bims-novged 2022-03-20 papers

on Non-viral vectors for gene delivery

Issue of 2022–03–20
ten papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Influence of Hydrophobicity in the Hydrophilic Region of Cationic Lipids on Enhancing Nucleic Acid Delivery and Gene Editing.
Microfluidic Fabrication of Lipid Nanoparticles for the Delivery of Nucleic Acids.
Poly(N-vinylpyrrolidone)-block-Poly(dimethylsiloxane)-block-Poly(N-vinylpyrrolidone) Triblock Copolymer Polymersomes for Delivery of PARP1 siRNA to Breast Cancers.
Nanodiamond Integration into Niosomes as an Emerging and Efficient Gene Therapy Nanoplatform for Central Nervous System Diseases.
Dual pH-Responsive and Tumor-Targeted Nanoparticle-Mediated Anti-Angiogenesis siRNA Delivery for Tumor Treatment.
Intratumoral delivery of IL-12 and IL-27 mRNA using lipid nanoparticles for cancer immunotherapy.
Effective mRNA Protection by Poly(l-ornithine) Synergizes with Endosomal Escape Functionality of a Charge-Conversion Polymer toward Maximizing mRNA Introduction Efficiency.
In vivo Evaluation of Non-viral NICD Plasmid-Loaded PLGA Nanoparticles in Developing Zebrafish to Improve Cardiac Functions.
Controllable Disulfide Exchange Polymerization of Polyguanidine for Effective Biomedical Applications by Thiol-Mediated Uptake.
A nanovaccine for enhancing cellular immunity via cytosolic Co-delivery of antigen and PolyIC RNA.

ACS Appl Bio Mater. 2022 Mar 17.

Influence of Hydrophobicity in the Hydrophilic Region of Cationic Lipids on Enhancing Nucleic Acid Delivery and Gene Editing.

Hithavani Rapaka, Shireesha Manturthi, Porkizhi Arjunan, Vigneshwaran Venkatesan, Saravanabhavan Thangavel, Srujan Marepally, Srilakshmi V Patri.

  Intracellular delivery of biomolecules using non-viral vectors critically depends on the vectors' ability to allow the escape and release of the contents from the endosomes. Prior findings demonstrated that aromatic/hydrophobic group-containing amino acids such as phenylalanine (F) and tryptophan (W) destabilize cellular membranes by forming pores in the lipid bilayer. Taking cues from these findings, we have developed four α-tocopherol-based cationic amphiphiles by varying the aromatic/hydrophobic amino acids such as glycine (G), proline (P), phenylalanine (F), and tryptophan (W) as head groups and triazole in the linker region to study their impact on endosomal escape for the enhanced transfection efficacy. The lipids tocopherol-triazole-phenylalanine (TTF) and tocopherol-triazole-tryptophan (TTW) exhibited similar potential to commercial transfecting reagents, Lipofectamine (LF) 3000 and Lipofectamine Messenger Max (LFMM), respectively, in transfecting plasmid DNA and messenger RNA in multiple cultured cell lines. The TTW liposome was also found to be effective in delivering Cas9 mRNA and demonstrated equal efficiency of gene editing AAVS1 locus compared to LFMM in CHO, Neuro-2a, and EA.HY926 cell lines. In this current investigation, it is shown that the synthesized cationic lipids with aromatic hydrophobic R group-containing amino acids are safe, economic, and actually more efficient in nucleic acid delivery and genome-editing applications. These findings can be further explored in the genome-editing approach for treating genetic disorders.

Keywords:  cationic lipids; endosomal escape; gene editing; non-viral nucleic acid delivery; α-tocopherol

DOI:  https://doi.org/10.1021/acsabm.1c01226
Adv Drug Deliv Rev. 2022 Mar 11. pii: S0169-409X(22)00087-4. [Epub ahead of print] 114197

Microfluidic Fabrication of Lipid Nanoparticles for the Delivery of Nucleic Acids.

Gyan Prakash, Ahmed Shokr, Niels Willemen, Showkeen Muzamil Bashir, Su Ryon Shin, Shabir Hassan.

  Gene therapy has emerged as a potential platform for treating several dreaded and rare diseases that would otherwise not be possible with traditional therapies. Due to their ability to transport genomes to cells, Viral vectors have been a platform of choice in gene delivery applications. However, since their delivery is not precision based, the application has led to off-target toxicities. As such, various strategies in the form of non-viral gene delivery vehicles have been explored and are being developed. In this review, we discuss the opportunities lipid nanoparticles (LNPs) present for gene delivery, efficiently and precisely. We also discuss synthesis strategies via microfluidics used for high throughput fabrication of such non-viral gene delivery vehicles. Finally, the application of these vehicles for the delivery of different genetic materials such as peptides and RNA for different diseases ranging from more common diseases to rare diseases are explored.

