bims-novged 2021-10-17 papers

on Non-viral vectors for gene delivery

Issue of 2021–10–17
eight papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Self-assembled PEGylated amphiphilic polypeptides for gene transfection.
Photocrosslinked Bioreducible Polymeric Nanoparticles for Enhanced Systemic siRNA Delivery as Cancer Therapy.
Boosting ionizable lipid nanoparticle-mediated in vivo mRNA delivery through optimization of lipid amine-head groups.
Kinetic Modeling to Accelerate the Development of Nucleic Acid Formulations.
ATP-Responsive Multifunctional Supramolecular Polymer as a Nonviral Vector for Boosting Cholesterol Removal from Lipid-Laden Macrophages.
siRNA delivery using intelligent chitosan-capped mesoporous silica nanoparticles for overcoming multidrug resistance in malignant carcinoma cells.
Notch Intracellular Domain Plasmid Delivery via Poly(Lactic-Co-Glycolic Acid) Nanoparticles to Upregulate Notch Pathway Molecules.
Chitosan-tripolyphosphate nanoparticles-mediated co-delivery of MTHFD1L shRNA and 5-aminolevulinic acid for combination photodynamic-gene therapy in oral cancer.

J Mater Chem B. 2021 Oct 13. 9(39): 8224-8236

Self-assembled PEGylated amphiphilic polypeptides for gene transfection.

Paul Klemm, Mira Behnke, Jana I Solomun, Colin Bonduelle, Sébastien Lecommandoux, Anja Traeger, Stephanie Schubert.

In the present study, three biodegradable block copolymers composed of a poly(ethylene glycol) block and a copolypeptide block with varying compositions of cationic L-lysine (L-Lys) and hydrophobic benzyl-L-glutamate (Bzl-L-Glu) were designed for gene delivery applications. The polypeptides were synthesized by ring opening polymerization (ROP) and after orthogonal deprotection of Boc-L-Lys side chains, the polymer exhibited an amphiphilic character. To bind or encapsulate plasmid DNA (pDNA), different formulations were investigated: a nanoprecipitation and an emulsion technique using various organic solvents as well as an aqueous pH-controlled formulation method. The complex and nanoparticle (NP) formations were monitored by dynamic light scattering (DLS), and pDNA interaction was shown by gel electrophoresis and subsequent controlled release with heparin. The polypeptides were further tested for their cytotoxicity as well as biodegradability. The complexes and NPs presenting the most promising size distributions and pDNA binding ability were subsequently evaluated for their transfection efficiency in HEK293T cells. The highest transfection efficiencies were obtained with an aqueous formulation of the polypeptide containing the highest L-Lys content and lowest proportion of hydrophobic, helical structures (P1*), which is therefore a promising candidate for efficient gene delivery by biodegradable gene delivery vectors.

DOI: https://doi.org/10.1039/d1tb01495a
Adv Funct Mater. 2021 Apr 22. pii: 2009768. [Epub ahead of print]31(17):

Photocrosslinked Bioreducible Polymeric Nanoparticles for Enhanced Systemic siRNA Delivery as Cancer Therapy.

Johan Karlsson, Stephany Y Tzeng, Shayan Hemmati, Kathryn M Luly, Olivia Choi, Yuan Rui, David R Wilson, Kristen L Kozielski, Alfredo Quiñones-Hinojosa, Jordan J Green.

  Clinical translation of polymer-based nanocarriers for systemic delivery of RNA has been limited due to poor colloidal stability in the blood stream and intracellular delivery of the RNA to the cytosol. To address these limitations, this study reports a new strategy incorporating photocrosslinking of bioreducible nanoparticles for improved stability extracellularly and rapid release of RNA intracellularly. In this design, the polymeric nanocarriers contain ester bonds for hydrolytic degradation and disulfide bonds for environmentally triggered small interfering RNA (siRNA) release in the cytosol. These photocrosslinked bioreducible nanoparticles (XbNPs) have a shielded surface charge, reduced adsorption of serum proteins, and enable superior siRNA-mediated knockdown in both glioma and melanoma cells in high-serum conditions compared to non-crosslinked formulations. Mechanistically, XbNPs promote cellular uptake and the presence of secondary and tertiary amines enables efficient endosomal escape. Following systemic administration, XbNPs facilitate targeting of cancer cells and tissue-mediated siRNA delivery beyond the liver, unlike conventional nanoparticle-based delivery. These attributes of XbNPs facilitate robust siRNA-mediated knockdown in vivo in melanoma tumors colonized in the lungs following systemic administration. Thus, biodegradable polymeric nanoparticles, via photocrosslinking, demonstrate extended colloidal stability and efficient delivery of RNA therapeutics under physiological conditions, and thereby potentially advance systemic delivery technologies for nucleic acid-based therapeutics.

