Efficient Targeting Research Articles

Improving Energy Efficiency in a Building Using Passive Energy-Saving Measures

Abstract We are going through a time when environmental, economic and social concerns, intensely amplified by climate change, by the depletion of resources, are growing. In this context, setting targets such as reducing energy consumption in the buildings sector, energy efficiency, and the use of renewable energies are becoming a priority, both at national and international level. Improving the energy efficiency of the existing building stock is important, not only for achieving national medium-term energy efficiency targets, but also for meeting the long-term objectives of the strategy on climate change and the transition to a competitive low-carbon economy by 2050 (https://energy.ec.europa.eu/system/files/2014-11/2014_article4_ro_romania_0.pdf). Currently, the building is considered as a structure in a continuous evolution, which in time through maintenance, rehabilitation and modernization works will meet the requirements expressed by the user. The case study presented in the paper presents an analysis from the point of view of improving the energy efficiency, reducing the energy requirement in a building using passive energy-saving measures, that is, the additional thermal insulation of an element of the building envelope. The materials selected for this research are: expanded polystyrene, basaltic mineral wool and cellulose fibres.

Discovery of Highly Efficient Novel Antifungal Lead Compounds Targeting Succinate Dehydrogenase: Pyrazole-4-carboxamide Derivatives with an N-Phenyl Substituted Amide Fragment.

Developing environmentally friendly fungicides is crucial to tackle the issue of rising pesticide resistance. In this study, a series of novel pyrazole-4-carboxamide derivatives containing N-phenyl substituted amide fragments were designed and synthesized. The structures of target compounds were confirmed by 1H NMR, 13C NMR, and HRMS, and the crystal structure of the most active compound N-(1-(4-(4-(tert-butyl)benzamido)phenyl)propan-2-yl)-3-(difluoromethyl)-N-methoxy-1-methyl-1H-pyrazole-4-carboxamide (U22) was further determined by X-ray single-crystal diffraction. The bioassay results indicated that the 26 target compounds possessed good in vitro antifungal activity against Sclerotinia sclerotiorum with EC50 values for compounds U12, U13, U15, U16, U18, U22, and U23 being 4.17 ± 0.46, 8.04 ± 0.71, 7.01 ± 0.71, 12.77 ± 1.00, 8.11 ± 0.70, 0.94 ± 0.11, and 9.48 ± 0.83 μg·mL-1, respectively, which were the similar to controls bixafen (6.70 ± 0.47 μg·mL-1), fluxapyroxad (0.71 ± 0.14 μg·mL-1), and pydiflumetofen (0.06 ± 0.01 μg·mL-1). Furthermore, in vivo antifungal activity results against S. sclerotiorum indicated that compounds U12 (80.6%) and U22 (89.9%) possessed excellent preventative efficacy at 200 μg·mL-1, which was the same as the control pydiflumetofen (82.4%). Scanning electron microscopy and transmission electron microscopy studies found that the compound U22 could destroy the hyphal morphology and damage mitochondria, cell membranes, and vacuoles. The results of molecular docking of compound U22 and pydiflumetofen with succinate dehydrogenase (SDH) indicated they interact well with the active site of SDH. This study validated our approach and design strategy to produce compounds with an enhanced biological activity as compared to the parent structure.

Engineered exosomes mediated targeted delivery of neuroprotective peptide NR2B9c for the treatment of traumatic brain injury

Neuroprotection is one of the core treatment strategies for brain injuries including traumatic brain injury (TBI). NR2B9c is a promising neuroprotective peptide but its clinical translation is limited because of poor brain penetrability. Exosomes are naturally occurring nanovesicles having therapeutic potential for TBI as well as an efficient drug delivery carrier to the brain. Here, we engineered exosomes with neuron targeting peptide rabies virus glycoprotein (RVG29) via bio-orthogonal click chemistry technique and loaded it with NR2B9c, developing RVG-ExoNR2B9c. RVG29 conjugated exosome had higher neuron targeting efficiency compared to naïve exosomes both in vivo and in vitro. RVG-ExoNR2B9c had great cytoprotective effect against oxygen glucose deprived Neuro2a cells. Intravenous administration of RVG-ExoNR2B9c significantly improved behavioral outcomes and reduced the lesion volume after TBI injury in a mice controlled cortical impact model. Due to their multifunctionality and significant efficacy, we anticipate that RVG-ExoNR2B9c have the potential to be translated both as therapeutic agent as well as cargo delivery system to the brain for the treatment of TBI.

