What is Nanomedicine?

A short overview by Prof. Patrick Hunziker, MD


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Publications digest

Ultrasound and nano/microbubbles for molecule delivery

Lymph nodes are important in responses of the immune system, antigen collection and in case of a solid tumor, the tumor cells are capable of migrating into the lymphatics of the primary tumor’s environment. Therefore, the lymphatic system can be considered as the delivery route for therapeutic molecules, vaccines and diagnostic reagents. Sentinel lymph nodes are likely involved in the spread of tumor cells and therefore the status of these nodes are seen as an independent additional factor in the prognosis in disease (1). Diagnostic techniques such as ultrasound, magnetic resonance imaging and histological confirmation (biopsy) are used to investigate a possible metastasis to the sentinel lymph nodes (2–4). In the case of head and neck cancer, where a metastasis is confirmed, the removal of the sentinel lymph node is the primary option to increase the survival rate of the patient (5). Often this is not without side effects such as nerve injuries or fibrosis of the scar. Animal models to investigate lymph node metastasis are limited and therefore treatment methods are not fully explored yet. In earlier work done by this group, animal models (mice; MXH10/Mo/lpr) were developed with lymph node sizes similar to those found in humans (10 mm in mice 2.5–3 months age) (6, 7). When tumor cells are injected into the subiliac lymph nodes, metasta- sis in the proper axillary lymph can be introduced (7). In their current work, Kato et al. used their mice model to develop a new lymphatic delivery method (8). Liposomes were generated by the reverse phase evaporation method (1,2 distearoyl-sn-glycero-phosphatidylcholine and 1,2 distearoyl-sn-glycero-3-phasphatidylehtanolamine- methoxy-polyethylene glycol [ratio 94:6 mol/mol]). The nano/microbubbles were generated from the liposomes by sonication and in the presence of C3F8 gas. The main diameter provided was 199±84.4 nm, and about 0.01% had diameters that exceed a few micrometers. Approximately 80% of these bubbles contained liquid alone and 20% contained both liquid and gas. The fluorophore used in this investigation was TOTO-3. A solution containing the nano/microbubbles and fluorophores was injected into a subiliac lymph node and delivered to the proper axillary lymph node through the lymphatic vessels, which was confirmed by applying a contrast enhanced high frequency ultrasound imaging system. After exposure to destructive ultrasound, the fluorophores were taken up by the lymphocytes. This new method paves the way towards new clinical treatments of lymph node metastasis.

 

Targeting mitochondria in cancer cells

Mitochondria are important membrane bound eukary- otic organelles involved in tasks like the supply of energy, signaling, the cell cycle, growth and cell death (9). When cells are exposed to ionizing radiation, the mitochondria exhibit an increased oxidative stress, an adjusted function and may even induce cell death (10, 11). Thus, the enhancement of mitochondria by ionizing radiation, leading to apoptosis may offer possibilities to cancer treatment. Gold nanoparticles have been used as a radio-sensitizing agent for a variety of radiation sources (MV-kV photon spectra) in a wide variety of cell lines [e.g. (12–14)]. Many tumors have an enhanced permeability and poor lymphatic drain- age that enables the accumulation of gold nanoparticles in the tumors’ microenvironment as presented by Upreti et al. (15), while in another study an uptake of 19:1 (tumor vs normal tissue) has been reported (16). The nanoparticles enter the cell via endocytosis and remain mainly in lysosomes, however, this depends on both size and coating properties of the nanoparticles (17, 18). Other investigations present the uptake of nanorods in lung carcinoma cells (19) and presence on or near the outer membrane of mitochondria (20). Thus, if enough gold nanoparticles are present in or near the mitochondria of cancer cells, the local dose enhancement arising from these particles may be investigated. Dose enhancement has been studied by others using Monte Carlo methods, however, not for mitochondria as presented by Kirby and Ghasroddashti (21). The investigators used the code developed by Salvat et al. (22). First, simple distributions were generated of localized dose enhancement around gold nanoparticles after the irradiation by individual monoenergetic photons. This was followed by the generation of models of mitochondria with gold nanoparticles attached to their outer membrane, which were than “exposed” to X-ray spectra from 6 MV to 90 kVp. As an example, the work of Karakatas et al (20) was used, who showed in their publication a mitochondrion covered with 565 gold nanoparticles (13 nm diameter). From the calculations, it was concluded that the dose enhancement ratios [1] were the highest in the kilovolt range, [2] depend on the dimensions of the mitochondrion, [3] the number of gold nanoparticles, and [4] the thickness of the gold nanoparticle coating. The presented work suggests further investigation of the role of the mitochondrium in cellular response to radiation and a novel possibility for radiation therapy where the mitochondria are the target.

