bims-novged 2022-02-20 papers

on Non-viral vectors for gene delivery

Issue of 2022–02–20
seventeen papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Incorporation of phosphatidylserine improves efficiency of lipid based gene delivery systems.
Co-delivery of doxorubicin and siRNA by all-trans retinoic acid conjugated chitosan-based nanocarriers for multiple synergistic antitumor efficacy.
Polycondensed Peptide Carriers Modified with Cyclic RGD Ligand for Targeted Suicide Gene Delivery to Uterine Fibroid Cells.
Loading of "cocktail siRNAs" into extracellular vesicles via TAT-DRBD peptide for the treatment of castration-resistant prostate cancer.
Delivery of siRNA based on engineered exosomes for glioblastoma therapy by targeting STAT3.
Poly-L-Lysine-Lactobionic Acid-Capped Selenium Nanoparticles for Liver-Targeted Gene Delivery.
Lung-selective mRNA delivery of synthetic lipid nanoparticles for the treatment of pulmonary lymphangioleiomyomatosis.
Successful Incorporation of Exosome-Capturing Antibody-siRNA Complexes into Multiple Myeloma Cells and Suppression of Targeted mRNA Transcripts.
Thermostable ionizable lipid-like nanoparticle (iLAND) for RNAi treatment of hyperlipidemia.
Bovine serum albumin-based biomimetic gene complexes with specificity facilitate rapid re-endothelialization for anti-restenosis.
Fast microwave heating-based one-step synthesis of DNA and RNA modified gold nanoparticles.
Hyaluronic acid-modified redox-sensitive hybrid nanocomplex loading with siRNA for non-small-cell lung carcinoma therapy.
Synergistic Silencing of Skp2 by siRNA Self-Assembled Nanoparticles as a Therapeutic Strategy for Advanced Prostate Cancer.
A targeting delivery system for effective genome editing in leukemia cells to reverse malignancy.
An mTOR siRNA-loaded Spermidine/DNA Tetrahedron Nanoplatform with a Synergistic Anti-Inflammatory Effect on Acute Lung Injury.
Micrometric DNA/PEI polyplexes correlate with higher transient gene expression yields in HEK 293 cells.
Intelligent Gold Nanoparticles with Oncogenic MicroRNA-dependent Activities to Manipulate Tumorigenic Environments for Synergistic Tumor Therapy.

Eur J Pharm Biopharm. 2022 Feb 15. pii: S0939-6411(22)00028-5. [Epub ahead of print]

Incorporation of phosphatidylserine improves efficiency of lipid based gene delivery systems.

Claudia Lotter, Claudio Luca Alter, Jan Stephan Bolten, Pascal Detampel, Cornelia G Palivan, Tomaž Einfalt, Jörg Huwyler.

  The essential homeostatic process of dead cell clearance (efferocytosis) is used by viruses in an act of apoptotic mimicry. Among others, virions leverage phosphatidylserine (PS) as an essential "eat me" signal in viral envelopes to increase their infectivity. In a virus-inspired biomimetic approach, we demonstrate that PS can be incorporated into non-viral lipid nanoparticle (LNP) pDNA/mRNA constructs to enhance cellular transfection. The inclusion of the bioactive PS leads to an increased ability of LNPs to deliver nucleic acids invitro to cultured HuH-7 hepatocellular carcinoma cells resulting in a 6-fold enhanced expression of a transgene. Optimal PS concentrations are in the range of 2.5 to 5% of total lipids. PS-decorated mRNA-LNPs show a 5.2-fold enhancement of invivo transfection efficiency as compared to mRNA-LNPs devoid of PS. Effects were less pronounced for PS-decorated pDNA-LNPs (3.2-fold increase). Incorporation of small, defined amounts of PS into gene delivery vectors opens new avenues for efficient gene therapy and can be easily extended to other therapeutic systems.

Keywords:  Lipid nanoparticles; lipid composition; nucleic acid delivery; phosphatidylserine; transfection efficiency; transfection potency

DOI:  https://doi.org/10.1016/j.ejpb.2022.02.007
Carbohydr Polym. 2022 May 01. pii: S0144-8617(22)00001-7. [Epub ahead of print]283 119097

Co-delivery of doxorubicin and siRNA by all-trans retinoic acid conjugated chitosan-based nanocarriers for multiple synergistic antitumor efficacy.

Chang Liu, Cui Tang, Chunhua Yin.

