bims-novged 2022-01-16 papers

on Non-viral vectors for gene delivery

Issue of 2022–01–16
fourteen papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Non-viral vectors for RNA delivery.
One-Step Production Using a Microfluidic Device of Highly Biocompatible Size-Controlled Noncationic Exosome-like Nanoparticles for RNA Delivery.
Serum Compatible Spermine-based Cationic Lipids with Non-identical Hydrocarbon Tails Mediate High Transfection Efficiency.
Small interfering RNAs based therapies for intracerebral hemorrhage: challenges and progress in drug delivery systems.
Metallohelix vectors for efficient gene delivery via cationic DNA nanoparticles.
Toward Gene Transfer Nanoparticles as Therapeutics.
Facile Formation of PAMAM Dendrimer Nanoclusters for Enhanced Gene Delivery and Cancer Gene Therapy.
Microfluidics for Development of Lipid Nanoparticles: Paving the Way for Nucleic Acids to the Clinic.
Development of a high-throughput platform for screening lipid nanoparticles for mRNA delivery.
Polyethylenimine-Functionalized Carbon Dots for Delivery of CRISPR/Cas9 Complexes.
Macrophage membrane- and cRGD-functionalized thermosensitive liposomes combined with CPP to realize precise siRNA delivery into tumor cells.
Role of siRNA-based nanocarriers for the treatment of neurodegenerative diseases.
Polyethylene Glycol-Mediated Fusion of Extracellular Vesicles with Cationic Liposomes for the Design of Hybrid Delivery Systems.
Runx1 Messenger RNA Delivered by Polyplex Nanomicelles Alleviate Spinal Disc Hydration Loss in a Rat Disc Degeneration Model.

J Control Release. 2022 Jan 08. pii: S0168-3659(22)00022-0. [Epub ahead of print]

Non-viral vectors for RNA delivery.

Yi Yan, Xiao-Yu Liu, An Lu, Xiang-Yu Wang, Lin-Xia Jiang, Jian-Cheng Wang.

  RNA-based therapy is a promising and potential strategy for disease treatment by introducing exogenous nucleic acids such as messenger RNA (mRNA), small interfering RNA (siRNA), microRNA (miRNA) or antisense oligonucleotides (ASO) to modulate gene expression in specific cells. It is exciting that mRNA encoding the spike protein of COVID-19 (coronavirus disease 2019) delivered by lipid nanoparticles (LNPs) exhibits the efficient protection of lungs infection against the virus. In this review, we introduce the biological barriers to RNA delivery in vivo and discuss recent advances in non-viral delivery systems, such as lipid-based nanoparticles, polymeric nanoparticles, N-acetylgalactosamine (GalNAc)-siRNA conjugate, and biomimetic nanovectors, which can protect RNAs against degradation by ribonucleases, accumulate in specific tissue, facilitate cell internalization, and allow for the controlled release of the encapsulated therapeutics.

Keywords:  Biological barrier; Control release; Gene therapy; Non-viral vector; RNA drugs

DOI:  https://doi.org/10.1016/j.jconrel.2022.01.008
ACS Appl Bio Mater. 2021 Feb 15. 4(2): 1783-1793

One-Step Production Using a Microfluidic Device of Highly Biocompatible Size-Controlled Noncationic Exosome-like Nanoparticles for RNA Delivery.

Niko Kimura, Masatoshi Maeki, Akihiko Ishida, Hirofumi Tani, Manabu Tokeshi.

  Size-controlled lipid nanoparticle (LNP)-based DNA/RNA delivery is a leading technology for gene therapies. For DNA/RNA delivery, typically, a cationic lipid is used to encapsulate DNA/RNA into LNPs. However, the use of the cationic lipid leads to cytotoxicity. In contrast, noncationic NPs, such as exosomes, are ideal nanocarriers for DNA/RNA delivery. However, the development of a simple one-step method for the production of size-controlled noncationic NPs encapsulating DNA/RNA is still challenging because of the lack of electrostatic interactions between the cationic lipid and negatively charged DNA/RNA. Herein, we report a microfluidic-based one-step method for the production of size-controlled noncationic NPs encapsulating small interfering RNA (siRNA). Our microfluidic device, named iLiNP, enables the efficient encapsulation of siRNA, as well as control over the NP size, by varying the flow conditions. We applied this method to produce size-controlled exosome-like NPs. The siRNA-loaded exosome-like NPs did not show in vitro cytotoxicity at a high siRNA dosage. In addition, we investigated the effect of the size of the exosome-like NPs on the target gene silencing and found that the 40-50 nm-sized NPs suppressed target protein expression at a dose of 20 nM siRNA. The iLiNP-based one-step production method for size-controlled noncationic-NP-encapsulated RNA is a promising method for the production of artificial exosomes and functionally modified exosomes for gene and cell therapies.

