Kejin Zhou

A nanoparticle-based strategy for the imaging of a broad range of tumours by nonlinear amplification of microenvironment signals

Stimuli-responsive nanomaterials are increasingly important in a variety of applications such as biosensing, molecular imaging, drug delivery and tissue engineering. For cancer detection, a paramount challenge still exists in search of methods that can illuminate tumors universally regardless of their genotypes and phenotypes. Here we capitalized on the acidic, angiogenic tumor microenvironment to achieve broad detection of tumor tissues in a wide variety of mouse cancer models. This was accomplished using ultra-pH sensitive fluorescent nanoprobes that have tunable, exponential fluorescence activation upon encountering subtle, physiologically relevant pH transitions. These nanoprobes were silent in the circulation, then dramatically activated (>300 fold) in response to neovasculature or to the low extracellular pH in tumors. Thus, we have established non-toxic, fluorescent nanoreporters that can non-linearly amplify tumor microenvironmental signals, permitting identification of tumor tissue independently of histological type or driver mutation, and detection of acute treatment responses much more rapidly than conventional imaging approaches.

Tunable, Ultrasensitive pH-Responsive Nanoparticles Targeting Specific Endocytic Organelles in Living Cells†

In recent years, multifunctional nanoparticles have received considerable attention in many applications such as biosensors, diagnostic nanoprobes and targeted drug delivery.[1] These efforts have been driven to a large extent by the need to improve biological specificity in diagnosis and therapy through the precise, spatio-temporal control of agent delivery. In order to achieve this goal, continuous efforts have been dedicated to develop stimuli-responsive nanoplatforms.[2] Environmental stimuli that were exploited include pH,[3] temperature,[4] enzymatic expression,[5] redox reaction[6] and light induction.[7] Among these activating signals, pH trigger is one of the most extensively studied stimuli based on two types of pH differences: (a) pathological (e.g. tumor) vs. normal tissues and (b) acidic intracellular compartments.[8] For example, due to the unusual acidity of the tumor extracellular microenvironment (pHe ≈ 6.5), several pHe-responsive nanosystems were reported to increase the sensitivity of tumor imaging or the efficacy of therapy.[9]

Non‐Viral CRISPR/Cas Gene Editing In Vitro and In Vivo Enabled by Synthetic Nanoparticle Co‐Delivery of Cas9 mRNA and sgRNA

CRISPR/Cas is a revolutionary gene editing technology with wide-ranging utility. The safe, non-viral delivery of CRISPR/Cas components would greatly improve future therapeutic utility. We report the synthesis and development of zwitterionic amino lipids (ZALs) that are uniquely able to (co)deliver long RNAs including Cas9 mRNA and sgRNAs. ZAL nanoparticle (ZNP) delivery of low sgRNA doses (15 nm) reduces protein expression by >90 % in cells. In contrast to transient therapies (such as RNAi), we show that ZNP delivery of sgRNA enables permanent DNA editing with an indefinitely sustained 95 % decrease in protein expression. ZNP delivery of mRNA results in high protein expression at low doses in vitro (<600 pM) and in vivo (1 mg kg-1 ). Intravenous co-delivery of Cas9 mRNA and sgLoxP induced expression of floxed tdTomato in the liver, kidneys, and lungs of engineered mice. ZNPs provide a chemical guide for rational design of long RNA carriers, and represent a promising step towards improving the safety and utility of gene editing.

Origin of hysteresis observed in association and dissociation of polymer chains in water.

By choosing poly(N,N-diethylacrylamide) which lacks the possibility to form intra- or inter-chain hydrogen bonds, we studied its chain association and dissociation in water by using laser light scattering (LLS), ultrasensitive differential scanning calorimetry (US-DSC) and Fourier transform infrared spectroscopy (FTIR). As the solution temperature increases, the average hydrodynamic radius (R(h)) and average radius of gyration (R(g)) decrease, indicating the intrachain shrinking. When the temperature is higher than its lower critical solution temperature (LCST, approximately 30 degrees C), the apparent weight-average molar mass (M(w,app)) increases, reflecting the interchain association. At the same time, FTIR study reveals that as the temperature increases, the area ratio of two absorption peaks, respectively, associated to one hydrogen bonded carbonyl >C=O...H-O-H and free carbonyl >C=O groups increases, while that related to two hydrated hydrogen bonded carbonyl groups decreases, indicating heating-induced dehydration. In the reversible cooling process, R(h), R(g), M(w,app) and area ratios of the absorption peak are similar to those in the heating process for each given temperature, indicating that there is no hysteresis in the interchain association and dissociation. This present study confirms that the hysteresis previously observed for a sister polymer, poly(N-isopropylacrylamide), is due to the formation of some additional hydrogen bonds in its collapsed state at temperatures higher than the LCST.