Efficient Combinatorial Adaptor-Mediated Targeting of Acute Myeloid Leukemia with CAR T-Cells

Introduction: Chimeric Antigen Receptor (CAR) T-cell therapies are remarkably efficient in treating B- and plasma-cell malignancies by targeting cell-of-origin antigens, eradicating both tumor cells as well as healthy B- and plasma-cell counterparts. Their loss can be compensated by immunoglobulin substitution until they regenerate from hematopoietic stem and progenitor cells (HSPCs). However, similar on-target, off-tumor cell-of-origin elimination would be detrimental in the context of HSPC-derived malignancies, such as acute myeloid leukemia (AML) or myelodysplastic neoplasia (MDS), as the CAR T-cell activity might lead to terminal ablation of hematopoiesis. In principle, CAR T-cells could be used to abrogate both, AML cells as well as HSPCs, followed by HSPC transplantation, ensuring hematopoietic recovery after termination of CAR T-cell activity and possibly without genotoxic preconditioning. This approach could be particularly beneficial for patients unable to tolerate radiation and high-dose chemotherapy. In order to address this challenge, we developed adaptor-mediated CAR T-cells displaying the single-chain variable fragment E2, AdFITC(E2), that binds to fluorescein conjugated to antigen-binding adaptors in diabody format (Db-FM). Such tagged adaptors, used as single linkers or in combination, would then act as safety switch, modulating CAR T-cell activation, proliferation, and lytic activity. Results: We generated second-generation AdFITC(E2)-CAR T-cells and multiple diabody-based adaptors against selected AML antigens. We then tested the diabodies targeting CD33 and CD117 with respect to their ability to mediate CAR T-cell biocidal activity as single adaptors or in combination. Combinatorial staining with CD117 Db-FM and CD33 Db-FM, each added at saturating concentration for the cognate antigen, resulted in higher fluorescein decoration of target cells, with more than two-fold increased fluorescence intensity detected by flow cytometry upon combined staining, thus enhancing AdFITC(E2)-CAR T-cell target density. In vitro cytotoxicity assays of healthy-donor-derived AdFITC(E2)-CAR T-cells, both against cell lines (MOLM14-CD117 +GFP +Luc +) as well as patient-derived AML blasts, revealed that dual adaptor use significantly improved tumor cell lysis compared to equimolar concentration of single adaptors. To further investigate the basis for increased killing by dual targeting, we performed live-cell imaging of effector-target cell interactions in microwells. The addition of both CD117 and CD33 Db-FM to the co-culture of MOLM14-CD117 +GFP +Luc + and AdFITC(E2)-CAR T-cells resulted in faster tumor cell lysis (measured by influx of PI) which correlated with a more rapid, durable engagement time of CAR T-cells with target cells. We next tested CAR T-cell biocidal activity in therapeutic xenogeneic mouse models engrafted with MOLM14-CD117 +GFP +Luc + where AdFITC(E2)-CAR T-cells in combination with CD117 or CD33 Db-FM administered as single adaptors were as efficient as direct CAR T-cells against the same antigens. Injection of AdFITC(E2)-CAR T-cells and both CD117 and CD33 Db-FM effectively inhibited tumor growth, outperforming monotherapies, and leading in some cases to full AML elimination in bone marrow and blood as shown by bioluminescence and terminal flow cytometry analysis (Figure 1). Conclusions: We here tested an adaptor CAR T-cell approach using multiple adaptors to on-off modulate and enhance CAR T-cell activity, focusing on AML as a relevant disease model with high clinical need for therapeutic improvement. The high heterogeneity in antigen expression on AML cells supports a combinatorial targeting strategy, individualized based on the respective AML immunophenotype. Due to their relatively small molecular weight (~55kDa), diabody-based adaptors are rapidly cleared from the body via the kidneys allowing for rapid control over AdFITC-CAR T-cell on-off activity prior to HSPC transplantation. We envision that this approach has the clinical potential to improve CAR T-cell safety profiles by enhancing the specificity towards target cells, while ameliorating the side-effects against healthy tissues expressing only one antigen of the combination.

Promising therapeutic targets for ischemic stroke identified from plasma and cerebrospinal fluid proteomes: a multicenter Mendelian randomization study.

