48 Medical Technology Volume 4 No. 1, 2007

MEDICAL TECHNOLOGY

Review of a Novel Gold Nanoshell Based Diagnostic and Therapeutic Technology for Future Cancer Treatment

Abhitej Rewari Syed Rahman, BHSc (Hon) Prateek Goyal

INTRODUCTION ing and therapeutic modalities that are increasingly safer and he mere thought of cancer can be sufficient to elicit more specific for their cancerous targets . It is important that images of pain, loss, and adverse side effects. With invasiveness, cost, and the patient’s quality of life are given cancer being Canada’s leading cause of premature or due consideration in these attempts. Recently, a series of early death, 1 the statistics are just as frightening. Recent exciting experiments revealed nanoparticles hold great Tincidence rates estimate that 38% of Canadian women and promise . In an effort to gain a comprehensive picture, this 44% of Canadian men will develop cancer at some point review paper highlights the pathophysiology of tumor devel - during their lifetimes .1 Although diagnostic and therapeutic opment, presents the many shortcomings in current drug advances have made it possible to detect cancer in earlier delivery systems, and subsequently discusses a promising stages and hence treat such cases more effectively, current gold nanoshell -based diagnostic and therapeutic technology diagnostic and treatment procedures are far from ideal and that can potentially be used for future cancer imaging and there is an urgent need to address the many shortcomings. treatment . Given cancer’s natural history, the time of diagnosis BACKGROUND heavily influences the prognosis, with early detection signif - 2 icantly reducing the risk of morbidity and mortality . Tumor Progression Simplified Unfortunately, the majority of cancer diagnoses are made only after significant anatomical and pathological changes A sound understanding of the mechanisms behind tumor have already developed - usually in the latter stages of the development, maintenance, and metastasis is of utmost disease .3 The use of novel contrast agents over convention - importance. A recent article effectively reviewed the mecha - al fluorescent dyes like indocyanine green (ICG) may be one nisms of tumor progression .5 Given that cancer cells divide way of solving the problem. Once the tumor has been char - much more rapidly than the healthy tissue that surrounds acterized, surgical excision is the treatment of choice. Yet in them, both tissues engage in fierce competition for a com - order for surgery to be successful, the tumor must be acces - mon nutrient supply and a drainage system whereby meta - sible, well defined , and located in non vital regions of the bolic waste products can be eliminated. This places great body. 4 Here, surgeons remove the primary tumor mass and strain on the pre-existent vasculature and when the tumor associated surrounding tissue to reduce the risk of recur - mass eventually out-competes healthy tissues , it grows rence . Chemotherapy or radiation therapy is usually given beyond its maximal size. The size of normal cells under nor - thereafter to keep the cancer from recurring .2 However, the mal physiological conditions would be limited by ineffective effectiveness of this chemotherapy or radiation therapy is diffusion of essential cellular metabolites over such large low if the tumor is located in vital regions of the body. distances . The ability of tumor cells to divide in spite of Alongside its high degree of invasiveness, this treatment lower than normal levels of nutrients can explain this phe - also results in significant morbidity, longer post surgery nomenon. Tumor cells on the outer edge of a mass have easy recovery time , and longer hospital stays. 4 The treatment is access to nutrients, while those at the center of the mass per - intrinsically toxic to the body and targets rapidly dividing ish. Eventually, the tumor mass develops into a mass of cells, such as those in a tumor. However, treatment is not invariable size with a central necrotic core. A steady rate of selective and rapidly dividing cells (i.e. hair, intestinal lin - proliferation and cell death maintains a steady tumor size, ing, and bone-marrow) are also killed in the process. A com - until angiogenesis creates new vasculature to further mon and debilitating side-effect of chemotherapy is that it promote tumor progression . This process is illustrated in results in hair loss, nausea and fatigue; thereby compromis - Figure 1. ing the patient’s quality of life .2 Such limitations warrant the need for novel cancer imag - MUMJ Medical Technology 49

