Effective delivery and selective insecticidal activity of double-stranded RNA via complexation with diblock copolymer varies with polymer block composition.

BACKGROUND: Chemical insecticides are an important tool to control damaging pest infestations. However, lack of species specificity, the rise of resistance and the demand for biological alternatives with improved ecotoxicity profiles means that chemicals with new modes of action are required. RNA interference (RNAi)-based strategies using double-stranded RNA (dsRNA) as a species-specific bio-insecticide offer an exquisite solution that addresses these issues. Many species, such as the fruit pest Drosophila suzukii, do not exhibit RNAi when dsRNA is orally administered due to degradation by gut nucleases and slow cellular uptake pathways. Thus, delivery vehicles that protect and deliver dsRNA are highly desirable. RESULTS: In this work, we demonstrate the complexation of D. suzukii-specific dsRNA for degradation of vha26 mRNA with bespoke diblock copolymers. We study the ex vivo protection of dsRNA against enzymatic degradation by gut enzymes, which demonstrates the efficiency of this system. Flow cytometry then investigates the cellular uptake of Cy3-labelled dsRNA, showing a 10-fold increase in the mean fluorescence intensity of cells treated with polyplexes. The polymer/dsRNA polyplexes induced a significant 87% decrease in the odds of survival of D. suzukii larvae following oral feeding only when formed with a diblock copolymer containing a long neutral block length (1:2 cationic block/neutral block). However, there was no toxicity when fed to the closely related Drosophila melanogaster. CONCLUSION: We provide evidence that dsRNA complexation with diblock copolymers is a promising strategy for RNAi-based species-specific pest control, but optimisation of polymer composition is essential for RNAi success. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Driving Diversity and Inclusion in Cancer Drug Development - Industry and Regulatory Perspectives, Current Practices, Opportunities, and Challenges.

In April 2022, the FDA issued draft guidance to help industry develop strategies to improve diversity in clinical trials. Historically, clinical trial sponsors have not systematically incorporated efforts to promote diversity, equity, and inclusion (DEI), particularly during the early design stages of clinical development plans and operational strategies. Unfortunately, a retrospective approach to DEI often results in clinical trial participants not being reflective of the diversity of patients intended to be treated with new therapies. A shift to prospective, intentional DEI strategies for clinical trials, including long-term engagement with diverse patients and communities throughout the development life cycle, is necessary to maximize the benefits and minimize the risks of new drugs and devices for all patients. Sponsors' current practices and opportunities for improving DEI address four major topics: institutional commitment, culture change, and governance; clinical development strategy; setting enrollment goals to ensure trial participant diversity; and development and implementation of the operational strategy. As DEI practices gain wider adoption in clinical trials, shared learning and collaboration among stakeholders on an ongoing and noncompetitive basis will lead to sustainable change. Prioritization of enrollment of diverse populations as an integral part of study start-up planning, clinical trial design, and recruitment capabilities will enhance the clinical development process for oncology therapies. Importantly, these efforts will help provide equitable access to clinical trials and innovative cancer therapies.

Electronic Nicotine Delivery Systems: An Updated Policy Statement From the American Association for Cancer Research and the American Society of Clinical Oncology.

