Symposium SM02—Progress in Supramolecular Nanotheranostics

Acquired drug resistance remains a challenge in chemotherapy. Here we discuss enzymatic, in-situ assembling of cholesterol derivatives for acting as imaging probes for reveal membrane dynamics and for develop polypharmaceuticals for selectively inducing death of cancer cells via multiple pathways and without inducing acquired drug resistance. We designed novel active and raft-affinitive probes consisting of an enzymatic trigger, a fluorophore, and a cholesterol. Being water soluble and as the substrate of ectophosphatase, the probes preferably and rapidly assemble in the membrane to exhibit strong fluorescence. Such cell compatible, active probes work at micromolar concentrations, require short incubation period, and easily achieve high resolution monitoring of nanoscale heterogeneity in live-cell membranes. In a related study, we developed a conjugate of tyrosine and cholesterol (TC), formed by enzyme catalyzed dephosphorylation of phosphorylate TC (pTC). TC self-assembles selectively on or in cancer cells. Acting as polypharmaceuticals, the assemblies of TC augment lipid rafts, aggregate extrinsic cell death receptors (e.g., DR5, CD95, or TRAILR), modulate the expression of oncoproteins (e.g., Src and Akt), disrupt the dynamics of cytoskeletons (e.g., actin filaments or microtubules), induce ER stress, and increase the production of reactive oxygen species (ROS), thus resulting in cell death and preventing acquired drug resistance. Moreover, the assemblies inhibit the growth of platinum-resistant ovarian cancer tumor in a murine model. These results illustrate the use of instructed-assembly (iA) in cellular environment to form polypharmaceuticals in-situ that not only interact with multiple proteins, but also modulate membrane dynamics for developing novel anticancer therapeutics.

In terms of the native 3D resolution of cell structures in all living species, the specific tracking of complexity and dynamics of cellular functions with precise resolution in intact cells or organisms will be important to understand the biological basis and to conduct related biomedical studies. Usually, within complex environment, biological specificity can be mediated by the accurate regulation of biomolecule functions in response to intrinsic development programs and extrinsic signals. In order to fully understand the biomolecule functions and further elucidate their interaction mechanisms, a simple and reliable assay with high sensitivity and fidelity will be essential to efficiently report the biological process of interest. Generally, optical imaging modalities enable such rapid, direct and sensitive visualization, mainly due to their high sensitivity, relative safety, and easily handling, and therefore have become robust and reliable tools in monitoring of subcellular protein dynamics and analysis of tumors or pathogen–host interactions. The systematic imaging investigation of enzymes or proteins activities in a complicated environment offers the great possibility for the in-depth understanding of the biological basis conferring diseases status, and importantly, for the facilitating of new discovery of effective theranostic approaches in vitro and in vivo. In our group, a series of simple and specific NIR based fluorescent/bioluminescent molecules or nano-probes have been developed to real-time visualize the intrinsic mechanisms of bacterial and tumor functions, more importantly, the unique strategies regarding site-specific localization of contrast agents have also been established for precise regulation of cell functions and preclinical theranostics.

Soft matters exhibiting stimuli-response under water-rich conditions are attractive because of their numerous potential bio-applications. My group recently makes efforts to design stimuli-responsive supramolecular hydrogels, inspired by natural living cells, an ultimately complex and intelligent soft material composed of numerous multiple components. By mimicking live cells, we hybridized our supramolecular with many synthetic and/or biological molecules bearing various structures and functions. We expected that controlling and facilitating the crosstalk between these multiple components could give biomolecule-response to the hybrid supramolecular hydrogels. One of the recent examples is self-sorted supramolecular nanofibers double network system. We firstly succeeded in in situ real-time imaging of self-sorted supramolecular nanofibers by using confocal laser scanning microscopy and super resolution imaging. Moreover, we are able to construct an adaptive hydrogel whose reological property can be modulated in both directions by distinct two extarnal stimuli.