Keywords:  exosomes-like nanoparticles; lipid nanoparticles; microfluidics; nanomedicines; noncationic lipids; siRNA delivery

DOI:  https://doi.org/10.1021/acsabm.0c01519
Chembiochem. 2022 Jan 09.

Serum Compatible Spermine-based Cationic Lipids with Non-identical Hydrocarbon Tails Mediate High Transfection Efficiency.

Chopaka Thongbamrer, Wanlapa Roobsoong, Jetsumon Sattabongkot, Praneet Opanasopit, Boon-Ek Yingyongnarongkul.

  Cationic lipids are widely used as non-viral synthetic vectors for gene delivery as a safer alternative to viral vectors. In this work, a library of L-shaped spermine-based cationic lipids with identical and non-identical hydrophobic chains having variable carbon length (from C10 to C18) was designed and synthesized. These lipids were characterized and the structure-activity relationships of these compounds were determined for DNA binding and transfection ability when formulated as cationic liposomes. The liposomes were then used successfully for the transfection of HEK293T, HeLa, PC3, H460, HepG2, SH-SY5Y and Calu'3 cell lines. The transfection efficiency of lipids with non-identical hydrocarbon chains was greater than the identical analog. These reagents exhibited superior efficiency to the commercial reagent, Lipofectamine3000, under both serum-free and 10-40% serum conditions in HEK293T, HeLa and H460 cell lines. The lipids were also not toxic to the tested cells. The results suggested that L-shaped spermine-based cationic lipids with non-identical hydrocarbon tails could serve as an efficient and safe non-viral vector gene carrier for further in vivo studies.

Keywords:  liposomes * cationic lipid * non-viral vector * DNA delivery * non-identical hydrophobic tails

DOI:  https://doi.org/10.1002/cbic.202100672
Neural Regen Res. 2022 Aug;17(8): 1717-1725

Small interfering RNAs based therapies for intracerebral hemorrhage: challenges and progress in drug delivery systems.

Daniyah A Almarghalani, Sai H S Boddu, Mohammad Ali, Akhila Kondaka, Devin Ta, Rayyan A Shah, Zahoor A Shah.

  Intracerebral hemorrhage (ICH) is a subtype of stroke associated with higher rates of mortality. Currently, no effective drug treatment is available for ICH. The molecular pathways following ICH are complicated and diverse. Nucleic acid therapeutics such as gene knockdown by small interfering RNAs (siRNAs) have been developed in recent years to modulate ICH's destructive pathways and mitigate its outcomes. However, siRNAs delivery to the central nervous system is challenging and faces many roadblocks. Existing barriers to systemic delivery of siRNA limit the use of naked siRNA; therefore, siRNA-vectors developed to protect and deliver these therapies into the specific-target areas of the brain, or cell types seem quite promising. Efficient delivery of siRNA via nanoparticles emerged as a viable and effective alternative therapeutic tool for central nervous system-related diseases. This review discusses the obstacles to siRNA delivery, including the advantages and disadvantages of viral and nonviral vectors. Additionally, we provide a comprehensive overview of recent progress in nanotherapeutics areas, primarily focusing on the delivery system of siRNA for ICH treatment.

Keywords:  intracerebral hemorrhage; lipid-based nanoparticle; nanoparticles; nanotechnology; nonviral vectors; peptide-mediated nanoparticle; polymer-based nanoparticle; siRNA therapeutics; siRNA-barriers; viral vectors

DOI:  https://doi.org/10.4103/1673-5374.332129
Nucleic Acids Res. 2022 Jan 08. pii: gkab1277. [Epub ahead of print]

Metallohelix vectors for efficient gene delivery via cationic DNA nanoparticles.

Jaroslav Malina, Hana Kostrhunova, Vojtech Novohradsky, Peter Scott, Viktor Brabec.

