Kuan-Ju Chen

Three-Dimensional Nanostructured Substrates toward Efficient Capture of Circulating Tumor Cells

During the progression of metastasis, cancer cells detach from the solid primary tumor, enter the blood stream, and travel to different tissues of the body. These breakaway cancer cells in the peripheral blood are known as circulating tumor cells (CTCs).[1] In addition to conventional diagnostic imaging and serum marker detection, quantification of CTCs in patient blood provides new and valuable information about managing cancer.[2–5] Over the past decade, CTC counting has been used for examining cancer metastasis, predicting patient prognosis, and monitoring the therapeutic outcomes of cancer.[6] However, isolation of CTCs has been technically challenging due to the extremely low abundance (a few to hundreds per milliliter) of CTCs among a large number of hematologic cells in the blood (109 mL−1).[4, 7, 8] Several technology platforms for isolating/counting CTCs have been developed with strategies that involve immunomagnetic beads or microfluidic devices.[3, 4,9, 10] The former utilizes capture-agent-coated magnetic beads to immunologically recognize CTCs in the blood, followed by magnetic isolation. However, these bead-based approaches are limited by their low CTC-capture yield and purity. Recently, a number of microfluidic technologies[9, 10] has been established for capturing viable CTCs from whole-blood samples with improved efficiency and selectivity compared to the bead-based approach.[3, 7] While different device architectures were applied in these CTC-sorting microchips, the improved CTC-capture efficiencies were achieved by increasing CTC/substrate contact frequency and duration.

Photothermal effects of supramolecularly assembled gold nanoparticles for the targeted treatment of cancer cells.

Noble-metal nanostructures with unique photophysical properties have been considered as prime candidate agents for the photothermal treatment of cancer.[1–4] Typically, the photothermal properties of these nanostructures can be controlled by manipulating their sizes and shapes.[4,5] Over the past decade, significant endeavors have been devoted to the production of a variety of gold nanostructures, such as nanoparticles,[6,7] nanoshells,[8–10] nanorods,[11,12] and nanocages,[5,13,14] which are able to overcome limitations of organic-dye-based photothermal agents,[7] such as low light absorption and undesired photobleaching. For sufficient energy to be harvested/generated to damage tumor cells, the size of these nanostructure-based agents are required in the range of tens to hundreds nm.[15] However, the relatively “large” size of the agents often leads to poor bioclearance (i.e., accumulation in the liver, spleen, and kidneys), which is a major obstacle to their in vivo application.[16–18] Alternatively, the photophysical properties of noble-metal nanostructures can be altered systematically by the formation of aggregates through self-assembly.[19–30] The antibody-assisted aggregation of Au nanoparticles on cell membranes or in intracellular environments led to the enhancement of photothermal performance[31] as a result of the collective effects[32,33] associated with the assembled structures. Therefore, the self-assembly of small noble-metal building blocks, that is, noble-metal colloids with diameters of less than 8 nm[16–18] (compatible with renal clearance) would be a promising approach toward a new class of noble-metal photothermal agents.

A rapid pathway toward a superb gene delivery system: programming structural and functional diversity into a supramolecular nanoparticle library.

Nanoparticles are regarded as promising transfection reagents for effective and safe delivery of nucleic acids into a specific type of cells or tissues providing an alternative manipulation/therapy strategy to viral gene delivery. However, the current process of searching novel delivery materials is limited due to conventional low-throughput and time-consuming multistep synthetic approaches. Additionally, conventional approaches are frequently accompanied with unpredictability and continual optimization refinements, impeding flexible generation of material diversity creating a major obstacle to achieving high transfection performance. Here we have demonstrated a rapid developmental pathway toward highly efficient gene delivery systems by leveraging the powers of a supramolecular synthetic approach and a custom-designed digital microreactor. Using the digital microreactor, broad structural/functional diversity can be programmed into a library of DNA-encapsulated supramolecular nanoparticles (DNA⊂SNPs) by systematically altering the mixing ratios of molecular building blocks and a DNA plasmid. In vitro transfection studies with DNA⊂SNPs library identified the DNA⊂SNPs with the highest gene transfection efficiency, which can be attributed to cooperative effects of structures and surface chemistry of DNA⊂SNPs. We envision such a rapid developmental pathway can be adopted for generating nanoparticle-based vectors for delivery of a variety of loads.

Delivery of Intact Transcription Factor by Using Self‐Assembled Supramolecular Nanoparticles

Protein delivery[1] has been considered as the most straightforward strategy for modulating cellular behavior without the safety concerns and expression performance issues associated with gene deliver approaches. Two major challenges remain to be overcome in order to enable practical applications in biology and medicine 1) how to foster cellular uptake of protein molecules and 2) how to retain their stabilities and functions[2] over the delivery process. Recently, attempts have been made to develop a variety of delivery vectors, including liposomes,[3] polymer micelles,[4] and nanoparticle,[5] to enhance the uptake of protein molecules in target cells, and at the same time, to stabilize the encapsulated proteins. Owing to the time-consuming procedures employed in optimization of delivery materials, significant endeavors have been made in search of better delivery systems, although there has been limited progress in the field to date. Alternatively, recombinant technology[6] can be utilized to conjugate cell-penetrating peptides[7] (CPPs) onto protein molecules, this is the most commonly used protein delivery system with improved delivery efficiency. In this case, the major bottlenecks associated with the complicated procedure of generating recombinant proteins and the lack of protection mechanism against protein denature need to be solved.