References
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Therapeutic delivery of proteins and nucleic acids into cells is a difficult goal. Of the many challenges in the delivery process, perhaps the most demanding is providing these biologics with access to the cytosol. Most delivery strategies employ endosomal uptake, requiring endosomal escape for the payload biologics to be effective. In our research, we have developed strategies to deliver proteins and nucleic acids directly to the cytosol through fusion of nanomaterials with the cell membrane.

We have developed two systems for delivering biologics into the cytosol. The first is nanoparticle-stabilized nanocapsules (NPSC), formed via assembly of arginine-functionalized gold nanoparticles (AuNP) around fatty acid nanodroplets. These systems have been used to deliver proteins1 and siRNA directly to the cytosol in vitro, and have been used for efficient delivery of siRNA targeting TNF-α in vivo, providing effective anti-inflammatory treatment.

Our second system uses co-engineering of nanoparticles and proteins to deliver proteins into the cytosol. This platform co-assembles arginine-functionalized AuNPs with proteins modified with a glutamate (E-tag) chain. This strategy has been applied to a wide range of proteins, and we have employed this system to co-deliver complete CRISPR machinery (Cas9 protein and guide RNA) for effective editing both in vitro and in vivo.

The kinetics of antigen availability following immunization impact follicular helper T cell priming, germinal center responses, and ultimate antibody production, but clinically-relevant methods to control the duration of antigen delivery to lymph nodes in subunit vaccines are lacking. We conjugated antigens derived from the gp140 HIV envelope trimer with a phosphoserine (pSer) peptide that binds tightly to the most common clinical adjuvant, aluminum hydroxide (Alhydrogel, or alum). Site specific modification of an engineered outer domain (eOD) gp120 or stabilized SOSIP trimer immunogen with varying numbers of pSer groups allowed self-assembly of these antigens on the surfaces of alum particles to be tuned and alum-bound antigens were presented from alum particle surfaces with a defined orientation. Tight binding to alum converted alum itself into a nanoparticle delivery vehicle, as alum nanocrystals released from the injection site trafficked to lymph nodes and were taken up by antigen-specific B cells. Ultimately, a 30-fold increase in germinal center responses and antibody titer relative to the unmodified protein was observed four weeks after primary immunization with both pSer-eOD and pSer-SOSIP conjugates, and long-lived plasma cells in bone marrow were doubled by immunization with pSer-modified immunogens. Overall, pSer-antigen conjugates elicited significant increases in antibody titers and altered specificity of the humoral response through controlling the display of antigen on the surface of alum.

Nanoscopic delivery vehicles capable of encapsulating drug molecules and releasing them in response to external stimuli are of great interest due to implications in therapeutic applications. However, the stability of encapsulation with self-assembled systems is limited during blood circulation and translating nanomedicines into clinical applications still remains a challenge due to the difficulty in regulating interactions on the interfaces between nanoparticles and biological systems. Thus, deliberate molecular design for stable encapsulation, targeting and triggered release is required. For this purpose, we have developed a facile synthetic method for highly stable, polymeric nanogels or polymer-caged hollow nanoparticles using a simple intra/inter-chain crosslinking reaction. We show a simple method for the preparation of biocompatible nanovehicles that provides the ability to encapsulate hydrophobic or hydrophilic drug molecules. Nano-carriers showed great stability to encapsulate drug molecules and drugs were only released inside cell. In addition, we showed a targeting strategy for nanoparticles incorporated with a supramolecularly pre-coated recombinant fusion protein in which HER2-binding affibody combines with glutathione-S-transferase. Once thermodynamically stabilized in preferred orientations on the nanoparticles, the adsorbed fusion proteins as a corona minimize interactions with serum proteins to prevent the clearance of nanoparticles by macrophages, while ensuring systematic targeting functions in vitroand in vivo. This study provides insight into the use of the supramolecularlybuilt protein corona shield as a targeting agent through regulating the interfaces between nanoparticles and biological systems.

