New tumor-targeted nanosized delivery carrier for oligonucleotides: characteristics in vitro and in vivo

Tianyang Zhou1,2, Xin Jia1, Huixiang Li3, Jin Wang3, Hongling Zhang1,2, Youmei A1,2, Zhenzhong Zhang1,21School of Pharmaceutical Sciences, 2Nanotechnology Research Center for Drugs, 3Department of Pathology, Medical School of Zhengzhou University, Zhengzhou, People’s Republic of ChinaBackground: The purpose of this study was to investigate the in vitro and in vivo characteristics of a new tumor-targeted nanosized delivery carrier for antisense oligonucleotide (ASON).Methods: Polyethylenimine (PEI) was used to condense ASON to form nanosized complexes (PEI/ASON), which were then modified using asparagine-glycine-arginine (NGR) peptide to obtain a tumor-targeted nanosized delivery carrier (NGR/PEI/ASON). The conditions required to form PEI/ASON were investigated.Results: A linear correlation between the natural logarithm of the N/P ratio (PEI to ASON) and the zeta potential of the PEI/ASON complexes was found, ranging from 1.5 to 5.0. The pH of the solution strongly influenced the zeta potential of the PEI/ASON complexes. PEI/ASON and NGR/PEI/ASON were stable in RPMI-1640 culture medium in the presence of Dextran 70. Incorporation of ASON into PEI/ASON and NGR/PEI/ASON complexes prevented degradation of ASON by DNase I.Conclusion: Both ASON/PEI and NGR/PEI/ASON complexes enhanced the uptake of ASON by EC9706 cells in vitro. In vivo, NGR/PEI/ASON complexes had the ability to target tumor tissues effectively.Keywords: nanosized delivery system, squamous cell carcinoma, antisense oligonucleotid

An optimized protocol for the generation and monitoring of conditional orthotopic lung cancer in the KP mouse model using an adeno-associated virus vector compatible with biosafety level 1.

BACKGROUND The inducible Kras/p53 lung adenocarcinoma mouse model, which faithfully recapitulates human disease, is routinely initiated by the intratracheal instillation of a virus-based Cre recombinase delivery system. Handling virus-based delivery systems requires elevated biosafety levels, e.g., biosafety level 2 (BSL-2). However, in experimental animal research facilities, following exposure to viral vectors in a BSL-2 environment, rodents may not be reclassified to BSL-1 according to standard practice, preventing access to small animal micro-computed tomography (micro-CT) scanners that are typically housed in general access areas such as BSL-1 rooms. Therefore, our goal was to adapt the protocol so that the Cre-induced KP mouse model could be handled under BSL-1 conditions during the entire procedure. RESULTS The Kras-Lox-STOP-Lox-G12D/p53 flox/flox (KP)-based lung adenocarcinoma mouse model was activated by intratracheal instillation of either an adenoviral-based or a gutless, adeno-associated viral-based Cre delivery system. Tumor growth was monitored over time by micro-CT. We have successfully substituted the virus-based Cre delivery system with a commercially available, gutless, adeno-associated, Cre-expressing vector that allows the KP mouse model to be handled and imaged in a BSL-1 facility. By optimizing the anesthesia protocol and switching to a microscope-guided vector instillation procedure, productivity was increased and procedure-related complications were significantly reduced. In addition, repeated micro-CT analysis of individual animals allowed us to monitor tumor growth longitudinally, dramatically reducing the number of animals required per experiment. Finally, we documented the evolution of tumor volume for different doses, which revealed that individual tumor nodules induced by low-titer AAV-Cre transductions can be monitored over time by micro-CT. CONCLUSION Modifications to the anesthesia and instillation protocols increased the productivity of the original KP protocol. In addition, the switch to a gutless, adeno-associated, Cre-expressing vector allowed longitudinal monitoring of tumor growth under BSL-1 conditions, significantly reducing the number of animals required for an experiment, in line with the 3R principles

Targeting lactate dehydrogenase B-dependent mitochondrial metabolism affects tumor initiating cells and inhibits tumorigenesis of non-small cell lung cancer by inducing mtDNA damage.