Combustible tobacco use has reached historic lows, demonstrating the importance of proven strategies to reduce smoking since publication of the 1964 Surgeon General's report. In contrast, the use of electronic nicotine delivery systems (ENDS), specifically e-cigarettes, has grown to alarming rates and threatens to hinder progress against tobacco use. A major concern is ENDS use by youth and adults who never previously used tobacco. While ENDS emit fewer carcinogens than combustible tobacco, preliminary evidence links ENDS use to DNA damage and inflammation, key steps in cancer development. Furthermore, high levels of nicotine can also increase addiction, raise blood pressure, interfere with brain development, and suppress the immune system. The magnitude of long-term health risks will remain unknown until longitudinal studies are completed. ENDS have been billed as a promising tool for combustible tobacco cessation, but further evidence is needed to assess their potential efficacy for adults who smoke. Of concern, epidemiological studies estimate that approximately 15%-42% of adults who use ENDS have never used another tobacco product, and another 36%-54% dual use both ENDS and combustible tobacco. This policy statement details advances in science related to ENDS and calls for urgent action to end predatory practices of the tobacco industry and protect public health. Importantly, we call for an immediate ban on all non-tobacco-flavored ENDS products that contain natural or synthetic nicotine to reduce ENDS use by youth and adults who never previously used tobacco. Concurrently, evidence-based treatments to promote smoking cessation and prevent smoking relapse to reduce cancer incidence and improve public health remain top priorities for our organizations. We also recognize there is an urgent need for research to understand the relationship between ENDS and tobacco-related disparities.

Electronic Nicotine Delivery Systems: An Updated Policy Statement from the American Association for Cancer Research and the American Society of Clinical Oncology.

Combustible tobacco use has reached historic lows, demonstrating the importance of proven strategies to reduce smoking since publication of the 1964 Surgeon General's report. In contrast, the use of electronic nicotine delivery systems (ENDS), specifically e-cigarettes, has grown to alarming rates and threatens to hinder progress against tobacco use. A major concern is ENDS use by youth and adults who never previously used tobacco. While ENDS emit fewer carcinogens than combustible tobacco, preliminary evidence links ENDS use to DNA damage and inflammation, key steps in cancer development. Furthermore, high levels of nicotine can also increase addiction, raise blood pressure, interfere with brain development, and suppress the immune system. The magnitude of long-term health risks will remain unknown until longitudinal studies are completed. ENDS have been billed as a promising tool for combustible tobacco cessation, but further evidence is needed to assess their potential efficacy for adults who smoke. Of concern, epidemiological studies estimate that approximately 15% to 42% of adults who use ENDS have never used another tobacco product, and another 36% to 54% "dual use" both ENDS and combustible tobacco. This policy statement details advances in science related to ENDS and calls for urgent action to end predatory practices of the tobacco industry and protect public health. Importantly, we call for an immediate ban on all non-tobacco-flavored ENDS products that contain natural or synthetic nicotine to reduce ENDS use by youth and adults who never previously used tobacco. Concurrently, evidence-based treatments to promote smoking cessation and prevent smoking relapse to reduce cancer incidence and improve public health remain top priorities for our organizations. We also recognize there is an urgent need for research to understand the relationship between ENDS and tobacco-related disparities.

Protection of Double-Stranded RNA via Complexation with Double Hydrophilic Block Copolymers: Influence of Neutral Block Length in Biologically Relevant Environments.

Interaction between the anionic phosphodiester backbone of DNA/RNA and polycations can be exploited as a means of delivering genetic material for therapeutic and agrochemical applications. In this work, quaternized poly(2-(dimethylamino)ethyl methacrylate)-block-poly(N,N-dimethylacrylamide) (PQDMAEMA-b-PDMAm) double hydrophilic block copolymers (DHBCs) were synthesized via reversible addition-fragmentation chain-transfer (RAFT) polymerization as nonviral delivery vehicles for double-stranded RNA. The assembly of DHBCs and dsRNA forms distinct polyplexes that were thoroughly characterized to establish a relationship between the length of the uncharged poly(N,N-dimethylacrylamide) (PDMA) block and the polyplex size, complexation efficiency, and colloidal stability. Dynamic light scattering reveals the formation of smaller polyplexes with increasing PDMA lengths, while gel electrophoresis confirms that these polyplexes require higher N/P ratio for full complexation. DHBC polyplexes exhibit enhanced stability in low ionic strength environments in comparison to homopolymer-based polyplexes. In vitro enzymatic degradation assays demonstrate that both homopolymer and DHBC polymers efficiently protect dsRNA from degradation by RNase A enzyme.