Keywords:  bioreducible; crosslinking; nanoparticles; siRNA; stimuli-responsive polymers

DOI:  https://doi.org/10.1002/adfm.202009768
Biomater Sci. 2021 Oct 14.

Boosting ionizable lipid nanoparticle-mediated in vivo mRNA delivery through optimization of lipid amine-head groups.

Feng Ding, Hongqian Zhang, Jiwei Cui, Qiang Li, Chuanxu Yang.

In vitro transcribed messenger RNA (IVT-mRNA) holds great promise for the development of novel therapeutics, such as immunotherapy and vaccination. However, the main obstacle towards clinical translation is the lack of effective delivery systems. Herein, we have synthesized a series of ionizable lipids by the addition of an alkyl-acrylate to amine-containing molecules (amine-head groups) as a key component of ionizable lipid nanoparticles (iLNPs) and thoroughly investigated the impact of the amine-head group on the transfection efficiency of iLNPs/mRNA lipoplexes both in vitro and in vivo. The top-performing iLNP (114-iLNP), composed of a lipid with spermine as the amine-head, demonstrated the strongest cellular uptake, membrane disruption and endosomal escape, and further achieved the highest protein expression in HeLa cells with more than 95% transfection efficiency. More importantly, intravenous injection of luciferase mRNA loaded 114-iLNP enables the most efficacious in vivo protein expression, predominantly in the liver. Biodistribution and biosafety evaluation of 114-iLNP/mRNA further demonstrated the liver-selective delivery capability and high biocompatibility. In addition, 114-iLNP facilitated efficient in vivo delivery of a therapeutic gene, human erythropoietin (hEPO) mRNA, and induced hEPO expression in a dose-dependent manner. Therefore, these results demonstrate that the amine-head group in the ionizable lipid significantly affects mRNA delivery efficacy and the leading candidate 114-iLNP composed of a lipid with spermine as the amine-head has great potential for mRNA therapeutics development.

DOI: https://doi.org/10.1039/d1bm00866h
ACS Nano. 2021 Oct 12.

Kinetic Modeling to Accelerate the Development of Nucleic Acid Formulations.

Esther H Roh, Thomas H Epps, Millicent O Sullivan.

  A critical hurdle in the clinical translation of nucleic acid drugs is the inefficiency in testing formulations for therapeutic potential. Specifically, the ability to quantitatively predict gene expression is lacking when transitioning between cell culture and animal studies. We address this challenge by developing a mathematical framework that can reliably predict short-interfering RNA (siRNA)-mediated gene silencing with as few as one experimental data point as an input, evaluate the efficacies of existing formulations in an expeditious manner, and ultimately guide the design of nanocarriers with optimized performances. The model herein consisted of only essential rate-limiting steps and parameters with easily characterizable values of the RNA interference process, enabling the easy identification of which parameters play dominant roles in determining the potencies of siRNA formulations. Predictions from our framework were in close agreement with in vitro and in vivo experimental results across a retrospective analysis using multiple published data sets. Notably, our findings suggested that siRNA dilution was the primary determinant of gene-silencing kinetics. Our framework shed light on the fact that this dilution rate is governed by different parameters, i.e., cell dilution (in vitro) versus clearance from target tissue (in vivo), highlighting a key reason why in vitro experiments do not always predict in vivo outcomes. Moreover, although our current effort focuses on siRNA, we anticipate that the framework can be modified and applied to other nucleic acids, such as mRNA, that rely on similar biological processes.

Keywords:  gene delivery; kinetic model; nanoparticle; nucleic acid therapeutics; siRNA

DOI:  https://doi.org/10.1021/acsnano.1c04555
ACS Biomater Sci Eng. 2021 Oct 14.

