bims-novged 2022-08-07 papers

bims-novged

Biomed News

on Non-viral vectors for gene delivery

Issue of 2022–08–07
ten papers selected by
the Merkel lab, Ludwig-Maximilians University

Engineered ionizable lipid siRNA conjugates enhance endosomal escape but induce toxicity in vivo.
Hyaluronate-coated perfluoroalkyl polyamine prodrugs as bioactive siRNA delivery systems for the treatment of peritoneal cancers.
Artificial primary-miRNAs as a platform for simultaneous delivery of siRNA and antisense oligonucleotide for multimodal gene regulation.
Delivery of Therapeutic miR-148b Mimic via Poly(β Amino Ester) Polyplexes for Post-transcriptional Gene Regulation and Apoptosis of A549 Cells.
VCAM-1-binding peptide targeted cationic liposomes containing NLRP3 siRNA to modulate LDL transcytosis as a novel therapy for experimental atherosclerosis.
Ultra-thin layered double hydroxide-mediated photothermal therapy combine with asynchronous blockade of PD-L1 and NR2F6 inhibit hepatocellular carcinoma.
Role of a "Magic" Methyl: 2'-Deoxy-2'-α-F-2'-β-C-methyl Pyrimidine Nucleotides Modulate RNA Interference Activity through Synergy with 5'-Phosphate Mimics and Mitigation of Off-Target Effects.
Poly(lipoic acid)-based nanoparticles as a new therapeutic tool for delivering active molecules.
The Effects of Serum Albumin Pre-Adsorption of Nanoparticles on Protein Corona and Membrane Interaction: A Molecular Simulation Study.
Customizing nano-chitosan for sustainable drug delivery.

J Control Release. 2022 Jul 30. pii: S0168-3659(22)00476-X. [Epub ahead of print]

Engineered ionizable lipid siRNA conjugates enhance endosomal escape but induce toxicity in vivo.

Annabelle Biscans, Socheata Ly, Nicholas McHugh, David A Cooper, Anastasia Khvorova.

  Lipid conjugation supports delivery of small interfering RNAs (siRNAs) to extrahepatic tissues, expanding the therapeutic potential of siRNAs beyond liver indications. However, siRNA silencing efficacy in extrahepatic tissues remains inferior to that routinely achieved in liver, partially due to the low rate of endosomal escape following siRNA internalization. Improving siRNA endosomal release into cytoplasm is crucial to improving efficacy of lipid-conjugated siRNAs. Given the ability of ionizable lipids to enhance endosomal escape in a context of lipid nanoparticles (LNP), here, we provide the first report on the effect of an ionizable lipid conjugate on siRNA endosomal escape, tissue distribution, efficacy, and toxicity in vivo. After developing a synthetic route to covalently attach the ionizable lipid, DLin-MC3-DMA, to siRNAs, we demonstrate that DLin-MC3-DMA enhances endosomal escape in cell culture without compromising siRNA efficacy. In mice, DLin-MC3-DMA conjugated siRNAs exhibit a similar overall tissue distribution profile to the similarly hydrophobic cholesterol-conjugated siRNA. However, only DLin-MC3-DMA conjugated siRNAs accumulated in vascular compartments, suggesting an effect of conjugate structure on intratissue distribution. Interestingly, we observed non-specific modulation of gene expression in tissues with high accumulation of DLin-MC3-DMA siRNAs (>20 pmol/mg of tissue) while limited non-specific gene modulation has been observed in tissues with lower siRNA accumulation. These findings suggest modulating the nature of the conjugate is a promising strategy to alter siRNA intratissue and intracellular trafficking. Fine-tuning the nature of the conjugate to optimize endosomal escape while minimizing toxicity will be critical for the progression of therapeutic siRNA applications beyond the liver.