 

Bone regeneration in periodontitis

Periodontitis is a disease caused by bacteria that form biofilms, which induce a chronic inflammation resulting in the destruction of the supporting tissues of the teeth and osteoclast mediated bone resorption (23). Leukocytes are important for, e.g. killing of bacteria, secretion of inflammation cytokines, activation of osteoclasts (24). However, in susceptible individuals the inflammation might be unable to resolve which will result in a chronic inflammation in the periodontium and may lead to bone loss (25). At present, there is urgent need for new therapeutics in regeneration due to our actual incapability to control the inflammation during regeneration (26). Microparticles, which are shed by cell membranes, can be used to generate nano-proresolving medicines. The incorporation of proresolving lipid mediators enables the targeting of specific tissues as was shown by an investigation of Dalli et al. (27). Here an increased survival in animal models of sepsis is found. Lipoxin A4 is such a lipid mediator.

In earlier investigations done by part of the authors of the work highlighted here, it is found that in rabbit periodontitis, the agonists of resolution of inflammation prevent periodontal bone loss and in addition, in case of existing periodontitis the regeneration of the periodontal bone occurs. Agonists include lipoxins and resolvins. In addition, it was found that lipoxins have direct anabolic actions on bone, which is in agreement with the increased actions that were observed in bone regeneration. The topical application of lipoxin stable analogs showed a dramatic reduction of the leukocyte infiltration, inflammation and bone loss (28).

Van Dyke et al. (26) generated nano-proresolving medicines containing the stable lipoxin analog benzo lipoxin A4. The nanomedicine was clinically evaluated in a large animal model of inflammation induced bone loss (Hanford miniature pig). Even though the number of animals used was not enough to generalize, the results are robust, valid and show evidence for a new regeneration mechanism of bone lost to inflammatory disease (e.g. periodontitis). The generated nano-proresolving medicine is transferred to the serum membrane receptors of bone cells and as well to the leukocytes regulating their responses to stimulate the formation of bone. While the bone is remodeled, the biofilm environment is controlled as a result of the cellular events, which are initiated by benzo lipoxin A4. This further results in an up regulation of the endogenous proresolving lipid mediators and direct actions on the bone cells which influences osteogenesis. The result is a significant regeneration of the connective tissue and bone and a recovery of the periodontal organ.

References

1. Hirakawa S, Detmar M, Kerjaschki D, Nagamatsu S, Matsuo K, Tanemura A, et al. Nodal lym- phangiogenesis and metastasis: role of tumor-induced lymphatic vessel activation in extramammary Paget’s disease. Am J Pathol 2009;175:2235–48.

2. Black D, Specht M, Lee JM, Dominguez F, Gadd M, Hughes K,et al. Detecting occult malignancy in prophylactic mastectomy: pre-operative MRI versus sentinel lymph node biopsy. Ann Surg Oncol 2007;14:2477–84.

3. Shigekawa T, Sugitani I, Takeuchi H, Misumi M, Nakamiya N, Sugiyama M, et al. Axillary ultrasound examination is useful for selecting patients optimally suited for sentinel lymph node biopsy after primary systemic chemotherapy. Am J Surg 2012;204:487–93.

4. Yamamoto S, Maeda N, Tamesa M, Nagashima Y, Suga K, Oka M. Sentinel lymph node detection in breast cancer patients by real-time virtual sonography constructed with three- dimensional computed tomography lymphography. Breast J 2010;16:4–8.