Rapid Synthesis of a Lipocationic Polyester Library via Ring-Opening Polymerization of Functional Valerolactones for Efficacious siRNA Delivery

The ability to control chemical functionality is an exciting feature of modern polymer science that enables precise design of drug delivery systems. Ring-opening polymerization of functional monomers has emerged as a versatile method to prepare clinically translatable degradable polyesters.1 A variety of functional groups have been introduced into lactones; however, the direct polymerization of tertiary amine functionalized cyclic esters has remained elusive. We report a strategy that enabled the rapid synthesis of >130 lipocationic polyesters directly from functional monomers without protecting groups. These polymers are highly effective for siRNA delivery at low doses in vitro and in vivo.

Modular degradable dendrimers enable small RNAs to extend survival in an aggressive liver cancer model

Significance Liver cancer is a leading cause of death and a global health problem. Unfortunately, five small-molecule drugs for hepatocellular carcinoma (HCC) recently failed phase III clinical trials partly because late-stage liver dysfunction amplifies drug toxicity. MicroRNAs (miRNAs) present a promising alternative treatment strategy but require development of delivery vehicles that can avoid this cancer-induced dysfunction, which exacerbates toxicity. We overcame this challenge by developing dendrimer nanoparticles that mediate miRNA delivery to late-stage liver tumors with low hepatotoxicity. An aggressive, MYC-driven transgenic liver cancer model was used to examine let-7g tumor suppressor efficacy, resulting in a significant survival benefit. These dendrimer carriers provide high potency in tumors without negatively affecting normal tissues, solving a critical issue in treating aggressive liver cancer. RNA-based cancer therapies are hindered by the lack of delivery vehicles that avoid cancer-induced organ dysfunction, which exacerbates carrier toxicity. We address this issue by reporting modular degradable dendrimers that achieve the required combination of high potency to tumors and low hepatotoxicity to provide a pronounced survival benefit in an aggressive genetic cancer model. More than 1,500 dendrimers were synthesized using sequential, orthogonal reactions where ester degradability was systematically integrated with chemically diversified cores, peripheries, and generations. A lead dendrimer, 5A2-SC8, provided a broad therapeutic window: identified as potent [EC50 < 0.02 mg/kg siRNA against FVII (siFVII)] in dose–response experiments, and well tolerated in separate toxicity studies in chronically ill mice bearing MYC-driven tumors (>75 mg/kg dendrimer repeated dosing). Delivery of let-7g microRNA (miRNA) mimic inhibited tumor growth and dramatically extended survival. Efficacy stemmed from a combination of a small RNA with the dendrimer’s own negligible toxicity, therefore illuminating an underappreciated complication in treating cancer with RNA-based drugs.

Functional polyesters enable selective siRNA delivery to lung cancer over matched normal cells.