Organelle targeting has emerged as a promising strategy in developing effective and specific cancer therapeutics by delivering a drug in its active form to the cellular compartment where it works. Among all the subcellular targets, endoplasmic reticulum (ER) targeting therapy has been little explored due to its complexity in cell signaling. As the largest cellular organelle, ER is responsible for crucial biosynthetic, sensing, and signaling functions in eukaryotic cells. Particularly, ER is responsible for the synthesis, folding, and posttranslational modifications of proteins destined for the secretory pathway, which amount to approximately 30% of the total proteome. Disturbing the protein-folding capacity of ER would result in ER stress, ultimately activating apoptotic signaling pathways and cell death. Therefore, selective disrupting ER function in cancer cells is a promising new strategy for anticancer therapies. However, current ER targeting small molecules, like tunicamycin and thapsigargin, lack cell selectivity and exhibit severe neurotoxicity, thus hindering their clinical applications. Therefore, it is necessary to develop novel ER targeting strategies that have high specificity against cancer cells. In this work, we employ enzyme-instructed self-assembly (EISA) to selectively target ER of cancer cells. Generated via enzymatic reactions on and in the cancer cells, the enzymatic assemblies interact with cellular membranes and disrupt plasma membrane integrity to enable the assemblies accumulate on the ER, thus inducing cancer cell death through ER stress. Utilizing enzymatic reactions and reduced diffusion of assembly, EISA enables spatiotemporal control of the generation and cellular distribution of the cytotoxic assemblies, thus providing a new strategy to regulate amyloid-like aggregates for treating cancer. This work, for the first time, demonstrates a reaction-based process for disrupting membranes in a spatiotemporally controlled manner, as well as subcellular organelle (i.e., ER) targeting, which illustrates a new concept in controlling cell fates via instructed-assembly.

Near-infrared (NIR) light has been intensively exploited in biomedical applications due to its unrivaled benefits. Although most studies are focused on the first NIR (NIR-I) window (650-950 nm), there is growing interest to extend the wavelength to the longer second NIR (NIR-II) window (1000-1700 nm). As compared with NIR-I window, NIR-II light benefits from reduced photon scattering in biological tissues and higher maximum permissible exposure limit. Despite the great advantage, reported nanoagents that absorb in NIR-II window are limited, which include copper sulfide nanoparticles, gold nanorods, phosphorus phthalocyanine, etc.
Recently, semiconducting polymer nanoparticles (SPNs) composed of π-conjugated polymer backbones have emerged as a new category of optical agents. Due to the benign nature, SPNs circumvent the concern of metal-ion induced toxicity and possess size- and morphology-independent optical properties. Moreover, bandgap engineering provides a way to impart SPNs with high photothermal conversion efficiency, allowing for photoacoustic (PA) imaging and cancer photothermal therapy (PTT). However, most SPN-based materials can only be responsive to visible or NIR-I light, and few have been used for NIR-II applications.
Herein, we design and synthesize the first organic imaging agent based on SPN (SPN-II) that absorbs both NIR-I and NIR-II light and apply it for NIR-II PA imaging. The broadband absorption of SPN-II allows us to directly compare NIR-II vs NIR-I PA imaging so as to find out the advantages of NIR-II light in PA imaging. Because of the weaker background PA signals from biological tissues in NIR-II window, the signal-to-noise ratio (SNR) of SPN-II resulted PA images at 1064 nm can be 1.4-times higher than that at 750 nm when comparing at the imaging depth of 3 cm. The proof-of-concept application of NIR-II PA imaging is demonstrated in in vivo imaging of brain vasculature in living rats, which showed 1.5-times higher SNR as compared with NIR-I PA imaging. Our study not only introduces the first broadband absorbing organic contrast agent that is applicable for PA imaging in both NIR-I and NIR-II windows but also reveals the advantages of NIR-II over NIR-I in PA imaging.
Furthermore, to reveal the advantages of NIR-II relative to NIR-I window in terms of cancer PTT, we also report the first organic SP-based photothermal nanoagent with the dual-peak absorption in both NIR-I and NIR-II windows. Such nanoagent consists of poly[(diketopyrrolopyrrole-alt-cyclopentadithiophene)-ran-(diketopyrrolopyrrole-alt-thiadiazoloquinoxaline)], which has two different segments showing absorption in NIR-I and NIR-II windows, respectively. Finetuning of the ratio between two segments leads to a final polymer with the nearly identical absorbance at 808 and 1064 nm. The resulted SPN_I-II not only allows for deep-tissue NIR-II PTT but also provides the opportunity to conduct a fair comparative study between NIR-I and NIR-II windows in order to reveal the superiority of NIR-II over NIR-I light for PTT. The proof-of-concept application of SPN_I-II is demonstrated in tumor xenograft mouse model to validate the advantage of NIR-II window over NIR-I window for deep-tissue PTT. At last, a NIR dye is doped into SPN_I-II to afford a fluorescent nanoparticle (SPNF_I-II), permitting NIR fluorescence imaging guided NIR-II PTT.