NANOSHELL MEDIATED CANCER DIAGNOSIS AND THERAPY

Cancer Imaging Redefined by Gold Nano Shells There is a pressing need to develop cost effective diag - nostic technology which provides high resolution images, and is yet minimally invasive to use. Dyes such as Indocyanine green, a Food and Drug Administration (FDA ) approved fluorescent dye, are commonly used at the time. Due to their vulnerability to photo bleaching and thermal and chemical damage, 8,9 gold nanoshells are a much more interesting option. As early as 3000 BC, Egyptian glass makers added small amounts of metal to glass crucibles to produce colored glass. This result is largely due to the ability of metal surfaces to produce vivid colors when excited by light. Metals can scat - ter and absorb light of different wavelengths due to surface plasmon resonance, which is a process wherein incident light causes free electrons in the metal to be excited and thereby oscillate. 4,10 Gold nanoshells consist of a spherical dielectric-silica core, covered by an ultra-thin sheet of gold. Varying the relative thicknesses of the core and outer shell Figure 1. Adapted from Brannon-Peppas et al (2005) 5 allows them to absorb and scatter light over a broad region Continued tumour development behond a maximal size that is limit - ed by diffusion of essential cellular metabolites (A). In (B), angiogenic of the electromagnetic spectrum (UV to Infrared). This activity has created an imbalance between growth factors and property is known as optical tunability and is illustrated in inhibitors, which eventually lead to endothelial cell proliferation and Figure 2. 3,4,10 migration. These endothelial cells progressively form a vessel which extends towards the tumour and provides nutrients to sustain cell pro - Photonic based imaging techniques such as optical coher - liferation (C). A fully vascularized tumour (D) is capable of continued ence tomography (OCT) utilize scattered light in the near growth with metastatic potential due to the proximity to the blood infrared region (NIR 800-1300nm) to image cancerous tis - stream. sue. OCT’s minimal degree of invasiveness, cost effective -

Therapeutically Exploitable Mechanisms Tumor physiology has given scientists three therapeuti - cally exploitable mechanisms . It is not uncommon for many tumors to over-express particular surface antigens which are not found on the surface of healthy cells .5 Identifying such markers would allow for tumor-specific targeting of particu - lar cells and not others. 5 Angiogenesis or the creation of new blood vessels, which plays a central role in tumor devel - opment, can also be disrupted for therapy. 5 For example, growth factors such as vascular endothelial growth factor (VEGF) which encourages angiogenesis can be inhibited so developing cancers do not obtain the necessary nutrients by means of these new blood vessels. 5 More importantly, the existence of a tumor mass also results in the enhanced per - meation and retention effect (EPR effect) .5 That is, greater spaces between endothelial cells of neovascularized tissues , Figure 2. Adapted from Loo et al (2004) 3, Hirsch et al resulting from rapid vascularization , and poor lymphatic (2003) 4, and Hirsch et al (2006) 10 drainage permit the easy accumulation of substances within Optical tunabillity is demonstrated for nanoshells with a 60 nm silica a tumor mass .5 This phenomenon could allow designer core radius and gold shells 5, 7, 10 and 20 nm thick. Observe that the plasmon resonance (extinction) of the particles red shifts with drugs to accumulate within tumors in significant concentra - decreasing thickness of the gold shell (or an increasing core:shell tions for maximal effectiveness .5 ratio). Nanoshells are easily fabricated with resonance in the NIR. Greater tunability can be achieved by also altering the core size, changing the composition of the core and shell, and forming multi - layered structures. 50 Medical Technology Volume 4 No. 1, 2007

