AMP coated SERS NanoTags with hydrophobic locking: Maximizing brightness, stability, and cellular targetability.

Developing innovative surface-enhanced Raman scattering (SERS) nanotags continues to attract significant attention due to their unparalleled sensitivity and specificity for in vitro diagnostic and in vivo tumor imaging applications. Here, we report a new class of bright and stable SERS nanotags using alkylmercaptan-PEG (AMP) polymers. Due to its amphiphilic structure and a thiol anchoring group, these polymers strongly absorb onto gold nanoparticles, leading to an inner hydrophobic layer and an outer hydrophilic PEG layer. The inner hydrophobic layer serves to "lock in" the Raman reporter molecules adsorbed on the particle surface via favorable hydrophobic interactions that also allow denser PEG coatings, which "lock out" other molecules from competitive binding or adsorbing to the gold surface, thereby providing superior colloidal and signal stability. The higher grafting densities of AMP polymers compared to conventional thiolated PEG also led to dramatic increases in cellular target selectivity, with specific-to-nonspecific binding ratios reaching beyond an order of magnitude difference. Experimental evaluations and theoretical considerations of dielectric polarization and light scattering indicate that the hydrophobic layer provides a more favorable dielectric environment with less plasmon dampening, greater particle scattering efficiency, and increased Raman reporter polarizability. Accordingly, SERS nanotags with AMP polymer coatings are observed to be considerably brighter (∼10-fold). Furthermore, the AMP-coated SERS nanotag's increased intensity and avidity can boost cellular detection sensitivity by nearly two orders of magnitude.

Alternating stealth polymer coatings between administrations minimizes toxic and antibody immune responses towards nanomedicine treatment regimens.

In efforts to achieve minimal systemic toxicity and high tumor delivery efficiencies in cancer therapy, various nanomedicine formulations having stealth polymer coatings have been developed for minimizing immune cell uptake and off-target macrophage phagocyte system (MPS) organ accumulation. Despite an initial reduction in immune cell uptake, stealth nanoparticles still initiate an antibody immune response. This response acts on subsequent administrations in treatment regimens resulting in accelerated blood clearance of particles into MPS organs, particularly the liver, where they are retained for prolonged periods. Consequently, doses after the first administration in treatment regimens have diminished tumor accumulation and increased MPS toxicity. Here, we present a strategy reducing antibody responses to each dose in a treatment regimen by alternating between polyethylene-glycol and polymethyloxazoline polymers as the nanoparticle coating between administrations. In a weekly dosing regimen, we find that the first dose of particles having either coating display similar favorable pharmacokinetics and biodistributions, thus allowing the polymers to be used interchangeably. However, when maintaining the same coating in subsequent administrations, we find that particles are in circulation at the height of the antibody immune response resulting in 50-60% decreases of circulation half-lives and tumor accumulation along with 50% increases in liver accumulation. By alternating the polymers used in the nanoparticle coating between administrations, we find each dose maintains favorable in vivo behaviors at the height of the antibody immune response to the previous administration. Furthermore, our strategy increases the clearance of particles uptaken by macrophages and hepatocytes, resulting in marked decreases in hepatotoxicity.

Probing the biological obstacles of nanomedicine with gold nanoparticles.

Despite massive growth in nanomedicine research to date, the field still lacks fundamental understanding of how certain physical and chemical features of a nanoparticle affect its ability to overcome biological obstacles in vivo and reach its intended target. To gain fundamental understanding of how physical and chemical parameters affect the biological outcomes of administered nanoparticles, model systems that can systematically manipulate a single parameter with minimal influence on others are needed. Gold nanoparticles are particularly good model systems in this case as one can synthetically control the physical dimensions and surface chemistry of the particles independently and with great precision. Additionally, the chemical and physical properties of gold allow particles to be detected and quantified in tissues and cells with high sensitivity. Through systematic biological studies using gold nanoparticles, insights toward rationally designed nanomedicine for in vivo imaging and therapy can be obtained. This article is categorized under: Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Method for Real-Time Tissue Quantification of Indocyanine Green Revealing Optimal Conditions for Near Infrared Fluorescence Guided Surgery.

Near infrared fluorescence guided surgery (NIRFGS) offers better distinction between cancerous and normal tissues compared to surgeries relying on a surgeon's senses of sight and touch. Because of the greater accuracy in determining tumor tissue margins, NIRFGS within clinics continues to grow. However, NIRFGS lacks standardization of the indocyanine green (ICG) dose and the preoperative period allowed after ICG administration. In an aim to find optimal doses and preoperative periods for NIRFGS standardization, we developed a method that quantitatively determines ICG levels within tissues in real-time. We find that not only do the dose and the preoperative periods influence tumor-to-background ratios (TBRs), but both also heavily influence subject-to-subject variances of these ratios. Optimal detection conditions are observed when larger than typical ICG doses are administered and longer than typical preoperative periods are allowed. Larger doses lead to increased TBRs, but longer preoperative periods are necessary to reduce TBR variances to those observed when using smaller doses. Our results suggest that a clinical investigation into maximum tolerable ICG doses and prolonging preoperative periods in NIRFGS is warranted.