Keywords:  CRISPR-delivery; Non-viral vectors; gene delivery; lipid nanoparticles; microfluidics

DOI:  https://doi.org/10.1016/j.addr.2022.114197
ACS Appl Bio Mater. 2022 Mar 16.

Poly(N-vinylpyrrolidone)-block-Poly(dimethylsiloxane)-block-Poly(N-vinylpyrrolidone) Triblock Copolymer Polymersomes for Delivery of PARP1 siRNA to Breast Cancers.

Yiming Yang, Veronika Kozlovskaya, Zhuo Zhang, Chuan Xing, Steve Zaharias, Maksim Dolmat, Shuo Qian, Jun Zhang, Jason M Warram, Eddy S Yang, Eugenia Kharlampieva.

  Nearly 20% of HER2-positive breast cancers develop resistance to HER2-targeted therapies requiring the use of advanced therapies. Silencing RNA therapy may be a powerful modality for treating resistant HER2 cancers due to its high specificity and low toxicity. However, the systemic administration of siRNAs requires a safe and efficient delivery platform because of siRNA's low stability in physiological fluids, inefficient cellular uptake, immunoreactivity, and rapid clearance. We have developed theranostic polymeric vesicles to overcome these hurdles for encapsulation and delivery of small functional molecules and PARP1 siRNA for in vivo delivery to breast cancer tumors. The 100 nm polymer vesicles were assembled from biodegradable and non-ionic poly(N-vinylpyrrolidone)14-block-poly(dimethylsiloxane)47-block-poly(N-vinylpyrrolidone)14 triblock copolymer PVPON14-PDMS47-PVPON14 using nanoprecipitation and thin-film hydration. We demonstrated that the vesicles assembled from the copolymer covalently tagged with the Cy5.5 fluorescent dye for in vivo imaging could also encapsulate the model drug with high loading efficiency (40%). The dye-loaded vesicles were accumulated in tumors after 18 h circulation in 4TR breast tumor-bearing mice via passive targeting. We found that PARP1 siRNA encapsulated into the vesicles was released intact (13%) into solution by the therapeutic ultrasound treatment as quantified by gel electrophoresis. The PARP1 siRNA-loaded polymersomes inhibited the proliferation of MDA-MB-361TR cells by 34% after 6 days of treatment by suppressing the NF-kB signaling pathway, unlike their scrambled siRNA-loaded counterparts. Finally, the treatment by PARP1 siRNA-loaded vesicles prolonged the survival of the mice bearing 4T1 breast cancer xenografts, with the 4-fold survival increase, unlike the untreated mice after 3 weeks following the treatment. These biodegradable, non-ionic PVPON14-PDMS47-PVPON14 polymeric nanovesicles capable of the efficient encapsulation and delivery of PARP1 siRNA to successfully knock down PARP1 in vivo can provide an advanced platform for the development of precision-targeted therapeutic carriers, which could help develop highly effective drug delivery nanovehicles for breast cancer gene therapy.

Keywords:  PARP1; RNA; anticancer drug delivery; in vivo imaging; poly(N-vinylpyrrolidone); polymersomes; triblock copolymer

DOI:  https://doi.org/10.1021/acsabm.2c00063
ACS Appl Mater Interfaces. 2022 Mar 15.

Nanodiamond Integration into Niosomes as an Emerging and Efficient Gene Therapy Nanoplatform for Central Nervous System Diseases.

Nuseibah Al Qtaish, Idoia Gallego, Alejandro J Paredes, Ilia Villate-Beitia, Cristina Soto-Sánchez, Gema Martínez-Navarrete, Myriam Sainz-Ramos, Tania B Lopez-Mendez, Eduardo Fernández, Gustavo Puras, José Luis Pedraz.

