Lipophilic siRNA targets albumin in situ and promotes bioavailability, tumor penetration, and carrier-free gene silencing

Clinical translation of therapies based on small interfering RNA (siRNA) is hampered by siRNA's comprehensively poor pharmacokinetic properties, which necessitate molecule modifications and complex delivery strategies. We sought an alternative approach to commonly used nanoparticle carriers by leveraging the long-lived endogenous serum protein albumin as an siRNA carrier. We synthesized siRNA conjugated to a diacyl lipid moiety (siRNA-L2), which rapidly binds albumin in situ. siRNA-L2, in comparison with unmodified siRNA, exhibited a 5.7-fold increase in circulation half-life, an 8.6-fold increase in bioavailability, and reduced renal accumulation. Benchmarked against leading commercial siRNA nanocarrier in vivo jetPEI, siRNA-L2 achieved 19-fold greater tumor accumulation and 46-fold increase in per-tumor-cell uptake in a mouse orthotopic model of human triple-negative breast cancer. siRNA-L2 penetrated tumor tissue rapidly and homogeneously; 30 min after i.v. injection, siRNA-L2 achieved uptake in 99% of tumor cells, compared with 60% for jetPEI. Remarkably, siRNA-L2 achieved a tumor:liver accumulation ratio >40:1 vs. <3:1 for jetPEI. The improved pharmacokinetic properties of siRNA-L2 facilitated significant tumor gene silencing for 7 d after two i.v. doses. Proof-of-concept was extended to a patient-derived xenograft model, in which jetPEI tumor accumulation was reduced fourfold relative to the same formulation in the orthotopic model. The siRNA-L2 tumor accumulation diminished only twofold, suggesting that the superior tumor distribution of the conjugate over nanoparticles will be accentuated in clinical situations. These data reveal the immense promise of in situ albumin targeting for development of translational, carrier-free RNAi-based cancer therapies.

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Abstract 33: Unlocking Reprogramming Capability: Silencing Antiplasticity Gene p63 Enhances the Reprogramming of Fibroblasts into Induced Cardiomyocytes

Circulation Research ◽

10.1161/res.119.suppl_1.33 ◽

2016 ◽

Vol 119 (suppl_1) ◽

Author(s):

Vivekkumar Patel ◽

Austin Cooney ◽

Elsa Flores ◽

Vivek Singh ◽

Megumi Mathison ◽

...

Keyword(s):

Transcription Factors ◽

Troponin T ◽

Cardiac Fibroblasts ◽

Fold Increase ◽

Cellular Reprogramming ◽

Embryonic Fibroblasts ◽

Reprogramming Efficiency ◽

Cardiac Transcription Factors

Objective: In situ cellular reprogramming of cardiac fibroblasts into (induced) cardiomyocytes (iCMs) represents a promising new potential intervention for the treatment of heart failure. Despite encouraging in vivo data in rodent myocardial infarction models, the relative resistance of human cells to reprogramming may be a significant barrier to the clinical application of this new therapy. We hypothesized that knockdown of the anti-plasticity gene p63 could therefore be used to enhance cellular reprogramming efficiency. Methods: p63 knockout (KO) murine embryonic fibroblasts (MEFs) and MEFs treated with p63 silencing shRNA were assessed for expression of the cardiomyocyte marker Cardiac Troponin T (cTnT) and pro-cardiogenic genes, with or without the treatment with known cardiac transcription factors Hand2 and Myocardin (HM). Results: After 3 wks in culture, expression of the cardiomyocyte marker cTnT (FACS) was significantly greater in p63 KO MEFs than in wild-type (WT) MEFs or WT MEFs treated with transcription factors Hand2 and Myocardin (39% ± 8%, 2.0% ± 1% and 2.7 ± 0.3%, respectively, p < 0.05). Treatment of p63 KO MEFs with Hand2 and Myocardin further increased cTnT expression up to 74% ± 3%. Treatment of WT MEFs with p63 shRNA likewise yielded a 20-fold increase in cTnT expression (qPCR) without HM and a 600-fold increase with HM when compared to non-silencing shRNA treated MEFs. Consistent with these findings, p63 KO or p63 shRNA-treated MEFs demonstrated increased expression (qPCR) of pro-cardiogenic genes Gata4, Mef2c and Tbx5 compared to naïve or non-silencing shRNA treated MEFs. After treatment with p63 shRNA, adult human epidermal cells also demonstrated increased expression of cTnT, myosin heavy chain and pro-cardiogenic genes when analyzed by qPCR. Conclusions: Downregulation of the anti-plasticity gene p63 enhances cellular reprogramming efficiency and iCM generation, as reflected in the increased expression of the cardiomyocyte marker cTnT and pro-cardiogenic genes Gata4, Mef2c and Tbx5. Use of such cellular plasticity enhancing strategies may be a useful strategy to overcome barriers to cellular reprogramming in the clinical arena.