Emergence of two near-infrared windows for in vivo and intraoperative SERS.

Two clear windows in the near-infrared (NIR) spectrum are of considerable current interest for in vivo molecular imaging and spectroscopic detection. The main rationale is that near-infrared light can penetrate biological tissues such as skin and blood more efficiently than visible light because these tissues scatter and absorb less light at longer wavelengths. The first clear window, defined as light wavelengths between 650nm and 950nm, has been shown to be far superior for in vivo and intraoperative optical imaging than visible light. The second clear window, operating in the wavelength range of 1000-1700nm, has been reported to further improve detection sensitivity, spatial resolution, and tissue penetration because tissue photon scattering and background interference are further reduced at longer wavelengths. Here we discuss recent advances in developing biocompatible plasmonic nanoparticles for in vivo and intraoperative surface-enhanced Raman scattering (SERS) in both the first and second NIR windows. In particular, a new class of 'broad-band' plasmonic nanostructures is well suited for surface Raman enhancement across a broad range of wavelengths allowing a direct comparison of detection sensitivity and tissue penetration between the two NIR window. Also, optimized and encoded SERS nanoparticles are generally nontoxic and are much brighter than near-infrared quantum dots (QDs), raising new possibilities for ultrasensitive detection of microscopic tumors and image-guided precision surgery.

Quantitative Examination of the Active Targeting Effect: The Key Factor for Maximal Tumor Accumulation and Retention of Short-Circulated Biopolymeric Nanocarriers.

Targeted and nontargeted biopolymeric nanoparticles with identical hydrodynamic sizes and surface charges were quantitatively examined in terms of the pharmacokinetic and biodistribution differences in detail. In adding cancer cell targeting folate molecules to the surface of the heparin nanocarriers, the amount of drug delivered to the tumor is doubled, and tumor growth inhibition is significantly enhanced. The folate-targeted heparin particles offered similar therapeutic potentials compared to their synthetic long-circulating analogues, thus presenting a viable alternative for drug-delivery vehicle construction using biological polymers, which are easier for the body to eliminate.

Functionalized, Long-Circulating, and Ultrasmall Gold Nanocarriers for Overcoming the Barriers of Low Nanoparticle Delivery Efficiency and Poor Tumor Penetration.

The development of sophisticated nanoplatforms for in vivo targeted delivery of therapeutic agents to solid tumors has the potential for not only improving therapeutic efficacy but also minimizing systemic toxicity. However, the currently low delivery efficiency (about 1% of the injected dose) and the limited tumor penetration of nanoparticles remain two major challenges. Here we report a class of functionalized, long-circulating, and ultrasmall gold nanocarriers (5 nm gold core and 20 nm overall hydrodynamic diameter) for improved drug delivery and deep tumor penetration. By using doxorubicin as a model drug, our design also includes a pH-sensitive hydrazone linkage that is stable at neutral or slightly basic pH but is rapidly cleaved in the acidic tumor microenvironments and intracellular organelles. With a circulation halftime of 1.6 days, the small particle size is an important feature not only for efficient extravasation and accumulation via the enhanced permeability and retention (EPR) effect, but also for faster nanoparticle diffusion and improved tumor penetration. In xenograft animal models, the results demonstrate that up to 8% of the injected nanoparticles can be accumulated at the tumor sites, among the highest nanoparticle delivery efficiencies reported in the literature. Also, histopathological and direct visual examinations reveal dark-colored tumors with deep nanoparticle penetration and distribution throughout the tumor mass. In comparison with pure doxorubicin which is known to cause considerable heart, kidney, and lung toxicity, in vivo animal data indicate that this class of functionalized and ultrasmall gold nanoparticles indeed provides better therapeutic efficacies with no apparent toxicity in vital organs.

Enhanced Detection Specificity and Sensitivity of Alzheimer's Disease Using Amyloid-ß-Targeted Quantum Dots.

Diagnostics of Alzheimer's disease (AD) commonly employ the use of fluorescent thioflavin derivatives having affinity for the amyloid-ß (Aß) proteins associated with AD progression. However, thioflavin probes have limitations in their diagnostic capabilities arising from a number of undesireable qualities, including poor photostability, weak emission intensity, and high emission overlap with the backgound tissue autofluorescence. To overcome such limitations, we have developed nanoformulated probes consisting of a red-emitting fluorescent quantum dot (QD) core encapsulated in a PEGylated shell with benzotriazole (BTA) targeting molecules on the surface (QD-PEG-BTA). The combination of strong red fluorescence, multivalent binding, and decreased backgound signal and nonspecific binding provided the ability of the QD-PEG-BTA probes to achieve detection sensitivites 4 orders of magnitude greater than those of conventional thioflavin derivatives. This study opens the door for the use of QDs in AD detection applications.

