bims-novged 2022-04-10 papers

on Non-viral vectors for gene delivery

Issue of 2022–04–10
eleven papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Structural Exploration of Polycationic Nanoparticles for siRNA Delivery.
Reactive Oxygen Species─Responsive Lipid Nanoparticles for Effective RNAi and Corneal Neovascularization Therapy.
Importance of Process Parameters Influencing the Mean Diameters of siRNA-Containing Lipid Nanoparticles (LNPs) on the in Vitro Activity of Prepared LNPs.
Macrophage-targeting gene silencing orchestrates myocardial microenvironment remodeling toward the anti-inflammatory treatment of ischemia-reperfusion (IR) injury.
Lactoferrin- and Dendrimer-Bearing Gold Nanocages for Stimulus-Free DNA Delivery to Prostate Cancer Cells.
The diffusion-driven microfluidic process to manufacture lipid-based nanotherapeutics with stealth properties for siRNA delivery.
Multistage pH-responsive codelivery liposomal platform for synergistic cancer therapy.
Poly(beta-amino ester)-Based Nanoparticles Enable Nonviral Delivery of Base Editors for Targeted Tumor Gene Editing.
Platelet pharmacytes for the hierarchical amplification of anti-tumor immunity in response to self-generated immune signals.
Amphipathic dendritic poly-peptides carrier to deliver antisense oligonucleotides against multi-drug resistant bacteria in vitro and in vivo.
Microfluidic Nanoparticles for Drug Delivery.

ACS Biomater Sci Eng. 2022 Apr 05.

Structural Exploration of Polycationic Nanoparticles for siRNA Delivery.

Yunfeng Yan, Guangliang Zhang, Chengfan Wu, Qidi Ren, Xiaomin Liu, Fangqian Huang, Yi Cao, Wenbo Ye.

  RNA interference (RNAi) is a promising approach to the treatment of genetic diseases by the specific knockdown of target genes. Functional polymers are potential vehicles for the effective delivery of vulnerable small interfering RNA (siRNA), which is required for the broad application of RNAi-based therapeutics. The development of methods for the facile modulation of chemical structures of polymeric carriers and an elucidation of detailed delivery mechanisms remain important areas of research. In this paper, we synthesized a series of methacrylate-based polymers with controllable structures and narrow distributions by atom transfer radical polymerization using various combinations of cationic monomers (2-dimethylaminoethyl methacrylate, 2-diethylaminoethyl methacrylate, and 2-dibutylaminoethyl methacrylate) and hydrophobic monomers (2-butyl methacrylate (BMA), cyclohexyl methacrylate, and 2-ethylhexyl methacrylate). These polymers exhibited varying hydrophobicities, charge densities, and pKa values, enabling the discovery of effective carriers for siRNA by in vitro delivery assays. For the polymers with BMA segments, 50% of cationic segments were beneficial to the formation of siRNA nanoparticles (NPs) and the in vitro delivery of siRNA. The optimal ratio varied for different combinations of cationic and hydrophobic segments. In particular, 20k PMB 0.5, PME 0.5, and PEB 1.0 showed >75% luciferase knockdown. Efficacious delivery was dependent on high siRNA binding, the small size of NPs, and balanced hydrophobicity and charge density. Cellular uptake and endosomal escape experiments indicated that carboxybetaine modification of 20k PMB 0.5 did not remarkably affect the internalization of corresponding NPs after incubation for 6 h but significantly reduced the endosomal escape of NPs, which leads to the notable decrease in delivery efficacy of polymers. These results provide insights into the mechanism of polymer-based siRNA delivery and may inspire the development of novel polymeric carriers.

Keywords:  cationic polymer; drug delivery; nanoparticle; siRNA delivery

DOI:  https://doi.org/10.1021/acsbiomaterials.2c00196
ACS Appl Mater Interfaces. 2022 Apr 05.