A worm gel-based 3D model to elucidate the paracrine interaction between multiple myeloma and mesenchymal stem cells.

Multiple myeloma (MM) is a malignancy of terminally-differentiated plasma cells that develops mainly inside the bone marrow (BM) microenvironment. It is well known that autocrine and paracrine signals are responsible for the progression of this disease but the precise mechanism and contributions from single cell remain largely unknown. Mesenchymal stem cells (MSC) are an important cellular component of the BM: they support MM growth by increasing its survival and chemo-resistance, but little is known about the paracrine signaling pathways. Three-dimensional (3D) models of MM-MSC paracrine interactions are much more biologically-relevant than simple 2D models and are considered essential for detailed studies of MM pathogenesis. Herein we present a novel 3D co-culture model designed to mimic the paracrine interaction between MSC and MM cells. MSC were embedded within a previously characterized thermoresponsive block copolymer worm gel that can induce stasis in human pluripotent stem cells (hPSC) and then co-cultured with MM cells. Transcriptional phenotyping of co-cultured cells indicated the dysregulation of genes that code for known disease-relevant factors, and also revealed IL-6 and IL-10 as upstream regulators. Importantly, we have identified a synergistic paracrine signaling pathway between IL-6 and IL-10 that plays a critical role in sustaining MM cell proliferation. Our findings indicate that this 3D co-culture system is a useful model to investigate the paracrine interaction between MM cells and the BM microenvironment in vitro. This approach has revealed a new mechanism that promotes the proliferation of MM cells and suggested a new therapeutic target.

Comparison of the different mechanisms of cytotoxicity induced by checkpoint kinase I inhibitors when used as single agents or in combination with DNA damage.

Inhibition of the DNA damage response is an emerging strategy to treat cancer. Understanding how DNA damage response inhibitors cause cytotoxicity in cancer cells is crucial to their further clinical development. This review focuses on three different mechanisms of cell killing by checkpoint kinase I inhibitors (CHK1i). DNA damage induced by chemotherapy drugs, such as topoisomerase I inhibitors, results in S and G2 phase arrest. Addition of CHK1i promotes cell cycle progression before repair is completed resulting in mitotic catastrophe. Ribonucleotide reductase inhibitors such as gemcitabine also arrest cells in S phase by preventing dNTP synthesis. Addition of CHK1i re-activates the DNA helicase to unwind DNA, but in the absence of dNTPs, this leads to excessive single-strand DNA that exceeds the protective capacity of the single-strand-binding protein RPA. Unprotected DNA is subjected to nuclease cleavage, resulting in replication catastrophe. CHK1i alone also kills a subset of cell lines through MRE11 and MUS81-mediated DNA cleavage in S phase cells. The choice of mechanism depends on the activation state of CDK2. Low level activation of CDK2 mediates helicase activation, cell cycle progression, and both replication and mitotic catastrophe. In contrast, high CDK2 activity is required for sensitivity to CHK1i as monotherapy. This high CDK2 activity threshold usually occurs late in the cell cycle to prepare for mitosis, but in CHK1i-sensitive cells, high activity can be attained in early S phase, resulting in DNA cleavage and cell death. This sensitivity to CHK1i has previously been associated with endogenous replication stress, but the dependence on high CDK2 activity, as well as MRE11, contradicts this hypothesis. The major unresolved question is why some cell lines fail to restrain their high CDK2 activity and hence succumb to CHK1i in S phase. Resolving this question will facilitate stratification of patients for treatment with CHK1i as monotherapy.

Inhibition of checkpoint kinase 1 following gemcitabine-mediated S phase arrest results in CDC7- and CDK2-dependent replication catastrophe.