  To achieve the co-delivery of doxorubicin (DOX) and small interfering RNA (siRNA) targeting B-cell lymphoma 2 (siBcl-2), lactose acid (LA) and all-trans retinoic acid (ATRA) double grafted N,N,N-trimethyl chitosan (TMC) nanoparticles (GTA NPs) were developed. The relative viability of QGY-7703 cells was decreased to 81.3% when the concentration of GTA NPs was 0.1 mg/mL, but no toxicity to normal cells was observed, indicating that the GTA NPs selectively inhibited the proliferation of tumor cells. With DOX loaded into the hydrophobic core and siRNA condensed onto the hydrophilic shell, GTA/DOX/siRNA NPs were prepared. The GTA/DOX/siRNA NPs possessed high cellular uptake via receptor-mediated endocytosis. Owing to multiple cooperative antitumor effects of DOX, siBcl-2, and GTA NPs, GTA/DOX/siRNA NPs had superior in vitro and in vivo antitumor efficiency to other formulations. These findings provide a guideline for the combined applications of multiple synergistic antitumor manners.

Keywords:  All-trans retinoic acid; B-cell lymphoma 2 siRNA; Chitosan; Doxorubicin; Synergistic antitumor treatment

DOI:  https://doi.org/10.1016/j.carbpol.2022.119097
Int J Mol Sci. 2022 Jan 21. pii: 1164. [Epub ahead of print]23(3):

Polycondensed Peptide Carriers Modified with Cyclic RGD Ligand for Targeted Suicide Gene Delivery to Uterine Fibroid Cells.

Anna Egorova, Sofia Shtykalova, Marianna Maretina, Alexander Selutin, Natalia Shved, Dmitriy Deviatkin, Sergey Selkov, Vladislav Baranov, Anton Kiselev.

  Suicide gene therapy was suggested as a possible strategy for the treatment of uterine fibroids (UFs), which are the most common benign tumors inwomen of reproductive age. For successful suicide gene therapy, DNAtherapeutics should be specifically delivered to UF cells. Peptide carriers are promising non-viral gene delivery systems that can be easily modified with ligands and other biomolecules to overcome DNA transfer barriers. Here we designed polycondensed peptide carriers modified with a cyclic RGD moiety for targeted DNA delivery to UF cells. Molecular weights of the resultant polymers were determined, and inclusion of the ligand was confirmed by MALDI-TOF. The physicochemical properties of the polyplexes, as well as cellular DNA transport, toxicity, and transfection efficiency were studied, and the specificity of αvβ3 integrin-expressing cell transfection was proved. The modification with the ligand resulted in a three-fold increase of transfection efficiency. Modeling of the suicide gene therapy by transferring the HSV-TK suicide gene to primary cells obtained from myomatous nodes of uterine leiomyoma patients was carried out. We observed up to a 2.3-fold decrease in proliferative activity after ganciclovir treatment of the transfected cells. Pro- and anti-apoptotic gene expression analysis confirmed our findings that the developed polyplexes stimulate UF cell death in a suicide-specific manner.

Keywords:  DNA delivery; gene therapy; integrins; peptide-based carriers; polycondensation; thymidine kinase; uterine fibroids

DOI:  https://doi.org/10.3390/ijms23031164
Cancer Biol Ther. 2022 Dec 31. 23(1): 163-172

Loading of "cocktail siRNAs" into extracellular vesicles via TAT-DRBD peptide for the treatment of castration-resistant prostate cancer.

Yanjun Diao, Gangqiang Wang, Bingbing Zhu, Zhuo Li, Shan Wang, Lijuan Yu, Rui Li, Weixiao Fan, Yue Zhang, Lei Zhou, Liu Yang, Xiaoke Hao, Jiayun Liu.