Keywords:  Endosomal escape; Ionizable lipid; Lipid conjugation; RNA interference; Therapeutic oligonucleotides; siRNA extrahepatic delivery

DOI:  https://doi.org/10.1016/j.jconrel.2022.07.041
Biomater Adv. 2022 May;pii: S2772-9508(22)00032-2. [Epub ahead of print]136 212755

Hyaluronate-coated perfluoroalkyl polyamine prodrugs as bioactive siRNA delivery systems for the treatment of peritoneal cancers.

Ao Yu, Siyuan Tang, Ling Ding, Jackson Foley, Weimin Tang, Huizhen Jia, Sudipta Panja, Cassandra E Holbert, Yu Hang, Tracy Murray Stewart, Lynette M Smith, Diptesh Sil, Robert A Casero, David Oupický.

  RNA interference (RNAi) is an emerging therapeutic modality for cancer, which remains in critical need of effective delivery vectors due to the unfavorable biopharmaceutical properties of small RNAs. Polyamines are essential for functioning of mammalian cells. Dysregulated polyamine metabolism is found in many cancers and has been an attractive therapeutic target in combination therapies. Combination therapies based on drugs that affect polyamine metabolism and nucleic acids promise to enhance anticancer activity due to a cooperative effect on multiple oncogenic pathways. Here, we report bioactive polycationic prodrug (F-PaP) based on an anticancer polyamine analogue bisethylnorspermine (BENSpm) modified with perfluoroalkyl moieties. Following encapsulation of siRNA, F-PaP/siRNA nanoparticles were coated with hyaluronic acid (HA) to form ternary nanoparticles HA@F-PaP/siRNA. The presence of perfluoroalkyl moieties and HA reduced cell membrane toxicity and improved stability of the particles with cooperatively enhanced siRNA delivery in pancreatic and colon cancer cell lines. We then tested a therapeutic hypothesis that combining BENSpm with siRNA silencing of polo-like kinase 1 (PLK1) would result in cooperative cancer cell killing. HA@F-PaP/siPLK1 induced polyamine catabolism and cell cycle arrest, leading to enhanced apoptosis in the tested cell lines. The HA-coated nanoparticles facilitated tumor accumulation and contributed to strong tumor inhibition and favorable modulation of the immune tumor microenvironment in orthotopic pancreatic cancer model.

Keywords:  Combination cancer therapy; Hyaluronic acid; Perfluoroalkyls; Polyamines; siRNA delivery

DOI:  https://doi.org/10.1016/j.bioadv.2022.212755
J Control Release. 2022 Aug 02. pii: S0168-3659(22)00479-5. [Epub ahead of print]

Artificial primary-miRNAs as a platform for simultaneous delivery of siRNA and antisense oligonucleotide for multimodal gene regulation.

Hyunsook Kim, Bora Jang, Dayoung Lee, S Chul Kwon, Hyukjin Lee.

  Self-assembled nucleic acid nanostructures have been widely explored for gene therapy applications due to their unique advantages. Their roles are not limited to offer intracellular delivery platforms but additionally provide a biological function to induce targeted gene regulation. Here, we report a self-assembled artificial primary-miRNA (pri-miRNA) for achieving simultaneous multimodal gene regulation. Artificial pri-miRNAs are designed to play a role as substrate RNAs to recruit and interact with Drosha/DGCR8 (Microprocessor). Incorporation of functional RNA motifs and site-specific chemical modification of the primary miRNA are utilized for the biogenesis of two individual gene-regulating oligonucleotides. Once they are cleaved by the endogenous Drosha/DGCR8 complex, basal strands and pre-miRNA can be generated inside of cells. In this study, we integrated basal strands with either SMN2 ASO or anti-miR21 to induce multimodal gene regulation. Microprocessing and subsequent gene regulation were first evaluated by measuring the activity of reporter pre-miRNA. Chemical modification on the primary miRNA was optimized through a series of in vitro Drosha cleavage tests and targeted gene silencing in cells. Primary miRNA with the basal ASO or anti-miR strands showed a successful in vitro activity and resulted in simultaneous multimodal gene regulation in cells. Artificial primary miRNA may offer synergistic therapeutic effects for treating various diseases, including spinal muscular atrophy and cancer.