Multi-drug resistant bacterial infections are responsible for 700,000 deaths each year world-wide, with more than 10 million deaths per year predicted by the year 2050. The majority of human bacterial infections (~80%) are associated with formation of biofilms on living-tissues. Early detection of biofilms is crucial for limiting infection-based damage. Imaging these biofilms is challenging: conventional imaging agents are unable to penetrate the dense matrix of the biofilm, and many are susceptible to false positive/negative responses due to phenotypical mutations of the constituent microbes. We have engineered nanomaterials to penetrate the extracellular polymeric substance (EPS) matrix of biofilms and to intrinsically target the acidic microenvironment of the biofilms. Here, we report the creation of pH-responsive nanoparticles with embedded transition metal catalysts (nanozymes) that effectively target the acidic microenvironment of biofilms. These pH-switchable nanozymes generate imaging agents through bioorthogonal activation of profluorophores inside biofilms. The specificity of these nanozymes for imaging biofilms in complex biosystems was demonstrated using biofilm-mammalian cell co-culture experiments.

The subjects of this talk are multifunctional mesoporous silica nanoparticles controlled by supramolecular nanomachines and caps for imaging and drug delivery in cells and in vivo. The nanoparticles are designed to 1) trap therapeutic molecules inside of the nanocarriers, 2) carry therapeutics to the site of the disease with no leakage, 3) release a high local concentration of drugs, 4) release only on command – either autonomous or external, and 5) kill the cancer or infectious organism. The most important functionality is the ability to trap molecules in the pores and release them in response to desired specific stimuli. Two types of external stimuli will be discussed: light and oscillating magnetic fields. Activation by internal biological stimuli such as pH changes, redox potential changes, antibodies and enzymes will also be presented. Molecular machines based on molecules that undergo large amplitude motion when attached to mesoporous silica - impellers, snap-tops and valves – will be described. Derivatized azobenzene molecules, attached to the interior pore walls function as impellers that can move other molecules through the pores. Nanoparticles containing anticancer drugs in the mesopores are taken up by cancer cells, and optical stimulation of the impellers drives out the toxic molecules and kills the cells. Snap-tops with cleavable stoppers release cargo molecules when the stopper is removed from the pore entrance. Nanovalves consisting of rotaxanes and pseudorotaxanes placed at pore entrances can trap and release molecules from the pores in response to stimuli. Both reusable and completely reversible nanovalves will be described. Activation of these nanodevices by the five types of stimuli in solution, in living cells, and in animal models will be discussed. Applications to treatments of cancers (including pancreatic and breast) and of intracellular infectious diseases (including tuberculosis and tularemia) will be presented.

Theranostic nanomaterials have shown potentials for molecular diagnostic tests and targeted therapeutics in modern medicine. To reach their full potentials for clinical applications, novel methods for spatial and temporal control of stimuli-responsive nanomaterials are required in order to elucidate detailed mechanisms of theranostic effects in vivo and to personalize the diagnostics and treatments in clinics. Toward this goal, we have been selecting, from a large DNA library of up to 10¹⁵ different sequences, functional DNA molecules, such as DNAzymes (DNA molecules with enzymatic activities) and DNA aptamers (DNA molecules that can bind targets selectively) that can bind a wide variety of targets, especially those small molecular biomarkers that antibodies have not been able to recognize [1]. When conjugated to nanomaterials such as gold nanoparticles, quantum dots, liposomes, iron oxide nanoparticles, upconversion nanoparticles and polymeric organic nanoparticles, these functional DNA molecules allow stimuli-responsive assembly or disassembly of these nanomaterials, resulting in target-dependent changes of not only optical and magnetic signals for diagnostics, but also controlled release of therapeutic drugs [2]. To realize the spatial and temporal control of these stimuli-responsive nanomaterials, we have added a caged group to the functional DNA molecules so that these DNA nanomaterials can be delivered into living cells, zebrafish and other animals until a decaging event at a specific location and a certain time is triggered [3]. Recent progress in this area will be presented.