Reactive Oxygen Species─Responsive Lipid Nanoparticles for Effective RNAi and Corneal Neovascularization Therapy.

Anqi Liu, Chunjing Liang, Ji Liu, Yifei Huang, Ming Wang, Liqiang Wang.

  Corneal neovascularization (CNV) is a common disease that affects the vision ability of more than 1 million people annually. Small interfering RNA (siRNA) delivery nanoparticle platforms are a promising therapeutic modality for CNV treatment. However, the efficient delivery of siRNA into cells and the effective release of siRNA from delivery vehicles in a particular cell type challenge effective RNAi clinical application for CNV suppression. This study reports the design of a novel reactive oxygen species (ROS)-responsive lipid nanoparticle for siRNA delivery into corneal lesions for enhanced RNAi as a potential CNV treatment. We demonstrated that lipid nanoparticles could efficiently deliver siRNA into human umbilical vein endothelial cells and release siRNA for enhanced gene silencing by using the upregulated ROS of CNV to promote lipid nanoparticle degradation. Moreover, the subconjunctival injection of siRNA nanocomplexes into corneal lesions effectively knocked down vascular endothelial growth factor expression and suppressed CNV formation in an alkali burn model. Thus, we believe that the strategy of using ROS-responsive lipid nanoparticles for enhanced RNAi in CNV could be further extended to a promising clinical therapeutic approach to attenuate CNV formation.

Keywords:  RNA interference; corneal neovascularization; drug delivery; lipid nanoparticles; reactive oxygen species

DOI:  https://doi.org/10.1021/acsami.1c23412
Biol Pharm Bull. 2022 ;45(4): 497-507

Importance of Process Parameters Influencing the Mean Diameters of siRNA-Containing Lipid Nanoparticles (LNPs) on the in Vitro Activity of Prepared LNPs.

Kazuya Nakamura, Keisuke Aihara, Tatsuhiro Ishida.

  Genetic drugs have the potential to treat a variety of diseases. Recently, lipid nanoparticles (LNPs) have attracted much attention among drug delivery systems for genetic drugs. LNPs have been practically used in small interfering RNA (siRNA) drugs and mRNA vaccines. Although LNPs are generally prepared by mixing nucleic acids in acidic aqueous buffer and lipid excipients in alcohol (i.e., ethanol), it is not well understood which process parameters in the LNPs formation affect the physicochemical properties and the functionality of LNPs. In this study, we used siRNA-containing LNPs as a model, and evaluated the effect that aqueous solution parameters (buffering agent type, salt concentration, and pH) and mixing parameters (ratio, speed, and temperature) exert on the physicochemical properties and in vitro gene-knockdown activity of LNPs. Among such parameters, the type of buffering agent, salt concentration (ionic strength), pH in acidic aqueous buffer, as well as the mixing ratio and speed significantly affected the mean particle diameter and in vitro gene-knockdown activity of LNPs. A strong correlation between the mean particle diameters and their in vitro gene-knockdown activities was observed. These observations suggest that the process parameters influencing the mean LNPs diameter are likely to be important in the formation of LNPs and also that these correlate with in vitro gene-knockdown activity. Because LNP systems are being further developed for future clinical applications of genetic drugs, information regarding the LNPs manufacturing process is of utmost importance. The results observed in this study will be useful for the manufacturing of optimal LNPs.

Keywords:  lipid nanoparticle; mRNA; manufacture; process parameter; small interfering RNA (siRNA)

DOI:  https://doi.org/10.1248/bpb.b21-01016
Bioact Mater. 2022 Nov;17 320-333

Macrophage-targeting gene silencing orchestrates myocardial microenvironment remodeling toward the anti-inflammatory treatment of ischemia-reperfusion (IR) injury.

Yao Wang, Mengying Hou, Shanzhou Duan, Ziyin Zhao, Xuejie Wu, Yongbing Chen, Lichen Yin.