Significance Ideal cancer therapeutics accurately hit tumors and avoid side effects on healthy cells. We used a patient-derived pair of matched cancer/normal cell lines to discover selective nanoparticles that could deliver a cytotoxic siRNA to kill cancer cells and not normal cells. The finding that cells respond differently to the same nanoparticle has profound implications for gene therapy because cell-type specificity of drug carriers in vivo could alter clinical patient outcomes. Our data suggest that selectivity is an underappreciated reality that should be carefully considered when evaluating drug carriers. The combination of both well-defined molecular targets and nanoparticle delivery to targeted cells is likely required to improve cancer drug accuracy in the clinic. Conventional chemotherapeutics nonselectively kill all rapidly dividing cells, which produces numerous side effects. To address this challenge, we report the discovery of functional polyesters that are capable of delivering siRNA drugs selectively to lung cancer cells and not to normal lung cells. Selective polyplex nanoparticles (NPs) were identified by high-throughput library screening on a unique pair of matched cancer/normal cell lines obtained from a single patient. Selective NPs promoted rapid endocytosis into HCC4017 cancer cells, but were arrested at the membrane of HBEC30-KT normal cells during the initial transfection period. When injected into tumor xenografts in mice, cancer-selective NPs were retained in tumors for over 1 wk, whereas nonselective NPs were cleared within hours. This translated to improved siRNA-mediated cancer cell apoptosis and significant suppression of tumor growth. Selective NPs were also able to mediate gene silencing in xenograft and orthotopic tumors via i.v. injection or aerosol inhalation, respectively. Importantly, this work highlights that different cells respond differentially to the same drug carrier, an important factor that should be considered in the design and evaluation of all NP carriers. Because no targeting ligands are required, these functional polyester NPs provide an exciting alternative approach for selective drug delivery to tumor cells that may improve efficacy and reduce adverse side effects of cancer therapies.

Precise let-7 expression levels balance organ regeneration against tumor suppression

The in vivo roles for even the most intensely studied microRNAs remain poorly defined. Here, analysis of mouse models revealed that let-7, a large and ancient microRNA family, performs tumor suppressive roles at the expense of regeneration. Too little or too much let-7 resulted in compromised protection against cancer or tissue damage, respectively. Modest let-7 overexpression abrogated MYC-driven liver cancer by antagonizing multiple let-7 sensitive oncogenes. However, the same level of overexpression blocked liver regeneration, while let-7 deletion enhanced it, demonstrating that distinct let-7 levels can mediate desirable phenotypes. let-7 dependent regeneration phenotypes resulted from influences on the insulin-PI3K-mTOR pathway. We found that chronic high-dose let-7 overexpression caused liver damage and degeneration, paradoxically leading to tumorigenesis. These dose-dependent roles for let-7 in tissue repair and tumorigenesis rationalize the tight regulation of this microRNA in development, and have important implications for let-7 based therapeutics. DOI: http://dx.doi.org/10.7554/eLife.09431.001

Chaotropic-anion-induced supramolecular self-assembly of ionic polymeric micelles.

Traditional micelle self-assembly is driven by the association of hydrophobic segments of amphiphilic molecules forming distinctive core-shell nanostructures in water. Here we report a surprising chaotropic-anion-induced micellization of cationic ammonium-containing block copolymers. The resulting micelle nanoparticle consists of a large number of ion pairs (≈60,000) in each hydrophobic core. Unlike chaotropic anions (e.g. ClO4(-)), kosmotropic anions (e.g. SO4(2-)) were not able to induce micelle formation. A positive cooperativity was observed during micellization, for which only a three-fold increase in ClO4(-) concentration was necessary for micelle formation, similar to our previously reported ultra-pH-responsive behavior. This unique ion-pair-containing micelle provides a useful model system to study the complex interplay of noncovalent interactions (e.g. electrostatic, van der Waals, and hydrophobic forces) during micelle self-assembly.

A comparative study of urea-induced aggregation of collapsed poly(N-isopropylacrylamide) and poly(N,N-diethylacrylamide) chains in aqueous solutions.

The urea-induced aggregation of poly(N-isopropylacrylamide) (PNIPAM) and poly(N,N-diethylacrylamide) (PDEAM) globules was studied by using a combination of static and dynamic light scattering. Our results have revealed that urea acting as a cross-linker via formation of two hydrogen bonds with the amide groups of PNIPAM and PDEAM in different globules causes the aggregation, and the aggregation of PNIPAM and PDEAM globules is a reaction-limited cluster-cluster aggregation (RLCA) process. The aggregates have a uniform sphere structure that may be due to the restructuring of the aggregates. The aggregation rate of PNIPAM globules is slower than that of PDEAM, which might mainly contribute to the reasons that the amides groups of PNIPAM have more chance to be inside the globules because of the formation of intra- and inter-hydrogen bonds and the smaller number density of the PNIPAM aggregates at the original time. When the aqueous urea solutions were cooled and reheated to 40 °C, the aggregation became faster than the first heating process, indicating that the urea molecules have replaced some water molecules binding to the amide groups at high temperature and some of the urea molecules remain interacting with the polymers even at the temperature lower than the cloud point temperature.