Photo-Thermal Destruction –Future Therapeutic ness, and ability to deliver high resolution images make Gold Standard ? them the current gold standard in cancer imaging. More specifically, “OCT detects the reflections of a low coherence In light of the many adverse side effects of radiation ther - light source directed into a tissue and determines at what apy, a more effective therapeutic intervention is needed. depth the reflections occurred. It is often described as an Photo-thermal destruction of cancerous tissue by means of optical analog to ultrasound.” 3 Since OCT uses NIR, scien - gold nanoshells may be the long awaited answer. In a typi - tists using this technology are able to obtain high quality cal treatment, a patient would receive intravenous gold images of such tissue at greater depths. Gold nanoshells can nanoshells , which have been modified to selectively target also be designed to scatter or absorb light in the NIR and tumor tissues. The nanoshells accumulate within the tumor hence are a promising candidate for use as contrasting over time, and after exposure to NIR radiation, emit heat. agents for OCT. 3,4,8,9,11 The increase in temperature results in irreversible damage Utilizing gold as the outer shell also endows several other to the tumor cells , whereas healthy tissues are spared .3,11 biologically relevant advantages. Gold is an inert metal with Since body tissue demonstrates the least absorption for NIR high resistance to corrosion and low toxicity in vivo – pri - radiations, this technology exhibits maximal tissue penetra - marily why gold is extensively utilized in dental prosthetics. tion, and minimally affects the tissues surrounding the It is also very easy to conjugate proteins to a surface made affected area .4 of gold . Disulfide and thiol groups are extensively found in Other advantages of using gold nanoshells for this pur - many proteins, which would explain proteins’ astonishing pose are similar for that of using gold nanoshells for imag - ability to self assemble on gold surface molecules. This has ing purposes. This includes the fact that gold particles are considerable immunological importance since different anti - optically tunable particles , and as such , they can be engi - bodies can be attached to gold nanoshells, making them very neered to strongly absorb NIR radiation. Moreover, when selective for particular antigens that are displayed on tissues Chen and colleagues 12 compared gold nanoshells against of interest .10 ICG (Indocyanine Green – a NIR fluorescent dye), they Recent experiments have also demonstrated a high found gold nanoshells to be more resistant to thermal and degree of efficacy for gold nanoshells to successfully image chemical damage. Their decreased susceptibility to photo - cancerous tissues. In one in vitro experiment, Loo et al bleaching (a process wherein prolonged exposure to light showed gold nanoshells to be extremely effective contrast - causes a material to lose absorbtivity) also provides extend - ing agents. 3,8,9 They treated intervention group breast car - ed functional times in vivo .4 Their ability to encounter an cinoma cells with gold nanoshells designed to scatter light in absorption event and convert the light into thermal energy the NIR region. Following incubation, the cells were imaged is a million times greater than conventional agents, which is with a darkfield microscope. Cells which had been incubat - exactly why they are also more efficient in photo thermal ed with anti-HER2 nanoshells demonstrated significantly therapy .3,4 increased scatter-based optical contrast when compared to Moreover, the increased efficacy of gold nanoshells as the two control cell groups 3,8,9 (See Figure 3). These results powerful tools in photo thermal therapy has been confirmed are, to say the least, highly encouraging. by several recent experiments. In vitro and in vivo experi - ments conducted by Hirsch and colleagues 4 and Loo and colleagues 8, 9 report such results in a set of brilliant exper - Scatter-based imaging iments. The in vitro experiment consisted of one treatment group and two control groups. The treatment group con - No Nanoshells Non Specific Antibody Anti-HER2 tained human breast carcinoma cells, which were incubated with gold nanoshells targeted for HER2. HER2 is an impor - tant cancer biomarker which is over -expressed by carcino - ma cells. The two control groups utilized breast cancer cells that were either incubated with gold nanoshells conjugated to a nonspecific antibody or to gold nanoshells without con - jugation to any antibody. Cells in all groups were subse - Figure 3. Adapted from Loo et al (2004), 3 Loo et al (2005), 8 quently exposed to NIR laser light (Coherent, 820 nm, and Loo et al (2005) 9 35W/cm 2) for seven minutes to induce photothermal cell Breast carcinoma cells incubated with gold nanoshells designed to damage. The results of calcien staining, which was used to scatter light in the NIR region. Cells were imaged with darkfield assess cell viability are shown in Figure 4. Only cells treat - microscopy. Cells which had been incubated with anti-HER2 ed with anti-HER2 nanoshells displayed a large and signif - nanoshells demonstrated significantly increased scatter-based optical icant amount of cell death, compared to no significant cell contrast when compared to the two control cell groups. death in the other two control groups. Additionally, the diameter of dark circular region matches that of the laser MUMJ Medical Technology 51

beam , indicating that cell death was solely confined to the area containing the Anti-HER2 nanoshells, leaving sur - rounding tissues undamaged. 4, 8, 9

Therapy

No Nanoshells Non-Specific Anti-HER2 Antibody

Figure 5. Adapted from Hirsch et al (2003) 4 Figure 4. Adapted from Hirsch et al (2003) 4 Loo et al Temporal plots of maximum temperature change of NIR-irradiated (2005) 8 and Loo et al (2005) 9 tumours with nanoshells (red) and without nanoshells (blue) at depths of (a) 2.5, (b) 3.75, (c) 5.0, and (d)7.3 nm beneath the apical tissue Calcien stains viable cells green, whereas cell death is indicated by surface. dark regions. Only cells treated with anti-HER2 nanoshells (right) dis - played a large and significant amount of cell death, compared to no significant cell death in the other two control groups (left and middle). Additionally, the diameter of dark circular region matches that of the laser beam, indicating that cell death was solely confined to the area containing the Anti-HER2 nanoshells – leaving surrounding tissues undamaged.