Once considered a waste product of anaerobic cellular metabolism, lactate has been identified as a critical regulator of tumorigenesis, maintenance, and progression. The putative primary function of lactate dehydrogenase B (LDHB) is to catalyze the conversion of lactate to pyruvate; however, its role in regulating metabolism during tumorigenesis is largely unknown. To determine whether LDHB plays a pivotal role in tumorigenesis, we performed 2D and 3D in vitro experiments, utilized a conventional xenograft tumor model, and developed a novel genetically engineered mouse model (GEMM) of non-small cell lung cancer (NSCLC), in which we combined an LDHB deletion allele with an inducible model of lung adenocarcinoma driven by the concomitant loss of p53 (also known as Trp53) and expression of oncogenic KRAS (G12D) (KP). Here, we show that epithelial-like, tumor-initiating NSCLC cells feature oxidative phosphorylation (OXPHOS) phenotype that is regulated by LDHB-mediated lactate metabolism. We show that silencing of LDHB induces persistent mitochondrial DNA damage, decreases mitochondrial respiratory complex activity and OXPHOS, resulting in reduced levels of mitochondria-dependent metabolites, e.g., TCA intermediates, amino acids, and nucleotides. Inhibition of LDHB dramatically reduced the survival of tumor-initiating cells and sphere formation in vitro, which can be partially restored by nucleotide supplementation. In addition, LDHB silencing reduced tumor initiation and growth of xenograft tumors. Furthermore, we report for the first time that homozygous deletion of LDHB significantly reduced lung tumorigenesis upon the concomitant loss of Tp53 and expression of oncogenic KRAS without considerably affecting the animal's health status, thereby identifying LDHB as a potential target for NSCLC therapy. In conclusion, our study shows for the first time that LDHB is essential for the maintenance of mitochondrial metabolism, especially nucleotide metabolism, demonstrating that LDHB is crucial for the survival and proliferation of NSCLC tumor-initiating cells and tumorigenesis

Experimental design.

Controlling the deformation rate is the key to improving the product quality of engineered wood flooring. In this work, the changes in the deformation rate of engineered wood flooring were in focus with cold-pressing, response surface methodology, and adaptive network-based fuzzy inference system were used to explore the relationship between deformation rate and processing parameters, including adhesive spreading rate, pressing time, and pressing pressure. According to the results, the deformation rate was positively related to pressing time, while it increased first and then decreased with both the increase of adhesive spreading rate and pressing pressure. Meanwhile, a mathematical model was developed, and the significant influence of each term on the deformation rate was analyzed. This model had high feasibility and can be used to describe the relationship between the deformation rate and processing parameters. Furthermore, an adaptive network-based fuzzy inference system model was established. It has higher accuracy than that of the response surface methodology model, and it can be used for predicting deformation rate and optimizing processing parameters. Finally, an optimal processing conditions with the lowest deformation rate was determined as follows: 147 g/m2 adhesive spreading rate, 12s pressing time, and 1.2 MPa pressing pressure, and it hope to be adopted in the industrial processing of engineered wood flooring with respective of the higher product quality and lower production costs.</div

ANIFIS model structure.

Controlling the deformation rate is the key to improving the product quality of engineered wood flooring. In this work, the changes in the deformation rate of engineered wood flooring were in focus with cold-pressing, response surface methodology, and adaptive network-based fuzzy inference system were used to explore the relationship between deformation rate and processing parameters, including adhesive spreading rate, pressing time, and pressing pressure. According to the results, the deformation rate was positively related to pressing time, while it increased first and then decreased with both the increase of adhesive spreading rate and pressing pressure. Meanwhile, a mathematical model was developed, and the significant influence of each term on the deformation rate was analyzed. This model had high feasibility and can be used to describe the relationship between the deformation rate and processing parameters. Furthermore, an adaptive network-based fuzzy inference system model was established. It has higher accuracy than that of the response surface methodology model, and it can be used for predicting deformation rate and optimizing processing parameters. Finally, an optimal processing conditions with the lowest deformation rate was determined as follows: 147 g/m2 adhesive spreading rate, 12s pressing time, and 1.2 MPa pressing pressure, and it hope to be adopted in the industrial processing of engineered wood flooring with respective of the higher product quality and lower production costs.</div

Fig 5 -

Three-dimension response surface (a-c) and contours (d-f) of two-level interactions on deformation rate.</p

Optimization and verification results for RSM and ANFIS models.

Optimization and verification results for RSM and ANFIS models.</p

Model fit statistics of deformation rate.

Controlling the deformation rate is the key to improving the product quality of engineered wood flooring. In this work, the changes in the deformation rate of engineered wood flooring were in focus with cold-pressing, response surface methodology, and adaptive network-based fuzzy inference system were used to explore the relationship between deformation rate and processing parameters, including adhesive spreading rate, pressing time, and pressing pressure. According to the results, the deformation rate was positively related to pressing time, while it increased first and then decreased with both the increase of adhesive spreading rate and pressing pressure. Meanwhile, a mathematical model was developed, and the significant influence of each term on the deformation rate was analyzed. This model had high feasibility and can be used to describe the relationship between the deformation rate and processing parameters. Furthermore, an adaptive network-based fuzzy inference system model was established. It has higher accuracy than that of the response surface methodology model, and it can be used for predicting deformation rate and optimizing processing parameters. Finally, an optimal processing conditions with the lowest deformation rate was determined as follows: 147 g/m2 adhesive spreading rate, 12s pressing time, and 1.2 MPa pressing pressure, and it hope to be adopted in the industrial processing of engineered wood flooring with respective of the higher product quality and lower production costs.</div