ATP-Responsive Multifunctional Supramolecular Polymer as a Nonviral Vector for Boosting Cholesterol Removal from Lipid-Laden Macrophages.

Cuiping Jiang, Yuan Wang, Peiyi Liang, Yao Chen, Ziming Zhuang, Lu Zhang, Yankui Yi, Li Liu, Qiang Liu.

  Specific delivery of NCEH1 plasmid is a promising approach to boost the cholesterol removal from lipid-laden macrophages for antiatherosclerosis. Polyethylenimine (PEI) is one of the most efficient gene carriers among nonviral vectors. However, the high transfection activity of PEI is always accompanied by profound cytotoxicity. To tackle the paradox between transfection efficiency and safety, we constructed a novel ATP-responsive multifunctional supramolecular polymer by cross-linking functionalized low-molecular-weight PEI via a boronic ester bond for NCEH1 plasmid delivery. The supramolecular polymer could condense NCEH1 plasmids to form stable nanosized polyplexes when the w/w ratios of the polymer and gene were higher than 2. ATP-triggered degradation of the polymer and pDNA release were characterized by a series of studies, including 1H NMR, 31P NMR, XPS, agarose gel electrophoresis, and ethidium bromide exclusion tests. In addition, the changes in particle size and morphology were observed in the presence of ATP. Interestingly, the supramolecular polymer showed broad spectrum antioxidant activities by measuring the elimination rates of different reactive oxygen species. In addition, the supramolecular polymer displayed a high buffering capability and good cytocompatibility as demonstrated by the results of the buffering capacity, a hemolysis assay, and a cytotoxicity test. Importantly, it was revealed that the supramolecular polymer/NCEH1 plasmid polyplex formulated at a w/w ratio of 20 was most effective in enhancing cholesterol removal from lipid-laden macrophages and reducing the accumulation of lipid droplets as evidenced by transfection study, cholesterol efflux assay, and oil red O staining studies. Collectively, the ATP-responsive multifunctional supramolecular polymer holds great potential for safe and efficient gene delivery for antiatherosclerosis.

Keywords:  atherosclerosis; cholesterol removal; gene delivery; nonviral vector; polyethylenimine

DOI:  https://doi.org/10.1021/acsbiomaterials.1c00919
Sci Rep. 2021 Oct 15. 11(1): 20531

siRNA delivery using intelligent chitosan-capped mesoporous silica nanoparticles for overcoming multidrug resistance in malignant carcinoma cells.

Razieh Heidari, Pegah Khosravian, Seyed Abbas Mirzaei, Fatemeh Elahian.

Although siRNA is a promising technology for cancer gene therapy, effective cytoplasmic delivery has remained a significant challenge. In this paper, a potent siRNA transfer system with active targeting moieties toward cancer cells and a high loading capacity is introduced to inhibit drug resistance. Mesoporous silica nanoparticles are of great potential for developing targeted gene delivery. Amino-modified MSNs (NH2-MSNs) were synthesized using a modified sol-gel method and characterized by FTIR, BET, TEM, SEM, X-ray diffraction, DLS, and 1H-NMR. MDR1-siRNA was loaded within NH2-MSNs, and the resulting negative surface was capped by functionalized chitosan as a protective layer. Targeting moieties such as TAT and folate were anchored to chitosan via PEG-spacers. The loading capacity of siRNA and the protective effect of chitosan for siRNA were determined by gel retardation assay. MTT assay, flow cytometry, real-time PCR, and western blot were performed to study the cytotoxicity, cellular uptake assay, targeting evaluation, and MDR1 knockdown efficiency. The synthesized NH2-MSNs had a particle size of ≈ 100 nm and pore size of ≈ 5 nm. siRNA was loaded into NH2-MSNs with a high loading capacity of 20% w/w. Chitosan coating on the surface of siRNA-NH2-MSNs significantly improved the siRNA protection against enzyme activity compared to naked siRNA-NH2-MSNs. MSNs and modified MSNs did not exhibit significant cytotoxicity at therapeutic concentrations in the EPG85.257-RDB and HeLa-RDB lines. The folate-conjugated nanoparticles showed a cellular uptake of around two times higher in folate receptor-rich HeLa-RDB than EPG85.257-RDB cells. The chitosan-coated siRNA-NH2-MSNs produced decreased MDR1 transcript and protein levels in HeLa-RDB by 0.20 and 0.48-fold, respectively. The results demonstrated that functionalized chitosan-coated siRNA-MSNs could be a promising carrier for targeted cancer therapy. Folate-targeted nanoparticles were specifically harvested by folate receptor-rich HeLa-RDB and produced a chemosensitized phenotype of the multidrug-resistant cancer cells.