  Ischemia-reperfusion (IR) injury represents a major cause of myocardial dysfunction after infarction and thrombolytic therapy, and it is closely related to the free radical explosion and overwhelming inflammatory responses. Herein, macrophage-targeting nanocomplexes (NCs) are developed to mediate efficient co-delivery of siRNA against MOF (siMOF) and microRNA-21 (miR21) into myocardial macrophages, cooperatively orchestrating the myocardial microenvironment against IR injury. Bioreducible, branched poly(β-amino ester) (BPAE-SS) is designed to co-condense siMOF and miR21 into NCs in a multivalency-reinforced approach, and they are surface-decorated with carboxylated mannan (Man-COOH) to shield the positive surface charges and enhance the serum stability. The final MBSsm NCs are efficiently internalized by myocardial macrophages after systemic administration, wherein BPAE-SS is degraded into small segments by intracellular glutathione to promote the siMOF/miR21 release, finally provoking efficient gene silencing. Thus, cardiomyocyte protection and macrophage modulation are realized via the combined effects of ROS scavenging, inflammation inhibition, and autophagy attenuation, which ameliorates the myocardial microenvironment and restores the cardiac function via positive cellular crosstalk. This study renders promising solutions to address the multiple systemic barriers against in vivo nucleic acid delivery, and it also offers new options for IR injury by manipulating multiple reciprocal bio-reactions.

Keywords:  Anti-inflammation; Microenvironment remodeling; Myocardial ischemia-reperfusion (IR) injury; Reduction-responsive branched poly(β-amino ester); siRNA/miRNA delivery

DOI:  https://doi.org/10.1016/j.bioactmat.2022.01.026
Int J Nanomedicine. 2022 ;17 1409-1421

Lactoferrin- and Dendrimer-Bearing Gold Nanocages for Stimulus-Free DNA Delivery to Prostate Cancer Cells.

Jamal Almowalad, Partha Laskar, Sukrut Somani, Jitkasem Meewan, Rothwelle J Tate, Christine Dufès.

   Background: The use of gene therapy to treat prostate cancer is hampered by the lack of effective nanocarriers that can selectively deliver therapeutic genes to cancer cells. To overcome this, we hypothesize that conjugating lactoferrin, a tumor-targeting ligand, and the diaminobutyric polypropylenimine dendrimer into gold nanocages, followed by complexation with a plasmid DNA, would enhance gene expression and anti-proliferation activity in prostate cancer cells without the use of external stimuli.
Methods: Novel gold nanocages bearing lactoferrin and conjugated to diaminobutyric polypropylenimine dendrimer (AuNCs-DAB-Lf) were synthesized and characterized. Following complexation with a plasmid DNA, their gene expression, cellular uptake and anti-proliferative efficacies were evaluated on PC-3 prostate cancer cells.
Results: AuNCs-DAB-Lf was able to complex DNA at conjugate: DNA weight ratios 5:1 onwards. Gene expression was at its highest after treatment with AuNCs-DAB-Lf at a weight ratio of 10:1, as a result of a significant increase in DNA uptake mediated by the conjugate at that ratio in PC-3 cells. Consequently, the anti-proliferative activity of AuNCs-DAB-Lf-DNA encoding TNFα was significantly improved by up to 9-fold compared with DAB dendriplex encoding TNFα.
Conclusion: Lactoferrin-bearing dendrimer-conjugated gold nanocages are highly promising gene delivery systems for the treatment of prostate cancer.

Keywords:  cancer targeting; dendrimer; gene therapy; gold nanocages; lactoferrin; prostate cancer; transfection efficacy

DOI:  https://doi.org/10.2147/IJN.S347574
Colloids Surf B Biointerfaces. 2022 Mar 23. pii: S0927-7765(22)00159-X. [Epub ahead of print]215 112476

The diffusion-driven microfluidic process to manufacture lipid-based nanotherapeutics with stealth properties for siRNA delivery.