In another important experiment performed in vivo , Hirsch and colleagues 4 demonstrated that gold nanoshells can affectively absorb NIR and result in significantly large temperature increases that are capable of causing irre - versible tissue damage to tumor-laden areas. The treatment and control groups were exposed to NIR light for seven min - utes at a coherent intensity of 820nm. PEG coated gold nanoshells were injected interstitially into the tumor within NAPT Treatment Sham Treatment Control the intervention group, while control groups were injected Figure 6. Adapted from O’Neal et al (2004) 11 with saline. After thirty minutes had elapsed, tumor tissues Mean tumour sizer measured on treatment day and 10 days later for of mice in both groups were exposed to NIR radiation for 25 tumours. All tumours treated using NAPT (Nanoshell Assisted four to six minutes . A resulting temperature profile was gen - Photothermal Therapy) showed complete necrosis by day 10. erated using Magnetic Resonance Thermal Imaging (MRTI). An average temperature increase of 37.4 ± 6.6°C was observed in tissues treated with nanoshells. Additionally, the tumor growth/regression as well as the animal survival time. controls experienced a temperature increase of 9.1 ± 4.7 °C, Subcutaneous tumors were grown in twenty-four mice and a level insufficient to cause irreversible damage. In other they were randomly divided into three groups. Mice in the experiments, it has been confirmed that a tumour tempera - treatment group were intravenously injected with PEG -coat - ture of 45°C or more for more than one minute, renders irre - ed gold nanoshells. The sham-treatment group received versible destructive effects on cells and eliminates the need saline injection and controls received no injection. Six hours for additional radiotherapy or cytostatic drug thera - subsequent to injection , the treatment and sham-treatment py .13,14,15 Thermal damage of tumor tissues was further group were exposed to NIR light . The control group confirmed with histological analysis. Figure 5 demonstrates received neither the injection nor NIR laser treatment. Note the temperature change versus time for several depths (2.5, that surface temperature measurement is a good indicator of 3.75, 5.0, and 7.3 mm) within the tumor during the treatment the temperature reached within the tumor. After thirty sec - regimen .4 onds of laser treatment, the temperature of the treated area The aforementioned experiments provide strong evidence within the treatment group was significantly higher than in in favor of nanoshell assisted photothermal therapy (NAPT). the sham-treatment group. To assess the effect of treatment These experiments paved the way for another in vivo exper - on surrounding healthy tissues, mice in the treatment group iment by Hirsch and colleagues. In these set of experiments , received NIR radiation at a healthy skin area near the tumor. the efficacy of gold nanoshells was assessed by measuring The effect on healthy tissue was similar to that in sham treat - 52 Medical Technology Volume 4 No. 1, 2007

