cadmium_sulfide_quantum_dots_on_cells_high_resThis is the sixth article in a review series on Nanomedicine. In Part I and Part II, we reviewed the major research and entrepreneurial development of nanomedicine and the relevant patent landscape. We then introduced specific fields in nanomedicine, such as Organs-on-a-chip (Part III), Nanoparticle drug delivery (Part IV), and drug delivery for cancer therapy (Part V). Here, we will continue to review the current progress of using nanoparticles as biomarkers for cancer diagnosis.

Cancer Diagnosis
A critical step to effectively fighting cancer is detecting it at a very early stage. Currently the clinical diagnosis of cancer mainly relies on imaging techniques such as X-ray, mammography, ultrasound, endoscopy, computed tomography (CT), magnetic resonance imaging (MRI) and histopathology [e.g., examination of a tissue biopsy under a microscope]). However, these techniques often cannot distinguish differences between healthy and diseased cells/tissues at the early stage of cancer, when the malignancy of tissues are not sufficiently visible, but the alternation of far more subtle protein and molecular markers due to the cancer have already presented. Although notable successful techniques have been developed in the molecular analysis such as enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and fluorescence in situ hybridization (FISH), these techniques are labor intensive, involving complex operational procedures, and requiring high stability of reagents. Therefore, the market is calling to develop new techniques and tools to enhance the biomarker detection at the very early stages of cancer.

Nanoparticle Biosensors
Nanoparticle biosensors have achieved great attention in the research development for molecular analysis, particularly in fluorescence imaging and surface enhanced Raman spectroscopy (SERS)—a technique to enhance Raman scattering by molecule adsorption on the nanostructured surfaces. The nanoparticle biosensors are comprised of an inner core, a surface layer outside the core, and an outer sensing layer outside the surface layer. The core is formed by nanomaterials, e.g., semiconductor nanoparticles, metallic nanoparticles, or polymer nanoparticles, having excellent optical properties for signal sensing. The sensing layer is immobilized by bioconjugated binding molecules to recognize the targeted molecules in the environment. The surface layer provides a barrier between the core from the environment and prevents the aggregation of nanoparticles. The dimensions and structures of nanoparticles provide unique optical behaviors, which are not present in their bulk form. These nanoparticles exhibit intense responses to external stimuli (e.g., incident light) and tunable emission wavelengths by interacting with targeted molecules. By varying the size of nanoparticles, broad absorption and emission spectra can be achieved, enabling the possibility of the simultaneous multiple detection (i.e., multiplex detection) of molecules at one test. In addition, nanoparticle biosensors have good photostability compared to organic dyes, due to their inert chemical properties. We will briefly discuss the applications of nanoparticle biosensors as fluorescence probes and SERS tags in the following.

Nanoparticles as Fluorescence Probes
The main nanoparticle fluorescence probes used in cancer diagnosis are: quantum dots (QDs) and gold nanoparticles (AuNPs). QDs are single crystal semiconductor nanocrystals, which have a broader excitation wavelength and a narrower emission wavelength compared to organic dyes. Dr. Cho’s group in Korea demonstrated the feasibility of using quantum dot conjugated microchip to probe prostate specific antigen (PSA), a FDA approved biomarker for prostate cancer in clinic. Dr. Hildebrandt in France demonstrated the feasibility of using a quantum-dot-based resonance energy transfer immunoassay for clinic diagnosis of PSA in patient serum samples. Dr. Swanson and his colleagues at Los Alamos National Laboratory also reported the application of quantum dot based biosensing assay to quantitatively detect carcinoembryonic antigen (CEA), a breast cancer biomarker (US 8,859,268). Dr. Xu and Dr. Liu’s group in China developed a quantum dot labeled microarray to detect carbohydrate antigen 19-9, a biomarker for pancreatic cancer. Dr. Lu’s group in China also reported the simultaneous detection of two lung cancer biomarkers, CEA and neuron-specific enolase (NSE). Dr. Nie at Emory University and Georgia Institute of Technology developed an in vivo cancer targeting and imaging method with QDs (US9,073,751). Quantum Dot Corporation successfully labeled the breast cancer marker, HER2, with semiconductor QDs on cancer cells (US6,274,323). To address the challenge of a large mismatch in absorption cross-section and fluorescence brightness across a series of colors, Dr. Smith at University of Illinois at Urbana-Champaign (UIUC) developed brightness equalized QDs based on colloidal multi-domain core/shell structures, alloyed cores with composition-tunable bandgaps and semiconductor shells with adjustable electronic properties, to precisely match the absorption cross-section and quantum yield (US 2016/0200974 A1, published US patent application).

Recently, another exotic shape nanomaterial, nanoplatelets (NPLs), has emerged as a quantum material with a variety of unusual optical properties. (Dr. Dubertret in France, US 9,011,715). NPLs are flexible semiconductor nanocrystals flat sheets with atomically precise thickness. Compared to QDs, NPLs have exceptionally narrow fluorescence emission bandwidth, higher light-collecting efficiency, and enhanced detection sensitivity, due to their polarity, atomically precise dimension and high surface-area-to-volume ratio. These unique optical properties of NPLs provide a great advantage for their application in biomolecular and cellular imaging, but it is still challenging to prevent the aggregation of NPLs in biological media. Dr. Smith at UIUC developed a NPL with phospholipid absorption around the plate surface and lipoprotein binding to the sharp edges to ensure long-term stability in biological buffer. These lipoproterin NPLs even exhibit a fast internalization into epidermoid cancer cells with brightly fluorescent emission.