Ischemic stroke (IS) is more common every year, the condition is serious, and have a poor prognosis. New, efficient, and safe therapeutic targets are desperately needed as early treatment especially prevention and reperfusion is the key to lowering the occurrence of poorer prognosis. Generally circulating proteins are attractive therapeutic targets, this study aims to identify potential pharmacological targets among plasma and cerebrospinal fluid (CSF) proteins for the prevention and treatment of IS using a multicenter Mendelian randomization (MR) approach. First, the genetic instruments of 734 plasma and 151 CSF proteins were assessed for causative connections with IS from MEGASTROKE consortium by MR to identify prospective therapeutic targets. Then, for additional validation, plasma proteins from the deCODE consortium and the Fenland consortium, as well as IS GWAS data from the FinnGen cohort, the ISGC consortium and UK biobank, were employed. A thorough evaluation of the aforementioned possible pharmacological targets was carried out using meta-analysis. The robustness of MR results was then confirmed through sensitivity analysis using several techniques, such as bidirectional MR analysis, Steiger filtering, and Bayesian colocalization. Finally, methods like Protein-Protein Interaction (PPI) Networking were utilized to investigate the relationship between putative drug targets and therapeutic agents. The authors discovered three proteins that may function as promising therapeutic targets for IS and meet the Bonferroni correction ( P <0.05/885=5.65×10 -5 ). Prekallikrein (OR=0.41, 95% CI: 0.27-0.63, P =3.61×10 -5 ), a protein found in CSF, has a 10-fold protective impact in IS, while the plasma proteins SWAP70 (OR=0.85, 95% CI: 0.80-0.91, P =1.64×10 -6 ) and MMP-12 (OR=0.92, 95% CI: 0.89-0.95, P =4.49×10 -6 ) of each SD play a protective role in IS. Prekallikrein, MMP-12, SWAP70 was replicated in the FinnGen cohort and ISGC database. MMP-12 (OR=0.93, 95% CI: 0.91-0.94, P <0.001), SWAP70 (OR=0.92, 95% CI: 0.90-0.94, P <0.001), and prekallikrein (OR=0.53, 95% CI: 0.33-0.72, P <0.001) may all be viable targets for IS, according to the combined meta-analysis results. Additionally, no evidence of reverse causality was identified, and Bayesian colocalization revealed MMP-12 (PPH 4 =0.995), SWAP70 (PPH 4 =0.987), and prekallikrein (PPH 4 =0.894) shared the same variant with IS, supporting the robustness of the aforementioned causation. Prekallikrein and MMP-12 were associated with the target protein of the current treatment of IS. Among them, Lanadelumab, a new drug whose target protein is a prekallikrein, may be a promising new drug for the treatment of IS. The prekallikrein, MMP-12, and SWAP70 are causally associated with the risk of IS. Moreover, MMP-12 and prekallikrein may be treated as promising therapeutic targets for medical intervention of IS.

Development and evaluation of redox-responsive alginate–SS–ibuprofen derivative based anti-tumor target drug delivery system for the controlled release of doxorubicin

Nowadays, self-assembled polymeric micelles from amphiphilic polymer conjugates are urgently needed to enhance drug targeting and release efficiency. On account of the excellent properties of alginate and the redox-responsiveness of disulfide bonds, a redox-sensitive alginate–SS–ibuprofen derivative (LSA–SS–IBU) based anti-tumor target drug delivery system was created by attaching the hydrophobic ibuprofen (IBU) onto the hydrophilic alginate molecular skeleton through disulfide linkages for glutathione (GSH) triggered delivery of doxorubicin hydrochloride (DOX). The structure and self-assembly behavior of the synthesized LSA–SS–IBU was systematically evaluated by FT-IR spectrometer, 1H NMR spectrometer, fluorescence spectrophotometer (FM), transmission electron microscope (TEM) and dynamic light scattering (DLS). Furthermore, the in vitro release of drugs from the LSA–SS–IBU micelles loaded with DOX was examined, along with the assessment of their cytotoxicity to HeLa cells. The resultant LSA–SS–IBU had a critical micelle concentration (CMC) value of 0.19 mg/mL that was able to self-assemble into the spherical micelles with the particle size and zeta potential of 148.2 ± 4.3 nm (PDI = 0.25) and −47.2 ± 0.4 mV, respectively. At a mass ratio of 3:10 for DOX/LSA–SS–IBU, the LSA–SS–IBU micelles achieved drug loading ratio (DLR) and encapsulation efficiency (EE) values of 12.2 % and 46.3 %, respectively. Furthermore, the in vitro drug release study indicated that the total drug release percentage of the drug-encapsulated LSA–SS–IBU micelles in PBS with the presence of 10 mmol/L GSH for 12 h exceeded 67.0 %, demonstrating a substantial increase compared to that in GSH-free PBS. The in vitro cytotoxicity findings indicated that LSA–SS–IBU had good biocompatibility, while the IC50 value and the cell viability of DOX-loaded LSA–SS–IBU micelles cultured with HeLa cells for 48 h was 2.2 μg/mL and less than 20.1 %, respectively, which could significantly inhibit the growth of HeLa cells. Therefore, redox-responsive LSA–SS–IBU micelles had potential applications in cancer therapy for hydrophobic anti-tumor drug delivery.

