Betty Y.S. Kim

Nanoparticle-mediated cellular response is size-dependent

Nanostructures of different sizes, shapes and material properties have many applications in biomedical imaging, clinical diagnostics and therapeutics. In spite of what has been achieved so far, a complete understanding of how cells interact with nanostructures of well-defined sizes, at the molecular level, remains poorly understood. Here we show that gold and silver nanoparticles coated with antibodies can regulate the process of membrane receptor internalization. The binding and activation of membrane receptors and subsequent protein expression strongly depend on nanoparticle size. Although all nanoparticles within the 2-100 nm size range were found to alter signalling processes essential for basic cell functions (including cell death), 40- and 50-nm nanoparticles demonstrated the greatest effect. These results show that nanoparticles should no longer be viewed as simple carriers for biomedical applications, but can also play an active role in mediating biological effects. The findings presented here may assist in the design of nanoscale delivery and therapeutic systems and provide insights into nanotoxicity.

Surface modification of nanoparticles enables selective evasion of phagocytic clearance by distinct macrophage phenotypes

Nanomedicine is a burgeoning industry but an understanding of the interaction of nanomaterials with the immune system is critical for clinical translation. Macrophages play a fundamental role in the immune system by engulfing foreign particulates such as nanoparticles. When activated, macrophages form distinct phenotypic populations with unique immune functions, however the mechanism by which these polarized macrophages react to nanoparticles is unclear. Furthermore, strategies to selectively evade activated macrophage subpopulations are lacking. Here we demonstrate that stimulated macrophages possess higher phagocytic activities and that classically activated (M1) macrophages exhibit greater phagocytic capacity than alternatively activated (M2) macrophages. We show that modification of nanoparticles with polyethylene-glycol results in decreased clearance by all macrophage phenotypes, but importantly, coating nanoparticles with CD47 preferentially lowers phagocytic activity by the M1 phenotype. These results suggest that bio-inspired nanoparticle surface design may enable evasion of specific components of the immune system and provide a rational approach for developing immune tolerant nanomedicines.

Biodegradable Quantum Dot Nanocomposites Enable Live Cell Labeling and Imaging of Cytoplasmic Targets

Semiconductor quantum dots (QDs) offer great promise as the new generation of fluorescent probes to image and study biological processes. Despite their superior optical properties, QDs for live cell monitoring and tracking of cytoplasmic processes remain limited due to inefficient delivery methods available, altered state or function of cells during the delivery process and the requirement of surface-functionalized QDs for specific labeling of subcellular structures. Here, we present a noninvasive method to image subcellular structures in live cells using bioconjugated QD nanocomposites. By incorporating antibody-coated QDs within biodegradable polymeric nanospheres, we have designed a bioresponsive delivery system that undergoes endolysosomal to cytosolic translocation via pH-dependent reversal of nanocomposite surface charge polarity. Upon entering the cytosol, the polymer nanospheres undergo hydrolysis thus releasing the QD bioconjugates. This approach facilitates multiplexed labeling of subcellular structures inside live cells without the requirement of cell fixation or membrane permeabilization. As compared to conventional intracellular delivery techniques, this approach allows the high throughput cytoplasmic delivery of QDs with minimal toxicity to the cell. More importantly, this development demonstrates an important rational strategy for the design of a multifunctional nanosystem for biological applications.

Remodeling Tumor Vasculature to Enhance Delivery of Intermediate-Sized Nanoparticles