[1] a) Hang Xing, Kevin Hwang, Ji Li, Seyed-Fakhreddin Torabi, and Yi Lu, Curr. Opin. Chem. Eng. 4, 79-87 (2014); b) Hang Xing, Kevin Hwang and Yi Lu, Theranostics 6, 1336-1352 (2016); c) Claire E. McGhee, Kang Yong Loh, Yi Lu, Curr. Opin. Biotech. 45, 191-201 (2017).
[2] a) Peiwen Wu, Kevin Hwang, Tian Lan, Yi Lu, J. Am. Chem. Soc. 135, 5254–5257 (2013); b) Hang Xing, et al., J. Mater. Chem. B 1, 5288-5297 (2013); c) Jingjing Zhang, Hang Xing and Yi Lu, Chem. Sci. 9, 3906-3910 (2018); d) Le-le Li and Yi Lu, J. Am. Chem. Soc. 137, 5272–5275 (2015); e) Jian Zhao, Jinhong Gao, Wenting Xue, Zhenghan Di, Hang Xing, Yi Lu, and Lele Li, J. Am. Chem. Soc. 140 578–581 (2018); f) Hang Xing, et al., J. Am. Chem. Soc. 139, 3623–3626 (2017).
[3] a) Kevin Hwang, Peiwen Wu, Taejin Kim, Lei Lei, Shiliang Tian, Yingxiao Wang, Yi Lu, Angew. Chemie Int.l Ed. 53: 13798–13802 (2014); b) Wenjing Wang, Nitya Sai Reddy Satyavolu, Zhenkun Wu, Jian-Rong Zhang, Jun-Jie Zhu, and Yi Lu, Angew. Chemie Int. Ed. 56, 6798–6802 (2017).

Optical agents with long-lasting luminescence after removal of light excitation are promising for ultrasensitivein vivoimaging due to minimized tissue background. However, such imaging agents are rare and generally limited to inorganic nanoparticles. In this study,we introduce a new generation of purely organic agents based on semiconducting polymer nanoparticles (SPNs) that can store photon energy via chemical defects and gradually emit near-infrared (NIR) afterglow luminescence at 780 nm after light irradiation.The in vivoafterglow of SPNs is more than two orders of magnitude brighter than that of concentration-matched inorganic agents, and its signal to background ratio (SBR) is more than two orders of magnitude higher than NIR fluorescence. Such a high sensitivity couple with the ideal biodistribution allow SPNs to rapidly detect xenograft tumors with the size as small as 1 mm³and tiny peritoneal metastatic tumors that are almost invisible to naked eye. Moreover, the temperature-correlated afterglow signal of SPNs permits real-time temperature monitoring during photothermal cancer therapy in living mice. The structural versatility of SPNs enables a smart activatable afterglow probe with activated signal towards biothiols, permitting excitation-free real-time imaging of drug-induced hepatotoxicity. This study provides the basis for an entirely new class of optical reporters for molecular imaging at a sensitivity level not achievable with conventional NIR fluorescence imaging.

