bims-novged 2021-05-16 papers

on Non-viral vectors for gene delivery

Issue of 2021–05–16
eleven papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Dose-Independent Transfection of Hydrophobized Polyplexes.
Multifunctional Liposomes Enable Active Targeting and Twinfilin 1 Silencing to Reverse Paclitaxel Resistance in Brain Metastatic Breast Cancer.
Efficient tumor-targeting delivery of siRNA via folate-receptor mediated biomimetic albumin nanoparticles enhanced by all-trans retinoic acid.
Protein corona precoating on redox-responsive chitosan-based nano-carriers for improving the therapeutic effect of nucleic acid drugs.
Two-Photon Near-Infrared AIE Luminogens as Multifunctional Gene Carriers for Cancer Theranostics.
Tumor-Derived Exosomes Enriched by miRNA-124 Promote Anti-tumor Immune Response in CT-26 Tumor-Bearing Mice.
Green surfactant-dendrimer aggreplexes: An ingenious way to launch dual attack on arch-enemy cancer.
Harnessing subcellular-resolved organ distribution of cationic copolymer-functionalized fluorescent nanodiamonds for optimal delivery of active siRNA to a xenografted tumor in mice.
Design and Synthesis of Piperazine-Based Compounds Conjugated to Humanized Ferritin as Delivery System of siRNA in Cancer Cells.
Intra-articular silencing of periostin via nanoparticle-based siRNA ameliorates post-traumatic osteoarthritis in mice.
Recruitment of solid lipid nanoparticles for the delivery of CRISPR/Cas9: primary evaluation of anticancer gene editing.

Adv Mater. 2021 May 14. e2102219

Dose-Independent Transfection of Hydrophobized Polyplexes.

Zhen Zhang, Nasha Qiu, Shuling Wu, Xin Liu, Zhuxian Zhou, Jianbin Tang, Yanpeng Liu, Ruhong Zhou, Youqing Shen.

  Cationic polymers dynamically complex DNA into complexes (polyplexes). So, upon dilution, polyplexes easily dissociate and lose transfection ability, limiting their in vivo systemic gene delivery. Herein, it is found that polyplex's stability and endocytosis pathway determine its transfection dose-dependence. The polyplexes of hydrophilic polycations have dose-dependent integrity and lysosome-trafficking endocytosis; at low doses, most of these polyplexes dissociate, and the remaining few are internalized and trapped in lysosomes, abolishing their transfection ability. In contrast, the polyplexes of the polycations with optimal hydrophobicity remain integrated even at low concentrations and enter cells via macropinocytosis directly into the cytosol evading lysosomes, so each polyplex can accomplish its infection process, leading to dose-independent DNA transfection like viral vectors. Furthermore, the tuned hydrophobicity balancing the affinity of anionic poly(γ-glutamic acid) (γ-PGA) to the polyplex surface enables γ-PGA to stick on the polyplex surface as a shielding layer but peel off on the cell membrane to release the naked polyplexes for dose-independent transfection. These findings may provide guidelines for developing polyplexes that mimick a viral vector's dose-independent transfection for effective in vivo gene delivery.

Keywords:  cationic polymer polyplexes; dose-independent transfection; hydrophobicity effect; macropinocytosis pathway; non-viral gene delivery

DOI:  https://doi.org/10.1002/adma.202102219
ACS Appl Mater Interfaces. 2021 May 13.

Multifunctional Liposomes Enable Active Targeting and Twinfilin 1 Silencing to Reverse Paclitaxel Resistance in Brain Metastatic Breast Cancer.

Jiawei Du, Yong Shao, Yue Hu, Yiwen Chen, Jiehui Cang, Xin Chen, Wenqin Pei, Fengqin Miao, Yuqing Shen, Mohd Muddassir, Ying Zhang, Jianqiong Zhang, Gaojun Teng.

  Paclitaxel (PTX) is a first-line chemotherapeutic drug for breast cancer, but PTX resistance often occurs in metastatic breast cancer. In addition, due to the poor targeting of chemotherapeutic drugs and the presence of the blood-brain barrier (BBB), it is hard to effectively treat brain metastatic breast cancer using paclitaxel. Thus, it is urgent to develop an effective drug delivery system for the treatment of brain metastatic breast cancer. The current study found that TWF1 gene, an epithelial-mesenchymal transition-associated gene, was overexpressed in brain metastatic breast cancer (231-BR) cells and was associated with the PTX resistance of 231-BR cells. Knockdown of TWF1 by small interference RNA (siRNA) in 231-BR cells could effectively increase the sensitivity of brain metastatic breast cancer cells to paclitaxel. Then, a liposome-based drug delivery system was developed for PTX delivery across BBB, enhancing PTX sensitivity and brain metastases targeting via BRBP1 peptide modification. The results showed that BRBP1-modified liposomes could effectively cross the BBB, specifically accumulate in brain metastases, and effectively interfere TWF1 gene expression in vitro and in vivo, and thus they enhanced proliferation inhibition, cell cycle arrest, and apoptosis induction, thereby inhibiting the formation and growth of brain metastases. In summary, our results indicated that BRBP1-modified and PTX- and TWF1 siRNA-loaded liposomes have the potential for the treatment of brain metastatic breast cancer, which lays the foundation for the development of a new targeted drug delivery system.