Restoration of dysfunctional tumor vasculature can reestablish the pressure gradient between intravascular and interstitial space that is essential for transporting nanomedicines into solid tumors. Morphologic and functional normalization of tumor vessels improves tissue perfusion to facilitate intratumoral nanoparticle delivery. However, this remodeling process also reduces tumor vessel permeability, which can impair nanoparticle transport. Although nanoparticles sized below 10 nm maximally benefited from tumor vessel normalization therapy for enhanced nanomedicine delivery, the small particle size severely limits its applicability. Here, we show that intermediate-sized nanoparticles (20-40 nm) can also benefit from tumor vasculature remodeling. We demonstrate that a window of opportunity exists for a two-stage transport strategy of different nanoparticle sizes. Overall, tumor vessel remodeling enhances the transvascular delivery of intermediate-size nanoparticles of up to 40 nm. Once within the tumor matrix, however, smaller nanoparticles experience a significantly lesser degree of diffusional hindrance, resulting in a more homogeneous distribution within the tumor interstitium. These findings suggest that antiangiogenic therapy and nanoparticle design can be combined in a multistage fashion, with two sets of size-inclusion criteria, to achieve optimal nanomedicine delivery into solid tumors.

Breaking Down the Barriers to Precision Cancer Nanomedicine

Nanomedicine offers unique advantages in treating human cancers. However, physiological and pathological barriers within normal and disease tissues, which are highly variable among individuals, often hinder its effectiveness. The body possesses specific innate responses to nanoparticles (NPs), which when combined with unique pathophysiological signatures in the tumor microenvironment, can severely limit the utility of nanomedicine in the oncological setting. Furthermore, with the successes of cancer immunotherapies, understanding nanoimmune interactions and developing immune-smart cancer nanomedicine that can take advantage of the bodys immune functions will increasingly become clinically relevant. Therefore, a better understanding of the important native and acquired biological processes that dictate the fate of nanomedicine is integral to developing more effective individualized platforms for treating cancer patients.

Assessing Near-Infrared Quantum Dots for Deep Tissue, Organ, and Animal Imaging Applications

Semiconductor quantum dots (Qdots) have emerged as novel ultrasensitive optical probes to target, detect, and image fundamental events occurring within the biological system. In particular, near-infrared (near-IR) Qdots holds great promise as in vivo contrast agents for real-time bioimaging capabilities. In this study, biocompatible near-IR Qdots are used to image organs, tissues, and cells. Compared to visible Qdots, we obtained a significant enhancement in signal detection sensitivity for imaging deep tissues and organs. In addition, biomolecules were used to target these optical contrast agents for multiplexed imaging of cells and organs in vivo. The ability to simultaneously distinguish emission profiles of multiple near-IR Qdots will likely emerge as important tools for addressing fundamental questions in molecular biology and in medical sciences.

Phylogenetic relationships among Hepatozoon species from snakes, frogs and mosquitoes of Ontario, Canada, determined by ITS-1 nucleotide sequences and life-cycle, morphological and developmental characteristics

The molecular biological characteristics of Hepatozoon species infecting various species of snakes, frogs and mosquitoes were investigated by determining the nucleotide sequences of the first internal transcribed spacer region. A phylogenetic analysis was performed on seven isolates of Hepatozoon infecting snakes, including Hepatozoon sipedon and four morphologically similar but not identical forms, and two isolates of Hepatozoon catesbianae infecting Green frogs (Rana clamitans melanota). This analysis, which utilised data from first internal transcribed spacer nucleotide sequences, morphological and morphometric features of gamonts, oocysts and sporocysts, and previously determined life-cycle and host-specificity characteristics, revealed that H. sipedon is a polymorphic species with a wide host and geographic range. Four synapomorphies. including two nucleotide substitutions and two morphological character state changes, supported a monophyletic group of six isolates of H. sipedon from the central region of Ontario which was the sister group for an isolate (HW1) from the southern part of the province. Based on the results of this study, an evaluation of which criteria are useful for describing species of Hepatozoon is presented, with the intent of curtailing the practice of naming species based on morphological features of gamonts or on incomplete life-cycle data.