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MNAT1 promotes proliferation and the chemo-resistance of osteosarcoma cell to cisplatin through regulating PI3K/Akt/mTOR pathway

BMC Cancer ◽

10.1186/s12885-020-07687-3 ◽

2020 ◽

Vol 20 (1) ◽

Author(s):

Chensheng Qiu ◽

Weiliang Su ◽

Nana Shen ◽

Xiaoying Qi ◽

Xiaolin Wu ◽

...

Keyword(s):

Tumor Growth ◽

Xenograft Model ◽

Mtor Pathway ◽

Regulation Mechanism ◽

Osteosarcoma Cell ◽

Migration Ability ◽

Mouse Xenograft Model

Abstract Background MNAT1 (menage a trois 1, MAT1), a cyclin-dependent kinase-activating kinase (CAK) complex, highly expressed in diverse cancers and was involved in cancer molecular pathogenesis. However, its deliverance profile and biological function in osteosarcoma (OS) remain unclear. Methods The expression of MNAT1 in OS was detected by western blot (WB) and immunohistochemistry (IHC). The potential relationship between MNAT1 molecular level expression and OS clinical expectations were analyzed according to tissues microarray (TMA). Proliferation potential of OS cells was evaluated in vitro based on CCK8 and OS cells colony formation assays, while OS cells transwell and in situ tissue source wound healing assays were employed to analyze the OS cells invasion and migration ability in vitro. A nude mouse xenograft model was used to detect tumor growth in vivo. In addition, ordinary bioinformatics analysis and experimental correlation verification were performed to investigate the underlying regulation mechanism of OS by MNAT1. Results In this research, we found and confirmed that MNAT1 was markedly over-expressed in OS tissue derived in situ, also, highly MNAT1 expression was closely associated with bad clinical expectations. Functional studies had shown that MNAT1 silencing could weaken the invasion, migration and proliferation of OS cells in vitro, and inhibit OS tumor growth in vivo. Mechanism study indicated that MNAT1 contributed to the progression of OS via the PI3K/Akt/mTOR pathway. We further verified that the MNAT1 was required in the regulation of OS chemo-sensitivity to cisplatin (DDP). Conclusions Taken together, the data of the present study demonstrate a novel molecular mechanism of MNAT1 involved in the formation of DDP resistance of OS cells.

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The coordinated action of G-CSF and ELR + CXC chemokines in neutrophil mobilization during acute inflammation

Blood ◽

10.1182/blood-2007-07-099648 ◽

2008 ◽

Vol 111 (1) ◽

pp. 42-49 ◽

Cited By ~ 144

Author(s):

Antje M. Wengner ◽

Simon C. Pitchford ◽

Rebecca C. Furze ◽

Sara M. Rankin

Keyword(s):