  Nanodiamonds (NDs) are promising materials for gene delivery because of their unique physicochemical and biological features, along with their possibility of combination with other nonviral systems. Our aim was to evaluate the biophysical performance of NDs as helper components of niosomes, named nanodiasomes, to address a potential nonviral gene delivery nanoplatform for therapeutic applications in central nervous system (CNS) diseases. Nanodiasomes, niosomes, and their corresponding complexes, obtained after genetic material addition at different ratios (w/w), were evaluated in terms of physicochemical properties, cellular uptake, intracellular disposition, biocompatibility, and transfection efficiency in HEK-293 cells. Nanodiasomes, niosomes, and complexes fulfilled the physicochemical features for gene therapy applications. Biologically, the incorporation of NDs into niosomes enhanced 75% transfection efficiency (p < 0.001) and biocompatibility (p < 0.05) to values over 90%, accompanied by a higher cellular uptake (p < 0.05). Intracellular trafficking analysis showed higher endocytosis via clathrins (p < 0.05) in nanodiaplexes compared with nioplexes, followed by higher lysosomal colocalization (p < 0.05), that coexisted with endosomal escape properties, whereas endocytosis mediated by caveolae was the most efficient pathway in the case of nanodiaplexes. Moreover, studies in CNS primary cells revealed that nanodiaplexes successfully transfected neuronal and retinal cells. This proof-of-concept study points out that ND integration into niosomes represents an encouraging nonviral nanoplatform strategy for the treatment of CNS diseases by gene therapy.

Keywords:  CNS diseases; cationic lipids; gene delivery; nanodiamonds; nanomedicine; niosomes

DOI:  https://doi.org/10.1021/acsami.2c02182
Int J Nanomedicine. 2022 ;17 953-967

Dual pH-Responsive and Tumor-Targeted Nanoparticle-Mediated Anti-Angiogenesis siRNA Delivery for Tumor Treatment.

Xiangyang Zhang, Bin Qin, Min Wang, Junyi Feng, Chenglin Zhang, Chengshen Zhu, Suqin He, Hao Liu, Yaohe Wang, Saadyah E Averick, Nga T N Vo, Lei Huang, Wentao Liu, Zhimin Wang.

   Purpose: In order to overcome the biological barriers at all levels and enhance the delivery efficiency of siRNA, we have prepared a multifunctional siRNA delivery system (CHCE/siRNA nanoparticles) through self-assembly of the carboxymethyl chitosan modified with histidine, cholesterol, and anti-EGFR antibody (CHCE).
Methods: The morphology of CHCE/siRNA NPs was detected by dynamic light scattering and scanning electron microscope. In vitro, we assessed the tumor-targeting, cellular uptake, and endosomal escape by flow cytometry and confocal laser scanning microscopy, confirming the CHCE/siRNA NPs functions in gene silencing and cell killing ability. In vivo, we examined the biodistribution of the CHCE/siRNA NPs by the IVIS imaging system and confirmed the therapeutic effect of NPs in the nude-mouse tumor model.
Results: The CHCE/siRNA NPs exhibited nanosized spherical with narrow size distribution. In vitro, the CHCE/siRNA NPs incorporated a dual capability of tumor targeting and pH response that could facilitate cellular bind, cellular uptake, and endosomal escape. The CHCE/siRNA NPs could effectively silence the vascular endothelial growth factor A (VEGFA) to cause cell apoptosis and inhibit proliferation. In vivo, the CHCE/siRNA NPs could target tumor sites to knock down VEGFA and achieve a better anti-tumor effect.
Conclusion: We successfully prepared a novel siRNA delivery system with the double capability of tumor targeting and pH response, which can break through the biological barriers to penetrate deep into tumors and achieve better therapeutic tumor effects, providing a new ideal delivery platform for siRNA.

Keywords:  anti-tumor therapy; endosomal escape; gene silencing; multifunctional carboxymethyl chitosan; targeting delivery

DOI:  https://doi.org/10.2147/IJN.S340926
J Control Release. 2022 Mar 14. pii: S0168-3659(22)00147-X. [Epub ahead of print]

Intratumoral delivery of IL-12 and IL-27 mRNA using lipid nanoparticles for cancer immunotherapy.

Jin-Qing Liu, Chengxiang Zhang, Xinfu Zhang, Jingyue Yan, Chunxi Zeng, Fatemeh Talebian, Kimberly Lynch, Weiyu Zhao, Xucheng Hou, Shi Du, Diana D Kang, Binbin Deng, David W McComb, Xue-Feng Bai, Yizhou Dong.