  Extracellular vesicles (EVs) are cell-derived, membranous nanoparticles that mediate intercellular communication by transferring biomolecules between cells. As natural vehicles, EVs may exhibit higher delivery efficiency, lower immunogenicity, and better compatibility than existing RNA carriers. A major limitation of their therapeutic use is the shortage of efficient, robust, and scalable methods to load siRNA of interest. Here, we report a novel strategy using polycationic membrane-penetrating peptide TAT to encapsulate siRNAs into EVs. Three TAT peptides were co-expressed with DRBD as 3TD fusion protein. The sequence-independent binding of DRBD facilitates multiplex genes targeting of mixed siRNAs. Functional assays for siRNA-mediated gene silencing of CRPC were performed after engineered EVs treatment. EVs were isolated using differential centrifugation from WPMY-1 cell culture medium. The increase of merged yellow fluorescence in the engineered EVs showed by TIRFM and the decrease in zeta potential absolute values certified the co-localization of siRNA with EVs, which indicated that siRNA had been successfully delivered into WPMY-1 EVs. qRT-PCR analysis revealed that the mRNA level of FLOH1, NKX3, and DHRS7 was dramatically decreased when cells were treated with engineered EVs loaded with siRNAs mixtures relative to the level of untreated cells. Western and flow cytometry results indicate that delivery of siRNA mixtures by engineered EVs can effectively downregulate AR expression and induce LNCaP-AI cell apoptosis. The uptake efficiency of the EVs and the significantly downregulated expression of three genes suggested the potential of TAT as efficient siRNA carriers by keeping the function of the cargoes.

Keywords:  Prostate cancer; extracellular vesicles; hormonal resistance; multi-targets therapy; siRNA delivery

DOI:  https://doi.org/10.1080/15384047.2021.2024040
Biomater Sci. 2022 Feb 18.

Delivery of siRNA based on engineered exosomes for glioblastoma therapy by targeting STAT3.

Shi-Fu Liang, Fang-Fang Zuo, Bin-Cheng Yin, Bang-Ce Ye.

Small interfering RNA (siRNA) therapy has been considered as a promising strategy for treatment of glioblastoma (GBM), which is an aggressive brain disease with poor prognosis. However, siRNA therapy for GBM is seriously hindered by a multitude of barriers including possible immunogenicity, poor cellular uptake, short blood circulation, poor blood stability and low blood-brain barrier (BBB) penetration. This paper reports Angiopep-2 (An2)-functionalized signal transducers and activators of transcription 3 (STAT3) siRNA-loaded exosomes (Exo-An2-siRNA) as potential therapeutic agents to improve GBM therapy. The experimental results indicate that Exo-An2-siRNA displays high blood stability, efficient cellular uptake, and outstanding BBB penetration ability. Exo-An2-siRNA also exhibits excellent in vitro anti-GBM therapeutic effects due to the exosomes for siRNA protection and An2 modification for GBM targeting and BBB penetration. Such superior properties of Exo-An2-siRNA are responsible for favorable inhibition of the proliferation of orthotopic U87MG xenografts with limited side effects, significantly enhancing the median survival time (MST) of U87MG-bearing nude mice. The developed siRNA therapy featuring An2-functionalized exosomes as nanoplatforms is a safe and effective GBM treatment strategy.

DOI: https://doi.org/10.1039/d1bm01723c
Int J Mol Sci. 2022 Jan 27. pii: 1492. [Epub ahead of print]23(3):

Poly-L-Lysine-Lactobionic Acid-Capped Selenium Nanoparticles for Liver-Targeted Gene Delivery.

Shaigan Naidoo, Aliscia Daniels, Saffiya Habib, Moganavelli Singh.

  Liver cancer is currently regarded as the second leading cause of cancer-related mortality globally and is the sixth most diagnosed malignancy. Selenium nanoparticles (SeNPs) have attracted favorable attention as nanocarriers for gene therapy, as they possess beneficial antioxidant and anticancer properties. This study aimed to design, functionalize and characterize SeNPs to efficiently bind, protect and deliver pCMV-Luc DNA to hepatocellular carcinoma (HepG2) cells. The SeNPs were synthesized by ascorbic acid reduction and functionalized with poly-L-lysine (PLL) to stabilize and confer positive charges to the nanoparticles. The SeNPs were further decorated with lactobionic acid (LA) to target the asialoglycoprotein receptors abundantly expressed on the surface of the hepatocytes. All SeNPs were spherical, in the nanoscale range (<130 nm) and were capable of successfully binding, compacting and protecting the pDNA against nuclease degradation. The functionalized SeNP nanocomplexes exhibited minimal cytotoxicity (<30%) with enhanced transfection efficiency in the cell lines tested. Furthermore, the targeted SeNP (LA-PLL-SeNP) nanocomplex showed significant (* p < 0.05, ** p < 0.01, **** p < 0.0001) transgene expression in the HepG2 cells compared to the receptor-negative embryonic kidney (HEK293) cells, confirming receptor-mediated endocytosis. Overall, these functionalized SeNPs exhibit favorable features of suitable gene nanocarriers for the treatment of liver cancer.

