using 0–3% isoflurane on a heating pad. Confirm surgical plane of anesthesia by the lack of corneal reflex and/or toe pinch response.

   NOTE: Alternatively, the nanodrug can be administered to a restrained awake mouse. All subsequent steps would be the same.

3. Prepare an insulin syringe with 29 G needle with 10 mg of Fe nanodrug/kg mouse body weight.

4. Submerge the animal’s tail in warm water (30–35 °C) for 30 s to dilate the tail veins.

5. Wipe excess water from the tail and clean the injection site with a 70% alcohol wipe.

6. Insert the needle bevel up in the lateral tail vein approximately halfway down the tail and pull back the plunger slightly to confirm placement with flashback of blood into the needle. If required, move the needle slightly forward or to a more superficial depth to achieve successful placement.

7. Upon successful insertion, inject the nanodrug steadily at a slow rate of approximately 5–10 s for a 40 μL injection. Confirm successful injection by lack of solution pooling under the skin of the tail near the injection site and by darkening of the vein (from the dark nanoparticle solution).

   NOTE: Values used for dosing calculations are summarized in Table 1. Clumped nanoparticle formulations may embolize to the lung. If respiratory distress is observed shortly after injection, gently perform chest compressions on the mouse. Immediate action always results in the successful recovery of the animal from this possible issue.

8. Hold pressure over the injection site with gauze and remove the needle, keeping pressure for approximately 30 s until bleeding stops.

9. Allow the animal to recover in a clean cage on a warming pad until fully ambulatory.

9. Collection of metastases for analysis

1. Image the mice by BLI as described in section 6.

   NOTE: In this study, 5 × 10³ radiance is considered indicative of metastasis and is generally observed at 5–7 weeks. A slight variance in time-to-metastasis is to be expected across mice.

2. Immediately after imaging, sacrifice the mice by cervical dislocation under heavy (5%) isoflurane. Prior to dissection, confirm death by lack of corneal reflex and/or toe pinch response.

3. Carefully collect the metastases. Lymph node metastases present as an enlarged, encapsulated mass. Lung metastases will generally be distributed throughout the lung parenchyma; hence, collect the whole lung.

4. Place the collected tissues in a Petri dish and image using the imaging system to confirm bioluminescence (indicating the presence of luciferase-expressing cancer cells) and fluorescence (indicating nanodrug accumulation). Use the same BLI acquisition settings as described in step 6.3.2. FLI acquisition settings are Exposure = Auto, Binning = Medium, FStop = 1, Excitation = 675 and Emission = 720 (default program for Cy5.5 dye), Lamp Level = High, FOV = D, Height 1.50. Image the mouse carcass to determine if there is remaining cancer tissue worth collecting.

5. Rinse the cancer tissues in PBS.

6. To collect tissues for microscopy or qRT-PCR, embed in OCT and store at −80 °C until ready for processing.

7. To collect tissues for inductively coupled plasma optical emission spectroscopy (ICP-OES), tare a scale using an empty 1.7 mL tube, place the tissue in the tube, and record its weight. Freeze the tissue and store at −80 °C until ready for processing.

10. Validation of nanodrug delivery by fluorescence microscopy

1. Cryosection the OCT-embedded fresh frozen samples onto microscopy slides at 10 μm thickness. Adjust the chamber and specimen holder temperatures according to the tissue type. Settings between −20 °C and −15 °C are appropriate for both lung and lymph tissue.

2. Fix the tissue sections onto slides by submerging the sections or whole slides in 4% paraformaldehyde solution for 15 min. Rinse carefully with PBS.

3. Mount coverslips onto the slides. Use a medium with 4’,6-diamidino-2-phenylindole (DAPI) for visualization of tissue architecture.

4. Use a fluorescence microscope to examine sections for Cy5.5 (excitation 683 nm/emission 703 nm), indicating nanodrug delivery. Confirm that the signal is not background by using a negative control sample (tissue from a non-injected animal).

11. Validation of nanodrug delivery by inductively coupled plasma optical emission spectroscopy (ICP-OES)

1. Transfer the samples stored at −80 °C to a 15 mL conical tube and incubate it in an oven with the cap removed at 37 °C to dry.

   NOTE: This process took 24 h for MDA-MB-231 metastasis samples but may take several days depending on the size and moisture content of the sample.

2. Record the dry weight using a balance.

3. Add 2 mL of 70% trace HNO₃ to the vessel and microwave digest the sample. The parameters used in this study are as follows: Power = 1030 – 1800 W, Ramp Time = 20:00 – 25:00, Hold Time = 15:00, Temperature = 200 °C, Cooling = 30 min.

   CAUTION: Exercise caution when working with nitric acid as it and the fumes generated when heating it are highly corrosive. Work should be completed in a well-ventilated space with full personal protective equipment, such as lab coat, goggles/face shield, and gloves compatible with acid work. The small volumes and lengthy cooling used here minimize risk somewhat, but care should always be taken.

