Scientific breakthroughs don’t always happen in labs. For Sophia and Richard Lunt, Michigan State University researchers, many of their breakthroughs happen during neighborhood walks.
The married couple’s step-by-step approach has revealed a new way to detect and attack cancer cells using technology traditionally reserved for solar power.
The results, published in the current issue of Scientific Reports, showcases dramatic improvements in light-activated fluorescent dyes for disease diagnosis, image-guided surgery and site-specific tumor treatment.
“We’ve tested this concept in breast, lung cancer and skin cancer cell lines and mouse models, and so far it’s all looking remarkably promising,” said Sophia, MSU biochemistry and molecular biologist.
While the cancer applications hold the most possibility, their findings have potential beyond the field of oncology, said Richard, the Johansen Crosby Endowed Professor of chemical engineering and materials science.
“This work has the potential to transform fluorescent probes for broad societal impact through applications ranging from biomedicine to photocatalysis – the acceleration of chemical reactions with light,” he said.
Prior to the Lunts’ combined effort, fluorescent dyes used for therapeutics and diagnostics, aka “theranostics,” had shortcomings, such as low brightness, high toxicity to cells, poor tissue penetration and unwanted side effects.
By optoelectronically tuning organic salt nanoparticles used as theranostics, the Lunts were able to control them in a range of cancer studies.
Coaxing the nanoparticles into the nontoxic zone resulted in enhanced imaging, while pushing them into the phototoxic—or light-activated—range produced effective on-site tumor treatment.
The key was learning to control the electronics of their photoactive molecules independently from their optical properties and then making the leap to apply this understanding in a new way to a seemingly unrelated field.
Richard had recently discovered the ability to electronically tune these salts from his work in converting photovoltaics into solar glass.
Sophia had long studied metabolic pathways unique to cancer cells. It was when the Lunts were discussing solar glass during a walk that they made the connection: Molecules active in the solar cells might also be used to more effectively target and kill cancer cells.
A journey of 1,000 miles
Their walks had rather unscientific beginnings. Shortly after the Lunts met at Princeton University, Richard moved to another university. To maintain their long-distance relationship, they scheduled daily phone calls. Upon their arrival at MSU, individual academic career demands replaced geographic distance as a challenge to their busy lives.
To connect daily, they take CEO-style walks together every evening. The two-mile saunters take place rain or shine, and they often engage in scientific discussions.
The three keys to their walks are intentional curiosity, perseverance and the merging of different fields and perspectives, Sophia said.
“We talk science, strategic plans for our careers and our various grants,” she said. “We ping ideas off each other. Our continual conversations brainstorming ideas on a particular topic or challenge often lead to those exciting ‘aha’ moments.”
Their walks have helped them push through many challenges.
“Our first experiments did not turn out as expected; I’m surprised that we didn’t give up given how crazy the idea seemed at first,” Richard said. “Figuring out how to do this research took many walks.”
Obviously, the results were worth the hike. Today, Richard designs the molecules; Babak Borhan, MSU chemist, synthesizes and improves them; and Sophia tests their photoactive inventions in cancer cell lines and mouse models.
Future research will work to improve the theranostics’ effectiveness, decrease toxicity and reduce side effects. The Lunts have applied for a patent for their work, and they’re looking forward to eventually pushing their photoactive molecule findings through clinical trials.
“Though that will take many more walks,” Richard said with a smile.
Today, 1 in 2 males and 1 in 3 females in the US will develop cancer at some point during their lifetimes, and 1 in 4 males and 1 in 5 females in the U.S. will die from the disease. New methods for detection and treatment have dramatically improved cancer care in the United States.
However, as improved detection and increasing exposure to carcinogens has led to higher rates of cancer incidence, clinicians and researchers have not balanced that increase with a similar decrease in cancer mortality rates.
This mismatch highlights a clear and urgent need for increasingly potent and selective methods with which to detect and treat cancers at their earliest stages.
Nanotechnology, the use of materials with structural features ranging from 1 to 100 nm in size, has dramatically altered the design, use, and delivery of cancer diagnostic and therapeutic agents.
