Safety evaluation of Nanodiamond-Doxorubicin Complexes in a Naïve Beagle Dog model

 

• March 17, 2023

Table of Contents

• 1. Doxorubicin (DOX)
• 2. Nanodiamonds (NDs)
• 3. Nanodiamond-Doxorubicin complexes (ND-DOX)
• 4. Naïve Beagle Dog
• 5. Using Naïve Beagle dogs to evaluate the safety of nanodiamond drug delivery platform to deliver DOX.
• 6. Conclusions

Nanodiamond-Doxorubicin complexes refer to a drug delivery system where tiny diamond particles, called nanodiamonds, deliver a chemotherapy drug called Doxorubicin to cancer cells. Research into nanodiamond-Doxorubicin complexes is ongoing, and early studies have shown promising results. However, more research is needed to fully understand the safety and effectiveness of this drug delivery system before it can be used in clinical settings.Naive Beagle Dogs(Naïve Beagle Dogs) are popular dogs. They are often used in scientific research for various purposes, including drug development, behavior studies, and genetics research.

This blog will discuss the safety evaluation of Nanodiamond-Doxorubicin Complexes in a Naive Beagle Dog model.

1. Doxorubicin (DOX)

Doxorubicin (DOX), an antibiotic derived from the Streptomyces peucetius bacterium, has had wide use as a chemotherapeutic agent since the 1960s. Doxorubicin is part of the anthracycline group of chemotherapeutic agents; other anthracyclines include Daunorubicin, Idarubicin, and Epirubicin. Doxorubicin is a highly effective anthracycline chemotherapy agent that treats a broad range of life-threatening malignancies but causes cardiotoxicity in many subjects.

Currently, rats or mice, rabbits, pigs, and beagle canines have been established to predict the cardiotoxicity of modified drugs and used to evaluate preclinical toxicity. Cell dysfunction, apoptosis, and molecular pathways related to the toxicity mechanism have also been improved—in-depth understanding.

2. Nanodiamonds (NDs)

Nanodiamonds (NDs) are among the most promising chemotherapy vectors. However, they tend to aggregate upon storage or when exposed to mild pH or ionic strength changes.

3. Nanodiamond-Doxorubicin complexes (ND-DOX)

As a valid and extensively used chemotherapeutic agent, Doxorubicin is narrowed in clinical application by dose-related toxicities, including cumulative cardiotoxicity and myelosuppression. Low dose or progressive release of Doxorubicin can prevent the significant risk factors from enabling long-term chemotherapy treatment. Nano-drug delivery systems can conquer toxicity challenges due to a lack of specificity and targeted delivery. Nanodiamonds (NDs) exhibit a high cell absorption ratio and minimum cytotoxicity. Furthermore, Doxorubicin can be reversibly loaded onto nanodiamonds by physisorption under primary conditions. Therefore, nanodiamonds are a promising strategy for designing highly efficient drug-delivery nanocarriers and therapeutic systems. NDs are widely used in biomedical applications.

Chemoresistance and drug efflux are two important limiting factors of cancer treatment. The nanodiamond delivery system can avoid transport protein consumption and retain sufficient drug exposure to the disease site.

Nanodiamond (ND)-based Doxorubicin (NDX) administration enhances treatment efficacy by reducing early mortality and inhibiting tumor growth, while the drug alone has no apparent efficacy. Furthermore, there is no evident myelosuppression. NDX improves apoptosis and tumor growth inhibition accompanied by a decrease in toxicity compared with conventional Doxorubicin therapy for liver tumor and mammary carcinoma models in murine. NDX delivery system inhibits the lung metastasis of breast cancer and metastatic hormone-refractory prostate cancer and aids in reducing the Doxorubicin dose necessary to attain a chemotherapeutic efficacy. The comprehensive evaluations of nanodiamond administration in vitro and in vivo models suggest that ND-drug can prolong drug release, accelerate tumor cell apoptosis and reduce systemic side effects such as myelosuppression.

Characterization of ND, DOX and NDX complex

In this study, researchers use large animals, Naïve Beagle dogs, to evaluate the safety of the nanodiamond drug delivery platform to deliver DOX.

4. Naïve Beagle Dog

The Naïve Beagle Dog(Naïve Beagle Canine) is often used in drug toxicology studies to assess new drugs or chemicals’ safety and potential risks. Naïve Beagle Dogs are given different doses of drugs or chemicals to determine their potential toxicity and side effects. Dogs were closely monitored for any adverse reactions, and results were used to assess the safety of the substance in question. The dogs were chosen for their breed, size, and age consistency, which helped control for experimental variables.

Beagle dogs are used in the study of pharmacokinetics and toxicokinetics, the blood sampling time and blood volume are accurate, and in the natural state, adverse reactions or toxic reactions can be directly observed; blood sampling does not require anesthesia, does not affect drug metabolism, and does not affect fluid replacement Blood concentration, in the bioavailability study, using its cross-control, can minimize the influencing factors.

5. Using Naïve Beagle dogs to evaluate the safety of nanodiamond drug delivery platform to deliver DOX.

Male Naïve Beagle dogs of groups 1–3 are injected with DOX, ND, and NDX intravenously. Female Naïve Beagle dogs of group 4 are injected with NDX through IV injection. Male Naïve Beagle dogs of group 5 are injected with NDX through hepatic portal vein injection (HPV). All the treatments are administered with a dose level of 3.648 mg/kg. After administration, the animals entered an observation phase to evaluate visible changes in their health. Meanwhile, blood and urine are obtained for comprehensive analysis before and after drug administration. Results indicate that Doxorubicin alone induces severe damage while NDX profoundly relieves Doxorubicin toxicity.