4. Transfer the digested samples to metal-free conical tubes; then, transfer 300 μL to a new tube. Dilute to 10 mL using 9.7 mL of ultrapure water, resulting in an HNO₃ concentration of 3% (v/v).

5. Prepare individual element Fe standards at concentrations of 1000, 100, 10, 1, 0.1, and 0 μg Fe/mL in 3% HNO₃ (v/v) and ultrapure water. Prepare individual element Y internal standard at a concentration of 1 μg/mL in 3% HNO₃ (v/v) in ultrapure water.

6. Analyze samples using ICP-OES. For both axial and radial modes, select the following emission lines for analysis of iron content. Fe (234.350 nm), Fe (238.204 nm), Fe (259.940 nm), and Y (371.029 nm) used for internal standardization.

7. Normalize the results to the amount of sample used for the input to calculate μg of Fe/g of tissue.

Representative Results

In our previous therapeutic in vivo studies, we treated mice with one dose of nanodrug (10 mg Fe nanodrug/kg mouse bodyweight) weekly for several weeks^3,7,8. For this demonstration, we sought to determine whether accumulation of nanodrug could be observed in lung metastases after one dose, 1 week later. The results of this study would guide the timeline for monitoring the nanodrug accumulation in future longitudinal studies. Ultimately, it can also serve as an indication of the nanodrug persistence in the tissue of interest.

We followed the protocol described above and induced orthotopic breast primary tumors. Mice were regularly imaged for the development of metastases. At 5–6 weeks, primary tumors were resected due to excessive size. Resection allowed mice to survive and metastases to continue to grow. Lymph node and/or lung metastases were observed in mice after 6–7 weeks post induction, and signal persistence and growth were monitored regularly using BLI. After visualization of persistent and widespread signals at metastatic sites (Figure 2A,B), one dose of nanodrug or PBS control was administered via the tail vein. After 7 days, mice were imaged one final time using BLI and sacrificed for sample collection. For mice with lung metastases, the lungs and heart were collected and imaged using BLI ex vivo to confirm that the lungs were the site of metastasis (Figure 3, top row).

Figure 2: Representative in vivo BLI.

Constitutive luciferase expression by the MDA-MB-231 cell line allows for BLI monitoring of metastasis formation in vivo. In this model, metastases are commonly seen at (A) the lungs or (B) lymph nodes. A minimum threshold of 5 × 10³ radiance was used for a positive signal during monitoring. Abbreviation: BLI = bioluminescence imaging.

Figure 3: Ex vivo BLI and FLI.

BLI (top row) and Cy5.5 FLI (bottom row) of heart and metastatic lungs from mice that received one dose of the nanodrug and control mice treated with PBS, collected after 1 week of treatment. Cy5.5 fluorescence in lung metastasis is only seen in animals injected with the nanodrug and not in control animals. N = BLI scale for nanodrug injected mice; C = BLI scale for control mice. Abbreviation: H = heart.

Samples were then imaged using fluorescence imaging using the excitation/emission wavelengths for Cy5.5, confirming that Cy5.5 from the nanodrug accumulated only in tissues harboring metastases (Figure 3, bottom row). A mouse with lung metastases that did not receive nanodrug treatment was used as a negative control (marked Control in Figure 3), showing bioluminescence but not Cy5.5 fluorescence in the lung tissue, demonstrating that Cy5.5 signal was independent from bioluminescence signal. Lung tissue was divided, and one part was embedded in OCT for sectioning and fluorescence microscopy and the other part was weighed and frozen for ICP-OES.

Fluorescence microscopy confirmed Cy5.5 signal in the lung metastases of nanodrug-treated mice but not in control mice (Figure 4), supporting the FLI results. ICP-OES revealed an increase of iron of more than 187 μg Fe/g tissue in lung metastases of nanodrug-treated mice (n = 2) compared to control mice (n = 2) with greater than 2.3-fold Fe concentration, indicating nanoparticle accumulation in the metastases of nanodrug-treated mice but not in control mice (Figure 5). Together, these results demonstrate that the nanodrug accumulated in the intended tissues, validating our methods as a means to monitor the delivery of nanotherapeutics.

Figure 4: Correlative fluorescence microscopy.

Fluorescence microscopy of metastatic lung sections from mice that received one dose of the nanodrug (top row) and control mice treated with PBS (bottom row), collected after 1 week of treatment. Cy5.5 fluorescence is only seen in metastatic tissues of mice injected with the nanodrug. Scale bar = 100 μm. Abbreviation: DAPI = 4’,6-diamidino-2-phenylindole.

Figure 5: Inductively coupled plasma optical emission spectroscopy results.

Iron concentrations in metastatic lungs from mice receiving one dose of nanodrug and control mice, collected after 1 week of treatment. Shown are mean ± SEM, statistical analysis performed using Welch’s t-test, n = 2 per condition.