An efficient targeted gene deletion approach for Cochliobolus heterostrophus using Agrobacterium tumefaciens-mediated transformation

Cochliobolus heterostrophus is a plant pathogenic fungus of southern corn leaf blight, which has been regarded as a model necrotrophic plant pathogen. Many methods have been developed to knock out targeted genes in C. heterostrophus, of which the most widely-used one is protoplast-mediated transformation. However, there are several problems of this method associated with protoplast preparation, DNA product, time consumption, or high cost. In this study, a highly efficient target gene deletion approach in C. heterostrophus was established and optimized, based on Agrobacterium tumefaciens-mediated transformation (ATMT); the transformation efficiency of this approach was 85–88 transformants per 105 conidia, and the homologous recombination efficiency was approximately 68.3%. Furthermore, six gene knockout mutants of C. heterostrophus were obtained using this ATMT method. The phenotypes of this fungus altered in the mutant strains, and the virulence of the mutants significantly reduced compared to of the wild type strain. Taken together, this ATMT system established in this study can be used as a genetic manipulation tool for C. heterostrophus, to better understand the functions of genes and its relation to virulence.

Multi-dimensional role of gangliosides in modulating cancer hallmarks and their prospects in targeted cancer therapy

Gangliosides are glycosphingolipids with prevalence in nervous tissue and their involvement in certain neuronal diseases have been widely known. Interestingly, many recent studies highlighted their importance in the development and progression of various cancers through orchestration of multiple attributes of tumorigenesis, i.e., promoting migration, invasion, escaping the host immune system, and influencing other cancer hallmarks. Therefore, the multidimensional role of gangliosides in different cancers has established them as potential cancer targets. However, the tremendous structural complexity and functional heterogeneity are the major challenges in ganglioside research. Moreover, despite numerous immunotherapeutic attempts to target different gangliosides, it has failed to yield consistent results in clinical trials owing to their poor immunogenicity, a broad range of cross-reactivity, severe side effects, lack of uniform expression as well as heterogeneity. The recent identification of selective O-acetylated ganglioside expression in cancer tissues, but not in normal tissues, has strengthened their potential as a better and specific target for treating cancer patients. It was further supported by reduced cross-reactivity and side effects in clinical trials, although poor immunogenicity remains a major concern. Therefore, in addition to characterization and identification of the biological importance of O-acetylated gangliosides, their specific and efficient targeting in cancer through engineered antibodies is an emerging area of glycobiology research. This review highlights the modulatory effect of select gangliosides on different hallmarks of cancer and presents the overall development of ganglioside targeted immunotherapies along with recent progress. Here, we have also discussed its potential for future modifications aimed towards improvement in ganglioside-based cancer therapies.

Packed to the Brim: Investigating the Impact of Highly Responsive Prefixes on Internet-wide Measurement Campaigns