Bone Marrow ◽

Intraperitoneal Injection ◽

Fold Increase ◽

In Situ Perfusion ◽

Acute Peritonitis ◽

Femoral Bone Marrow ◽

Prior Administration

In this study, we have identified a unique combinatorial effect of the chemokines KC/MIP-2 and the cytokine granulocyte colony-stimulating factor (G-CSF) with respect to the rapid mobilization of neutrophils from the bone marrow in a model of acute peritonitis. At 2 hours following an intraperitoneal injection of thioglycollate, there was a 4.5-fold increase in blood neutrophil numbers, which was inhibited 84% and 72% by prior administration of blocking mAbs against either the chemokines KC/MIP-2 or G-CSF, respectively. An intraperitoneal injection of G-CSF acted remotely to stimulate neutrophil mobilization, but did not elicit recruitment into the peritoneum. Further, in vitro G-CSF was neither chemotactic nor chemokinetic for murine neutrophils, and had no priming effect on chemotaxis stimulated by chemokines. Here, we show that, in vitro and in vivo, G-CSF induces neutrophil mobilization by disrupting their SDF-1α–mediated retention in the bone marrow. Using an in situ perfusion system of the mouse femoral bone marrow to directly assess mobilization, KC and G-CSF mobilized 6.8 × 106 and 5.4 × 106 neutrophils, respectively, while the infusion of KC and G-CSF together mobilized 19.5 × 106 neutrophils, indicating that these factors act cooperatively with respect to neutrophil mobilization.

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Biodegradable nanoparticles as siRNA carriers for in vivo gene silencing and pancreatic cancer therapy

Journal of Materials Chemistry B ◽

10.1039/c6tb03116a ◽

2017 ◽

Vol 5 (18) ◽

pp. 3327-3337 ◽

Cited By ~ 9

Author(s):

Guimiao Lin ◽

Chih-Kuang Chen ◽

Feng Yin ◽

Chengbin Yang ◽

Jinglin Tian ◽

...

Keyword(s):

Pancreatic Cancer ◽

Cancer Therapy ◽

Tumor Growth ◽

Gene Silencing ◽

Xenograft Model ◽

Biodegradable Nanoparticles ◽

Pancreatic Cancer Therapy

Biodegradable charged polyester-based vectors (BCPVs) were utilized for efficiently delivering mutatedK-Ras-targeting siRNA and successfully inhibiting tumor growth in a pancreatic xenograft modelin vivo.

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Optimization of Preparation and Preclinical Pharmacokinetics of Celastrol-Encapsulated Silk Fibroin Nanoparticles in the Rat

Molecules ◽

10.3390/molecules24183271 ◽

2019 ◽

Vol 24 (18) ◽

pp. 3271 ◽

Cited By ~ 3

Author(s):

Onyeabor ◽

Paik ◽

Kovvasu ◽

Ding ◽

Lin ◽

...

Keyword(s):

Silk Fibroin ◽

High Performance ◽

Water Solubility ◽

Fold Increase ◽

Bioactive Compound ◽

Rat Plasma ◽

Limit Of Quantification ◽

Pharmacokinetic Properties ◽

Precision And Accuracy

Celastrol (CL), a bioactive compound isolated from Tripterygium wilfordii, has demonstrated bioactivities against a variety of diseases including cancer and obesity. However, its poor water solubility and rapid in vivo clearance limit its clinical applications. To overcome these limitations, nanotechnology has been employed to improve its pharmacokinetic properties. Nanoparticles made of biological materials offer minimal adverse effects while maintaining the efficacy of encapsulated therapeutics. Silk fibroin (SF) solution was prepared successfully by extraction from the cocoons of silkworms, and a final concentration of 2 mg/mL SF solution was used for the preparation of CL-loaded SF nanoparticles (CL-SFNP) by the desolvation method. A stirring speed of 750 rpm and storage time of 20 h at −20 °C resulted in optimized product yield. A high-performance liquid chromatography (HPLC) method was developed and validated for the analysis of CL in rat plasma in terms of selectivity, linearity, intra-/inter-day precision and accuracy, and recovery. No interference was observed in rat plasma. Linearity in the concentration range of 0.05–5 µg/mL was observed with R2 of 0.999. Precision and accuracy values were below the limit of acceptance criteria, i.e., 15% for quality control (QC) samples and 20% for lower limit of quantification (LLOQ) samples. Rats were given intravenous (IV) administration of 1 mg/kg of pure CL in PEG 300 solution or CL-SFNP. The pharmacokinetic profile was improved with CL-SFNP compared to pure CL. Pure CL resulted in a maximum concentration (Cmax) value of 0.17 µg mL−1 at 5 min following administration, whereas that for CL-SFNP was 0.87 µg mL−1 and the extrapolated initial concentrations (C0) were 0.25 and 1.09 µg mL−1, respectively, for pure CL and CL-SFNP. A 2.4-fold increase in total area under the curve (AUC0-inf) (µg h mL−1) was observed with CL-SFNP when compared with pure CL. CL-SFNP demonstrated longer mean residence time (MRT; 0.67 h) than pure CL (0.26 h). In conclusion, the preparation of CL-SFNP was optimized and the formulation demonstrated improved pharmacokinetic properties compared to CL in solution following IV administration.