The unique and newly discovered properties of these structures can enhance the specificities with which biomedical agents are delivered, complementing their efficacy or diminishing unintended side effects. Gold (and silver) nanotechnologies afford a particularly unique set of physiological and optical properties which can be leveraged in applications ranging from in vitro/vivo therapeutics and drug delivery to imaging and diagnostics, surgical guidance, and treatment monitoring.
Nanoscale diagnostic and therapeutic agents have been in use since the development of micellar nanocarriers and polymer-drug nanoconjugates in the mid-1950s, liposomes by Bangham and Watkins the mid-1960s, and the introduction of polymeric nanoparticles by Langer and Folkman in 1976.
Since then, nanoscale constructs such as dendrimers, protein nanoconjugates, and inorganic nanoparticles have been developed for the systemic delivery of agents to specific disease sites.
Today, more than 20 FDA-approved diagnostic or therapeutic nanotechnologies are in clinical use with roughly 250 others are in clinical development. The global market for nano-enabled medical technologies is expected to grow to $70-160 billion by 2015, rivaling the current market share of biologics worldwide.
In this Account, we explore the emerging applications of noble metal nanotechnologies in cancer diagnostics and therapeutics carried out by our group and by others.
Many of the novel biomedical properties associated with gold and silver nanoparticles arise from confinement effects:
i) the confinement of photons within the particle which can lead to dramatic electromagnetic scattering and absorption (useful in sensing and heating applications, respectively);
ii) the confinement of molecules around the nanoparticle (useful in drug delivery); and
iii) the cellular/subcellular confinement of particles within malignant cells (such as selective, nuclear-targeted cytotoxic DNA damage by gold nanoparticles).
We then describe how these confinement effects relate to specific aspects of diagnosis and treatment such as:
i) laser photothermal therapy, optical scattering microscopy, and spectroscopic detection,
ii) drug targeting and delivery, and iii) the ability of these structures to act as intrinsic therapeutic agents which can selectively perturb/inhibit cellular functions such as division. We intend to provide the reader with a unique physical and chemical perspective on both the design and application of these technologies in cancer diagnostics and therapeutics. We also suggest a framework for approaching future research in the field.
Photon Confinement: Imaging Probes, Photothermal Therapy, and Spectroscopic Detection
Gold nanoparticles are particularly attractive platforms for targeted diagnostics and therapeutics due to their unique optical and electronic properties. These structures can be conjugated with upwards of 1014 ligands per cm2 (102 – 103 times higher than that typically attainable with liposomal or polymeric nanoparticles, respectively)1 and the selective accumulation of gold nanoparticles at solid tumors can facilitate highly efficient photothermal ablation via non-invasive near-infrared (NIR) laser exposure2–6 (Figure 1a), high-Z enhanced X-ray computed tomography/radiotherapy,7,8 non-invasive photoacoustic imaging/cytometry6,9,10 (Figure 1b), contrast-enhanced optical coherence tomographic imaging11 (OCT, Figure 1c), and the electromagnetic enhancement in non-invasive spectroscopic biomarker detection schemes both in vitro12 and in vivo13 (Figure 1d).
Gold nanoparticles are also highly useful probes for microscopic imaging-based applications. For example, the resonant optical scattering intensity from a single 80 nm gold nanoparticle15 is equivalent to the emission intensity of 500,000 of the most efficient Alexa Fluor® dyes or 2,000 of the most efficient Qdot® 800 quantum dots.16
While gold nanoparticles have been used for decades as labels in immunohistochemical and electron microscopic analysis of tissue sections,17 Sokolov et al. first demonstrated in 2003 that the resonant scattering from gold nanoparticles could be used to image subcellular cancer biomarkers (EGFR) in vitro using confocal reflectance microscopy of immunolabeled gold nanoparticles (ca. 12 nm core diameter) for potential cancer diagnostics, staging, and treatment monitoring.
