Cliff R. Wong

Multistage nanoparticle delivery system for deep penetration into tumor tissue

Current Food and Drug Administration-approved cancer nanotherapeutics, which passively accumulate around leaky regions of the tumor vasculature because of an enhanced permeation and retention (EPR) effect, have provided only modest survival benefits. This suboptimal outcome is likely due to physiological barriers that hinder delivery of the nanotherapeutics throughout the tumor. Many of these nanotherapeutics are ≈100 nm in diameter and exhibit enhanced accumulation around the leaky regions of the tumor vasculature, but their large size hinders penetration into the dense collagen matrix. Therefore, we propose a multistage system in which 100-nm nanoparticles “shrink” to 10-nm nanoparticles after they extravasate from leaky regions of the tumor vasculature and are exposed to the tumor microenvironment. The shrunken nanoparticles can more readily diffuse throughout the tumors interstitial space. This size change is triggered by proteases that are highly expressed in the tumor microenvironment such as MMP-2, which degrade the cores of 100-nm gelatin nanoparticles, releasing smaller 10-nm nanoparticles from their surface. We used quantum dots (QD) as a model system for the 10-nm particles because their fluorescence can be used to demonstrate the validity of our approach. In vitro MMP-2 activation of the multistage nanoparticles revealed that the size change was efficient and effective in the enhancement of diffusive transport. In vivo circulation half-life and intratumoral diffusion measurements indicate that our multistage nanoparticles exhibited both the long circulation half-life necessary for the EPR effect and the deep tumor penetration required for delivery into the tumors dense collagen matrix.

Compact high-quality CdSe–CdS core–shell nanocrystals with narrow emission linewidths and suppressed blinking

High particle uniformity, high photoluminescence quantum yields, narrow and symmetric emission spectral lineshapes and minimal single-dot emission intermittency (known as blinking) have been recognized as universal requirements for the successful use of colloidal quantum dots in nearly all optical applications. However, synthesizing samples that simultaneously meet all these four criteria has proven challenging. Here, we report the synthesis of such high-quality CdSe-CdS core-shell quantum dots in an optimized process that maintains a slow growth rate of the shell through the use of octanethiol and cadmium oleate as precursors. In contrast with previous observations, single-dot blinking is significantly suppressed with only a relatively thin shell. Furthermore, we demonstrate the elimination of the ensemble luminescence photodarkening that is an intrinsic consequence of quantum dot blinking statistical ageing. Furthermore, the small size and high photoluminescence quantum yields of these novel quantum dots render them superior in vivo imaging agents compared with conventional quantum dots. We anticipate these quantum dots will also result in significant improvement in the performance of quantum dots in other applications such as solid-state lighting and illumination.

Compact Biocompatible Quantum Dots via RAFT-Mediated Synthesis of Imidazole-Based Random Copolymer Ligand

We present a new class of polymeric ligands for quantum dot (QD) water solubilization to yield biocompatible and derivatizable QDs with compact size (approximately 10-12 nm diameter), high quantum yields (>50%), excellent stability across a large pH range (pH 5-10.5), and low nonspecific binding. To address the fundamental problem of thiol instability in traditional ligand exchange systems, the polymers here employ a stable multidentate imidazole binding motif to the QD surface. The polymers are synthesized via reversible addition-fragmentation chain transfer-mediated polymerization to produce molecular weight controlled monodisperse random copolymers from three types of monomers that feature imidazole groups for QD binding, polyethylene glycol (PEG) groups for water solubilization, and either primary amines or biotin groups for derivatization. The polymer architecture can be tuned by the monomer ratios to yield aqueous QDs with targeted surface functionalities. By incorporating amino-PEG monomers, we demonstrate covalent conjugation of a dye to form a highly efficient QD-dye energy transfer pair as well as covalent conjugation to streptavidin for high-affinity single molecule imaging of biotinylated receptors on live cells with minimal nonspecific binding. The small size and low serum binding of these polymer-coated QDs also allow us to demonstrate their utility for in vivo imaging of the tumor microenvironment in live mice.

Avidin as a model for charge driven transport into cartilage and drug delivery for treating early stage post-traumatic osteoarthritis.

Local drug delivery into cartilage remains a challenge due to its dense extracellular matrix of negatively charged proteoglycans enmeshed within a collagen fibril network. The high negative fixed charge density of cartilage offers the unique opportunity to utilize electrostatic interactions to augment transport, binding and retention of drug carriers. With the goal of developing particle-based drug delivery mechanisms for treating post-traumatic osteoarthritis, our objectives were, first, to determine the size range of a variety of solutes that could penetrate and diffuse through normal cartilage and enzymatically treated cartilage to mimic early stages of OA, and second, to investigate the effects of electrostatic interactions on particle partitioning, uptake and binding within cartilage using the highly positively charged protein, Avidin, as a model. Results showed that solutes having a hydrodynamic diameter ≤10 nm can penetrate into the full thickness of cartilage explants while larger sized solutes were trapped in the tissues superficial zone. Avidin had a 400-fold higher uptake than its neutral same-sized counterpart, NeutrAvidin, and >90% of the absorbed Avidin remained within cartilage explants for at least 15 days. We report reversible, weak binding (K(D) ~ 150 μM) of Avidin to intratissue sites in cartilage. The large effective binding site density (N(T) ~ 2920 μM) within cartilage matrix facilitates Avidins retention, making its structure suitable for particle based drug delivery into cartilage.