Combining DNA-damaging drugs with DNA checkpoint inhibitors is an emerging strategy to manage cancer. Checkpoint kinase 1 inhibitors (CHK1is) sensitize most cancer cell lines to DNA-damaging drugs and also elicit single-agent cytotoxicity in 15% of cell lines. Consequently, combination therapy may be effective in a broader patient population. Here, we characterized the molecular mechanism of sensitization to gemcitabine by the CHK1i MK8776. Brief gemcitabine incubation irreversibly inhibited ribonucleotide reductase, depleting dNTPs, resulting in durable S phase arrest. Addition of CHK1i 18 h after gemcitabine elicited cell division cycle 7 (CDC7)- and cyclin-dependent kinase 2 (CDK2)-dependent reactivation of the replicative helicase, but did not reinitiate DNA synthesis due to continued lack of dNTPs. Helicase reactivation generated extensive single-strand (ss)DNA that exceeded the protective capacity of the ssDNA-binding protein, replication protein A. The subsequent cleavage of unprotected ssDNA has been termed replication catastrophe. This mechanism did not occur with concurrent CHK1i plus gemcitabine treatment, providing support for delayed administration of CHK1i in patients. Alternative mechanisms of CHK1i-mediated sensitization to gemcitabine have been proposed, but their role was ruled out; these mechanisms include premature mitosis, inhibition of homologous recombination, and activation of double-strand break repair nuclease (MRE11). In contrast, single-agent activity of CHK1i was MRE11-dependent and was prevented by lower concentrations of a CDK2 inhibitor. Hence, both pathways require CDK2 but appear to depend on different CDK2 substrates. We conclude that a small-molecule inhibitor of CHK1 can elicit at least two distinct, context-dependent mechanisms of cytotoxicity in cancer cells.

Differential Sensitivity to CDK2 Inhibition Discriminates the Molecular Mechanisms of CHK1 Inhibitors as Monotherapy or in Combination with the Topoisomerase I Inhibitor SN38.

DNA damage activates checkpoints to arrest cell cycle progression in S and G2 phases, thereby providing time for repair and recovery. The combination of DNA-damaging agents and inhibitors of CHK1 (CHK1i) is an emerging strategy for sensitizing cancer cells. CHK1i induce replication on damaged DNA and mitosis before repair is complete, and this occurs in a majority of cell lines. However, ∼15% of cancer cell lines are hypersensitive to single-agent CHK1i. As both abrogation of S phase arrest and single-agent activity depend on CDK2, this study resolved how activation of CDK2 can be essential for both replication and cytotoxicity. S phase arrest was induced with the topoisomerase I inhibitor SN38; the addition of CHK1i rapidly activated CDK2, inducing S phase progression that was inhibited by the CDK2 inhibitor CVT-313. In contrast, DNA damage and cytotoxicity induced by single-agent CHK1i in hypersensitive cell lines were also inhibited by CVT-313 but at 20-fold lower concentrations. The differential sensitivity to CVT-313 is explained by different activity thresholds required for phosphorylation of CDK2 substrates. While the critical CDK2 substrates are not yet defined, we conclude that hypersensitivity to single-agent CHK1i depends on phosphorylation of substrates that require high CDK2 activity levels. Surprisingly, CHK1i did not increase SN38-mediated cytotoxicity. In contrast, while inhibition of WEE1 also abrogated S phase arrest, it more directly activated CDK1, induced premature mitosis, and enhanced cytotoxicity. Hence, while high activity of CDK2 is critical for cytotoxicity of single-agent CHK1i, CDK1 is additionally required for sensitivity to the drug combination.

Disulfide-Based Diblock Copolymer Worm Gels: A Wholly-Synthetic Thermoreversible 3D Matrix for Sheet-Based Cultures.