Keywords:  gene expression; hepatocellular carcinoma; lactobionic acid; nanomedicine; poly-L-lysine; selenium nanoparticles

DOI:  https://doi.org/10.3390/ijms23031492
Proc Natl Acad Sci U S A. 2022 Feb 22. pii: e2116271119. [Epub ahead of print]119(8):

Lung-selective mRNA delivery of synthetic lipid nanoparticles for the treatment of pulmonary lymphangioleiomyomatosis.

Min Qiu, Yan Tang, Jinjin Chen, Rachel Muriph, Zhongfeng Ye, Changfeng Huang, Jason Evans, Elizabeth P Henske, Qiaobing Xu.

  Safe and efficacious systemic delivery of messenger RNA (mRNA) to specific organs and cells in vivo remains the major challenge in the development of mRNA-based therapeutics. Targeting of systemically administered lipid nanoparticles (LNPs) coformulated with mRNA has largely been confined to the liver and spleen. Using a library screening approach, we identified that N-series LNPs (containing an amide bond in the tail) are capable of selectively delivering mRNA to the mouse lung, in contrast to our previous discovery that O-series LNPs (containing an ester bond in the tail) that tend to deliver mRNA to the liver. We analyzed the protein corona on the liver- and lung-targeted LNPs using liquid chromatography-mass spectrometry and identified a group of unique plasma proteins specifically absorbed onto the surface that may contribute to the targetability of these LNPs. Different pulmonary cell types can also be targeted by simply tuning the headgroup structure of N-series LNPs. Importantly, we demonstrate here the success of LNP-based RNA therapy in a preclinical model of lymphangioleiomyomatosis (LAM), a destructive lung disease caused by loss-of-function mutations in the Tsc2 gene. Our lung-targeting LNP exhibited highly efficient delivery of the mouse tuberous sclerosis complex 2 (Tsc2) mRNA for the restoration of TSC2 tumor suppressor in tumor and achieved remarkable therapeutic effect in reducing tumor burden. This research establishes mRNA LNPs as a promising therapeutic intervention for the treatment of LAM.

Keywords:  lipid nanoparticles; lung-targeted delivery; lymphangioleiomyomatosis; mRNA; tuberous sclerosis complex

DOI:  https://doi.org/10.1073/pnas.2116271119
Cancers (Basel). 2022 Jan 23. pii: 566. [Epub ahead of print]14(3):

Successful Incorporation of Exosome-Capturing Antibody-siRNA Complexes into Multiple Myeloma Cells and Suppression of Targeted mRNA Transcripts.

Emi Soma, Asako Yamayoshi, Yuki Toda, Yuji Mishima, Shigekuni Hosogi, Eishi Ashihara.

  Nucleic acid medicines have been developed as new therapeutic agents against various diseases; however, targeted delivery of these reagents into cancer cells, particularly hematologic cancer cells, via systemic administration is limited by the lack of efficient and cell-specific delivery systems. We previously demonstrated that monoclonal antibody (mAb)-oligonucleotide complexes targeting exosomal microRNAs with linear oligo-D-arginine (Arg) linkers were transferred into solid cancer cells and inhibited exosomal miRNA functions. In this study, we developed exosome-capturing anti-CD63 mAb-conjugated small interfering RNAs (siRNAs) with branched Arg linkers and investigated their effects on multiple myeloma (MM) cells. Anti-CD63 mAb-conjugated siRNAs were successfully incorporated into MM cells. The incorporation of exosomes was inhibited by endocytosis inhibitors. We also conducted a functional analysis of anti-CD63 mAb-conjugated siRNAs. Ab-conjugated luciferase+ (luc+) siRNAs significantly decreased the luminescence intensity in OPM-2-luc+ cells. Moreover, treatment with anti-CD63 mAb-conjugated with MYC and CTNNB1 siRNAs decreased the mRNA transcript levels of MYC and CTNNB1 to 52.5% and 55.3%, respectively, in OPM-2 cells. In conclusion, exosome-capturing Ab-conjugated siRNAs with branched Arg linkers can be effectively delivered into MM cells via uptake of exosomes by parental cells. This technology has the potential to lead to a breakthrough in drug delivery systems for hematologic cancers.