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Pharmacodynamic and Pharmacokinetic Properties of Full Phosphorothioate Small Interfering RNAs for Gene Silencing In Vivo

Nucleic Acid Therapeutics ◽

10.1089/nat.2020.0852 ◽

2020 ◽

Author(s):

Christian Berk ◽

Gianluca Civenni ◽

Yuluan Wang ◽

Christian Steuer ◽

Carlo V. Catapano ◽

...

Keyword(s):

Gene Silencing ◽

Small Interfering Rnas ◽

Pharmacokinetic Properties

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In vivo evaluation of CD38 and CD138 as targets for nanoparticle-based drug delivery in multiple myeloma

Journal of Hematology & Oncology ◽

10.1186/s13045-020-00965-4 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

David T. Omstead ◽

Franklin Mejia ◽

Jenna Sjoerdsma ◽

Baksun Kim ◽

Jaeho Shin ◽

...

Keyword(s):

Multiple Myeloma ◽

Tumor Suppression ◽

Synthetic Methods ◽

Cell Uptake ◽

In Vivo Evaluation ◽

Targeted Nanoparticles ◽

Liposomal Nanoparticles ◽

Tumor Accumulation

Abstract Background Drug-loaded nanoparticles have established their benefits in the fight against multiple myeloma; however, ligand-targeted nanomedicine has yet to successfully translate to the clinic due to insufficient efficacies reported in preclinical studies. Methods In this study, liposomal nanoparticles targeting multiple myeloma via CD38 or CD138 receptors are prepared from pre-synthesized, purified constituents to ensure increased consistency over standard synthetic methods. These nanoparticles are then tested both in vitro for uptake to cancer cells and in vivo for accumulation at the tumor site and uptake to tumor cells. Finally, drug-loaded nanoparticles are tested for long-term efficacy in a month-long in vivo study by tracking tumor size and mouse health. Results The targeted nanoparticles are first optimized in vitro and show increased uptake and cytotoxicity over nontargeted nanoparticles, with CD138-targeting showing superior enhancement over CD38-targeted nanoparticles. However, biodistribution and tumor suppression studies established CD38-targeted nanoparticles to have significantly increased in vivo tumor accumulation, tumor cell uptake, and tumor suppression over both nontargeted and CD138-targeted nanoparticles due to the latter’s poor selectivity. Conclusion These results both highlight a promising cancer treatment option in CD38-targeted nanoparticles and emphasize that targeting success in vitro does not necessarily translate to success in vivo.

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Determining androgen inactivation status in prostate cancer by [18F] DHT PET.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.7_suppl.191 ◽

2019 ◽

Vol 37 (7_suppl) ◽

pp. 191-191

Author(s):

Ziqi Zhu ◽

Yoon-Mi Chung ◽

Olga Sergeeva ◽

Vladimir Kepe ◽

Michael Berk ◽

...