  Cytokines are important immunotherapeutics with approved drugs for the treatment of human cancers. However, systemic administration of cytokines often fails to achieve adequate concentrations to immune cells in tumors due to dose-limiting toxicity. Thus, developing localized therapy that directly delivers immune-stimulatory cytokines to tumors may improve the therapeutic efficacy. In this study, we generated novel lipid nanoparticles (LNPs) encapsulated with mRNAs encoding cytokines including IL-12, IL-27 and GM-CSF, and tested their anti-tumor activity. We first synthesized ionizable lipid materials containing di-amino groups with various head groups (DALs). The novel DAL4-LNP effectively delivered different mRNAs in vitro to tumor cells and in vivo to tumors. Intratumoral injection of DAL4-LNP loaded with IL-12 mRNA was most potent in inhibiting B16F10 melanoma tumor growth compared to IL-27 or GM-CSF mRNAs in monotherapy. Furthermore, intratumoral injection of dual DAL4-LNP-IL-12 mRNA and IL-27 mRNA showed a synergistic effect in suppressing tumor growth without causing systematic toxicity. Most importantly, intratumoral delivery of IL-12 and IL-27 mRNAs induced robust infiltration of immune effector cells, including IFN-γ and TNF-α producing NK and CD8+ T cells into tumors. Thus, intratumoral administration of DAL-LNP loaded with IL-12 and IL-27 mRNA provides a new treatment strategy for cancer.

Keywords:  Cancer immunotherapy; Cytokines; Diamino lipid-derived nanoparticles (DAL-LNPs); mRNA therapeutics

DOI:  https://doi.org/10.1016/j.jconrel.2022.03.021
Macromol Rapid Commun. 2022 Mar 14. e2100754

Effective mRNA Protection by Poly(l-ornithine) Synergizes with Endosomal Escape Functionality of a Charge-Conversion Polymer toward Maximizing mRNA Introduction Efficiency.

Anjaneyulu Dirisala, Satoshi Uchida, Junjie Li, Joachim F R Van Guyse, Kotaro Hayashi, Sai V C Vummaleti, Sarandeep Kaur, Yuki Mochida, Shigeto Fukushima, Kazunori Kataoka.

  For efficient delivery of messenger (m)RNA, delivery carriers need two major functions: protecting mRNA from nucleases and translocating mRNA from endolysosomes to the cytoplasm. Herein, these two complementary functionalities are integrated into a single polyplex by fine-tuning the catiomer chemical structure and incorporating the endosomal escape modality. The effect of the methylene spacer length on the catiomer side chain is evaluated by comparing poly(l-lysine) (PLL) with a tetramethylene spacer and poly(L-ornithine) (PLO) with a trimethylene spacer. Noteworthily, the nuclease stability of the mRNA/catiomer polyplexes is largely affected by the difference in one methylene group, with PLO/mRNA polyplex showing enhanced stability compared to PLL/mRNA polyplex. To introduce the endosomal escape function, the PLO/mRNA polyplex is wrapped with a charge-conversion polymer (CCP), which is negatively charged at extracellular pH but turns positive at endosomal acidic pH to disrupt the endosomal membrane. Compared to the parent PLO/mRNA polyplex, CCP facilitated the endosomal escape of the polyplex in cultured cells to improve the protein expression efficiency from mRNA by approximately 80-fold. Collectively, this system synergizes the protective effect of PLO against nucleases and the endosomal escape capability of CCP in mRNA delivery.

Keywords:  mRNA delivery; poly(l-ornithine); polyion complexes; stimuli-sensitive polymers; supramolecular systems

DOI:  https://doi.org/10.1002/marc.202100754
Front Physiol. 2022 ;13 819767

In vivo Evaluation of Non-viral NICD Plasmid-Loaded PLGA Nanoparticles in Developing Zebrafish to Improve Cardiac Functions.

Victoria L Messerschmidt, Uday Chintapula, Fabrizio Bonetesta, Samantha Laboy-Segarra, Amir Naderi, Kytai T Nguyen, Hung Cao, Edward Mager, Juhyun Lee.

  In the era of the advanced nanomaterials, use of nanoparticles has been highlighted in biomedical research. However, the demonstration of DNA plasmid delivery with nanoparticles for in vivo gene delivery experiments must be carefully tested due to many possible issues, including toxicity. The purpose of the current study was to deliver a Notch Intracellular Domain (NICD)-encoded plasmid via poly(lactic-co-glycolic acid) (PLGA) nanoparticles and to investigate the toxic environmental side effects for an in vivo experiment. In addition, we demonstrated the target delivery to the endothelium, including the endocardial layer, which is challenging to manipulate gene expression for cardiac functions due to the beating heart and rapid blood pumping. For this study, we used a zebrafish animal model and exposed it to nanoparticles at varying concentrations to observe for specific malformations over time for toxic effects of PLGA nanoparticles as a delivery vehicle. Our nanoparticles caused significantly less malformations than the positive control, ZnO nanoparticles. Additionally, the NICD plasmid was successfully delivered by PLGA nanoparticles and significantly increased Notch signaling related genes. Furthermore, our image based deep-learning analysis approach evaluated that the antibody conjugated nanoparticles were successfully bound to the endocardium to overexpress Notch related genes and improve cardiac function such as ejection fraction, fractional shortening, and cardiac output. This research demonstrates that PLGA nanoparticle-mediated target delivery to upregulate Notch related genes which can be a potential therapeutic approach with minimum toxic effects.