The design of efficient and safe gene delivery vehicles remains a major challenge for the application of gene therapy. Of the many reported gene delivery systems, metal complexes with high affinity for nucleic acids are emerging as an attractive option. We have discovered that certain metallohelices-optically pure, self-assembling triple-stranded arrays of fully encapsulated Fe-act as nonviral DNA delivery vectors capable of mediating efficient gene transfection. They induce formation of globular DNA particles which protect the DNA from degradation by various restriction endonucleases, are of suitable size and electrostatic potential for efficient membrane transport and are successfully processed by cells. The activity is highly structure-dependent-compact and shorter metallohelix enantiomers are far less efficient than less compact and longer enantiomers.

DOI: https://doi.org/10.1093/nar/gkab1277
Adv Healthc Mater. 2022 Jan 10. e2102145

Toward Gene Transfer Nanoparticles as Therapeutics.

Erin W Kavanagh, Jordan J Green.

  Genetic medicine has great potential to treat the underlying causes of many human diseases with exquisite precision, but the field has historically been stymied by delivery as the central challenge. Nanoparticles, engineered constructs the size of natural viruses, are being designed to more closely mimic the delivery efficiency of viruses, while enabling the advantages of increased safety, cargo carrying flexibility, specific targeting, and ease in manufacturing. The speed in which non-viral gene transfer nanoparticles are making progress in the clinic is accelerating, with clinical validation of multiple non-viral nucleic acid delivery nanoparticle formulations recently FDA approved for both expression and for silencing of genes. While much of this progress has been with lipid nanoparticle formulations, significant development is being made with other nanomaterials for gene transfer as well, with favorable attributes such as biodegradability, scalability, and cell targeting. This review highlights the state of the field, current challenges in delivery, and opportunities for engineered nanomaterials to meet these challenges, including enabling long-term therapeutic gene editing. Delivery technology utilizing different kinds of nanomaterials and varying cargos for gene transfer (DNA, mRNA and ribonucleoproteins) are discussed. Clinical applications are presented, including for the treatment of genetic diseases such as cystic fibrosis. This article is protected by copyright. All rights reserved.

Keywords:  CRISPR; DNA; cystic fibrosis; gene editing; gene therapy; mRNA; nanoparticle

DOI:  https://doi.org/10.1002/adhm.202102145
ACS Appl Bio Mater. 2021 Sep 20. 4(9): 7168-7175

Facile Formation of PAMAM Dendrimer Nanoclusters for Enhanced Gene Delivery and Cancer Gene Therapy.

Shewaye Lakew Mekuria, Jin Li, Cong Song, Yue Gao, Zhijun Ouyang, Mingwu Shen, Xiangyang Shi.

  Preparation of versatile and safe nanovectors for efficient cancer gene therapy remains to be challenging in the current nanomedicine. Herein, we report the formation of dendrimer nanoclusters for enhanced gene delivery toward gene therapy of cancer. Here, poly(amidoamine) (PAMAM) dendrimers of generation 3 (G3) were cross-linked with 4,4'-dithiodibutryic acid (DA) to form nanoclusters (NCs) through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride-induced covalent bonding. The synthesized G3-DA NCs having a hydrodynamic size of 219.3 nm possess good colloidal stability and can condense pDNA, encoding both enhanced green fluorescent protein and tumor suppressor p53 gene to form polyplexes with good cytocompatibility. Strikingly, the created NCs/pDNA polyplexes enable 2.3 and 2.1 times higher gene transfection to cancer cells than the counterpart materials of single G3 and G5 PAMAM dendrimers, respectively, under the same conditions. Furthermore, polyplex-treated cancer cells have upregulated p53 and p21 protein and mRNA expression levels and downregulated Cyclin-D1 and CDK-4 protein and mRNA expressions, thus arresting the cell cycle to the G1 phase in vitro to achieve cancer cell gene therapy. The gene delivery efficiency of the polyplexes was further validated through the in vivo tumor therapy without systemic toxicity. The synthesized highly efficient dendrimer NC-based vector system with low cytotoxicity may be extended to tackle various types of diseases related to genetic disorders.

Keywords:  PAMAM dendrimers; cancer cells; gene delivery; gene therapy; nanoclusters

DOI:  https://doi.org/10.1021/acsabm.1c00743
ACS Appl Bio Mater. 2021 Sep 08.