THE LIFE HISTORY AND HOST SPECIFICITY OF HEPATOZOON CLAMATAE (APICOMPLEXA: ADELEORINA) AND ITS-1 NUCLEOTIDE SEQUENCE VARIATION OF HEPATOZOON SPECIES OF FROGS AND MOSQUITOES FROM ONTARIO

The life cycle of an intraerythrocytic hemogregarine, Hepatozoon clamatae, was studied in green frogs (Rana clamitans melanota), bullfrogs (Rana catesbeiana), northern leopard frogs (Rana pipiens), and in the mosquito, Culex territans. Gametogenesis, fertilization, and sporogony occurred within cells of the Malpighian tubules of laboratory-reared Cx. territans that had fed on naturally infected frogs. Mature oocysts containing hundreds of sporocysts were observed in mosquitoes 30 days postfeeding. Each sporocyst enclosed 4 sporozoites. Merozoites appeared in the peripheral circulation of laboratory-reared bullfrogs, green frogs and leopard frogs that had been fed sporocysts 35-70 days previously. Attempts to infect American toads (Bufo americanus) and blue-spotted salamanders (Ambystoma laterale) were not successful. Gamonts of this parasite induced nuclear fragmentation or segmentation in host erythrocytes. The life cycle, morphological, and morphometric features of H. clamatae are compared with H. catesbianae, a similar species that also infects ranids. Nucleotide sequence analysis of the internal transcribed spacer region (ITS-1) of these sympatric species revealed that only 6 nucleotide sites of the 129 base pairs of this region were variable among 4 isolates of H. clamatae and 2 isolates of H. catesbianae. A redescription of H. clamatae is presented based on data from this study and from the original description by Stebbins in 1905.

INTRAERYTHROCYTIC DEVELOPMENT OF SPECIES OF HEPATOZOON INFECTING RANID FROGS: EVIDENCE FOR CONVERGENCE OF LIFE CYCLE CHARACTERISTICS AMONG APICOMPLEXANS

Intraerythrocytic development of the adeleorin apicomplexans Hepatozoon clamatae and Hepatozoon catesbianae were investigated in the bullfrog, Rana catesbeiana, the green frog, Rana clamitans melanota, and the Northern leopard frog, Rana pipiens. Merozoites emerging from hepatic meronts penetrated erythrocytes and underwent 1–3 rounds of binary fission to produce 2–8 merozoites. Following their release from infected erythrocytes, individual merozoites entered new cells and transformed into gamonts. Although this is the first report of intraerythrocytic development for a fully described species of Hepatozoon, a phylogenetic reanalysis of 11 species of Hepatozoon, 6 species representative of the 5 other hemogregarine taxa, 2 species of dactylosomatids, and 2 species of piroplasms, indicates that asexual reproduction of parasites within blood cells of vertebrates has arisen at least 3 times in the apicomplexan lineage that includes adeleorins and piroplasms. This method of asexual development, which is also observed in species of hemospororin genera such as Plasmodium, is discussed in the context of the evolution of apicomplexan life cycles. In addition to supporting the paraphyly of the genus Hepatozoon determined in an earlier study, this phylogenetic analysis featured a monophyletic group, consisting of the sister taxa Hemolivia and Karyolysus, that was the sister group to a clade consisting of the more derived hemogregarines, the dactylosomatids, and the piroplasms.

Multivalent bi-specific nanobioconjugate engager for targeted cancer immunotherapy

Tumour-targeted immunotherapy offers the unique advantage of specific tumouricidal effects with reduced immune-associated toxicity. However, existing platforms suffer from low potency, inability to generate long-term immune memory and decreased activities against tumour-cell subpopulations with low targeting receptor levels. Here we adopted a modular design approach that uses colloidal nanoparticles as substrates to create a multivalent bi-specific nanobioconjugate engager (mBiNE) to promote selective, immune-mediated eradication of cancer cells. By simultaneously targeting the human epidermal growth factor receptor 2 (HER2) expressed by cancer cells and pro-phagocytosis signalling mediated by calreticulin, the mBiNE stimulated HER2-targeted phagocytosis and produced durable antitumour immune responses against HER2-expressing tumours. Interestingly, although the initial immune activation mediated by the mBiNE was receptor dependent, the subsequent antitumour immunity also generated protective effects against tumour-cell populations that lacked the HER2 receptor. Thus, the mBiNE represents a new targeted, nanomaterial-immunotherapy platform to stimulate innate and adaptive immunity and promote a universal antitumour response.