5. Liauw SL, Mancuso AA, Amdur RJ, Morris CG, Villaret DB, Werning JW, et al. Postradiotherapy neck dissection for lymph node-positive head and neck cancer: the use of computed tomography to manage the neck. J Clin Oncol 2006;24: 1421–27.

6. Li L, Mori S, Kodama M, Sakamoto M, Takahashi S, Kodama T. Enhanced sonographic imaging to diagnose lymph node metastasis: Importance of blood vessel volume and density. Cancer Res 2013;73:2082–92.

7. Shao L, Mori S, Yagishita Y, Okuno T, Hatakeyama Y, Sato T, et al. Lymphatic mapping of mice with systemic lymphoproliferative disorder: usefulness as an interlymph node metastasis model of cancer. J Immunol Methods 2013;389:69–78.

8. Kato S, Shirai Y, Kanzaki H, Sakamoto M, Mori S, Kodama T. Delivery of molecules to the lymph node via lymphatic vessels using ultrasound and nano/microbubbles. Ultrasound Med Biol 2015;41:1411–21.

9. McBride HM, Neuspiel M, Wasiak S. Mitochondria: more than just a powerhouse. Curr Biol 2006;16:R551–60.

10. Fang F, Gong PS, Zhao HG, Bi YJ, Zhao G, Gong SL, et al. Mitochondrial modulation of apoptosis induced by low- dose radiation in mouse testicular cells. Biomed Environ Sci 2013;26:820–30.

11. Kam WW, Banati RB. Effects of ionizing radiation on mitochon- dria. Free Radical Bio Med 2013;65C:607–19.

12. Kong T, Zeng J, Wang X, Yang X, Yang J, McQuarrie S, et al. Enhancement of radiation cytotoxicity in breast-cancer cells by localized attachment of gold nanoparticles. Small 2008;4: 1537–43.

13. Berbeco RI, Korideck H, Ngwa W, Kumar R, Patel J, Sridhar S, et al. DNA damage enhancement from gold nanoparticles for clinical MV photon beams. Radiat Res 2012;178:604–8.

14. Khoshgard K, Hashemi B, Arbabi A, Rasaee MJ, Soleimani M. Radiosensitization effect of folate-conjugated gold nanoparti- cles on HeLa cancer cells under orthovoltage superficial radio- therapy techniques. Phys Med Biol 2014;59:2249–63.

15. Upreti M, Jyoti A, Sethi P. Tumor microenvironment and nano- therapeutics. Transl Cancer Res 2013;2:309–19.

16. Hainfeld JF, Smilowitz HM, O’Connor MJ, Dilmanian FA, Slatkin DN. Gold nanoparticle imaging and radiotherapy of brain tumors in mice. Nanomedicine (Lond) 2013;8:1601–9.

17. Chithrani DB. Intracellular uptake transport and processing of gold nano-structures. Mol Membr Biol 2010;27:299–311.

18. Shukla R, Bansal V, Chaudhary M, Basu A, Bhonde RR, Sastry M. Biocompatibility of gold nanoparticles and their endocytotic fate inside the cellular compartment: a microscopic overview. Langmuir 2005;21:10644–54.

19. Wang L, Liu Y, Li W, Jiang X, Ji Y, Wu X, et al. Selective targeting of gold nanorods at the mitochondria of cancer cells: Implications for cancer therapy. Nano Lett 2010;11:772–80.

20. Karatas OF, Sezgin E, Aydin O, Culha M. Interaction of gold nano- particles with mitochondria. Colloids Surf B 2009;71:315–8.

21. Kirby C, Ghasroddashti E. Targeting mitochondria in cancer cells using gold nanoparticle enhanced radiotherapy: a Monte Carlo study. Med Phys 2015;41: 1119–28.

22. Salvat F, Fernandez-Varea JM, Sempau J. Penelope-2008: a code system for Monte Carlo simulation of electron and photon transport. In Workshop Proceedings (OECD nuclear Energy Agency, Barcelona, Spain, 2009).

23. Taubman MA, Valverde P, Han X, Kawai T. Immune response: the key to bone resorption in periodontal disease. J Periodontol 2005;76(Suppl. 11):2033–41.