Control of the carrier type in InAs nanocrystal films by predeposition incorporation of Cd.

Nanocrystal (NC) films have been proposed as an alternative to bulk semiconductors for electronic applications such as solar cells and photodetectors. One outstanding challenge in NC electronics is to robustly control the carrier type to create stable p-n homojunction-based devices. We demonstrate that the postsynthetic addition of Cd to InAs nanocrystals switches the resulting InAs:Cd NC films from n-type to p-type when operating in a field effect transistor. This method presents a stable, facile way to control the carrier type of InAs nanocrystals prior to deposition. We present two mechanisms to explain the observed switch in carrier type. In mechanism 1, Cd atoms are incorporated at In sites in the lattice and act as acceptor defects, forming a partially compensated p-type semiconductor. In mechanism 2, Cd atoms passivate donor-type InAs surface states and create acceptor-type surface states. This work represents a critical step toward the creation of p-n homojunction-based NC electronics.

Multistage Nanoparticles for Improved Delivery into Tumor Tissue

The enhanced permeability and retention (EPR) effect has been a key rationale for the development of nanoscale carriers to solid tumors. As a consequence of EPR, nanotherapeutics are expected to improve drug and detection probe delivery, have less adverse effects than conventional chemotherapy, and thus result in improved detection and treatment of tumors. Physiological barriers posed by the abnormal tumor microenvironment, however, can hinder the homogeneous delivery of nanomedicine in amounts sufficient to eradicate cancer. To effectively enhance the therapeutic outcome of cancer patients by nanotherapeutics, we have to find ways to overcome these barriers. One possibility is to exploit the abnormal tumor microenvironment for selective and improved delivery of therapeutic agents to tumors. Recently, we proposed a multistage nanoparticle delivery system as a potential means to enable uniform delivery throughout the tumor and improve the efficacy of anticancer therapy. Here, we describe the synthesis of a novel multistage nanoparticle formulation that shrinks in size once it enters the tumor interstitial space to optimize the delivery to tumors as well as within tumors. Finally, we provide detailed experimental methods for the characterization of such nanoparticles.

Abstract 548: Multistage nanoparticle delivery system for deep penetration into solid tumor

Background: Through the enhanced permeation and retention (EPR) effect, current FDA-approved cancer nanotherapeutics accumulate preferentially around leaky regions of the tumor vasculature due to their large size – typically ∼100 nm in diameter. However, these extravasated large nanocarriers localize mostly at the tumor periphery and might not be able to effectively distribute throughout the tumor due to the dense collagen matrix in the tumor interstitum. The resulting heterogeneous distribution of therapeutic are likely responsible for the modest survival benefits of current nanomedicine. Methods: To overcome these physiological barriers to drug delivery in tumors, we have developed a multistage nanoparticle delivery system (QDGelNP) in which 100-nm nanoparticles “shrink” to 10-nm nanoparticles after they extravasate from the tumor vasculature and are exposed to the tumor microenvironment, allowing enhanced penetration into the tumor parenchyma. This “shrinkage” is preferentially triggered in the tumor by proteases, such as MMP-2, which are highly expressed and/or activated in the tumor microenvironment. These proteases degrade the cores of 100-nm gelatin nanoparticles, releasing smaller 10-nm nanoparticles from their surfaces. We used quantum dots (QD) as a model system for the 10-nm particles because their fluorescence can be monitored in vivo to test the validity of our approach. Results: In vivo blood circulation half-life measurement indicates that our QDGelNPs exhibited the long circulation half-life (22.0 ± 3.4 hours) necessary for the EPR effect. In vitro MMP-2 activation of QDGelNPs was characterized using gel filtration chromatography, fluorescence correlation spectroscopy, and collagen gel diffusion; these experiments revealed that the size change was efficient (50% activation in 1.5 hrs using 0.16 μM MMP-2) and effective in the enhancement of diffusive transport in dense collagen matrices (∼1 mm penetration in 12 hrs, D = ∼2.3 × 10−7 cm2s−1). To test whether tumor secreted MMP-2 can change the size of QDGelNPs in vivo, we intratumorally administered QDGelNPs into MMP-2 expressing tumors (HT-1080) grown in mouse dorsal skin chamber transparent window models and monitored interstitial distribution of QDs by intravital microscopy. At 6 hrs post-injection, the QDGelNPs penetrated up to ∼300 μm into the surrounding tumor tissue (Deff = ∼2.2 × 10-8 cm2s−1), whereas 100-nm single-stage control nanoparticles were confined mostly at the injection site. These data confirmed our QDGelNPs’ capacity to penetrate the tumor9s dense collagen matrix for delivery deep into solid tumors. Conclusion: We have successfully developed a multistage nanoparticle delivery system. Such delivery systems provide a promising approach to improving the delivery of anticancer agents into solid tumors and, as a result, reduction of the likelihood for tumor regression and enhancement of the drug9s therapeutic efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 548. doi:10.1158/1538-7445.AM2011-548