Gold nanoparticles have also been used in fluorescence assays for cancer detection as fluorescence quenchers or fluorophore labels due to their strong and size dependent absorption in the ultraviolet-visible range. Dr. Rotello’s group at the University of Massachusetts, Amherst, utilized gold nanoparticle-fluorescent polymer conjugates to detect and quantify two cancer biomarkers: acid phosphatase and alkaline phosphatase, which are up-regulated in various malignant organs (US 8,021,891).  Dr. Chang’s group in China used a similar structure, but aptamer-gold nanoparticle conjugates, to detect platelet derived growth factor (PDGF), which is up-regulated in various cancers. (Aptamer is single-stranded DNA or RNA molecule that can bind to pre-selected targets, such as proteins and peptides, with high affinity and specificity.) Dr. Song’s group in China also developed hyaluronic acid functionalized fluorescent gold nanoparticles used for the urine sample screening from bladder cancer patients. Dr. Lee’s group at the University of California at Berkeley reported the application of surface enhanced fluorescence between gold nanoparticles and Cyanine 3B (Cy3B) in the detection of vascular endothelial growth factor (VEGF), which is associated with tumor angiogenesis (US 8,379,212). Fluorophore-labeled nanoparticles have also been used for cancer biomarker detection. Dr. Rubinstein in Israel fabricated a microarray to use boradiazaindacene labeled nanoparticles to probe alpha 1-antitrypsin precursor (AIAT), a biomarker for gastric cancer.

Nanoparticles as SERS Tags
The efficient Raman enhancement of nanoparticles is achieved by two mechanisms: (1) a long-range electromagnetic (EM) effect, which relies on the surface plasmon resonance in metal nanoparticles, leading to a significant enhancement of the EM field strength on the surface; (2) a short-range chemical effect, which is raised from the surface absorption of molecules, leading to electronic structural changes on the surface. In contrast to fluorescence, SERS has more stable signals against photodegradation and a shaper peak emission spectrum. Also SERS is highly effective in the near-infrared (NIR), which shows low autofluorescence from tissues and little interference from water, and thus is suited for biological imaging. Dr. Lipert and Dr. Porter at Iowa State University developed an immunoassay based on SERS gold nanoparticles to detect prostate specific antigen (PSA) (US 7,829,348). Dr. Wu at West Virginia University utilized gold nanostar SERS tags and gold nanotriangle arrays to increase the sensitivity of SERS-based ELISAs and used it to probe the VEGF from clinical blood samples. Dr. Nie at Emory University developed an in vivo tumor targeting and spectroscopic detection technique with SERS nanoparticle tags, especially on tumor markers such as epidermal growth factor receptor (EGFR) (US7,588,827). Gambhir’s group at Stanford University demonstrated the multiplexing ability of SERS tags in in vivo imaging by distinguishing 10 different SERS tags spontaneously in a living mouse (US 8,795,628).

Summary
Nanoparticle biosensors have drawn intense attention in scientific research and have promising applications in medicine. For example, earlier this year, Luminex, a molecular diagnosis and life sciences research tools firm, acquired Nanosphere, a nanoparticle based nucleic acid and protein detection company, for $58 M. To further advance the applications of nanoparticle sensors as research analytical tools, intracellular sensors, and in vivo imaging at real-time, there are still many challenges to conquer. Besides reducing the long-term toxicity, it is particularly crucial to develop techniques to scale up the synthesis of reproducible high-quality nanoparticles. Because of the importance of the technology and the high financial stakes involved, this has been an area of intense patent activity.

Patent Number Title Assignee Inventor
8,859,268 Quantitative multiplex detection of pathogen biomarkers Los Alamos National Security, LLC Harshini Mukundan; Hongzhi Xie; Basil I. Swanson; Jennifer Martinez; Wynne K. Grace

 
8,021,891 Methods and compositions for protein detection using nanoparticle-fluorescent polymer complexes University of Massachusetts Vincent Rotello; Uwe Bunz; Chang-Cheng You; Oscar Miranda; Ik-Bum Kim

 
8,379,212 Plasmonic droplet, method and apparatus for preparing the same, detection method using plasmonic droplet Industry-University Cooperation Foundation Sogang University;

University of California, Berkeley

 Taewook Kang; Luke P. Lee; Yeonho Choi; Younggeun Park

 
9,073,751 Quantum dots, methods of making quantum dots, and methods of using quantum dots

 

 EMORY UNIVERSITY Shuming Nie; Andrew M. Smith; Brad A. Kairdolf

 
6,274,323 Method of detecting an analyte in a sample using semiconductor nanocrystals as a detectable label Quantum Dot Corporation

 

 Marcel P. Bruchez; R. Hugh Daniels; Stephen A. Empedocles; Vince E. Phillips; Edith Y. Wong; Donald A. Zehnder

 
9,011,715 Process for manufacturing colloidal materials, colloidal materials and their uses Nexdot Benoit Dubertret; Sandrine

Ithurria
7,829,348 Raman-active reagents and the use thereof Iowa State University Research Foundation, Inc.

 

 Marc D. Porter; Jing Ni; Robert J. Lipert; G. Brent Dawson

 
7,588,827 Surface enhanced Raman spectroscopy (SERS)-active composite nanoparticles, methods of fabrication thereof, and methods of use thereof Emory University Shuming Nie; William Doering

 
8,795,628

 

 

 

 Molecular imaging of living subjects using Raman spectroscopy and labeled Raman nanoparticles The Board of Trustees of the Leland Stanford Junior University Sanjiv S. Gambhir; Shay Keren; Ian Walton; David Guagliardo

 
2016/0200974 A1 Brightness equalized quantum dots The Board of Trustees of the University of Illinois Andrew Smith, Sung Jun Lim

– Jing Zhou, PhD and Anthony D. Sabatelli, PhD, JD

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