Ismail Eş, Antonio A Malfatti-Gasperini, Lucimara Gaziola de la Torre.

  Our study investigated the manufacturing of lipid-based nanotherapeutics with stealth properties for siRNA delivery by employing a diffusion-driven microfluidic process in one or two-steps strategies to produce siRNA-loaded lipid nanocarriers and lipoplexes, respectively. In the one-step synthesis, siRNA in the aqueous phase is introduced from one inlet, while phospholipids dispersed in anhydrous ethanol are introduced from other inlets, generating the lipid nanocarriers. In the two-steps strategies, the pre-formed liposomes are complexed with siRNA. The process configuration with an aqueous diffusion barrier exerts a significant effect on the nanoaggregates synthesis. Dynamic light scattering data showed that lipid nanocarriers had a bigger particle diameter (298 ± 24 nm) and surface charge (43 ± 6 mV) compared to lipoplexes (194 ± 7 nm and 37.0 ± 0.4 mV). Moreover, DSPE-PEG(2000) was included in the formulation to synthesize lipid-based nanotherapeutics containing siRNA with stealth characteristics. The inclusion of PEG-lipid resulted in an increase in the surface charge of lipoplexes (from 33.7 ± 4.4-54.3 ± 1.6 mV), while a significant decrease was observed in the surface charge of lipid nanocarriers (30.3 ± 8.7 mV). The different structural assemblies were identified for lipoplex and lipid nanocarriers using Synchrotron SAXS. Lipid nanocarriers present a lower amount of multilayers than lipoplexes. Lipid-PEG insertion significantly influenced lipid nanocarriers' characteristics, drastically decreasing the number of multilayers. This effect was not observed in lipoplexes. The association between process configuration, lipid composition, and its effect on the characteristics of lipid-based vector systems can generate fundamental insights, contributing to gene-based nanotherapeutics development.

Keywords:  Diffusion; Gene delivery; Lipid nanocarriers; Lipoplex; Microfluidics; SiRNA

DOI:  https://doi.org/10.1016/j.colsurfb.2022.112476
J Nanobiotechnology. 2022 Apr 02. 20(1): 177

Multistage pH-responsive codelivery liposomal platform for synergistic cancer therapy.

Ting Zhao, Ce Liang, Yanrong Zhao, Xiangdong Xue, Zhao Ma, Jinlong Qi, Haitao Shen, Shaokun Yang, Jia Zhang, Qingzhong Jia, Qing Du, Deying Cao, Bai Xiang, Hailin Zhang, Xianrong Qi.

   BACKGROUND: Small interfering RNA (siRNA) is utilized as a potent agent for cancer therapy through regulating the expression of genes associated with tumors. While the widely application of siRNAs in cancer treatment is severely limited by their insufficient biological stability and its poor ability to penetrate cell membranes. Targeted delivery systems hold great promise to selectively deliver loaded drug to tumor site and reduce toxic side effect. However, the elevated tumor interstitial fluid pressure and efficient cytoplasmic release are still two significant obstacles to siRNA delivery. Co-delivery of chemotherapeutic drugs and siRNA represents a potential strategy which may achieve synergistic anticancer effect. Herein, we designed and synthesized a dual pH-responsive peptide (DPRP), which includes three units, a cell-penetrating domain (polyarginine), a polyanionic shielding domain (ehG)n, and an imine linkage between them. Based on the DPRP surface modification, we developed a pH-responsive liposomal system for co-delivering polo-like kinase-1 (PLK-1) specific siRNA and anticancer agent docetaxel (DTX), D-Lsi/DTX, to synergistically exhibit anti-tumor effect.
RESULTS: In contrast to the results at the physiological pH (7.4), D-Lsi/DTX lead to the enhanced penetration into tumor spheroid, the facilitated cellular uptake, the promoted escape from endosomes/lysosomes, the improved distribution into cytoplasm, and the increased cellular apoptosis under mildly acidic condition (pH 6.5). Moreover, both in vitro and in vivo study indicated that D-Lsi/DTX had a therapeutic advantage over other control liposomes. We provided clear evidence that liposomal system co-delivering siPLK-1 and DTX could significantly downregulate expression of PLK-1 and inhibit tumor growth without detectable toxic side effect, compared with siPLK-1-loaded liposomes, DTX-loaded liposomes, and the combinatorial administration.
CONCLUSION: These results demonstrate great potential of the combined chemo/gene therapy based on the multistage pH-responsive codelivery liposomal platform for synergistic tumor treatment.