Supramolecular systems have attracted great scientific interests in recent few decades due to their great potential in biomedical applications. Researchers can obtain different elaborate supramolecular systems with multiple on-demand functionalities using various noncovalent interactions, such as hydrophilic hydrophobic interaction, hydrogen bonding, host-guest interaction, p-p interaction, metal coordination, etc. Although the formation or responsive process of these assemblies can be monitored by NMR, IR, MS and electron microscopy, etc., a problem associated with these methods is that they are either invisible to our naked eye.
Fluorescence is a perfect technique in the preparation and application of supramolecules, in the light of its advantages including superior sensitivity, fast responsiveness, high signal to noise ratio. More importantly, it can be applied for in situ visualization of bioanalytes at the molecular level and monitoring complex biological processes in real time. Comparing to most traditional fluorophores which suffer severe photobleaching and aggregation-caused quenching (ACQ) problems, luminogens with aggregation-induced emission (AIEgens) provide a unique light-up fluorescent signal. They are nearly non-luminescent in the isolated state but emit strongly in the aggregate/clustered state. The AIE aggregates exhibit large absorptivity, robust luminosity, strong photobleaching resistance, no random blinking and excellent biocompatibility. Based on different AIEgens, various supramolecular systems have been fabricated and present fascinating applications in biosensing, imaging and therapeutics, such as biomolecular analysis, micro-environment sensing, organelle or cellular imaging, pathogen detection and killing, as well as multimodal imaging-guided diseases therapy.

Selected references
Peng, H.-Q; Tang, B. Z. et.al. J. Am. Chem. Soc. 2017, 139, 10150–10156.
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Transition metal catalysts (TMCs) can catalyze a wide variety of chemical transformations, making them potential tools for bioorthogonal catalysis. We recently demonstrated that TMCs can be encapsulated in the surface monolayer of nanoparticles (NP), generating water-soluble “nanozymes”. These nanozymes demonstrate catalysis reminiscent of enzymes but can also catalyze a variety of bioorthogonal processes. We report a rational approach to control the localization and kinetics of nanozymes by fine-tuning their surface chemistry. We demonstrated intra-extracellular catalysis by surface engineering of the nanozymes. We used membrane-penetrating cationic nanoparticles for catalysis inside and ‘stealth’ zwitterionic particles to limit catalysis to outside of cancer cells. Specific localization of nanozyme activity was demonstrated through profluorophore activation. Therapeutic efficacy was demonstrated through intra- and extracellular activation of a prodrug. Further, we fabricated nanozymes possessing different surface hydrophobicity to regulate their interaction with a library of profluorophore substrates. We determined that nanozyme catalysis efficiency was driven by supramolecular interactions between the nanozymes and substrates. This study of nanozyme surface engineering provides the nanozymes with specific localization and tunable catalysis reminiscent of their enzyme prototypes for imaging and drug delivery.

Nanomedicines have achieved several breakthroughs in cancer treatment over the past decades, however their potential immunotoxicity are ignored, which results in serious adverse effects and greatly reduces the potential in clinical translation. Herein, we innovatively develop a theranostic supramolecular polymer using β-cyclodextrin as the host and camptothecin (CPT) as the guest linked by a glutathione-cleavable disulfide bond. The supramolecular polymerization remarkably increases the solubility of CPT by a factor of 232 and effectively inhibits its lactone ring opening in physiological environment, which is favorable for intravenous formulation and maintenance of the therapeutic efficacy. Supramolecular nanoparticles can be prepared through orthogonal self-assembly driven by π-π stacking interaction, host−guest complexation, and hydrogen bonds. The sophisticated nanomedicine constructed from the obtained supramolecular polymer can be specifically delivered to tumor sites and rapidly excreted from body after drug release, thus effectively avoiding systemic toxicity, especially long-term immunotoxicity. In vivo investigations demonstrate this supramolecular nanomedicine possesses superior anti-tumor performance and anti-metastasis capability. This pioneering example integrating the advantages of the dynamic nature of supramolecular chemistry and nanotechnology provides a promising platform for cancer theranostics.