Keywords:  Artificial primary miRNA; Multimodal gene regulation; Nucleic acid nanostructures; RNA motif

DOI:  https://doi.org/10.1016/j.jconrel.2022.07.043
Langmuir. 2022 Aug 02.

Delivery of Therapeutic miR-148b Mimic via Poly(β Amino Ester) Polyplexes for Post-transcriptional Gene Regulation and Apoptosis of A549 Cells.

Nick A Alden, Julien H Arrizabalaga, Yiming Liu, Shantu Amin, Krishne Gowda, Shun Yao, Marco Archetti, Adam B Glick, Daniel J Hayes.

In this study, we utilized selectively modified, biodegradable polymer-based polyplexes to deliver custom, exogenous miR-148b mimics to induce apoptosis in human lung cancer (A549) cells. The gene regulatory effects of the payload miRNA mimics (miR-148b-3p) were first evaluated through bioinformatic analyses to uncover specific gene targets involved in critical carcinogenic pathways. Hyperbranched poly(β amino ester) polyplexes (hPBAE) loaded with custom miR-148b mimics were then developed for targeted therapy. When evaluated in vitro, these hPBAE-based polyplexes sustained high intracellular uptake, low cytotoxicity, and efficient escape from endosomes to deliver functionally intact miRNA mimics to the cytosol. High-resolution confocal microscopy revealed successful intracellular uptake, cell viability was assessed through qualitative fluorescence microscopy and fluorescence-based DNA quantification, and successful cytosolic delivery of intact miRNA mimics was evaluated using real-time polymerase chain reaction (RT-PCR) to demonstrate target gene knockdown. The hPBAE-miRNA mimic polyplexes were shown to induce apoptosis among A549 cells through direct modulation of intracellular protein expression, targeting multiple potential carcinogenic pathways at the gene level. These results indicated that spatially controlled miR-148b mimic delivery can promote efficient cancer cell death in vitro and may lead to an enhanced therapeutic design for in vivo application.

DOI: https://doi.org/10.1021/acs.langmuir.2c00913
Metabolism. 2022 Jul 30. pii: S0026-0495(22)00152-4. [Epub ahead of print]135 155274

VCAM-1-binding peptide targeted cationic liposomes containing NLRP3 siRNA to modulate LDL transcytosis as a novel therapy for experimental atherosclerosis.

Xiong Jia, Xiangli Bai, Xiaoyan Yang, Ling Wang, Yajing Lu, Lin Zhu, Ying Zhao, Wenzhuo Cheng, Meng Shu, Qiyong Mei, Si Jin.

   BACKGROUND: Activation of NLRP3 inflammasome accelerates the formation of atherosclerotic plaques. Here, we evaluated the effects of inflammation on the expression of the NLRP3 inflammasome in endothelial cells (ECs).
METHODS: The effect of TNF-α on transcytosis of LDL was measured. VCAM-1 binding peptide targeting cationic liposomes (PCLs) were prepared as siRNA vectors. Methylated NLRP3 siRNA was encapsulated into the PCLs to knock down NLRP3 in vitro and in vivo. In rats with partial carotid ligation, TNF-α-induced LDL retention in the carotid artery endothelium was observed. In ApoE-/- mice, NLRP3 siRNA-PCLs were injected intravenously to observe their effect on the formation of atherosclerosis.
RESULTS: Our results showed that TNF-α upregulated NLRP3 in ECs, promoting the assembly of the NLRP3 inflammasome and processing of pro-IL-1β into IL-1β. Moreover, TNF-α accelerated LDL transcytosis in ECs. Knockdown of NLRP3 prevented TNF-α-induced NLPR3 inflammasome/IL-1β signaling and LDL transcytosis. Using optimized cationic liposomes to encapsulate methylated NLRP3 siRNA, resulting in targeting of VCAM-1-expressing ECs, to knockdown NLRP3, TNF-α-induced NLRP3 inflammasome activation and LDL transcytosis were prevented. Using the partial carotid ligation as an atherosclerosis rat model, we found that local administration of NLRP3 siRNA-PCLs efficiently knocked down NLPR3 expression in the carotid endothelium and dramatically attenuated the deposition of atherogenic LDL in carotid ECs in TNF-α-challenged rats. Furthermore, NLRP3 siRNA-PCLs were injected intravenously in ApoE-/- mice, resulting in reduced plaque formation.
CONCLUSION: These findings established a novel strategy for targeting the NLRP3 inflammasome using NLRP3 siRNA-PCLs to interrupt LDL transcytosis, representing a potential novel therapy for atherosclerosis.