ment group, indicating that nanoshells preferentially accu - mining the technology’s safety, and then its efficacy in mulated in the tumor via the EPR effect, in turn preventing humans. Research will also be needed to dictate which any observable damage to surrounding healthy tissue. tumor types are the best candidates for this therapy. We hope Tumor size and survival times were monitored for ninety this review successfully highlights an exciting development days following treatment and the complete necrosis of the wherein nanotechnology and cancer biology have merged to tumor was reported within ten days of treatment in the treat - create a powerful tool with the potential to save millions of ment group. All the mice in this group remained healthy and lives. tumor free for more than ninety days after treatment. On the REFERENCES other hand, tumors in the sham-treatment and control groups grew rapidly. The results of the tumor growth and regression 1. September 1, 2006. Cancer Statistics. National Cancer Institute of Canada. Retrieved from: http://www.ncic.cancer.ca/vgn/images/portal/cit_86751114/ are displayed in Figure 6. The mean survival times of the 31/23/935505938cw_2006stats_en.pdf.pdf. mice in control and sham-treatment group were 10.1 and 2. Cancer - Surgery [online]. Wikipedia Encyclopedia [cited Jul]. Available from: 12.5 days respectively .11 URL: http://en.wikipedia.org/wiki/Cancer#Surgery. 3. Loo C, Lin A, Hirsch L, Lee M, Barton J, Drezek R, et al. Nanoshell-Enabled The future of nanoshells assisted photothermal therapy in Photonics-Based Imaging and Therapy of Cancer. Technology in Cancer cancer treatment seems very promising. Its ability to com - Research & Treatment.2004.Feb; 3(1). bine two benign moieties (nanoshells and NIR light) makes 4. Hirsch L, Stafford R, Bankson J, Sershen S, Rivera B, Price R , et al. Nanoshell-mediated near-infrared thermal therapy of tumors under magnetic this technique highly effective and minimally invasiveness – resonance guidance. Proceedings of the National Acedmy of Sciences of the the perfect traits for a successful therapeutical successor in United States of America. 2003. Nov 11; 100 (23):13549-13554. our fight against cancer . 5. Brannon-Peppas L, Blanchette J. Nanoparticle and Targeted Systems for Cancer Therapy. Advanced Drug Delivery Reviews.2005; 56: 1649-1659. CONCLUSION 6. Kawasaki E, Player A. Nanotechnology, Nanomedicine, and the development of new, effective therapies for cancer. Nanomedicine.2005: 101-109. There is no doubt that current cancer diagnostic and ther - 7. Langer R. Drug Delivery: Drugs on target. Science.2001.Jul6;293(5527):58-59. 8. Loo C, Lowery A, Halas N, West J, Drezek R. Immunotargeted Nanoshells for apeutical methods, though effective, have many inherent Integrated Cancer Imaging and Therapy .Nano Letters.2005; 5(4): 709-711. limitations. A review of the literature suggests that gold 9. Loo C, Hirsch L, Lee M, Chang E, West J, Halas N, et al. Gold nanoshell bio - nanoshells hold great potential, both as an imaging and ther - conjugates for molecular imaging in living cells. Optics Letters. 2005. May 11; 30 (9). apeutical agent. This is particularly useful as this one tech - 10. Hirsch L, Gobin A, Lowery A, Tam F, Drezek R, Halas N, et al. Metal nology eliminates the need for two different agents to be Nanoshells. Annals of Biomedical Engineering.2006. Jan; 34(1):15-22. used in diagnostics and therapy. Several experiments illus - 11. O’Neal D, Hirsch L, Halas N, Payne J, West J. Photo-thermal tumor ablation in mice using near infrared-absorbing nanoparticles. Cancer Letters. 2004: 171-6. trate that Nanoshells Assisted Photothermal Therapy 12. Chen, W. R., Adams, R. L., Higgins, A. K., Bartels, K. E. & Nordquist, R. E. (NAPT) can provide a cost effective and minimally invasive (1996) Cancer Lett. (Shannon, Irel.) 98, 169–173. way of reducing tumor tissue, without damaging intervening 13. Journée-de Korver JG, Verburg-van der Marel EH, Oosterhuis JA, van Best JA, de Wolff-Rouendaal D. Tumoricidal effect of hyperthermia by near infrared irra - tissues. Gold nanoshell -based contrast agents have advan - diation on pigmented hamster melanoma. Lasers Light Ophthalmol 1992;4:175- tages over the conventional NIR dyes and early diagnosis 180. can significantly reduce morbidity and mortality. More 14. Oosterhuis JA, Journée-de Korver JG, Kakebeeke-Kemme HM, Bleeker JC. Transpupillary thermotherapy in choroidal melanomas. Arch Ophthalmol importantly, nanoshell based techniques hold the potential to 1995;113:315-321. dramatically improve the quality of life for cancer patients. 15. Wheatley DN, Kerr C, Gregory DW. Heat-induced damage to HeLa-S3 cells: At this stage, further research is needed to validate this tech - correlation of viability, permeability, osmosensitivity, phase-contrast light-, scanning electron- and transmission electron-microscopical findings. Int J nique in order to ethically allow for its application to phase Hyperthermia 1989; 5:145-162. 1 trials involving human subjects. This involves first deter -

Author Biographies Abhitej Rewari is a fourth year student of the Bachelor of Health Sciences (Honours) Program at McMaster University. Syed Rahman completed his Bachelor of Health Sciences (Honours) at McMaster University and is presently a first year student at the Michael G. DeGroote School of Medicine, McMaster University. Prateek Goyal is in his fourth year of the Life Sciences (Honours) Program at McMaster University.