Fluorescent upconversion nanoparticles (UCNPs) are one of the most preferred class of nanoparticles for use in near infrared (NIR) light-based theranostics. NIR light has better tissue penetration capabilities and NIR-based imaging drastically reduces the background autofluorescence, thus becoming the ideal choice in comparison to UV/visible light which has low tissue penetration capabilities and high phototoxicity. UCNPs have the unique ability of converting NIR light to a range of UV/visible/NIR wavelengths, which could be exploited for imaging and light-based therapeutic appllications. The UCNPs developed so far can only be used to perform imaging and therapy simultaneously but this is not desirable as the therapeutic process activated during imaging might cause unwanted side effects and does not offer any control over the whole process. For example, tumor imaging should be performed first to identify the location of the tumor before performing the therapeutic intervention and if the UCNPs are activated in areas other than the tumor during imaging, it would cause unwanted cell killing in normal tissues. To overcome this problem, we have designed a new UCNP-nanocluster which can be used for performing temporally separated imaging and photodynamic therapy. These nanoclusters were tested in-vitro and was shown to perform imaging and photodynamic therapy without any overlap. There was no cytotoxicity observed in cells which were used for imaging but significant cytotoxicity was observed in cells subjected to photodynamic therapy using the same nanocluster. These UCNP nanoclusters offer excellent control over theranostic applications and is foreseen to be a game-changer in this field.

Imaging-guided drug delivery has recently attracted much attention in nanomedicine where imaging probes are used to indicate the delivery and distribution of the drugs. For such an application of nanostructures, the blood circulation times and effective delivery and monitoring the drugs remain as major challenges in order to reduce toxicity and enhance drug or diagnostic efficiency. In this presentation, we present a state of art approach to forming nanoclusters by crosslinking ultrasmall iron oxide nanoparticles with bovine serum albumin. This novel design not only maintains the T1 performance of the ultrasmall nanoparticles, but also significantly increases their blood circulation times from 15 minutes to over two hours. Our breast tumor model study also exhibited enhanced localization and retention of these nanoclusters at tumor sites for more than 24 hours. The ability of maintaining the T1 performance of the ultrasmall nanoparticles is significant, because previous studies have shown complete T1 loss or signal decrease upon polymer encapsulation. This design also shows great potential in encapsulating model drug molecules, which will greatly benefit the field of imaging-guided drug delivery.

Employing the property that self-assembly of probes can enhance imaging signals, we have conducted sensitive analyses on several important biomarker-instructed self-assembly processes. 1) By rational design of a system of one enzyme (alkaline phosphatase) two substrates, for the first time, we have successfully used chemiluminescence imaging to precisely analyze the simultaneous enzyme-instructed self-assembly process. 2) Using cryo transmission electron microscopy imaging analysis, we have differentiated several nanofibers which were obtained from different biomolecule-instructed self-assemblies at angstrom scale. 3) By designing small molecular probe (or drug), we have conducted real time fluorescence imaging (or magnetic resonance imaging, etc) analyses on the intracellular enzyme-instructed self-assembling processes of nanoprobes (or drugs).

Signal transducer and activator of transcription factor 3 (STAT 3) is known to be overexpressed in cancer stem cells. Poor solubility and variable drug absorption are linked to low bioavailability and decreased efficacy. Many of the drugs regulating STAT 3 expression lack aqueous solubility; hence hindering efficient bioavailability. A theranostics nanoplatform based on luminescent carbon particles decorated with cucurbit[6]uril is introduced for enhancing the solubility of niclosamide, a STAT 3 inhibitor. The host–guest chemistry between cucurbit[6]uril and niclosamide makes the delivery of the hydrophobic drug feasible while carbon nanoparticles enhance cellular internalization. Extensive physicochemical characterizations confirm successful synthesis. Subsequently, the host–guest chemistry of niclosamide and cucurbit[6]uril is studied experimentally and computationally. In vitro assessments in human breast cancer cells indicate approximately twofold enhancement in IC₅₀ of drug. Fourier transform infrared and fluorescence imaging demonstrate efficient cellular internalization. Furthermore, the catalytic biodegradation of the nanoplatforms occur upon exposure to human myeloperoxidase in short time. In vivo studies on athymic mice with MCF 7 xenograft indicate the size of tumor in the treatment group is half of the controls after 40 d. Immunohistochemistry corroborates the downregulation of STAT 3 phosphorylation. Overall, the host–guest chemistry on nanocarbon acts as a novel arsenal for STAT 3 inhibition.