Keywords:  Atherosclerosis; Cationic liposome; LDL transcytosis; NLRP3 Inflammasome; Targeted delivery

DOI:  https://doi.org/10.1016/j.metabol.2022.155274
J Nanobiotechnology. 2022 Jul 30. 20(1): 351

Ultra-thin layered double hydroxide-mediated photothermal therapy combine with asynchronous blockade of PD-L1 and NR2F6 inhibit hepatocellular carcinoma.

Yuan-Fei Lu, Jia-Ping Zhou, Qiao-Mei Zhou, Xiao-Yan Yang, Xiao-Jie Wang, Jie-Ni Yu, Jin-Guo Zhang, Yong-Zhong Du, Ri-Sheng Yu.

   BACKGROUND: The efficacy of immune checkpoint blockade (ICB), in the treatment of hepatocellular carcinoma (HCC), is limited due to low levels of tumor-infiltrating T lymphocytes and deficient checkpoint blockade in this immunologically "cool" tumor. Thus, combination approaches are needed to increase the response rates of ICB and induce synergistic antitumor immunity.
METHODS: Herein, we designed a pH-sensitive multifunctional nanoplatform based on layered double hydroxides (LDHs) loaded with siRNA to block the intracellular immune checkpoint NR2F6, together with the asynchronous blockade surface receptor PD-L1 to induce strong synergistic antitumor immunity. Moreover, photothermal therapy (PTT) generated by LDHs after laser irradiation modified an immunologically "cold" microenvironment to potentiate Nr2f6-siRNA and anti-PD-L1 immunotherapy. Flow cytometry was performed to assess the immune responses initiated by the multifunctional nanoplatform.
RESULTS: Under the slightly acidic tumor extracellular environment, PEG detached and the re-exposed positively charged LDHs enhanced tumor accumulation and cell uptake. The accumulated siRNA suppressed the signal of dual protumor activity in both immune and H22 tumor cells by silencing the NR2F6 gene, which further reduced the tumor burden and enhanced systemic antitumor immunity. The responses include enhanced tumor infiltration by CD4+ helper T cells, CD8+ cytotoxic T cells, and mature dendritic cells; the significantly decreased level of immune suppressed regulator T cells. The therapeutic responses were also attributed to the production of IL-2, IFN-γ, and TNF-α. The prepared nanoparticles also exhibited potential magnetic resonance imaging (MRI) ability, which could serve to guide synergistic immunotherapy treatment.
CONCLUSIONS: In summary, the three combinations of PTT, NR2F6 gene ablation and anti-PD-L1 can promote a synergistic immune response to inhibit the progression of primary HCC tumors and prevent metastasis. This study can be considered a proof-of-concept for the targeting of surface and intracellular immune checkpoints to supplement the existing HCC immunotherapy treatments.

Keywords:  Hepatocellular carcinoma; NR2F6; PD-L1 blockade; Photothermal therapy

DOI:  https://doi.org/10.1186/s12951-022-01565-9
J Am Chem Soc. 2022 Aug 03.