Microfluidics for Development of Lipid Nanoparticles: Paving the Way for Nucleic Acids to the Clinic.

Moustafa S Ali, Nasrin Hooshmand, Mostafa El-Sayed, Hagar I Labouta.

  Nucleic acid therapeutics hold an unprecedented promise toward treating many challenging diseases; however, their use is hampered by delivery issues. Microfluidics, dealing with fluids in the microscale dimensions, have provided a robust means to screening raw materials for development of nano delivery vectors, in addition to controlling their size and minimizing their polydispersity. In this mini-review, we are briefly highlighting the different types of nucleic acid therapies with emphasis on the delivery requirement for each type. We provide a thorough review of available methods for the development of nanoparticles, especially lipid nanoparticles (LNPs) that resulted in FDA approval of the first ever nucleic acid nanomedicine. We then focus on recent research attempts for how microfluidic synthesis of lipid nanoparticles and discuss the various parameters required for successful formulation of LPNs including chip design, flow regimes, and lipid composition. We then identify key areas of research in microfluidics and related fields that require attention for future success in clinical translation of nucleic acid nanomedicines.

Keywords:  mRNA, lipid nanoparticles; microfluidics; nanomedicine; nucleic acid therapies

DOI:  https://doi.org/10.1021/acsabm.1c00732
Nanoscale. 2022 Jan 13.

Development of a high-throughput platform for screening lipid nanoparticles for mRNA delivery.

Lili Cui, Sara Pereira, Silvia Sonzini, Sally van Pelt, Steven M Romanelli, Lihuan Liang, David Ulkoski, Venkata R Krishnamurthy, Emily Brannigan, Christopher Brankin, Arpan S Desai.

mRNA lipid nanoparticles (LNPs) are at the forefront of nucleic acid intracellular delivery, as exemplified by the recent emergency approval of two mRNA LNP-based COVID-19 vaccines. The success of an LNP product largely depends on the systematic optimisation of the four lipidic components, namely the ionisable lipid, PEG lipid, structural and helper lipids. However, the in vitro screening of novel lipidic components and LNP compositions is limited by the low-throughput of LNP preparation. To address these issues, we herein present an automated high-throughput screening platform to select novel ionisable lipids and corresponding LNPs encapsulating mRNA in vitro. This high-throughput platform employs a lab-based automated liquid handling system, amenable to high-throughput (up to 384 formulations per plate and several plates per run) and allows precise mixing and reproducible mRNA LNP preparation which ensures a direct head-to-head comparison of hundreds and even thousands of novel LNPs. Most importantly, the robotic process has been successfully applied to the screening of novel LNPs encapsulating mRNA and has identified the same novel mRNA LNP leads as those from microfluidics-mixing technology, with a correlation coefficient of 0.8751. This high-throughput platform can facilitate to narrow down the number of novel ionisable lipids to be evaluated in vivo. Moreover, this platform has been integrated into a fully-automated workflow for LNP property control, physicochemical characterisation and biological evaluation. The high-throughput platform may accelerate proprietary lipid development, mRNA LNP lead optimisation and candidate selection to advance preclinical mRNA LNP development to meet urgent global needs.

DOI: https://doi.org/10.1039/d1nr06858j
ACS Appl Bio Mater. 2021 Nov 15. 4(11): 7979-7992

Polyethylenimine-Functionalized Carbon Dots for Delivery of CRISPR/Cas9 Complexes.

Iman Hashemzadeh, Akbar Hasanzadeh, Fatemeh Radmanesh, Beheshteh Khodadadi Chegeni, Elaheh Sadat Hosseini, Jafar Kiani, Ali Shahbazi, Marzieh Naseri, Yousef Fatahi, Helena Nourizadeh, Behjat Kheiri Yeghaneh Azar, Amir R Aref, Yong Liu, Michael R Hamblin, Mahdi Karimi.