Our group18,19 has shown that simpler, less expensive dark-field optics can also be used to obtain high-contrast scattering images from immunolabeled gold nanoparticles in vitro, in this case in true-color, to achieve the identification and selective (photothermal) labeling of malignant cells (Figure 2a). Currently, the method is in wide use for both the imaging and spectroscopy of nanoparticles and their labeling of living cells and tissues.20,21
While mild hyperthermic cancer treatments have been in clinical use since the early 1980s,22 laser phothermal therapy (or ablation) using plasmonic nanoparticles was first demonstrated in 2003 by locally injecting so-called gold nanoshells (silica-gold core-shell nanoparticles) directly into the tumor interstitium and later by the passive accumulation of systemically delivered nanoshells.23
Our group2 was the first to show that increasingly efficient3 gold nanorods could be used as contrast agents for in vitro and in vivo near-infrared (NIR) laser photothermal therapy to achieve selective tumor cell ablation and resorption/remission in vivo (Figure 3). In the latter, NIR-absorbing PEGylated gold nanorods were systemically or intratumorally administered in mice bearing head and neck tumor models (squamous cell carcinoma). Subsequent exposure of the nanoparticle-loaded tumors for just 10 min was found to result in upwards of 20 °C temperature increases at the tumor center, with minimal damage to surrounding tissues.
Complete tumor resorption was observed in more than 50% of the group directly administered gold nanorods and 25% of those systemically administered at two weeks post-laser treatment.2 Subsequent studies by our group24 exploring active targeting of these nanorods by
i) a single-chain variable fragment (ScFv) peptide that recognizes the epidermal growth factor receptor (EGFR),
ii) an amino terminal fragment (ATF) peptide that recognizes the urokinase plasminogen activator receptor (uPAR), or
iii) a cyclic RGD peptide that recognizes the avb3 integrin receptor were also performed (hydrodynamic diameter 68-81 nm, zeta potential −5 to −25 mV).
As expected, active targeting significantly improved the cellular accumulation of these nanoconjugates in vitro (A549 lung cancer cells). The blood half life of PEGylated gold nanorods was reduced by 25 – 48% upon co-conjugation with these active targeting ligands and the tumor accumulation (24 h) of ATF- and ScFV EGFR-targeted gold nanorods was enhanced ca. 67 and 46% relative to passively targeted PEGylated gold nanorods administered in mice models (Figure 4).
Surprisingly, the tumor accumulation of cyclic RGD-targeted gold nanorods was significantly diminished (ca. −57 % relative to PEGylated gold nanorods), suggesting that laser photothermal therapy using this specific formulation may be best suited to intratumoral injection schemes. The use of gold nanocage25 and hollow gold nanoparticles26 as contrast agents in preclinical laser photothermal cancer therapy have also been subsequently explored by Xia and Li, respectively, and human pilot studies investigating the treatment of refractory and/or recurrent head and neck tumors using plasmonic laser photothermal therapy are currently ongoing.27
The unique photophysical properties of gold nanostructures can be further leveraged to enhance a number of spectroscopically-relevant processes28–32 with applications in biodiagnostics and treatment monitoring.
These processes include two-photon luminescence (TPL) imaging,33 metal enhanced fluorescence (MEF), optical coherence tomography (OCT),11 photoacoustic tomography/cytometry,6,9,10 surface enhanced infrared absorption (SEIRA), and most notably, surface enhanced Raman scattering (SERS).34
In an early study demonstrating the utility of SERS for in vitro diagnostics, our group35 showed that conjugation of gold nanorods with a nuclear localization sequence (NLS) peptide greatly promotes their uptake by both malignant and non-malignant cells (Figure 5a-b).
High resolution Raman microscopy found that spectral responses from the cells exhibited increasing contributions from peptides, nucleic acids, DNA backbone vibrations, chromatin, and histone structures upon transfection. Moreover, malignant cells were also found to selectively exhibit increasing contributions from adenine nucleobases and a characteristic band at 398 cm−1, both of which may serve as potential diagnostic cancer markers. Prior research by our group36 has also indicated that cell-surface labeling can be useful in identifying malignant and premalignant cells based on their characteristic overexpression of epidermal growth factor receptor (EGFR, Figure 5c-e).