Stimuli-responsive nanoparticles for targeting the tumor microenvironment.

One of the most challenging and clinically important goals in nanomedicine is to deliver imaging and therapeutic agents to solid tumors. Here we discuss the recent design and development of stimuli-responsive smart nanoparticles for targeting the common attributes of solid tumors such as their acidic and hypoxic microenvironments. This class of stimuli-responsive nanoparticles is inactive during blood circulation and under normal physiological conditions, but is activated by acidic pH, enzymatic up-regulation, or hypoxia once they extravasate into the tumor microenvironment. The nanoparticles are often designed to first "navigate" the body's vascular system, "dock" at the tumor sites, and then "activate" for action inside the tumor interstitial space. They combine the favorable biodistribution and pharmacokinetic properties of nanodelivery vehicles and the rapid diffusion and penetration properties of smaller drug cargos. By targeting the broad tumor habitats rather than tumor-specific receptors, this strategy has the potential to overcome the tumor heterogeneity problem and could be used to design diagnostic and therapeutic nanoparticles for a broad range of solid tumors.

An unusual role of folate in the self-assembly of heparin-folate conjugates into nanoparticles.

Tumor targeting agents including antibodies, peptides, and small molecules, are often used to improve the delivery efficiency of nanoparticles. Despite numerous studies investigating the abilities of targeting agents to increase the accumulation of nanosized therapeutics within diseased tissues, little attention has been focused on how these ligands can affect the self-assembly of the nanoparticle's modified polymer constituents upon chemical conjugation. Here we present an actively tumor targeted nanoparticle constructed via the self-assembly of a folate modified heparin. Folate conjugation unexpectedly allowed the self-assembly of heparin, where a majority of the folate molecules (>80%) resided inside the core of the nanoparticle. The folate-heparin nanoparticles could also physically encapsulate lipophilic fluorescent dyes, enabling the use of the constructs as activatable fluorescent probes for targeted in vivo tumor imaging.

Physical chemistry of nanomedicine: understanding the complex behaviors of nanoparticles in vivo.

Nanomedicine is an interdisciplinary field of research at the interface of science, engineering, and medicine, with broad clinical applications ranging from molecular imaging to medical diagnostics, targeted therapy, and image-guided surgery. Despite major advances during the past 20 years, there are still major fundamental and technical barriers that need to be understood and overcome. In particular, the complex behaviors of nanoparticles under physiological conditions are poorly understood, and detailed kinetic and thermodynamic principles are still not available to guide the rational design and development of nanoparticle agents. Here we discuss the interactions of nanoparticles with proteins, cells, tissues, and organs from a quantitative physical chemistry point of view. We also discuss insights and strategies on how to minimize nonspecific protein binding, how to design multistage and activatable nanostructures for improved drug delivery, and how to use the enhanced permeability and retention effect to deliver imaging agents for image-guided cancer surgery.

Compact and blinking-suppressed quantum dots for single-particle tracking in live cells.

Quantum dots (QDs) offer distinct advantages over organic dyes and fluorescent proteins for biological imaging applications because of their brightness, photostability, and tunability. However, a major limitation is that single QDs emit fluorescent light in an intermittent on-and-off fashion called "blinking". Here we report the development of blinking-suppressed, relatively compact QDs that are able to maintain their favorable optical properties in aqueous solution. Specifically, we show that a linearly graded alloy shell can be grown on a small CdSe core via a precisely controlled layer-by-layer process, and that this graded shell leads to a dramatic suppression of QD blinking in both organic solvents and water. A substantial portion (>25%) of the resulting QDs does not blink (more than 99% of the time in the bright or "on" state). Theoretical modeling studies indicate that this type of linearly graded shell not only can minimize charge carrier access to surface traps but also can reduce lattice defects, both of which are believed to be responsible for carrier trapping and QD blinking. Further, we have evaluated the biological utility of blinking-suppressed QDs coated with polyethylene glycol (PEG)-based ligands and multidentate ligands. The results demonstrate that their optical properties are largely independent of surface coatings and solvating media, and that the blinking-suppressed QDs can provide continuous trajectories in live-cell receptor tracking studies.

Mapping the spatial distribution of charge carriers in quantum-confined heterostructures.

Quantum-confined nanostructures are considered 'artificial atoms' because the wavefunctions of their charge carriers resemble those of atomic orbitals. For multiple-domain heterostructures, however, carrier wavefunctions are more complex and still not well understood. We have prepared a unique series of cation-exchanged Hg(x)Cd(1-x)Te quantum dots (QDs) and seven epitaxial core-shell QDs and measured their first and second exciton peak oscillator strengths as a function of size and chemical composition. A major finding is that carrier locations can be quantitatively mapped and visualized during shell growth or cation exchange simply using absorption transition strengths. These results reveal that a broad range of quantum heterostructures with different internal structures and band alignments exhibit distinct carrier localization patterns that can be used to further improve the performance of optoelectronic devices and enhance the brightness of QD probes for bioimaging.