Keywords:  breast cancer brain metastasis; drug resistance; liposome nanoparticles; paclitaxel; siRNA; twinfilin 1

DOI:  https://doi.org/10.1021/acsami.1c02822
Mater Sci Eng C Mater Biol Appl. 2021 Feb;pii: S0928-4931(20)33501-3. [Epub ahead of print]119 111583

Efficient tumor-targeting delivery of siRNA via folate-receptor mediated biomimetic albumin nanoparticles enhanced by all-trans retinoic acid.

Dandan Wang, Hui Li, Weiliang Chen, Han Yang, Yang Liu, Bengang You, Xuenong Zhang.

  Acute myeloid leukemia (AML) is the most universal type and fatal disease of hematological malignancy, with poor outcomes despite chemotherapy and bone marrow transplantations. Benefited from the narrow tissue specificity of folate receptor β (FRβ) aberrantly expressed on hematological linage cell lines, NPs modified with folate acid (FA) has been widely applied for crossing cell membrane barriers in FR-targeted therapies for AML. Thus, the biomimetic nanoparticles (NPs) mediated by FRβ were conducted by an albumin modifier as previously synthesized and cationic liposomes. However, how to further enhance the tumor-targeting and cellular uptake of NPs have been great challenges in cancer therapy. It was reported that FRβ could be selectively augmented by all-trans retinoic acid (ATRA). Herein, we demonstrated the enhanced active tumor-targeting of FA-modified siRNA-loaded biomimetic albumin NPs (Lip-S@FBH) could be achieved by upregulating FRβ expression via ATRA NPs. And the systematic administration of ATRA NPs significantly promoted endocytosis and thereby increased the intracellular concentration of Lip-S@FBH. This strategy combined the FRβ amplification effect with the effective delivery of siRNA, is mostly desirable for the AML-targeting therapy.

Keywords:  Acute myeloid leukemia; All-trans retinoic acid; Biomimetic albumin nanoparticles; Folate receptor up-regulation; siRNA

DOI:  https://doi.org/10.1016/j.msec.2020.111583
Carbohydr Polym. 2021 Aug 01. pii: S0144-8617(21)00458-6. [Epub ahead of print]265 118071

Protein corona precoating on redox-responsive chitosan-based nano-carriers for improving the therapeutic effect of nucleic acid drugs.

Han Yang, Tingting Liu, Yan Xu, Gaoxing Su, Tianqing Liu, Yanyan Yu, Bohui Xu.

  Spontaneous formation of protein corona on chitosan-based nano-carriers is inevitable once they enter the blood, which is considered to be an important factor that weakens the delivery efficiency and therapeutic effect of nucleic acid drugs. For this, cyclic RGDyK peptide (cRGD) modified bovine serum albumin (BSA) was designed as a corona to precoat on redox-responsive chitosan-based nano-carriers (TsR NPs) before administration. The effects of the precoating corona on the pharmaceutical properties and delivery efficiency of the nano-carriers and the therapeutic effect of model siRNA (siVEGF) were investigated. The results showed that BSA-cRGD formed steady corona around TsR NPs, which enhanced targeting ability to cancer cells and reduced serum proteins adsorption. The Bc corona improved the stability and biocompatibility of TsR NPs, increased the intracellular uptake, facilitated the lysosomal escape and maintained their redox-sensitive responsiveness, resulting in enhanced gene silencing efficiency and anti-tumor proliferation effects both in vitro and in vivo.

Keywords:  BSA-cRGD; Chitosan; Precoating; Protein corona; Redox-responsive; siRNA

DOI:  https://doi.org/10.1016/j.carbpol.2021.118071
ACS Appl Mater Interfaces. 2021 May 13.

Two-Photon Near-Infrared AIE Luminogens as Multifunctional Gene Carriers for Cancer Theranostics.

Fang Tang, Jin-Yu Liu, Cheng-Yan Wu, Ya-Xuan Liang, Zhong-Lin Lu, Ai-Xiang Ding, Ming-Di Xu.