Keywords:  Notch signaling; PLGA nanoparticles; gene delivery; non-viral transfection; toxicity; zebrafish

DOI:  https://doi.org/10.3389/fphys.2022.819767
Angew Chem Int Ed Engl. 2022 Mar 18.

Controllable Disulfide Exchange Polymerization of Polyguanidine for Effective Biomedical Applications by Thiol-Mediated Uptake.

Fu-Jian Xu, Yiwen Zhu, Mengyu Lin, Wenting Hu, Junkai Wang, Zhi-Guo Zhang, Kai Zhang, Bingran Yu.

  New preparation methods of vectors are the key to developing next generation of biomacromolecule delivery systems. In this study, a controllable disulfide exchange polymerization was established to obtain low-toxicity and efficient bioreducible polyguanidines (mPEG225-b-PSSn, n = 13, 26, 39, 75, 105) by regulating the concentration of activated nucleophiles and reaction time under mild reaction conditions. The relationship between the degrees of polymerization and biocompatibility was studied to identify the optimal polyguanidine mPEG225-b-PSS26. Such polyguanidine exhibited good in virto performance in delivering different functional nucleic acids. The impressive therapeutic effects of mPEG225-b-PSS26 were further verified in the 4T1 tumor-bearing mice as well as the mice with full-thickness skin defects. Controllable disulfide exchange polymerization provides an attractive strategy for the construction of new biomacromolecule delivery systems.

Keywords:  Wound Healing; antibacterial; antitumor; controllable disulfide exchange polymerization; gene delivery

DOI:  https://doi.org/10.1002/anie.202200535
J Control Release. 2022 Mar 14. pii: S0168-3659(22)00146-8. [Epub ahead of print]

A nanovaccine for enhancing cellular immunity via cytosolic Co-delivery of antigen and PolyIC RNA.

Carcia S Carson, Kyle W Becker, Kyle M Garland, Hayden M Pagendarm, Payton T Stone, Karan Arora, Lihong Wang-Bishop, Jessalyn J Baljon, Lorena D Cruz, Sebastian Joyce, John T Wilson.

  Traditional approaches to cancer vaccines elicit weak CD8+ T cell responses and have largely failed to meet clinical expectations. This is in part due to inefficient antigen cross-presentation, inappropriate selection of adjuvant and its formulation, poor vaccine pharmacokinetics, and/or suboptimal coordination of antigen and adjuvant delivery. Here, we describe a nanoparticle vaccine platform for facile co-loading and dual-delivery of antigens and nucleic acid adjuvants that elicits robust antigen-specific cellular immune responses. The nanovaccine design is based on diblock copolymers comprising a poly(ethylene glycol)-rich first block that is functionalized with reactive moieties for covalent conjugation of antigen via disulfide linkages, and a pH-responsive second block for electrostatic packaging of nucleic acids that also facilitates endosomal escape of associated vaccine cargo to the cytosol. Using polyIC, a clinically-advanced nucleic acid adjuvant, we demonstrated that endosomolytic nanoparticles promoted the cytosolic co-delivery of polyIC and protein antigen, which acted synergistically to enhance antigen cross-presentation, co-stimulatory molecule expression, and cytokine production by dendritic cells. We also found that the vaccine platform increased the accumulation of antigen and polyIC in the local draining lymph nodes. Consequently, dual-delivery of antigen and polyIC with endsomolytic nanoparticles significantly enhanced the magnitude and functionality of CD8+ T cell responses relative to a mixture of antigen and polyIC, resulting in inhibition of tumor growth in a mouse tumor model. Collectively, this work provides a proof-of-principle for a new cancer vaccine platform that strongly augments anti-tumor cellular immunity via cytosolic co-delivery of antigen and nucleic acid adjuvant.

Keywords:  Cancer vaccine; Endosomal escape; Nucleic acid adjuvant; PolyIC; T cells; pH-responsive

DOI:  https://doi.org/10.1016/j.jconrel.2022.03.020