Keyword(s):

Prostate Cancer ◽

Retention Rate ◽

Xenograft Model ◽

Treatment Resistance ◽

Fold Increase ◽

Standard Uptake Value ◽

Retention Rates ◽

Castration Resistant Prostate Cancer ◽

Pet Ct

191 Background: Castration-resistant prostate cancer occurs in part due to increased tumor tissue testosterone (T) and dihydrotestosterone (DHT) that sustain tumor growth. T and DHT are converted to inactive T- and DHT-glucuronide (T/DHT-G) by uridine 5'-diphospho-glucuronosyltransferase family genes (UGT2B15 and UGT2B17) in glucuronidation-competent cells and excreted, but not in glucuronidation-deficient cells. Thus, low glucuronidation activity enables prostate tumors to preserve androgens, which increases hormone treatment resistance and may be detectable by functional imaging. Methods: We knocked out (KO) UGT2B15 and 17 in LNCaP cells and tested the DHT retention rate in the cell lines by pulse-chase using [3H]DHT as a probe. Free and DHT-G retention rates were separately determined after 5 to 60 min. To increase the signal difference between control and KO cells, we screened several ATP-binding cassette transporter inhibitors to block DHT-G excretion. We performed [18F]DHT PET/CT in castrated mice having a control and a KO xenograft on contralateral flanks ( n = 3). The ratio of the standard uptake value (SUV) in control to KO xenografts in each mouse was calculated. To increase the ratio between control and KO tumors, 50µg cyclosporin A (CSA) was injected 30 min before injecting [18F]DHT. Results: After 5 minutes of chase, control cells retained twice the DHT of KO cells. In control cells, 50%-70% DHT was glucuronidated. Almost no DHT-G was detected in KO cells, and free DHT was similar to control. Of the inhibitors, only CSA increased DHT-G (but not free DHT) in control cells, resulting in a 3-4-fold increase in overall signal. In vivo PET/CT showed control xenografts had higher peak SUV but also a higher elution rate. CSA increased the SUV ratio by 1.5-2. Conclusions: We developed a PET/CT modality to detect androgen inactivation in a prostate cancer xenograft model. Androgen-glucuronidation-proficient tumors give off a stronger signal that is increased by ATP transporter inhibition. Our method can provide a noninvasive means of determining androgen metabolism status and therefore could possibly predict effectiveness of potential therapies in a subgroup of tumors predisposed to androgen deprivation resistance.

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Co-expression With Replicating Vector Overcoming Competitive Effects Derived by a Companion Protease Inhibitor in Plants

Frontiers in Plant Science ◽

10.3389/fpls.2021.699442 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jiexue Ma ◽

Xiangzhen Ding ◽

Zhiying Li ◽

Sheng Wang

Keyword(s):

Protease Inhibitor ◽

Molecular Farming ◽

Expression System ◽

Fold Increase ◽

Protective Effects ◽

Competitive Effects ◽

Inoculum Dose ◽

Plant Expression Systems

Plant-based expression platforms are currently gaining acceptance as a viable alternative for the production of recombinant proteins (RPs), but the degradation of RPs by proteases in cells hinders their superb potentials. Co-expression of a protease inhibitor (PI) shows promise as a strategy to prevent RP from proteolytic degradation in plants. However, competitive effects behind the PI-RP co-expression system may mask or obfuscate the in situ protective effects of a companion PI. Here, we explored the competitive effects by co-expressing reteplase (rPA) with three unrelated PIs, namely NbPR4, HsTIMP, and SlCYS8, in Nicotiana benthamiana leaves. Remarkably, the accumulation of rPA was significantly repressed by each of the three PIs, suggesting that the competitive effects may be common among the PIs. The repression can be attenuated by reducing the PI inoculum dose in the co-inoculation mixtures, showing a negative correlation between the PI abundance of the PI-RP system and competitive effects. Interestingly, when a replicating vector was used to modulate the relative abundance of PI and RP in vivo, rPA was still boosted even at the maximal testing dose of PI, indicating that the competitive effects reduced to an ignorable level by this in vivo approach. Furthermore, a 7- to 12-fold increase of rPA was achieved, proving that it is a useful way for stimulating the potentials of a companion PI by overcoming competitive effects. And, this approach can be applied to molecular farming for improving the RP yields of plant expression systems.

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