Keywords:  Cancer therapy; Co-delivery system; Docetaxel; Liposome; Small interfering RNA; pH-responsive

DOI:  https://doi.org/10.1186/s12951-022-01383-z
Biomacromolecules. 2022 Apr 07.

Poly(beta-amino ester)-Based Nanoparticles Enable Nonviral Delivery of Base Editors for Targeted Tumor Gene Editing.

Qimingxing Chen, Lili Su, Xiaoyan He, Jinwei Li, Yan Cao, Qingxia Wu, Jianchao Qin, Zongxing He, Xingxu Huang, Huiying Yang, Jianfeng Li.

Base editing is an emerging genome editing technology with the advantages of precise base corrections, no double-strand DNA breaks, and no need for templates, which provides an alternative treatment option for tumors with point mutations. However, effective nonviral delivery systems for base editors (BEs) are still limited. Herein, a series of poly(beta-amino esters) (PBAEs) with varying backbones, side chains, and end caps were synthesized to deliver plasmids of BEs and sgRNA. Efficient transfection and base editing were achieved in HEK-293T-sEGFP and U87-MG-sEGFP reporter cell lines by using lead PBAEs, which were superior to PEI and lipo3k. A single intratumor injection of PBAE/pDNA nanoparticles induced the robust conversion of stopped-EGFP into EGFP in mice bearing xenograft glioma tumors, indicating successful gene editing by ABEmax-NG. Overall, these results demonstrated that PBAEs can efficiently deliver BEs for tumor gene editing both in vitro and in vivo.

DOI: https://doi.org/10.1021/acs.biomac.2c00137
Adv Mater. 2022 Apr 06. e2109517

Platelet pharmacytes for the hierarchical amplification of anti-tumor immunity in response to self-generated immune signals.

Jing Yan, Xun Liu, Fan Wu, Chenglong Ge, Huan Ye, Xingye Chen, Yuansong Wei, Renxiang Zhou, Shanzhou Duan, Rongying Zhu, Yiran Zheng, Lichen Yin.

  Systemic immunosuppression mediated by tumor-derived exosomes is an important cause for the resistance of immune checkpoint blockade (ICB) therapy. Herein, self-adaptive platelet (PLT) pharmacytes were engineered to mediate cascaded delivery of exosome-inhibiting siRNA and anti-PD-L1 (aPDL1) towards synergized anti-tumor immunity. In the pharmacytes, polycationic nanocomplexes (NCs) assembled from Rab27 siRNA (siRab) and a membrane-penetrating polypeptide were encapsulated inside the open canalicular system of PLTs, and cytotoxic T lymphocytes (CTLs)-responsive aPDL1 nanogels (NGs) were covalently backpacked on the PLT surface. Upon systemic administration, the pharmacytes enabled prolonged blood circulation and active accumulation to tumors, wherein PLTs were activated to liberate siRab NCs that efficiently transfected tumor cells, silenced Rab27a, and inhibited exosome secretion. The immunosuppression was thus relieved, leading to activation, proliferation, and tumoral infiltration of cytotoxic T cells which triggered latent aPDL1 release. As such, the competitive aPDL1 exhaustion by PD-L1-expressing exosomes was minimized to sensitize ICB. Synergistically, siRab and aPDL1 induced strong anti-tumor immunological response and memory against syngeneic murine melanoma. This study reports a bio-inspired mechanism to resolve the blood circulation/cell internalization contradiction of polycationic siRNA delivery systems, and renders an enlightened approach for the spatiotemporal enhancement of anti-tumor immunity. This article is protected by copyright. All rights reserved.