Internet-wide scans are an important tool to evaluate the deployment of services. To enable large-scale application layer scans, a fast, stateless port scan (e.g., using ZMap) is often performed ahead of time to collect responsive targets. It is a common expectation that port scans on the entire IPv4 address space provide a relatively unbiased view as they cover the complete address space. Previous work, however, has found prefixes where all addresses share particular properties. In IPv6, aliased prefixes and fully responsive prefixes, i.e., prefixes where all addresses are responsive, are a well-known phenomenon. However, there is no such in-depth analysis for prefixes with these responsiveness patterns in IPv4. This paper delves into the underlying factors of this phenomenon in the context of IPv4 and evaluates port scans on a total of 161 ports (142 TCP &amp; 19 UDP ports) from three different vantage points. To account for packet loss and other scanning artifacts, we propose the notion of a new category of prefixes, which we call highly responsive prefixes (HRPs). Our findings show that the share of HRPs can make up 70% of responsive addresses on selected ports. Regarding specific ports, we observe that CDNs contribute to the largest fraction of HRPs on TCP/80 and TCP/443, while TCP proxies emerge as the primary cause of HRPs on other ports. Our analysis also reveals that application layer handshakes to targets outside HRPs are, depending on the chosen service, up to three times more likely to be successful compared to handshakes with targets located in HRPs. To improve future scanning campaigns conducted by the research community, we make our study's data publicly available and provide a tool for detecting HRPs. Furthermore, we propose an approach for a more efficient, ethical, and sustainable application layer target selection. We demonstrate that our approach has the potential to reduce the number of TLS handshakes by up to 75% during an Internet-wide scan while successfully obtaining 99 % of all unique certificates.

MVSSC: Meta-reinforcement learning based visual indoor navigation using multi-view semantic spatial context

In Visual Indoor Navigation (VIN), Deep Reinforcement Learning (DRL) is commonly used by agents to achieve end-to-end mapping from vision to action when navigating toward a target based on observation. However, current DRL-based work suffers from two challenges: partial observability resulting from using solely a single first-person view and poor generalization in the case of unknown scenes and unknown objects. In light of these issues, this paper introduces the integration of multi-view as an expansion of observability and meta-learning as a primary generalization technique into the DRL framework and presents the meta-reinforcement learning method that leverages Multi-View Semantic Spatial Context (MVSSC). Specifically, aiming to explore the informative multi-view context for better efficiency in searching for and navigating to the target, we model the objects’ relationship from two aspects, multi-view semantic context (MVSEC) and multi-view spatial context (MVSPC). MVSEC enables agents to encode prior semantic relationships adaptively via a multi-view modulated graph. Meanwhile, MVSPC enhances the spatial representation of target-related objects’ correlation through similarity grids of multi-view. After adaptively fusing the multi-view and context information under the meta-reinforcement learning framework, our method can encourage efficient target search and robust navigation with stronger generalization performance to unknown scenes and unknown objects. Extensive experimental results on the AI2-THOR simulator demonstrate that our method outperforms the current state-of-the-art approaches.

3D-DNA walking nanomachine based on catalytic hairpin assembly and copper nanoclusters for sensitive detection of hepatitis C virus

Rapid and accurate detection of the hepatitis C virus (HCV) is essential for early diagnosis and prevention of virus transmission. This study presents a novel approach that combines the three-dimensional (3D)-DNA walking nanomachine with catalytic hairpin assembly (CHA) and copper nanoclusters (CuNCs). By integrating CHA with the 3D DNA walking nanomachine, efficient target amplification on 3D surfaces was achieved, leading to improved reaction speed and detection performance. Terminal deoxynucleotidyl transferase (TdT) was utilized to generate T-rich DNA sequences. These sequences served as templates for the formation of CuNCs, which functioned as the readout signal. The optimized 3D-DNA walking nanomachine exhibited excellent sensitivity in detecting HCV, with a detection limit of 42.4 pM and a linear range of 100 pM to 2 nM. The biosensor demonstrated excellent selectivity and reproducibility, with a recovery rate ranging from 94% to 108% for the detection of real samples. This design holds great potential for sensitive, label-free, and reliable detection of HCV in clinical settings. Furthermore, the versatility of this approach allows for the customization of target sequences, thereby facilitating the detection of various nucleic acid targets. Therefore, this method has the potential to advance personalized medicine, disease management, and genetic analysis in the field of molecular diagnosis.

Macrophage-hitchhiking interleukin-10 plasmid DNA delivery system modulates rheumatoid arthritis microenvironment via the re-polarization of macrophages