It is well-known that 3D in vitro cell cultures provide a much better model than 2D cell cultures for understanding the in vivo microenvironment of cells. However, significant technical challenges in handling and analyzing 3D cell cultures remain, which currently limits their widespread application. Herein, we demonstrate the application of wholly synthetic thermoresponsive block copolymer worms in sheet-based 3D cell culture. These worms form a soft, free-standing gel reversibly at 20-37 °C, which can be rapidly converted into a free-flowing dispersion of spheres on cooling to 5 °C. Functionalization of the worms with disulfide groups was found to be essential for ensuring sufficient mechanical stability of these hydrogels to enable long-term cell culture. These disulfide groups are conveniently introduced via statistical copolymerization of a disulfide-based dimethacrylate under conditions that favor intramolecular cyclization and subsequent thiol/disulfide exchange leads to the formation of reversible covalent bonds between adjacent worms within the gel. This new approach enables cells to be embedded within micrometer-thick slabs of gel with good viability, permits cell culture for at least 12 days, and facilitates recovery of viable cells from the gel simply by incubating the culture in buffer at 4 °C (thus, avoiding the enzymatic degradation required for cell harvesting when using commercial protein-based gels, such as Matrigel).

RAFT aqueous dispersion polymerization yields poly(ethylene glycol)-based diblock copolymer nano-objects with predictable single phase morphologies.

A poly(ethylene glycol) (PEG) macromolecular chain transfer agent (macro-CTA) is prepared in high yield (>95%) with 97% dithiobenzoate chain-end functionality in a three-step synthesis starting from a monohydroxy PEG113 precursor. This PEG113-dithiobenzoate is then used for the reversible addition-fragmentation chain transfer (RAFT) aqueous dispersion polymerization of 2-hydroxypropyl methacrylate (HPMA). Polymerizations conducted under optimized conditions at 50 °C led to high conversions as judged by (1)H NMR spectroscopy and relatively low diblock copolymer polydispersities (M(w)/M(n) < 1.25) as judged by GPC. The latter technique also indicated good blocking efficiencies, since there was minimal PEG113 macro-CTA contamination. Systematic variation of the mean degree of polymerization of the core-forming PHPMA block allowed PEG113-PHPMA(x) diblock copolymer spheres, worms, or vesicles to be prepared at up to 17.5% w/w solids, as judged by dynamic light scattering and transmission electron microscopy studies. Small-angle X-ray scattering (SAXS) analysis revealed that more exotic oligolamellar vesicles were observed at 20% w/w solids when targeting highly asymmetric diblock compositions. Detailed analysis of SAXS curves indicated that the mean number of membranes per oligolamellar vesicle is approximately three. A PEG113-PHPMA(x) phase diagram was constructed to enable the reproducible targeting of pure phases, as opposed to mixed morphologies (e.g., spheres plus worms or worms plus vesicles). This new RAFT PISA formulation is expected to be important for the rational and efficient synthesis of a wide range of biocompatible, thermo-responsive PEGylated diblock copolymer nano-objects for various biomedical applications.

Nile Blue-based nanosized pH sensors for simultaneous far-red and near-infrared live bioimaging.

Diblock copolymer vesicles are tagged with pH-responsive Nile Blue-based labels and used as a new type of pH-responsive colorimetric/fluorescent biosensor for far-red and near-infrared imaging of live cells. The diblock copolymer vesicles described herein are based on poly(2-(methacryloyloxy)ethyl phosphorylcholine-block-2-(diisopropylamino)ethyl methacrylate) [PMPC-PDPA]: the biomimetic PMPC block is known to facilitate rapid cell uptake for a wide range of cell lines, while the PDPA block constitutes the pH-responsive component that enables facile vesicle self-assembly in aqueous solution. These biocompatible vesicles can be utilized to detect interstitial hypoxic/acidic regions in a tumor model via a pH-dependent colorimetric shift. In addition, they are also useful for selective intracellular staining of lysosomes and early endosomes via subtle changes in fluorescence emission. Such nanoparticles combine efficient cellular uptake with a pH-responsive Nile Blue dye label to produce a highly versatile dual capability probe. This is in marked contrast to small molecule dyes, which are usually poorly uptaken by cells, frequently exhibit cytotoxicity, and are characterized by intracellular distributions invariably dictated by their hydrophilic/hydrophobic balance.