Keywords:  CD63; antibody; drug delivery systems; exosome; hematologic malignancy; multiple myeloma; nucleic acid medicine; siRNA

DOI:  https://doi.org/10.3390/cancers14030566
Sci Adv. 2022 Feb 18. 8(7): eabm1418

Thermostable ionizable lipid-like nanoparticle (iLAND) for RNAi treatment of hyperlipidemia.

Bo Hu, Bo Li, Kun Li, Yuanyuan Liu, Chunhui Li, Lulu Zheng, Mengjie Zhang, Tongren Yang, Shuai Guo, Xiyu Dong, Tian Zhang, Qing Liu, Abid Hussain, Yuhua Weng, Ling Peng, Yongxiang Zhao, Xing-Jie Liang, Yuanyu Huang.

Small interfering RNA (siRNA) therapeutic is considered to be a promising modality for the treatment of hyperlipidemia. Establishment of a thermostable clinically applicable delivery system remains a most challenging issue for siRNA drug development. Here, a series of ionizable lipid-like materials were rationally designed; 4 panels of lipid formulations were fabricated and evaluated on the basis of four representative structures. The lead lipid (A1-D1-5) was stable at 40°C, and the optimized formulation (iLAND) showed dose and time dual-dependent gene silencing pattern with median effective dose of 0.18 mg/kg. In addition, potent and durable reduction of serum cholesterol and triglyceride were achieved by administering siRNAs targeting angiopoietin-like 3 or apolipoprotein C3 (APOC3) in high-fat diet-fed mice, db/db mice, and human APOC3 transgenic mice, respectively, accompanied by displaying ideal safety profiles. Therefore, siRNA@iLAND prepared with thermostable A1-D1-5 demonstrates substantial value for siRNA delivery, hyperlipidemia therapy, and prevention of subsequent metabolic diseases.

DOI: https://doi.org/10.1126/sciadv.abm1418
Acta Biomater. 2022 Feb 10. pii: S1742-7061(22)00081-2. [Epub ahead of print]

Bovine serum albumin-based biomimetic gene complexes with specificity facilitate rapid re-endothelialization for anti-restenosis.

Xuefang Hao, Weiwei Gai, Feng Ji, Jiadi Zhao, Dandan Sun, Fan Yang, Haixia Jiang, Yakai Feng.

  Re-endothelialization is a critical problem to inhibit postoperative restenosis, and gene delivery exhibits great potential in rapid endothelialization. Unfortunately, the therapeutic effect is enormously limited by inefficient specificity, poor biocompatibility and in vivo stability owing largely to the complicated in vivo environment. Herein, we developed a series of platelet membrane (PM) cloaked gene complexes based on natural bovine serum albumin (BSA) and polyethyleneimine (PEI). The gene complexes aimed to accelerate re-endothelialization for anti-restenosis via pcDNA3.1-VEGF165 (VEGF) plasmid delivery. Based on BSA and PM coating, these gene complexes exhibited good biocompatibility, stability with serum and robust homing to endothelium-injured site inherited from platelets. Besides, they enhanced the expression of VEGF protein by their high internalization and nucleus accumulation efficiency, and also substantially promoted migration and proliferation of vascular endothelial cells. The biological properties were further optimized via altering PEI and PM content. Finally, rapid recovery of endothelium in a carotid artery injured mouse model (79% re-endothelialization compared with model group) was achieved through two weeks' treatment by the PM cloaked gene complexes. High level of expressed VEGF in vivo was also realized by the gene complexes. Moreover, neointimal hyperplasia (IH) was significantly inhibited by the gene complexes according to in vivo study. The results verified the great potential of the PM cloaked gene complexes in re-endothelialization for anti-restenosis. STATEMENT OF SIGNIFICANCE: Rapid re-endothelialization is a major challenge to inhibit postoperative restenosis. Herein, a series of biodegradable and biocompatible platelet membrane (PM) cloaked gene complexes were designed to accelerate re-endothelialization for anti-restenosis via pcDNA3.1-VEGF165 (VEGF) plasmid delivery. The PM cloaked gene complexes provided high VEGF expression in vascular endothelial cells (VECs), rapid migration and proliferation of VECs and robust homing to endothelium-injured site. In a carotid artery injured mouse model, PM cloaked gene complexes significantly promoted VEGF expression in vivo, accelerated re-endothelialization and inhibited neointimal hyperplasia due to their good biocompatibility and superior specificity. Overall, the optimized PM cloaked gene complexes overcomes multiple obstacles in gene delivery for re-endothelialization and can be a promising candidate for gene delivery and therapy of postoperative restenosis.