Here, SERS from malignant and non-malignant cells was interrogated following labeling with polystyrene-coated gold nanorods electrostatically conjugated with EGFR antibodies. Optical dark-field scattering microscopy and high-resolution microabsorption spectroscopy found that labeling of the malignant cells was roughly two-fold greater than the non-malignant cell line.
Raman analysis found that 90% of the malignant cells exhibited detectible SERS response while only 20% of the non-malignant cells exhibited detectible SERS. Moreover, due to the high density and orientation of these nanorods about the surfaces of the malignant cells, SERS signal was detected in a polarization-dependent manner, serving as another potential diagnostic marker for cancer. Natan, Gambhir, and Nie have also explored the in vivo use of gold nanoparticles decorated with so-called Raman reporter molecules for non-invasive imaging diagnostics,37 surgical guidance,38 and multiplexed in vivo labeling application.39
Molecular Confinement: Drug Delivery
Gold nanoparticles exhibit a range of properties which make them particularly amenable to systemic delivery applications. Due to their relatively large size, these constructs can preferentially accumulate at tumor sites due to the enhanced permeability and retention (EPR) effect40,41 (Figure 6a) and because of the high density of atoms on their surfaces, their receptor binding affinity can easily tuned over several orders of magnitude.42
Because multivalent nanoconjugates are large enough to occupy multiple, adjacent cellular binding sites,43 enhanced avidity can greatly improve the selectivity of their targeting, uptake, and/or delivery (Figure 6b).
These platforms protect and facilitate the delivery of physiologically unstable diagnostic/therapeutic agents or those which exhibit poor intracellular penetration (e.g. hydrophobic chemotherapeutics (Figure 6c),44 enzymatically-degradable siRNA (Figure 6d),45 etc.).
One of the earliest reports involving the use of gold nanoparticles in drug delivery applications came in 1954, when Root et al. investigated the clinical use of colloidal Au198 particles for radiotherapy treatment of liver cancer and leukemia, both with minimal success.48 In 2001, Paciotti and Tamarkin began research studying the use of gold nanoparticles as a platform for the delivery of tumor necrosis factor α (TNFα) to solid tumor models in mice.49,50 Subsequent Phase I clinical trials found that the PEGylated 27 nm diameter gold nanoparticles were well tolerated in patients and able to safely deliver TNFα at dosages 3-fold higher than the previously reported maximum tolerable dose for the lone drug with no serious adverse side effects; Phase II trials are currently pending.51
Our group3–5,52 is currently developing technologies for the treatment of hormone-dependent malignancies such as breast and prostate cancer using gold nanostructures which incorporate derivatives of small molecule receptor antagonists that can serve as combined targeting and therapeutic agents. 60-70% of breast cancers express estrogen receptor53 and 80-90% of prostate cancers express androgen receptor.54
Classically, these proteins function as intracellular gene transcription factors, that is, hormones passively diffuse across the cell membrane, engage the receptor, and the two translocate to the nucleus where they bind DNA and transcribe proteins associated with malignant growth and cancer progression.
Over the past 15 years, researchers have become increasingly aware that these hormone receptors are also expressed in large quantities on the plasma membrane following their post-translational modification.55
Because these membrane hormone receptors still maintain binding affinity for their endogenous hormones and antibodies, these receptors can serve as targets for tissue-selective drug delivery to breast, prostate, and other hormone-dependent cancers. Or lab has shown that a derivative of the breast cancer treatment drug tamoxifen can be used to selectively deliver our PEGylated gold nanoparticle platform to breast cancer cells in a receptor- and ligand-dependent manner, and can do so with accompanying potency at concentrations more than 10,000-fold higher than the lone drug (Figure 7).52
Moreover, we’ve shown that in vitro laser photothermal therapy can be used to selectively induce phototoxicity of these drug conjugates to breast cancer cells at otherwise sub-lethal concentrations,5 providing opportunities for subsequent radiotherapy enhancement, treatment monitoring by contrast-enhanced x-ray CT, photoacoustic imaging, phase-contrast OCT, and/or non-invasive SERS analysis.