  Construction of multifunctional nonviral gene vectors to execute defined tasks holds great potential for the precise and effective treatment of gene-associated diseases. Herein, we have developed four large π-conjugation triphenylamine derivatives bearing two polar [12]aneN3 heads and a lipophilic tail for applications in gene delivery, one/two-photon-triggered near-infrared (NIR) fluorescence bioimaging, and combined photodynamic therapy (PDT) and gene therapy of cancer. These compounds possess typical NIR aggregation-induced emission characteristics, mega Stokes shifts, strong two-photon excitation fluorescence, and excellent DNA condensation abilities. Among them, vector 4 with a tail of n-hexadecane realized a transfection efficiency as high as 6.7 times that of the commercial transfection agent Lipofectamine 2000 in HEK293T cell lines. Using vector 4 as an example, transfection process tracking and ex vivo/in vivo tumoral imaging and retention with high resolution, high brightness, deep tissue penetration, and good biosafety were demonstrated. In addition, efficient singlet oxygen (1O2) generation by the DNA complex formed by vector 4 (4/DNA) resulted in effective PDT. Combined with anticancer gene therapy, collaborative cancer treatment with a dramatically enhanced cancer cell-killing effect was achieved. The development of this "three birds, one stone" approach suggests a new and promising strategy for better cancer treatment and real-time tracking of gene delivery.

Keywords:  bioimaging; gene therapy; near-infrared fluorescence; nonviral gene vector; photodynamic therapy

DOI:  https://doi.org/10.1021/acsami.1c02600
Front Med (Lausanne). 2021 ;8 619939

Tumor-Derived Exosomes Enriched by miRNA-124 Promote Anti-tumor Immune Response in CT-26 Tumor-Bearing Mice.

Ramazan Rezaei, Kaveh Baghaei, Seyed Mahmoud Hashemi, Mohammad Reza Zali, Hossein Ghanbarian, Davar Amani.

  Exosomes have been introduced as a new alternative delivery system for the transmission of small molecules. Tumor-derived exosomes (TEXs) not only contain tumor-associated antigens to stimulate antitumor immune responses but also act as natural carriers of microRNAs. The aim of the current study was to evaluate the efficacy of miR-124-3p-enriched TEX (TEXomiR) as cell-free vaccine in the induction of antitumor immune responses in a mouse model of colorectal cancer. Briefly, the exosomes were isolated from cultured CT-26 cell line, and modified calcium chloride method was used to deliver miR-124-3p mimic into the exosomes. We used a CT-26-induced BALB/c mouse model of colorectal cancer and analyzed the effect of TEXomiR on survival, tumor size, spleen and tumor-infiltrated lymphocytes, and splenocyte proliferation. Furthermore, intra-tumor regulatory T cells, cytotoxic activity of the splenocytes, and cytokine secretion was also evaluated to describe the anti-tumor immune response. When the tumor size reached 100 mm3, the mice were injected with TEXomiR, TEX, and/or phosphate-buffered saline (PBS) subcutaneously three times with 3-day interval, and then tumor size was monitored every 2 days. The in vitro results indicated that TEXs could efficiently deliver functional miR-124-3p mimic. The in vivo evaluation in tumor-bearing mice showed that treatment with TEXomiR can elicit a stronger anti-tumor immune response than unloaded TEX and PBS. Significant tumor growth inhibition and increased median survival time was achieved in tumor-bearing mice treated with TEXomiR. A significant decrease in CD4/CD8 and Treg/CD8 ratio in tumor tissue was demonstrated. Moreover, increased cytotoxicity and proliferation of splenocytes in the TEXomiR group compared to the TEX and PBS groups were identified. Taken together, our data demonstrated that tumor-derived exosomes efficiently deliver miR-124-3p mimic, and TEXomiR promotes anti-tumor immune responses.

Keywords:   CT-26; MiR-124-3p; anti-tumor immune response; colon cancer; immunotherapy; tumor-derived exosomes

DOI:  https://doi.org/10.3389/fmed.2021.619939
Colloids Surf B Biointerfaces. 2021 May 05. pii: S0927-7765(21)00265-4. [Epub ahead of print]204 111821

Green surfactant-dendrimer aggreplexes: An ingenious way to launch dual attack on arch-enemy cancer.

Nilesh Malavia, Rohan Ghadi, Kaushik Kuche, Tushar Date, Nallamothu Bhargavi, Dasharath Chaudhari, Rajan Swami, Parmeshwar B Katare, Sanjay K Banerjee, Sanyog Jain.