A small library of DNA-encapsulated supramolecular nanoparticles for targeted gene delivery

We demonstrated a convenient, flexible and modular synthetic approach for preparation of a small library of DNA-encapsulated supramolecular nanoparticles SNPs superset DNA and RGD-SNPs superset DNA with different sizes and RGD target ligand coverage for targeted gene delivery.

Pretargeted Positron Emission Tomography Imaging That Employs Supramolecular Nanoparticles with in Vivo Bioorthogonal Chemistry

A pretargeted oncologic positron emission tomography (PET) imaging that leverages the power of supramolecular nanoparticles with in vivo bioorthogonal chemistry was demonstrated for the clinically relevant problem of tumor imaging. The advantages of this approach are that (i) the pharmacokinetics (PKs) of tumor-targeting and imaging agents can be independently altered via chemical alteration to achieve the desired in vivo performance and (ii) the interplay between the two PKs and other controllable variables confers a second layer of control toward improved PET imaging. In brief, we utilized supramolecular chemistry to synthesize tumor-targeting nanoparticles containing transcyclooctene (TCO, a bioorthogonal reactive motif), called TCO⊂SNPs. After the intravenous injection and subsequent concentration of the TCO⊂SNPs in the tumors of living mice, a small molecule containing both the complementary bioorthogonal motif (tetrazine, Tz) and a positron-emitting radioisotope ((64)Cu) was injected to react selectively and irreversibly to TCO. High-contrast PET imaging of the tumor mass was accomplished after the rapid clearance of the unreacted (64)Cu-Tz probe. Our nanoparticle approach encompasses a wider gamut of tumor types due to the use of EPR effects, which is a universal phenomenon for most solid tumors.

Molecular recognition enables nanosubstrate-mediated delivery of gene-encapsulated nanoparticles with high efficiency.

Substrate-mediated gene delivery is a promising method due to its unique ability to preconcentrate exogenous genes onto designated substrates. However, many challenges remain to enable continuous and multiround delivery of the gene using the same substrates without depositing payloads and immobilizing cells in each round of delivery. Herein we introduce a gene delivery system, nanosubstrate-mediated delivery (NSMD) platform, based on two functional components with nanoscale features, including (1) DNA⊂SNPs, supramolecular nanoparticle (SNP) vectors for gene encapsulation, and (2) Ad-SiNWS, adamantane (Ad)-grafted silicon nanowire substrates. The multivalent molecular recognition between the Ad motifs on Ad-SiNWS and the β-cyclodextrin (CD) motifs on DNA⊂SNPs leads to dynamic assembly and local enrichment of DNA⊂SNPs from the surrounding medium onto Ad-SiNWS. Subsequently, once cells settled on the substrate, DNA⊂SNPs enriched on Ad-SiNWS were introduced through the cell membranes by intimate contact with individual nanowires on Ad-SiNWS, resulting in a highly efficient delivery of exogenous genes. Most importantly, sequential delivery of multiple batches of exogenous genes on the same batch cells settled on Ad-SiNWS was realized by sequential additions of the corresponding DNA⊂SNPs with equivalent efficiency. Moreover, using the NSMD platform in vivo, cells recruited on subcutaneously transplanted Ad-SiNWS were also efficiently transfected with exogenous genes loaded into SNPs, validating the in vivo feasibility of this system. We believe that this nanosubstrate-mediated delivery platform will provide a superior system for in vitro and in vivo gene delivery and can be further used for the encapsulation and delivery of other biomolecules.

A High-Throughput Platform for Formulating and Screening Multifunctional Nanoparticles Capable of Simultaneous Delivery of Genes and Transcription Factors

Simultaneous delivery of multiple genes and proteins (e.g., transcription factors; TFs) is an emerging issue surrounding therapeutic research due to their ability to regulate cellular circuitry. Current gene and protein delivery strategies, however, are based on slow batch synthesis, which is ineffective, poorly controlled, and incapable of simultaneous delivery of both genes and proteins with synergistic functions. Consequently, advances in this field have been limited to in vitro studies. Here, by integrating microfluidic technologies with a supramolecular synthetic strategy, we present a high-throughput approach for formulating and screening multifunctional supramolecular nanoparticles (MFSNPs) self-assembled from a collection of functional modules to achieve simultaneous delivery of one gene and TF with unprecedented efficiency both in vitro and in vivo. We envision that this new approach could open a new avenue for immunotherapy, stem cell reprogramming, and other therapeutic applications.

Supramolecular Nanosubstrate-Mediated Delivery for Reprogramming and Transdifferentiation of Mammalian Cells

Supramolecular nanosubstrate-mediated delivery (SNSMD) leverages the power of molecular self-assembly and a nanostructured substrate platform for the low toxicity, highly efficient co-delivery of biological factors encapsulated in a nanovector. Human fibroblasts are successfully reprogrammed into induced pluripotent stems and transdifferentiated into induced neuronal-like cells.