Role of a "Magic" Methyl: 2'-Deoxy-2'-α-F-2'-β-C-methyl Pyrimidine Nucleotides Modulate RNA Interference Activity through Synergy with 5'-Phosphate Mimics and Mitigation of Off-Target Effects.

Dale C Guenther, Shohei Mori, Shigeo Matsuda, Jason A Gilbert, Jennifer L S Willoughby, Sarah Hyde, Anna Bisbe, Yongfeng Jiang, Saket Agarwal, Mimouna Madaoui, Maja M Janas, Klaus Charisse, Martin A Maier, Martin Egli, Muthiah Manoharan.

Although 2'-deoxy-2'-α-F-2'-β-C-methyl (2'-F/Me) uridine nucleoside derivatives are a successful class of antiviral drugs, this modification had not been studied in oligonucleotides. Herein, we demonstrate the facile synthesis of 2'-F/Me-modified pyrimidine phosphoramidites and their subsequent incorporation into oligonucleotides. Despite the C3'-endo preorganization of the parent nucleoside, a single incorporation into RNA or DNA resulted in significant thermal destabilization of a duplex due to unfavorable enthalpy, likely resulting from steric effects. When located at the terminus of an oligonucleotide, the 2'-F/Me modification imparted more resistance to degradation than the corresponding 2'-fluoro nucleotides. Small interfering RNAs (siRNAs) modified at certain positions with 2'-F/Me had similar or better silencing activity than the parent siRNAs when delivered via a lipid nanoparticle formulation or as a triantennary N-acetylgalactosamine conjugate in cells and in mice. Modification in the seed region of the antisense strand at position 6 or 7 resulted in an activity equivalent to the parent in mice. Additionally, placement of the antisense strand at position 7 mitigated seed-based off-target effects in cell-based assays. When the 2'-F/Me modification was combined with 5'-vinyl phosphonate, both E and Z isomers had silencing activity comparable to the parent. In combination with other 2'-modifications such as 2'-O-methyl, the Z isomer is detrimental to silencing activity. Presumably, the equivalence of 5'-vinyl phosphonate isomers in the context of 2'-F/Me is driven by the steric and conformational features of the C-methyl-containing sugar ring. These data indicate that 2'-F/Me nucleotides are promising tools for nucleic acid-based therapeutic applications to increase potency, duration, and safety.

DOI: https://doi.org/10.1021/jacs.2c01679
Nanomedicine. 2022 Jul 27. pii: S1549-9634(22)00079-X. [Epub ahead of print]45 102593

Poly(lipoic acid)-based nanoparticles as a new therapeutic tool for delivering active molecules.

Chiara Castellani, Claudia Maria Radu, Lucia Morillas-Becerril, Ilaria Barison, Federica Menato, Tomaz Michele Do Nascimento, Marny Fedrigo, Alessia Giarraputo, Grazia Maria Virzì, Paolo Simioni, Cristina Basso, Emanuele Papini, Regina Tavano, Fabrizio Mancin, Giorgio Vescovo, Annalisa Angelini.

  Pluronic-coated polylipoic acid-based nanoparticles (F127@PLA-NPs) have great potential as biodegradable nanovectors for delivering active molecules to different organs in complex diseases. In this study we describe the in vivo biodistribution, safety and ability to deliver molecules of F127@PLA-NPs in healthy rats following intravenous administration. Adult rats were injected with 10 mg/kg of rhodamine B-labeled F127@PLA-NPs, and NPs fluorescence and MFI rate were measured by confocal microscopy in whole collected organs. The NPs accumulation rate was maximal in the heart, compared to the other organs. At the cellular level, myocytes and kidney tubular cells showed the highest NPs uptake. Neither histopathological lesion nor thrombogenicity were observed after NPs injection. Finally, F127@PLA-NPs were tested in vitro as miRNAs delivery nanosystem, and they showed good ability in targeting cardiomyocytes. These results demonstrated that our F127@PLA-NPs constitute a biological, minimally invasive and safe delivery tool targeting organs and cells, such as heart and kidney.