  Combination therapy, which combines anti-cancer drugs with different oligonucleotides, have shown potential in cancer treatment. However, delivering a hydrophobic anti-cancer drug and a hydrophilic oligonucleotide simultaneously is a herculean task. This study takes advantage of interactions between histidine-lauric acid-based green surfactant and poly(amidoamine) dendrimers to achieve this aim. The green surfactant was synthesized by carbodiimide chemistry and characterized by FTIR, 1H-NMR, and mass spectroscopy. Further, green surfactant-dendrimer aggregates encapsulating DTX and complexing SIRT 1 shRNA i.e., "aggreplexes" were developed and characterized. The term "aggreplexes" signifies complexes which are formed between green-surfactant-dendrimer aggregates and SIRT-1 shRNA via electrostatic interaction. The aggreplexes displayed particle size of 262.33 ± 3.87 nm, PDI of 0.25 and entrapment efficiency of 70.56 %. The TEM images revealed spherical shape of aggreplexes with irregular outer surface and corroborated particle size obtained from zetasizer. The in-vitro release study revealed biphasic release patterns of DTX from aggreplexes and were compatible for intravenous administration. Further, aggreplexes augmented cellular uptake in MDA-MB-231 cells by ∼1.87-fold compared to free DTX. Also, EGFP expression revealed significantly higher transfection of aggreplexes compared to naked shRNA and Superfect™ complexes. Further, aggreplexes showed higher cytotoxicity in MDA-MB-231 cells and ∼4.16-fold reduction in IC50 value compared to free DTX. Finally, apoptosis-index observed in case of aggreplexes was ∼3.57-fold higher than free DTX. These novel aggreplexes showed increased drug loading capacity and superior gene transfection potential. Thus, they open new avenues for co-delivery of hydrophobic anti-cancer drugs and hydrophilic therapeutic genes for improving current standards of cancer therapy.

Keywords:  Aggreplexes; Dendrimer; Docetaxel; Green surfactant; shRNA

DOI:  https://doi.org/10.1016/j.colsurfb.2021.111821
Nanoscale. 2021 May 13.

Harnessing subcellular-resolved organ distribution of cationic copolymer-functionalized fluorescent nanodiamonds for optimal delivery of active siRNA to a xenografted tumor in mice.

Sandra Claveau, Marek Kindermann, Alexandre Papine, Zamira V Díaz-Riascos, Xavier Délen, Patrick Georges, Roser López-Alemany, Òscar Martínez Tirado, Jean-Rémi Bertrand, Ibane Abasolo, Petr Cigler, François Treussart.

Diamond nanoparticles (nanodiamonds) can transport active drugs in cultured cells as well as in vivo. However, in the latter case, methods allowing the determination of their bioavailability accurately are still lacking. A nanodiamond can be made fluorescent with a perfectly stable emission and a lifetime ten times longer than that of tissue autofluorescence. Taking advantage of these properties, we present an automated quantification method of fluorescent nanodiamonds (FND) in histological sections of mouse organs and tumors, after systemic injection. We use a home-made time-delayed fluorescence microscope comprising a custom pulsed laser source synchronized on the master clock of a gated intensified array detector. This setup allows ultra-high-resolution images (120 Mpixels in size) of whole mouse organ sections to be obtained, with subcellular resolution and single-particle sensitivity. As a proof-of-principle experiment, we quantified the biodistribution and aggregation state of new cationic FNDs capable of transporting small interfering RNA inhibiting the oncogene responsible for Ewing sarcoma. Image analysis showed a low yield of nanodiamonds in the tumor after intravenous injection. Thus, for the in vivo efficacy assay, we injected the nanomedicine into the tumor. We achieved a 28-fold inhibition of the oncogene. This method can readily be applied to other nanoemitters with ≈100 ns lifetime.

DOI: https://doi.org/10.1039/d1nr00146a
Bioconjug Chem. 2021 May 12.

Design and Synthesis of Piperazine-Based Compounds Conjugated to Humanized Ferritin as Delivery System of siRNA in Cancer Cells.

Natalia Pediconi, Francesca Ghirga, Cristina Del Plato, Giovanna Peruzzi, Constantinos M Athanassopoulos, Mattia Mori, Maria Elisa Crestoni, Davide Corinti, Franco Ugozzoli, Chiara Massera, Alessandro Arcovito, Bruno Botta, Alberto Boffi, Deborah Quaglio, Paola Baiocco.