Keywords:  Anti-restenosis; Gene complexes; Platelet membrane; Re-endothelialization

DOI:  https://doi.org/10.1016/j.actbio.2022.02.005
Nat Commun. 2022 Feb 18. 13(1): 968

Fast microwave heating-based one-step synthesis of DNA and RNA modified gold nanoparticles.

Mengqi Huang, Erhu Xiong, Yan Wang, Menglu Hu, Huahua Yue, Tian Tian, Debin Zhu, Hong Liu, Xiaoming Zhou.

DNA/RNA-gold nanoparticle (DNA/RNA-AuNP) nanoprobes have been widely employed for nanobiotechnology applications. Here, we discover that both thiolated and non-thiolated DNA/RNA can be efficiently attached to AuNPs to achieve high-stable spherical nucleic acid (SNA) within minutes under a domestic microwave (MW)-assisted heating-dry circumstance. Further studies show that for non-thiolated DNA/RNA the conjugation is poly (T/U) tag dependent. Spectroscopy, test strip hybridization, and loading counting experiments indicate that low-affinity poly (T/U) tag mediates the formation of a standing-up conformation, which is distributed in the outer layer of SNA structure. In further application studies, CRISPR/Cas9-sgRNA (136 bp), SARS-CoV-2 RNA fragment (1278 bp), and rolling circle amplification (RCA) DNA products (over 1000 bp) can be successfully attached on AuNPs, which overcomes the routine methods in long-chain nucleic acid-AuNP conjugation, exhibiting great promise in biosensing and nucleic acids delivery applications. Current heating-dry strategy has improved traditional DNA/RNA-AuNP conjugation methods in simplicity, rapidity, cost, and universality.

DOI: https://doi.org/10.1038/s41467-022-28627-8
Drug Deliv. 2022 Dec;29(1): 574-587

Hyaluronic acid-modified redox-sensitive hybrid nanocomplex loading with siRNA for non-small-cell lung carcinoma therapy.

Daoyuan Chen, Peng Zhang, Minghui Li, Congcong Li, Xiaoyan Lu, Yiying Sun, Kaoxiang Sun.

  A novel hyaluronic acid (HA)-modified hybrid nanocomplex HA-SeSe-COOH/siR-93C@PAMAM, which could efficiently deliver siRNA into tumor cells via a redox-mediated intracellular disassembly, was constructed for enhanced antitumor efficacy. Thereinto, siR-93C (siRNA) and positive PAMAM were firstly mixed into the electrostatic nano-intermediate, and then diselenide bond (-SeSe-)-modified HA was coved to shield excessive positive charges. This hybrid nanocomplex displayed uniform dynamic sizes, high stability, controlled zeta potential and narrow PDI distribution. Moreover, the -SeSe- linkage displayed GSH/ROS dual responsive properties, improving intracellular trafficking of siRNA. In vitro assays in A549 cell line presented that HA-SeSe-COOH/siR-93C@PAMAM has low cytotoxicity, rapid lysosomal escape and significant transfection efficiency; besides, an efficient proliferation inhibition ability and enhanced apoptosis. Furthermore, in animal studies, this negative-surfaced hybrid nanocomplex showed a prolonged circulation in blood and improved inhibition of tumor growth. All these results verified our hypothesis in this study that diselenide bonds-modified HA could promote not only stability and safety of nanoparticles in vivo but also intracellular behavior of siRNA via redox-dual sensitive properties; furthermore, this hybrid nanocomplex provided a visible potential approach for siRNA delivery in the antitumor field.

Keywords:  Non-viral gene vector; antitumor; redox-sensitive nanocomplex; safety; siRNA

DOI:  https://doi.org/10.1080/10717544.2022.2032874
Small. 2022 Feb 19. e2106046

Synergistic Silencing of Skp2 by siRNA Self-Assembled Nanoparticles as a Therapeutic Strategy for Advanced Prostate Cancer.

Hong Liang, Fangming Zhang, Yannv Hong, Yue Wu, Huanzhang Xie, Chen Zhang, Zonghua Wang, Zhonglei Lu, Huanghao Yang.