(1) Naïve beagle dog dosing protocol

The animal studies were performed with the Medicilon Preclinical Research (Shanghai). 13 Beagle dogs were divided into five groups. They administered 1 mg/mL Doxorubicin (Group 1), 1 mg/mL nanodiamond (Group 2), and final NDX suspension (Groups 3-5) in sterilized water via a single bolus intravenous injection (IV) (Groups 1-4) or HPV (Group 5).

The animals of Group 5 underwent HPV cannulation surgery and were allowed a few days of recovery before being placed on study. The dose for each test animal was given 3.648 mg/kg according to individual body weight. Body weight was measured on the day of dose administration and recorded. The animals had free access to drinking water during the next five days but were fasted overnight (at least ten h) before blood sample collection at study termination.

(2) Histological analysis

Dogs treated with Doxorubicin, nanodiamond, and NDX were conducted for histopathology analysis, and tissues from the liver, spleen, kidney, lung, and heart were stained with haematoxylin and eosin. Researchers found profound differences between Doxorubicin and NDX groups using IV injection.

The analysis of liver histopathology exhibited evidence of more significant damage in dogs treated with DOX. The liver-derived structure from the dead dog was disordered, with bleeding, obvious degeneration of liver cells, and inflammatory cells. The surviving dog treated with Doxorubicin also had denatured and inflammatory cells in the liver. There were also some minor degenerations and structural disorders in the NDXHPV group. Liver structure changes were slight or almost normal in ND-IV and NDX-IV.

For the spleen, dogs treated with Doxorubicin showed a disorder of structure, and that treated with nanodiamond was basically red pulp with obvious degeneration. Moreover, dogs treated with NDX-HPV exhibited spleen blood sinus expansion and degeneration. The spleen of NDX-IV groups changed mildly.

The kidney histological sections showed that dogs treated with Doxorubicin displayed blood vessels with blood stasis and inflammatory cells. The renal of dogs treated with nanodiamond was mild to moderately denatured, and interstitial fibres appeared in proliferation. The tubules were irregular in the NDX-HPV group. Only minor changes were observed in the kidney of dogs treated with NDX-IV.

Tissue sections of the lungs further revealed many exudates in the alveoli, haemorrhage, and stasis in the pulmonary capillaries in dogs treated with DOX. The alveolar wall had inflammatory degeneration and alveolar epithelial hyperplasia in the NDXHPV group, whereas the lung of dogs treated with ND-IV and NDXIV had only light lesions.

No gross abnormality was seen in the heart of dogs treated with NDX-IV. In contrast, dogs treated with DOX-IV, ND-IV, and NDX-HPV showed evidence of thinning of myocardial cells, interstitial edema, and myocarditis. In conclusion, the results based on histomorphology showed that for dogs treated with NDX-IV, the lesions in each organ were mild. The liver, spleen, kidney, lung, and heart of the other treatment groups showed more significant damage to a certain extent.

Histopathology of dogs treated with ND, NDX and DOX. Representative

(3) Hematology analysis

Hematology analysis showed that the ND-IV-M, NDX-IV-M, NDX-IV-F, and NDX-HPV-M did not cause dramatic changes to white blood cells, platelet count, neutrophils, and lymphocytes. Meanwhile, nanodiamonds or NDX did not cause subacute inflammation. Mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration analysis indicated that DOX, ND, and NDX groups showed slight fluctuation within the normal range.

These results suggest no apparent indications of the destruction of blood cells in nanodiamond and NDX groups for IV and HPV injection.

Complete hematology profiles of dogs treated with ND, NDX and DOX.

(4) Urinalysis analysis

Urine samples of all dogs were tested before and after administration. Urine pH is an indicator of the kidney’s adjustment of acid-base balance.

Data showed that urine pH and urine specific gravity did not fluctuate significantly before and after administration. However, the urine protein of the NDXM group increased significantly, suggesting the occurrence of nephritis. The urine obtained from the dogs injected with nanodiamond solution after 36 and 60 h exhibited the same variation trend. The heat release peak areas increased over time, indicating that more NDs were excreted.

Moreover, researchers observed NDs particle by transmission electron microscopy (TEM), confirming that nanodiamonds could be excreted in the urine.

Urinalysis analysis.

6. Conclusions

The study evaluated a dual-gender Naïve Beagle dogs model with DOX, ND, and NDX treatments. In addition, HPV and IV injections among dogs of the same sex are validated.

The collective findings from the different drug administrations indicate that Doxorubicin alone can damage dogs’ liver, spleen, lung, and kidney organs.

Histomorphology results indicate that NDX-IV groups show no apparent organ damage. NDX-HPV group suggests minor but observable side effects on the liver and spleen from histomorphology and blood analysis.

Furthermore, NDs can be detected in urine, indicating that their metabolic pathway could be through the urinary system. These studies provide a significant foundation for the preclinical safety assessment of NDX therapy and show large animal evidence that NDX alleviates the toxic effects. Thus, using NDX remains a promising clinical alternative to DOX treatment of cancer patients.

References:

[1] Raven El Khoury , et al. Demonstration of doxorubicin’s cardiotoxicity and screening using a 3D bioprinted spheroidal droplet-based system. RSC Adv. 2023 Mar 13;13(12):8338-8351. doi: 10.1039/d3ra00421j. eCollection 2023 Mar 8.

[2] Moustafa S Ali, et al. Towards more efficient inhalable chemotherapy: Fabrication of nanodiamonds-releasing microspheres. Int J Pharm. 2022 Oct 15;626:122169. doi: 10.1016/j.ijpharm.2022.122169.

[3] Liping Wang, et al. Safety evaluation of nanodiamond-doxorubicin complexes in a Naïve Beagle canine model using hematologic, histological, and urine analysis. Nano Research. 2022.15: 3356–3366.

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