Rheumatoid arthritis (RA) is a common chronic inflammatory disease and the current treatments for RA are unsatisfactory due to the buffer barrier from the RA microenvironment. Interleukin-10 (IL-10) was found to be able to modulate rheumatoid arthritis microenvironment (RAM) by the metabolic reprogramming of macrophages. However, its clinical application has proven challenging mainly due to its short half-life, non-ideal targeting efficiency via systemic administration and poor tissue penetration. Taking advantage of the recruitment of macrophages, we constructed an innovative macrophage-hitchhiking IL-10 pDNA delivery system using the peptides bearing nuclear localization signal (NLS) and tuftsin-modified disulfide-crosslinked polymers (bPEI-SS-PEG-T) which was responsive to the intracellular glutathione concentration. Firstly, we formed NLS/DNA complexes via electrostatic interactions to enhance the nuclear entry efficiency. Then NLS/DNA complexes were combined with bPEI-SS-PEG-T to construct bPEI-SS-PEG-T/NLS/DNA NPs with sizes of 168.4 nm and zeta potentials of + 10.9 mV. As the bPEI-SS-PEG-T was biodegradable and the tuftsin peptides enhanced macrophage-mediated phagocytosis, the nanoparticles exhibited lower cytotoxicity and better cellular uptake in addition to higher nuclear entry efficiency, leading to better IL-10 plasmid transfection. In addition, these macrophage-hitchhiking nanoparticles could effectively accumulate at inflammatory sites of RA via intra-peritoneal injection due to the macrophage migration to the inflammatory joints and alleviate symptoms of inflammation in vivo by inducing the re-polarization of macrophages, which might be mediated by the inhibition of mammalian target of rapamycin (mTOR) and the induction of arginase-2 (Arg2). As a result, this macrophage-hitchhiking gene delivery system demonstrated excellent inflammation targeting ability, good nuclear entry ability, high IL-10 plasmid transfection efficiency and great therapeutic efficacy in vivo, which is promising for the treatment of rheumatoid arthritis. Besides, this delivery system also provides a novel strategy for the gene therapy and the gene delivery system design for rheumatoid arthritis and other similar inflammatory diseases from the perspective of the inflammatory microenvironment.

Lipid-Centric Design of Plasma Membrane-Mimicking Nanocarriers for Targeted Chemotherapeutic Delivery.

The plasma membranes (PM) of mammalian cells contain diverse lipids, proteins, and carbohydrates that are important for systemic recognition and communication in health and disease. Cell membrane coating technology that imparts unique properties of natural plasma membranes to the surface of encapsulated nanoparticles is thus becoming a powerful platform for drug delivery, immunomodulation, and vaccination. However, current coating methods fail to take full advantage of the natural systems because they disrupt the complex and functionally essential features of PMs, most notably the chemical diversity and compositional differences of lipids in two leaflets of the PM. Herein, a new lipid coating approach is reported in which the lipid composition is optimized through a combination of biomimetic and systematic variation approaches for the custom design of nanocarrier systems for precision drug delivery. Nanocarriers coated with the optimized lipids offer unique advantages in terms of bioavailability and efficiency in tumor targeting, tumor penetration, cellular uptake, and drug release. This pilot study provides new insight into the rational design and optimization of nanocarriers for cancer chemotherapeutic drugs and lays the foundation for further customization of cell membrane-mimicking nanocarriers through systematic incorporation of other components.

Peptide-modified PAMAM-based bone-targeting RNA delivery system

BackgroundOsteoporosis, among other bone diseases, has become a prevalent cause of decreased quality of life in older and postmenopausal women. Traditional anti-osteoporotic therapies, though widely prescribed, are limited by a lack of cell- or tissue-specific targeting ability and effectiveness without side effects. Gene therapy is rapidly replacing traditional therapeutics, primarily because of its specific targeting ability and efficiency. Among viral- and non-viral-based gene therapies, the latter is often preferred over the former due to lower cytotoxicity, immunogenicity, and ease of modification with different molecules to improve efficiency and extend gene expression. We designed and synthesized a multifunctional bone-targeting ribonucleic acid (RNA) delivery system based on polyamidoamine (PAMAM). PAMAM was modified with the serine-aspartate-serine-serine-aspartate (SDSSD) peptide to deliver antagomir 138-5p to osteoblasts (MC3T3-E1 cell line) in vitro and in vivo using the ovariectomized (OVX) mouse model.ResultsThe results showed that this system was less cytotoxic than polyethylenimine (PEI) and could bind to RNA favorably while maintaining gene delivery ability in vitro. In vivo data showed that the distal tibia and femur of the mice in the PAMAM-SDSSD (PS) + RNA group had improved bone mineral density (BMD), bone mineral content (BMC), and bone volume compared to those in the PS + Negative Control (NC) or OVX groups. Moreover, the femurs of the PS + RNA group mice demonstrated a higher breaking point, stress, stiffness, and elasticity than those of the PS + NC or OVX mice, suggesting improved femur strength in the OVX mice treated with RNA delivered through SDSSD-modified PAMAM.ConclusionThis study shows that SDSSD modification of PAMAM not only improves gene delivery capacity but also enhances the cell targeting efficiency of nanoparticles towards osteoblasts. The successful delivery of antagomir 138-5p to osteoblasts demonstrates cell-specificity and gene delivery to alleviate osteoporotic symptoms.