  Advanced prostate cancer, harboring multiple mutations of tumor suppressor genes, is refractory to conventional therapies. Knockout of the Skp2 gene blocks pRb/p53 doubly deficient prostate cancer in mice, which inspired the authors to develop an approach for delivering siRNA that would efficiently silence Skp2 (siSkp2) in vivo. Here, a facile strategy is reported to directly assemble siSkp2 with the natural compound quercetin (Que) into supramolecular nanoparticles (NPs). This carrier-free siSkp2 delivery system could effectively protect siSkp2 from degradation in serum and enhance its cellular internalization. Furthermore, the siSkp2/Que NPs exhibit synergistic effects in Skp2 silencing, because they can degrade the mRNA and protein of Skp2 simultaneously. Indeed, siSkp2/Que NPs remarkably diminish the Skp2 abundance and further inhibit the proliferation and migration of TMU cells (RB1/TP53/KRAS triple mutations) in vitro. The in vivo results further show that i.v. administration of siSkp2/Que NPs efficiently accumulates in tumor sites and strongly inhibits the growth of TMU tumors in nude mice. Importantly, the siSkp2/Que NPs do not induce any abnormality in the treated mice, which suggests satisfactory biocompatibility. Collectively, this study describes a tractable siRNA self-assembled strategy for Skp2 silencing, which might be a promising nanodrug to cure multitherapy-resistant advanced prostate cancer.

Keywords:  Skp2; advanced prostate cancer; nanodrugs; self-assembly; siRNA delivery

DOI:  https://doi.org/10.1002/smll.202106046
J Control Release. 2022 Feb 11. pii: S0168-3659(22)00086-4. [Epub ahead of print]

A targeting delivery system for effective genome editing in leukemia cells to reverse malignancy.

Xiao-He Ren, Chang Xu, Li-Li Li, Yan Zuo, Di Han, Xiao-Yan He, Si-Xue Cheng.

  Therapy resistance associated with relapse is a main cause of death in acute myeloid leukemia (AML). To address this issue, a dual-targeting CRISPR-Cas9 genome editing nanosystem was constructed for CXCR4 knockout to reverse the malignancy of leukemia cells. The surface of the dual-targeting nanosystem is composed of MUC1 specific aptamer incorporated alginate (MUC1 aptamer-alginate) and T22-NLS peptide with T22 sequence targeting CXCR4; the core of the nanosystem consists of protamine complexed with CRISPR-Cas9 plasmid. The in vitro study shows that the nanosystem mediated genome editing induces cell apoptosis, cell cycle arrest, as well as inhibited cell migration and adhesion in edited THP-1 cells after CXCR4 knockout. Further, the unprocessed peripheral blood from acute myeloid leukemia (AML) patients was directly used to carry out ex vivo study. The results show genome editing nanosystem can effectively knock out CXCR4 in leukemia cells, leading to attenuated CXCR4 protein as studied by antibody labeling and reduced CXCR4 mRNA as probed by a molecular beacon delivery system. In addition to developing a promising delivery vector for gene therapy on AML, this study also provides an effective strategy to evaluate the therapeutic efficiency of particular treatments by peripheral blood-based ex vivo studies.

Keywords:  Acute myeloid leukemia; CRISPR-Cas9; CXCR4; Delivery vector; Genome editing; Leukemia targeting

DOI:  https://doi.org/10.1016/j.jconrel.2022.02.012
Adv Healthc Mater. 2022 Feb 15. e2200008

An mTOR siRNA-loaded Spermidine/DNA Tetrahedron Nanoplatform with a Synergistic Anti-Inflammatory Effect on Acute Lung Injury.

Chaowang Huang, Qianyi You, Jing Xu, Di Wu, Huaping Chen, Yuhang Guo, Jiancheng Xu, Mingdong Hu, Hang Qian.

  Acute lung injury (ALI) is characterized by severe inflammation and damage to the lung air-blood barrier, resulting in respiratory function damage and life-threatening outcomes. Macrophage polarization plays an essential role in the occurrence, development, and outcome of ALI. As drug carriers, self-assembled DNA nanostructures can potentially overcome the drawbacks and limitations of traditional anti-inflammatory agents owing to their nontoxicity, programmability, and excellent structural control at the nanoscale. In this study, we proposed and constructed an siRNA and drug dual therapy nanoplatform to combat ALI. The nanoplatform consisted of a spermidine-assembled DNA tetrahedron and four mTOR siRNAs. Spermidine serves as a mediator of drug delivery vehicle synthesis and a drug that alters macrophage polarization. Both spermidine and siRNA exerted anti-inflammatory effects in vitro and in vivo by regulating the macrophage phenotype. More importantly, these factors exhibited a synergistic anti-inflammatory effect by promoting macrophage autophagy. For the first time, an anti-inflammatory dual therapy strategy that uses self-assembled DNA nanostructures as nontoxic, programmable delivery vehicles has been proposed and demonstrated through this work. Future work on utilizing DNA nanostructures for the treatment of noncancerous diseases such as ALI is highly promising and desirable. This article is protected by copyright. All rights reserved.