  Carbon dots (CDs) have become the focus of many studies due to their outstanding optical properties and good biocompatibility. We investigated their potential application to produce a smart and highly efficient yet nontoxic nanovector for gene delivery. This was achieved by conjugating PEI1.8k-functionalized CDs (synthesized by one-step microwave-assisted pyrolysis) with arginine-disulfide linkers to produce CD-PEI1.8k-Arg nanoparticles. This nanovector could deliver p-CRISPR (9.3 kb) into different types of cell lines with higher efficiency compared to native PEI1.8k or PEI25k. CD-PEI1.8k-Arg also maintained its outstanding transfection efficiency at a high serum concentration and low p-CRISPR dose, compared to PEI25k, which was ineffective under those conditions. Additionally, CD-PEI1.8k-Arg could knock out the GFP gene with great efficiency by delivering the required components of CRISPR/Cas9, including a plasmid encoding Cas9, sgRNA targeting GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) into the HEK 293T-GFP cells. Moreover, the nanoparticles showed potential for the local delivery of p-CRISPR into brain tissue. The remarkable properties of CD-PEI1.8k-Arg could enable the development of a safe, highly efficient gene-delivery nanovector for the treatment of various diseases in the near future.

Keywords:  CRISPR; brain delivery; carbon dots; gene delivery; smart nanoparticles

DOI:  https://doi.org/10.1021/acsabm.1c00890
Mol Ther Nucleic Acids. 2022 Mar 08. 27 349-362

Macrophage membrane- and cRGD-functionalized thermosensitive liposomes combined with CPP to realize precise siRNA delivery into tumor cells.

Jingxue Nai, Jinbang Zhang, Jiaxin Li, Hui Li, Yang Yang, Meiyan Yang, Yuli Wang, Wei Gong, Zhiping Li, Lin Li, Chunsheng Gao.

  Despite the success of small interfering RNAs (siRNAs) in clinical settings, their fast clearance and poor delivery efficiency to target cells still hinder their therapeutic effect. Herein, a new treatment system was constructed by combining thermosensitive liposomes with the macrophage membrane, tumor-targeting cyclic Arg-Gly-Asp peptide, a cell-penetrating peptide, and thermotherapy. The constructed system was found to be thermosensitive and stable; the proteins were inherited from the macrophage membrane. This new system combined with thermotherapy displayed the least uptake by macrophages, the greatest uptake by HepG2 cells, the most obvious HepG2 cell apoptosis, and the strongest inhibition of Bcl-2 mRNA and Bcl-2 protein in HepG2 cells. Moreover, 24 h after system administration in tumor-bearing mice, the most prominent distribution of siRNA was observed in tumors, while almost no siRNA was found in other organs. The strongest inhibition of Bcl-2 mRNA, Bcl-2 protein, and tumors was found in mice that had received the proposed system. In summary, when using the constructed system both in vitro and in mice, less uptake by the reticuloendothelial system, greater accumulation in tumor cells, and improved therapeutic efficacy were observed. Therefore, this new system can deliver siRNA selectively and efficiently, and it is a promising therapeutic candidate for precise tumor-targeted therapy.

Keywords:  CPP; cRGD; macrophage membrane; siRNA; thermosensitive liposomes; tumor targeting

DOI:  https://doi.org/10.1016/j.omtn.2021.12.016
Drug Discov Today. 2022 Jan 10. pii: S1359-6446(22)00003-4. [Epub ahead of print]

Role of siRNA-based nanocarriers for the treatment of neurodegenerative diseases.

Neeraj Mishra, Sumel Ashique, Ashish Garg, Vineet Kumar Rai, Kamal Dua, Amit Goyal, Shvetank Bhatt.

  Neurodegenerative disorders (NDs) lead to the progressive degeneration of the structural and physiological functions of the central and peripheral nervous systems, resulting in lifelong cognitive and motor dysfunction. Although comprehensive treatment of NDs is lacking, small interfering (si)RNA has shown therapeutic utility in the form of cellular nuclease-driven downregulation of mRNA levels. Various nanotechnologies have been used to modulate crucial physicochemical and biopharmaceutical properties of siRNA to provide protection and to enhance biomembrane interactions, residence times, tissue absorption, and cellular internalization for improved cytoplasm and/or nucleus interactions. In this review, we highlight advances in, and the role of, siRNA-based nanocarriers for the treatment of various NDs.

Keywords:  Liposomes; Nanoparticles; Nanostructured lipid carriers; Neurodegenerative disorders; Small interfering RNA; Solid lipid nanoparticle

DOI:  https://doi.org/10.1016/j.drudis.2022.01.003
ACS Appl Bio Mater. 2021 Dec 20. 4(12): 8259-8266

Polyethylene Glycol-Mediated Fusion of Extracellular Vesicles with Cationic Liposomes for the Design of Hybrid Delivery Systems.