Epigenetic profiles guide improved CRISPR/Cas9-mediated gene knockout in human T cells.

Genetic modification of specific genes is emerging as a useful tool to enhance the functions of antitumor T cells in adoptive immunotherapy. Current advances in CRISPR/Cas9 technology enable gene knockout during in vitro preparation of infused T-cell products through transient transfection of a Cas9-guide RNA (gRNA) ribonucleoprotein complex. However, selecting optimal gRNAs remains a major challenge for efficient gene ablation. Although multiple in silico tools to predict the targeting efficiency have been developed, their performance has not been validated in cultured human T cells. Here, we explored a strategy to select optimal gRNAs using our pooled data on CRISPR/Cas9-mediated gene knockout in human T cells. The currently available prediction tools alone were insufficient to accurately predict the indel percentage in T cells. We used data on the epigenetic profiles of cultured T cells obtained from transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Combining the epigenetic information with sequence-based prediction tools significantly improved the gene-editing efficiency. We further demonstrate that epigenetically closed regions can be targeted by designing two gRNAs in adjacent regions. Finally, we demonstrate that the gene-editing efficiency of unstimulated T cells can be enhanced through pretreatment with IL-7. These findings enable more efficient gene editing in human T cells.

The Influence of PEG Chain Length on the in vivo Tumor Accumulation of Self-assembled Amphiphilic Drug-PEG Polymeric Micelles

The integration of a drug and polyethylene glycol (PEG) into an amphiphilic polymer to form self-assembled micelles with high drug loading for self-delivery to tumors is recently reported, which has showed high clinical availability. However, the effect of PEG chain length on cell internalization efficiency and tumor targeting efficiency in this PEG-drug conjugate micelle self-delivery system has yet to be studied. Herein, pyropheophorbide-a (PhA) was used as a model drug and a fluorescent tracer to synthesize amphiphilic polymers with varying PEG chain lengths, which self-assembled into PhA-PEG2000 NPs and PhA-PEG5000 NPs with high drug loading (21.2% and 9.7%, respectively, according to calculation). The results showed that PhA-PEG2000 NPs had higher cell internalization in vitro than PhA-PEG5000 NPs, but much lower tumor accumulation in vivo than PhA-PEG5000 NPs. This was attributed to the longer in vivo blood circulation time of the latter. Furthermore, the in vitro and in vivo examinations revealed negligible side effects, indicating the intrinsic safety of the micelles. This study provides insight into the influence of PEG chain length on biodistribution of organs and tumor accumulation after intravenous administration of PEG-drug conjugate micelle and guides the design of drug incorporated amphiphilic polymer-based micelles.

Efficient and Targeted siRNA Delivery to M2 Macrophages by Smart Polymer Blends for M1 Macrophage Repolarization as a Promising Strategy for Future Cancer Treatment.

Cancer remains an issue on a global scale. It is estimated that nearly 10 million people succumbed to cancer worldwide in 2020. New treatment options are urgently needed. A promising approach is a conversion of tumor-promoting M2 tumor-associated macrophages (TAMs) as part of the tumor microenvironment to tumor-suppressive M1 TAMs by small interfering RNA (siRNA). In this work, we present a well-characterized polymeric nanocarrier system capable of targeting M2 TAMs by a ligand-receptor interaction. Therefore, we developed a blended PEI-based polymeric nanoparticle system conjugated with mannose, which is internalized after interaction with macrophage mannose receptors (MMRs), showing low cytotoxicity and negligible IL-6 activation. The PEI-PCL-PEI (5 kDa-5 kDa-5 kDa) and Man-PEG-PCL (2 kDa-2 kDa) blended siRNA delivery system was optimized for maximum targeting capability and efficient endosomal escape by evaluation of different polymer and N/P ratios. The nanoparticles were formulated by surface acoustic wave-assisted microfluidics, achieving a size of ∼80 nm and a zeta potential of approximately +10 mV. Special attention was given to the endosomal escape as the so-called bottleneck of RNA drug delivery. To estimate the endosomal escape capability of the nanocarrier system, we developed a prediction method by evaluating the particle stability via the inflection temperature. Our predictions were then verified in an in vitro setting by applying confocal microscopy. For cellular experiments, however, human THP-1 cells were polarized to M2 macrophages by cytokine treatment and validated through MMR expression. To show the efficiency of the nanoparticle system, GAPDH and IκBα knockdown was performed in the presence or absence of an MMR blocking excess of mannan. Cellular uptake, GAPDH knockdown, and NF-κB western blot confirmed efficient mannose targeting. Herein, we presented a well-characterized nanoparticle delivery system and a promising approach for targeting M2 macrophages by a mannose-MMR interaction.