Keywords:  Immune checkpoint blockade (ICB); cytotoxic T lymphocytes (CTLs)-responsive; platelets (PLTs); siRNA delivery; tumor-derived exosomes

DOI:  https://doi.org/10.1002/adma.202109517
J Nanobiotechnology. 2022 Apr 02. 20(1): 180

Amphipathic dendritic poly-peptides carrier to deliver antisense oligonucleotides against multi-drug resistant bacteria in vitro and in vivo.

Zhou Chen, Yue Hu, Xinggang Mao, Dan Nie, Hui Zhao, Zheng Hou, Mingkai Li, Jingru Meng, Xiaoxing Luo, Xiaoyan Xue.

   BACKGROUND: Outbreaks of infection due to multidrug-resistant (MDR) bacteria, especially Gram-negative bacteria, have become a global health issue in both hospitals and communities. Antisense oligonucleotides (ASOs) based therapeutics hold a great promise for treating infections caused by MDR bacteria. However, ASOs therapeutics are strangled because of its low cell penetration efficiency caused by the high molecular weight and hydrophilicity.
RESULTS: Here, we designed a series of dendritic poly-peptides (DPP1 to DPP12) to encapsulate ASOs to form DSPE-mPEG2000 decorated ASOs/DPP nanoparticles (DP-AD1 to DP-AD12) and observed that amphipathic DP-AD2, 3, 7 or 8 with a positive charge ≥ 8 showed great efficiency to deliver ASOs into bacteria, but only the two histidine residues contained DP-AD7 and DP-AD8 significantly inhibited the bacterial growth and the targeted gene expression of tested bacteria in vitro. DP-AD7anti-acpP remarkably increased the survival rate of septic mice infected by ESBLs-E. coli, exhibiting strong antibacterial effects in vivo.
CONCLUSIONS: For the first time, we designed DPP as a potent carrier to deliver ASOs for combating MDR bacteria and demonstrated the essential features, namely, amphipathicity, 8-10 positive charges, and 2 histidine residues, that are required for efficient DPP based delivery, and provide a novel approach for the development and research of the antisense antibacterial strategy.

Keywords:  Antibacterial strategy; Antisense; Delivery; Dendritic poly-peptides; Multidrug-resistant bacteria; Nanoparticles; Oligonucleotide

DOI:  https://doi.org/10.1186/s12951-022-01384-y
Small. 2022 Apr 09. e2106580

Microfluidic Nanoparticles for Drug Delivery.

Yun Liu, Guangze Yang, Yue Hui, Supun Ranaweera, Chun-Xia Zhao.

  Nanoparticles (NPs) have attracted tremendous interest in drug delivery in the past decades. Microfluidics offers a promising strategy for making NPs for drug delivery due to its capability in precisely controlling NP properties. The recent success of mRNA vaccines using microfluidics represents a big milestone for microfluidic NPs for pharmaceutical applications, and its rapid scaling up demonstrates the feasibility of using microfluidics for industrial-scale manufacturing. This article provides a critical review of recent progress in microfluidic NPs for drug delivery. First, the synthesis of organic NPs using microfluidics focusing on typical microfluidic methods and their applications in making popular and clinically relevant NPs, such as liposomes, lipid NPs, and polymer NPs, as well as their synthesis mechanisms are summarized. Then, the microfluidic synthesis of several representative inorganic NPs (e.g., silica, metal, metal oxide, and quantum dots), and hybrid NPs is discussed. Lastly, the applications of microfluidic NPs for various drug delivery applications are presented.

Keywords:  drug delivery; lipids; microfluidics; nanomedicine; nanoparticles; polymers

DOI:  https://doi.org/10.1002/smll.202106580