We are currently further exploring the applications of this technology in treating estrogen-dependent malignancies and also investigating an increasingly selective and potent antiandrogen nanoparticle construct for the treatment of hormone-insensitive prostate cancers (which comprise nearly all prostate cancers 2-3 years following initial endocrine treatments).56
Mirkin,57 Rotello,58 and Xia59 have also extensively explored the use of gold nanotechnologies in drug delivery applications for the treatment of solid tumors; interested readers are directed to Dreaden et al. for further reading.3,4,6
Cellular Confinement: Intrinsic Pharmacodynamic Properties of Nanoparticles
Like other nanoscale materials, gold nanoparticles are capable of perturbing normal physiological functions depending on their location within the cell and the extent with which they interact with various biomolecules (Table 1).60
For example, Fe3O4 nanoparticles can interfere with electronic and/or ion transport processes, CeO2 nanoparticles can induce protein fibrillation, and dendrimers can cause damage to cell membranes (vide infra). Our group21,61 is exploring the possibility of exploiting such deleterious cellular interactions to selectively treat malignant cells (which are increasingly susceptible to damage). Gold nanoparticles have been shown capable of modifying protein charge, size, and hydrophobicity,60 as well as thermal stability.62
These particles have also demonstrated preferential binding to single-stranded DNA versus double-stranded DNA (which can interfere with cell division), G2/M cell cycle arrest for radiotherapy enhancement,63 and direct association with the heparin-binding domain of VEGF-165 to inhibit its ability to associate with cell surface receptors necessary for cancer-related angiogenesis.64
Subcellular Confinement: Selectively Localizing Nanoparticles at Cancer Cell Nuclei
We hypothesized that by using peptides to direct the accumulation of gold nanoparticles (and other nanomaterials) at discrete intracellular locations, that we can selectively perturb malignant cell growth and associated disease progression.
In a recent investigation using real-time, live-cell, dark-field scattering videography (which also makes use of the photon confinement properties of nanoscale noble metal nanoparticles), our group21 showed that spherical gold nanoparticles decorated with cell-penetrating RGD and nuclear localization sequence (NLS) peptides can impair cell division and induce cytokinesis arrest in a concentration-dependent manner (with no such effects observed for nanoparticles conjugated with either peptide alone) (Figure 9, Supporting Video S1).
G2/M cell cycle arrest, binucleate cell formation, apoptosis, and histone cleavage was observed in malignant cells treated with the RGD/NLS gold nanoparticles, while the cell cycle of non-malignant cells was not significantly affected at therapeutically-relevant concentrations (0.4 nM). In a more recent, but related, study investigating cellular effects from RGD/NLS silver nanoparticles, our group61 observed that apoptotic induction in malignant cells by these particles led to the attraction of neighboring cells and their subsequent engulfment by these cells (Figure 9, Supporting Video S2).
We also found that increases in particle concentration beyond this apoptotic threshold (0.05 nM) further increased cell death, also believed to be attributable to oxidative stress, but decreased cell clustering which we concluded occurred due to rapid apoptotic induction and a loss of capacity for intercellular signaling.61
In summary, we have shown that gold nanoparticles can serve as highly multifunctional platforms for the targeted diagnosis and treatment of solid tumors. These structures exhibit a particularly unique combination of physical, chemical, optical, and electronic properties distinct from all other biomedical nanotechnologies and provide a multimodal platform with which i) to image and diagnose cancers with increasingly sensitivity and selectivity, ii) to preferentially deliver therapeutic agents and electromagnetic radiation to malignant cells with increasing potency, and iii) to selectively target, sensitize and exert intrinsic pharmacodynamic effects on malignant cells. The studies discussed herein attempt, in particular, shed light on newly emerging applications of gold nanotechnologies in anticancer diagnostics and therapeutics and emphasize some of the novel aspects of their photophysical properties, pharmacodynamic profiles, and fundamental biophysical interactions.
More information:Scientific Reports (2019). DOI: 10.1038/s41598-019-51593-z , https://www.nature.com/articles/s41598-019-51593-z
Journal information: Scientific Reports
Provided by Michigan State University