Discussion

Nanoparticles have great potential for cancer treatment. Here, we showed that a Cy5.5-conjugated MNP carrier can reach cancer tissues to deliver therapeutic oligonucleotides in a murine model of metastatic breast cancer. The ability to administer the nanodrug systemically while still achieving considerable accumulation in cancer tissues offers tremendous advantages over many existing ASO delivery methods, which commonly require local and often invasive administration. As target specificity is imperative to patient safety and drug efficacy, these methods are valuable for establishing the feasibility of translation to the clinic. Additionally, the iron oxide core and Cy5.5 offer monitoring capabilities that other carriers or conventional therapeutics cannot.

In the protocol, there are several critical steps worth reiterating. When preparing cells for implantation, it is imperative that the stock is prepared in chilled PBS and liquefied Matrigel (e.g., placed in the fridge 24 h prior to preparation) that is kept on ice during the entire process up until implantation. As Matrigel solidifies at too-cool or too-warm temperatures, slight deviations in temperature may make drawing up and injecting cells difficult and inconsistent. If issues persist despite strict temperature control, Matrigel concentrations may be reduced to 25%. During BLI, consider the time dependency of luciferin metabolism. After luciferin administration, signal intensity will gradually increase, plateau, and then decrease. It is valuable to identify when the signal plateaus and include this time in the acquisition/exposure window, and similarly, to be as consistent as possible with when the acquisition is initiated. Failure to do so may result in an inaccurate assessment of tumor growth or lack thereof.

When waiting for spontaneous metastasis to occur, an important consideration is that primary tumors may ulcerate prior to the visualization of the metastases. These ulcers may cause pain or blood loss and are at risk of infection. If this occurs, monitor the mice carefully, addressing possible issues with veterinary staff if available. Lanolin and triple antibiotic ointment can be used to reduce these risks, but resection prior to metastasis may be requested by veterinary staff. If primary tumors must be resected prior to metastasis, continue to monitor the mice, as the cells may have already metastasized but not yet colonized or reached a size detectable by BLI. Another possible complication is extensive vascularization and, thus, the risk of greater blood loss on resection. A cautery tool may be useful to quickly stop excessive bleeding. Saline can also be administered subcutaneously to replenish volume if needed. When collecting metastases from a sacrificed mouse, work carefully but quickly, as the BLI signal used to guide collection of metastatic tissues will lose intensity. Transfer OCT-embedded samples to dry ice or −80 °C promptly to preserve proteins and nucleic acids.

The techniques shown here utilize orthotopic implants of human breast cancer cells in immunocompromised mice. They are, however, adaptable to other models of cancer such as glioblastoma¹³ and have been used by our laboratory in immunocompetent models^8,9. Protocols specific to the mouse model of cancer for induction of tumor should be used; for example, Matrigel is commonly used to assist with cancer cell implantation¹⁴ but is not necessary for all cell lines¹⁵. Use of cell lines that express luciferase or another reporter is recommended to allow imaging of mouse tumors in vivo and confirmation of the presence of cancer tissues ex vivo.

Nanodrug delivery in our study was validated using two methods: fluorescence microscopy and ICP-OES. Validation by fluorescence microscopy is made possible by conjugating Cy5.5 dye to MNP. If Cy5.5 (or other fluorescent dye that is conjugated to the nanoparticle) is not detected under fluorescence microscopy, confirm the dye was successfully conjugated and is able to enter cells by treating cells in vitro for 24–48 h. Afterward, change the medium to remove nanoparticles that were not taken up by cells and then examine under a fluorescent microscope. If the dye was successfully conjugated to the nanoparticle, punctate fluorescence should be seen within the cell cytoplasm. The signal should be strong; a weak signal may be indicative of low efficiency in conjugation or cellular uptake. Validation by ICP-OES requires careful measurement of “dry” sample weight, as iron content should be normalized by sample weight. It is also important to compare samples of the same tissue type/organ when evaluating iron uptake to control for differences in baseline iron content (i.e., iron that is not from the nanoparticle) among tissue types. For example, when evaluating metastatic lungs by ICP-OES, lung tissue (ideally also with metastases) should be used as a negative control to compare results. Accumulation of iron can also be seen using MRI, as has been demonstrated previously⁵, allowing for in vivo monitoring.

Nanoparticles offer unprecedented customizability compared to traditional therapeutics. Although our studies utilize the platform for inhibition of a microRNA using an antisense oligomer, the methods shown here apply to the delivery of other therapeutic moieties using the platform, such as small interfering RNAs (siRNA). By adding functional components such as Cy5.5, drug delivery in preclinical models can be monitored in ways previously not possible. These insights can be used to optimize drug delivery and efficacy, advancing these experimental therapeutics toward translation to the clinic.