Gene expression regulation by small interfering RNA (siRNA) holds promise in treating a wide range of diseases through selective gene silencing. However, successful clinical application of nucleic acid-based therapy requires novel delivery options. Herein, to achieve efficient delivery of negatively charged siRNA duplexes, the internal cavity of "humanized" chimeric Archaeal ferritin (HumAfFt) was specifically decorated with novel cationic piperazine-based compounds (PAs). By coupling these rigid-rod-like amines with thiol-reactive reagents, chemoselective conjugation was efficiently afforded on topologically selected cysteine residues properly located inside HumAfFt. The capability of PAs-HumAfFt to host and deliver siRNA molecules through human transferrin receptor (TfR1), overexpressed in many cancer cells, was explored. These systems allowed siRNA delivery into HeLa, HepG2, and MCF-7 cancer cells with improved silencing effect on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression with respect to traditional transfection methodologies and provided a promising TfR1-targeting system for multifunctional siRNA delivery to therapeutic applications.

DOI: https://doi.org/10.1021/acs.bioconjchem.1c00137
Arthritis Rheumatol. 2021 May 13.

Intra-articular silencing of periostin via nanoparticle-based siRNA ameliorates post-traumatic osteoarthritis in mice.

Xin Duan, Lei Cai, Christine T N Pham, Yousef Abu-Amer, Hua Pan, Robert H Brophy, Samuel A Wickline, Muhammad Farooq Rai.

   OBJECTIVE: Recent evidence delineates an emerging role of Periostin (Postn) in osteoarthritis (OA) as its expression subsequent to knee injury is detrimental to the articular cartilage. We hypothesize that intra-articular knockdown of Postn in a murine model of post-traumatic OA would ameliorate OA.
METHODS: Post-traumatic OA was induced in 10-week-old male C57BL/6J mice (n=24) by destabilization of the medial meniscus (DMM) and analyzed 8-week post-surgery. Intra-articular Postn was inhibited by siRNA using a novel peptide-nucleotide polyplex. Cartilage degeneration (OARSI score) and synovitis were assessed histologically. Bone changes were measured by μCT. The effect and mechanism of Postn silencing were investigated in human chondrocytes treated with IL-1β with or without IKK2 inhibitor, SC-514.
RESULTS: Peptide-siRNA nanoplatform significantly abolished Postn expression. OARSI score was significantly less in mice receiving Postn siRNA (10.94±0.66) compared to both untreated (22.38±1.30,P=0.002) and scrambled siRNA (22.69±0.87,P=0.002) treatment. No differences were observed in synovitis. Subchondral bone sclerosis, BV/TV, vBMD, and heterotopic ossification were significantly low in Postn siRNA treatment. Immunostaining of cartilage revealed that Postn knockdown reduced the DMM-induced MMP-13 intensity, phosphorylation of p65, and immunoreactivity of aggrecan neoepitope, DIPEN. Postn knockdown also suppressed IL-1β-induced MMP-13 and ADAMTS-4 in chondrocytes. Mechanistically, Postn-induced MMP-13 was abrogated by SC-514 demonstrating a link between Postn and NF-κB.
CONCLUSION: Intra-articular delivery of Postn siRNA nanocomplex represents a promising clinical approach to mitigate the severity of joint degeneration and provides an unequivocal scientific rationale for longitudinal studies. Employing a cartilage-specific gene knockout strategy will further illuminate the functional role of Postn in OA.

Keywords:  MMP-13; NF-κB; Periostin; chondrocytes; osteoarthritis

DOI:  https://doi.org/10.1002/art.41794
Nanomedicine (Lond). 2021 May 10.

Recruitment of solid lipid nanoparticles for the delivery of CRISPR/Cas9: primary evaluation of anticancer gene editing.

Hasan Akbaba, Gulsah Erel-Akbaba, Serif Senturk.

  Aim: The CRISPR/Cas9 system is a promising gene-editing tool for various anticancer therapies; however, development of a biocompatible, nonviral and efficient delivery of CRISPR/Cas9 expression systems remains a challenge. Materials & methods: Solid lipid nanoparticles (SLNs) were produced based on pseudo and 3D ternary plots. Obtained SLNs and their complexes with PX458 plasmid DNA were characterized and evaluated in terms of cytotoxicity and transfection efficiency. Results: SLNs were found to be nanosized, monodispersed, stable and nontoxic. Furthermore, they revealed similar transfection efficiency as the positive control. Conclusion: Overall, we have achieved a good SLN basis for CRISPR/Cas9 delivery and have the potential to produce SLNs with targeted anticancer properties by modifying production parameters and components to facilitate translating CRISPR/Cas9 into preclinical studies.

Keywords:  3D ternary plot; CRISPR/Cas9; PX458; gene delivery; solid lipid nanoparticle; transfection

DOI:  https://doi.org/10.2217/nnm-2020-0412