Molecular Hybridization of Alkaloids Using 1,2,3-Triazole-Based Click Chemistry.

Alkaloids found in multiple species, known as 'driver species', are more likely to be included in early-stage drug development due to their high biodiversity compared to rare alkaloids. Many synthetic approaches have been employed to hybridize the natural alkaloids in drug development. Click chemistry is a highly efficient and versatile reaction targeting specific areas, making it a valuable tool for creating complex natural products and diverse molecular structures. It has been used to create hybrid alkaloids that address their limitations and serve as potential drugs that mimic natural products. In this review, we highlight the recent advancements made in modifying alkaloids using click chemistry and their potential medicinal applications. We discuss the significance, current trends, and prospects of click chemistry in natural product-based medicine. Furthermore, we have employed computational methods to evaluate the ADMET properties and drug-like qualities of hybrid molecules.

Enhanced Mitochondrial Targeting and Inhibition of Pyroptosis with Multifunctional Metallopolyphenol Nanoparticles in Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IVDD) is a significant contributor to low back pain, characterized by excessive reactive oxygen species generation and inflammation-induced pyroptosis. Unfortunately, there are currently no specific molecules or materials available to effectively delay IVDD. This study develops a multifunctional full name of PG@Cu nanoparticle network (PG@Cu). A designed pentapeptide, bonded on PG@Cu nanoparticles via a Schiff base bond, imparts multifunctionality to the metal polyphenol particles (PG@Cu-FP). PG@Cu-FP exhibits enhanced escape from lysosomal capture, enabling efficient targeting of mitochondria to scavenge excess reactive oxygen species. The scavenging activity against reactive oxygen species originates from the polyphenol-based structures within the nanoparticles. Furthermore, Pyroptosis is effectively blocked by inhibiting Gasdermin mediated pore formation and membrane rupture. PG@Cu-FP successfully reduces the activation of the nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 inflammasome by inhibiting Gasdermin protein family (Gasdermin D, GSDMD) oligomerization, leading to reduced expression of Nod-like receptors. This multifaceted approach demonstrates higher efficiency in inhibiting Pyroptosis. Experimental results confirm that PG@Cu-FP preserves disc height, retains water content, and preserves tissue structure. These findings highlight the potential of PG@Cu-FP in improving IVDD and provide novel insights for future research in IVDD treatments.

Gold mineralized “hybrid nanozyme bomb” for NIR-II triggered tumor effective permeation and cocktail therapy

Lung cancer is one of the most common malignant tumors with the fastest increase in the incidence rate and mortality. Even after maximum tumor resection assistance with a radiotherapy and chemotherapy combination, the recurrence of non-small cell lung cancer is still inevitable. In addition, low targeting efficiency and poor permeability of drug delivery systems strongly affect the therapeutic efficiency of anti-cancer drugs on non-small cell lung cancer. Here we designed a gemcitabine (GEM) loaded arginine-glycine-aspartic acid-cysteine (RGDc)-modified gold mineralization “hybrid nanozyme bomb” (RGTG) to overcome those obstacles. RGDc modification improved the active targeting of liposomes to the tumor tissues with the second near-infrared (NIR-II)-triggered gold-shell disruption and GEM release. The collapsed gold-shell particles with a smaller size could penetrate the tumor solid barrier and act as photothermal therapy (PTT) agents to improve PTT therapy and starvation therapy via generating gluconic acid and reactive oxygen species (ROS). Moreover, the resting reversal effect of gold particles on tumor fibroblasts can achieve accelerating tumor penetration of gold particles and GEM. Compared to monotherapy, RGTG showed significant improvement in tumor inhibition, with a tumor volume reduction of 83% compared to the control group, which provides a promising tumor treatment platform for non-small cell lung cancer (NSCLC).