Dipanjan Mukherjee, Debashish Paul, Sushmita Sarker, Md Nur Hasan, Ria Ghosh, Sujanthi Easwara Prasad, Praveen K Vemula, Ranjan Das, Arghya Adhikary, Samir Kumar Pal, Tatini Rakshit.

  To realize a customizable biogenic delivery platform, herein we propose combining cell-derived extracellular vesicles (EVs) derived from breast cancer cell line MCF-7 with synthetic cationic liposomes using a fusogenic agent, polyethylene glycol (PEG). We performed a fluorescence resonance energy transfer (FRET)-based lipid-mixing assay with varying PEG 1000 concentrations (0%, 15%, and 30%) correlated with flow cytometry-based analysis and supported by dimensional analysis by dynamic light scattering (DLS), transmission electron microscopy (TEM), and atomic force microscopy (AFM) to validate our fusion strategy. Our data revealed that these hybrid vesicles at a particular concentration of PEG (∼15%) improved the cellular delivery efficiency of a model siRNA molecule to the EV parental breast cancer cells, MCF-7, by factors of 2 and 4 compared to the loaded liposome and EV precursors, respectively. The critical rigidity/pliability balance of the hybrid systems fused by PEG seems to be playing a pivotal role in improving their delivery capability. This approach can provide clinically viable delivery solutions using EVs.

Keywords:  FRET; PEG; extracellular vesicle; flow cytometry; fusion; liposome; siRNA

DOI:  https://doi.org/10.1021/acsabm.1c00804
Int J Mol Sci. 2022 Jan 05. pii: 565. [Epub ahead of print]23(1):

Runx1 Messenger RNA Delivered by Polyplex Nanomicelles Alleviate Spinal Disc Hydration Loss in a Rat Disc Degeneration Model.

Cheng-Chung Chang, Hsi-Kai Tsou, Hsu-Hsin Chang, Long Yi Chan, Guan-Yu Zhuo, Tomoji Maeda, Chin-Yu Lin.

  Vertebral disc degenerative disease (DDD) affects millions of people worldwide and is a critical factor leading to low back and neck pain and consequent disability. Currently, no strategy has addressed curing DDD from fundamental aspects, because the pathological mechanism leading to DDD is still controversial. One possible mechanism points to the homeostatic status of extracellular matrix (ECM) anabolism, and catabolism in the disc may play a vital role in the disease's progression. If the damaged disc receives an abundant amount of cartilage, anabolic factors may stimulate the residual cells in the damaged disc to secrete the ECM and mitigate the degeneration process. To examine this hypothesis, a cartilage anabolic factor, Runx1, was expressed by mRNA through a sophisticated polyamine-based PEG-polyplex nanomicelle delivery system in the damaged disc in a rat model. The mRNA medicine and polyamine carrier have favorable safety characteristics and biocompatibility for regenerative medicine. The endocytosis of mRNA-loaded polyplex nanomicelles in vitro, mRNA delivery efficacy, hydration content, disc shrinkage, and ECM in the disc in vivo were also examined. The data revealed that the mRNA-loaded polyplex nanomicelle was promptly engulfed by cellular late endosome, then spread into the cytosol homogeneously at a rate of less than 20 min post-administration of the mRNA medicine. The mRNA expression persisted for at least 6-days post-injection in vivo. Furthermore, the Runx1 mRNA delivered by polyplex nanomicelles increased hydration content by ≈43% in the punctured disc at 4-weeks post-injection (wpi) compared with naked Runx1 mRNA administration. Meanwhile, the disc space and ECM production were also significantly ameliorated in the polyplex nanomicelle group. This study demonstrated that anabolic factor administration by polyplex nanomicelle-protected mRNA medicine, such as Runx1, plays a key role in alleviating the progress of DDD, which is an imbalance scenario of disc metabolism. This platform could be further developed as a promising strategy applied to regenerative medicine.

Keywords:  gene therapy; mRNA medicine; molecular imaging; nanomaterials; polyplex nanomicelle; tissue engineering

DOI:  https://doi.org/10.3390/ijms23010565