Keywords:  Biodistribution; Confocal microscopy; Heart disease; Kidney disease; Poly (lipoic acid)-based nanoparticles

DOI:  https://doi.org/10.1016/j.nano.2022.102593
J Mol Biol. 2022 Aug 02. pii: S0022-2836(22)00373-4. [Epub ahead of print] 167771

The Effects of Serum Albumin Pre-Adsorption of Nanoparticles on Protein Corona and Membrane Interaction: A Molecular Simulation Study.

Lingxiao Li, Yuanyuan Yang, Lin Wang, Feng Xu, Yuan Li, Xiaocong He.

  As a platform to deliver imaging and therapeutic agents to targeted sites in vivo, nanoparticles (NPs) have widespread applications in diagnosis and treatment of cancer. However, the poor in vivo delivery efficiency of nanoparticles limits its potential for further application. Once enter the physiological environment, nanoparticles immediately interact with proteins and form protein corona, which changes the physicochemical properties of nanoparticle surface and further affects their transport. In this study, we performed molecular dynamics simulations to study the adsorption mechanism of nanoparticles with various surface modifications and different proteins (e.g., human serum albumin, complement protein C3b), and their interactions with cell membrane. The results show that protein human serum albumin prefers to interact with hydrophobic and positively charged nanoparticles, while the protein C3b prefers the hydrophobic and charged nanoparticles. The pre-adsorption of human serum albumin on the nanoparticle surface obviously decreases the interaction of nanoparticle with C3b. Furthermore, the high amount of protein pre-adsorption could decrease the probability of nanoparticle-membrane interaction. These results indicate that appropriate modification of nanoparticles with protein provides nanoparticles with better capability of targeting, which could be used to guide nanoparticle design and improve transport efficiency.

Keywords:  nanoparticle-membrane interaction; nanoparticles; pre-adsorption; protein corona

DOI:  https://doi.org/10.1016/j.jmb.2022.167771
J Control Release. 2022 Jul 29. pii: S0168-3659(22)00473-4. [Epub ahead of print]

Customizing nano-chitosan for sustainable drug delivery.

Mostafa Saeedi, Omid Vahidi, Mohammadreza Moghbeli, Sepideh Ahmadi, Mohsen Asadnia, Omid Akhavan, Farzad Seidi, Mohammad Rabiee, Mohammad Reza Saeb, Thomas J Webster, Rajender S Varma, Esmaeel Sharifi, Ali Zarrabi, Navid Rabiee.

  Chitosan is a natural polymer with acceptable biocompatibility, biodegradability, and mechanical stability; hence, it has been widely appraised for drug and gene delivery applications. However, there has been no comprehensive assessment to tailor-make chitosan cross-linkers of various types and functionalities as well as complex chitosan-based semi- and full-interpenetrating networks for drug delivery systems (DDSs). Herein, the various fabrication methods developed for chitosan hydrogels are deliberated, including chitosan crosslinking with and without diverse cross-linkers. Tripolyphosphate, genipin and multi-functional aldehydes, carboxylic acids, and epoxides are common cross-linkers used in developing biomedical chitosan for DDSs. Methods deployed for modifying the properties and performance of chitosan hydrogels, via their composite production (semi- and full-interpenetrating networks), are also cogitated here. In addition, recent advances in the fabrication of advanced chitosan hydrogels for drug delivery applications such as oral drug delivery, transdermal drug delivery, and cancer therapy are discussed. Lastly, thoughts on what is needed for the chitosan field to continue to grow is also debated in this comprehensive review article.

Keywords:  Chitosan polymer; Composite hydrogels; Cross-linkers; Drug delivery; Hydrogels

DOI:  https://doi.org/10.1016/j.jconrel.2022.07.038