Keywords:  Acute lung injury; DNA nanostructure; mTOR siRNA; macrophage; spermidine

DOI:  https://doi.org/10.1002/adhm.202200008
N Biotechnol. 2022 Feb 10. pii: S1871-6784(22)00012-7. [Epub ahead of print]68 87-96

Micrometric DNA/PEI polyplexes correlate with higher transient gene expression yields in HEK 293 cells.

Irene González-Domínguez, Eduard Puente-Massaguer, Jesús Lavado-García, Laura Cervera, Francesc Gòdia.

  DNA delivery with polyethylenimine (PEI) has been widely used in the last three decades for the transfection of mammalian cells. Advances in novel characterization techniques at the nanometric scale offer new opportunities to revisit the physicochemical properties of DNA/PEI polyplexes that lead to efficient transfection. In this work, these properties are tuned by studying the synergies between simple parameters such as NaCl concentration, pH and incubation time in the DNA/PEI polyplex preparation protocol by means of Design of Experiments (DoE). By doing so, a model is obtained where an optimal NaCl concentration of 125 mM and an incubation time of 11 min provided the highest transfection yields. Correlation analyses between the physicochemical properties of DNA/PEI polyplexes and the predicted model responses revealed the existence of an optimal degree of aggregation in the pre-complexing solution to attain the highest transfection efficiencies. The presence of these micrometric DNA/PEI polyplex aggregates was confirmed by several nanoparticle characterization techniques including cryo-TEM, DLS and flow virometry. The findings provide a better understanding of the role of DNA/PEI aggregates in transient gene expression approaches, in particular considering that similar complexation protocols and saline solutions are widely used for the transfection of mammalian cell cultures.

Keywords:  Cryogenic transmission electron microscopy (Cryo-TEM); DNA delivery; Design of Experiments (DoE); Flow virometry; Polyethylenimine (PEI); Polyplexes

DOI:  https://doi.org/10.1016/j.nbt.2022.02.002
Adv Mater. 2022 Feb 15. e2110219

Intelligent Gold Nanoparticles with Oncogenic MicroRNA-dependent Activities to Manipulate Tumorigenic Environments for Synergistic Tumor Therapy.

Xiangdong Wang, Tianfeng Yang, Zhi Yu, Tao Liu, Ronghua Jin, Lin Weng, Yongkang Bai, J Justin Gooding, Yanmin Zhang, Xin Chen.

  Tumorigenic environments, especially aberrantly over-expressed oncogenic microRNAs, play critical role in various activities of tumor progression. However, developing strategies to effectively utilize and manipulate these oncogenic microRNAs for tumor therapy is still a challenge. To address this challenge, we have fabricated spherical nucleic acids (SNAs) consisting of gold nanoparticles in the core and antisense oligonucleotides as the shell. Hybridized to the oligonucleotide shell was a DNA sequence to which doxorubicin was conjugated (DNA-DOX). The oligonucleotides shell was designed to capture over-expressed miR-21/miR-155 and inhibit the expression of these oncogenic miRNAs in tumor cells after tumor accumulation to manipulate genetic environment for accurate gene therapy. This process would further induce the aggregation of these SNAs, which not only generated photothermal agents to achieve on-demand photothermal therapy in situ, but enlarged the size of SNAs to enhance the retention time in tumor for sustained therapy. The capturing of the relevant miRNAs simultaneously triggered the intracellular release of the DNA-DOX from the SNAs to deliver tumor-specific chemotherapy. Both in vivo and in vitro results indicated that this combination strategy has excellent tumor inhibition properties with high survival rate of tumor-bearing mice, which could be a promising candidate for effective tumor treatment. This article is protected by copyright. All rights reserved.

Keywords:  Capture and Inhibition of Oncogenic MicroRNAs (miR-155/miR-21); Intelligent Gold Nanoparticles; Utilization and Manipulation of Tumorigenic Environments; miR-155 Triggered Chemotherapy; miR-21 Dependent Photothermal Therapy

DOI:  https://doi.org/10.1002/adma.202110219