DOI: https://doi.org/10.1038/s41598-021-00085-0
Front Cardiovasc Med. 2021 ;8 707897

Notch Intracellular Domain Plasmid Delivery via Poly(Lactic-Co-Glycolic Acid) Nanoparticles to Upregulate Notch Pathway Molecules.

Victoria L Messerschmidt, Uday Chintapula, Aneetta E Kuriakose, Samantha Laboy, Thuy Thi Dang Truong, LeNaiya A Kydd, Justyn Jaworski, Zui Pan, Hashem Sadek, Kytai T Nguyen, Juhyun Lee.

  Notch signaling is a highly conserved signaling system that is required for embryonic development and regeneration of organs. When the signal is lost, maldevelopment occurs and leads to a lethal state. Delivering exogenous genetic materials encoding Notch into cells can reestablish downstream signaling and rescue cellular functions. In this study, we utilized the negatively charged and FDA approved polymer poly(lactic-co-glycolic acid) to encapsulate Notch Intracellular Domain-containing plasmid in nanoparticles. We show that primary human umbilical vein endothelial cells (HUVECs) readily uptake the nanoparticles with and without specific antibody targets. We demonstrated that our nanoparticles are non-toxic, stable over time, and compatible with blood. We further demonstrated that HUVECs could be successfully transfected with these nanoparticles in static and dynamic environments. Lastly, we elucidated that these nanoparticles could upregulate the downstream genes of Notch signaling, indicating that the payload was viable and successfully altered the genetic downstream effects.

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

DOI:  https://doi.org/10.3389/fcvm.2021.707897
Photodiagnosis Photodyn Ther. 2021 Oct 11. pii: S1572-1000(21)00402-6. [Epub ahead of print] 102581

Chitosan-tripolyphosphate nanoparticles-mediated co-delivery of MTHFD1L shRNA and 5-aminolevulinic acid for combination photodynamic-gene therapy in oral cancer.

Jian Wang, Ke Wang, Jin Liang, Jianqiu Jin, Xing Wang, Shu Yan.

   BACKGROUND: Rationally designed nanostructured materials can produce ideal drug carriers that plays an increasingly important role in cancer treatment. In comparison with conventional drug combination approaches, using co-delivery systems of multiple drugs achieves sophisticated targeting strategies and multifunctionality.
METHODS: First, a nano-co-delivery of chitosan/tripolyphosphate (CS-TPP) was synthesized and characterized combining 5-aminolevulinic acid photodynamic therapy (ALA-PDT) with methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) shRNA. In this report, we investigated the efficacy of the simultaneous delivery of shRNA/photosensitizer on the gene expression of oral squamous cell carcinoma (OSCC) cells. The efficacy of CS-TPP-(shMTHFD1L-ALA)-PDT in inducing apoptosis and in generating of reactive oxygen species (ROS) in vitro was then assessed by Annexin V-PI and DCFH-DA assays respectively. In vivo therapeutic experiments were conducted in well-established orthotopic animal models of HNSCC.
RESULTS: The results showed that the CS-TPP-(shMTHFD1L-ALA) nanoparticles (NPs) were approximately 145 nm in size. The cytotoxicity of OSCC cells was significantly increased by co-delivery of MTHFD1L shRNA and ALA-PDT compared with other groups. Furthermore, individual and combined therapies revealed remarkable pro-apoptotic, ROS and anti-tumorigenesis effects, and CS-TPP-(shMTHFD1L-ALA)-PDT had additive effects in vitro and in vivo.
CONCLUSION: These observations indicate that CS-TPP-(shMTHFD1L-ALA) NPs may be an ideal candidate for gene/photosensitizer delivery.

Keywords:  chitosan; gene therapy; nanoparticles; oral squamous cell carcinoma; photodynamic therapy

DOI:  https://doi.org/10.1016/j.pdpdt.2021.102581