bims-novged 2021-06-06 papers

on Non-viral vectors for gene delivery

Issue of 2021–06–06
eleven papers selected by
the Merkel lab, Ludwig-Maximilians University and Benjamin Winkeljann, Ludwig-Maximilians University

Influence of the Modulation of the Protein Corona on Gene Expression Using Polyethylenimine (PEI) Polyplexes as Delivery Vehicle.
Cationic Polyporphyrins as siRNA Delivery Vectors for Photodynamic and Gene Synergistic Anticancer Therapy.
In Vivo Follow-Up of Gene Inhibition in Solid Tumors Using Peptide-Based Nanoparticles for siRNA Delivery.
Peptide-Based Nanoparticles for Therapeutic Nucleic Acid Delivery.
Development of a Rapid-Onset, Acid-Labile Linkage Polyplex-Mixed Micellar System for Anticancer Therapy.
Targeted RNAi of BIRC5/Survivin Using Antibody-Conjugated Poly(Propylene Imine)-Based Polyplexes Inhibits Growth of PSCA-Positive Tumors.
Size- and Surface- Dual Engineered Small Polyplexes for Efficiently Targeting Delivery of siRNA.
Liposomal Delivery of MicroRNA-7 Targeting EGFR to Inhibit the Growth, Invasion, and Migration of Ovarian Cancer.
A size-shrinkable matrix metallopeptidase-2-sensitive delivery nanosystem improves the penetration of human programmed death-ligand 1 siRNA into lung-tumor spheroids.
PEGylation of mRNA by Hybridization of Complementary PEG-RNA Oligonucleotides Stabilizes mRNA without Using Cationic Materials.
Delivery of antisense oligonucleotides using multi-layer coated gold nanoparticles to methicillin-resistant S. aureus for combinatorial treatment.

Adv Healthc Mater. 2021 Jun 04. e2100125

Influence of the Modulation of the Protein Corona on Gene Expression Using Polyethylenimine (PEI) Polyplexes as Delivery Vehicle.

Dingcheng Zhu, Huijie Yan, Zhuxian Zhou, Jianbin Tang, Xiangrui Liu, Raimo Hartmann, Wolfgang J Parak, Youqing Shen, Neus Feliu.

  The protein corona can significantly modulate the physicochemical properties and gene delivery of polyethylenimine (PEI)/DNA complexes (polyplexes). The effects of the protein corona on the transfection have been well studied in terms of averaged gene expression in a whole cell population. Such evaluation methods give excellent and reliable statistics, but they in general provide the final transfection efficiency without reflecting the dynamic process of gene expression. In this regard the influence of bovine serum albumin (BSA) on the gene expression of PEI polyplexes also on a single cell level via live imaging is analyzed. The results reveal that although the BSA corona causes difference in the overall gene expression and mRNA transcription, the gene expression behavior on the level of individual cell is similar, including the mitosis-dependent expression, distributions of onset time, expression pattern in two daughter cells, and expression kinetics in successfully transfected cells. Comparison of single cell and ensemble data on whole cell cultures indicate that the protein corona does not alter the transfection process after nuclear entry, including cell division, polyplex dissociation, and protein expression. Its influence on other steps of in vitro gene delivery before nuclear entry shall render the difference in the overall transfection.

Keywords:  gene delivery; protein corona; single cell tracking

DOI:  https://doi.org/10.1002/adhm.202100125
ACS Appl Mater Interfaces. 2021 Jun 04.

Cationic Polyporphyrins as siRNA Delivery Vectors for Photodynamic and Gene Synergistic Anticancer Therapy.

Nan Zheng, Xiaoqin Luo, Zhiyi Zhang, Aiguo Wang, Wangze Song.

  Successful gene therapy is highly dependent on the efficiency of gene delivery, which is mostly achieved by the carrier. Current gene carriers are generally nontherapeutic and take over most of the proportion in the delivery systems. Therefore, a library of polymerized and cationic photosensitive drugs (polyphotosensitizers, pPSs) with HIF-1α siRNA delivery capability is constructed to realize using "drug" to deliver "gene". The pPS component acts as both a therapeutic carrier for intracellular HIF-1α siRNA delivery and a photosensitive drug with photodynamic therapy (PDT). A reactive oxygen species (ROS)-cleavable linker is used to polymerize PS, allowing the successful segregation of PS monomers in space, avoiding the undesired aggregation-caused quenching (ACQ) effect and enhancing the in vitro and in vivo PDT effect. The complexes formed by pPSs and HIF-1α siRNA exhibited desired siRNA condensation and serum stability at the optimal conditions (pPSs with guanidines/siRNA weight ratio of 15), efficient intracellular internalization, and gene-silencing efficiency (60%) compared with commercial available transfection reagents (40%), as well as synergistic in vitro and in vivo phototoxicity for the combination PDT-gene therapy toward cancer treatment. This study provides a promising paradigm for the design of both the gene delivery carrier and the photosensitizer, as well as for broad utilities in the combination therapy toward cancer treatment.

Keywords:  PDT−gene combination therapy; cationic polyphotosensitizers; gene carrier; photodynamic therapy; synergistic anticancer treatment

DOI:  https://doi.org/10.1021/acsami.1c07662
Pharmaceutics. 2021 May 19. pii: 749. [Epub ahead of print]13(5):

In Vivo Follow-Up of Gene Inhibition in Solid Tumors Using Peptide-Based Nanoparticles for siRNA Delivery.

Isabel Ferreiro, Coralie Genevois, Karidia Konate, Eric Vivès, Prisca Boisguérin, Sébastien Deshayes, Franck Couillaud.

  Small interfering RNA (siRNA) exhibits a high degree of specificity for targeting selected genes. They are efficient on cells in vitro, but in vivo siRNA therapy remains a challenge for solid tumor treatment as siRNAs display difficulty reaching their intracellular target. The present study was designed to show the in vivo efficiency of a new peptide (WRAP5), able to form peptide-based nanoparticles (PBN) that can deliver siRNA to cancer cells in solid tumors. WRAP5:siRNA nanoparticles targeting firefly luciferase (Fluc) were formulated and assayed on Fluc-expressing U87 glioblastoma cells. The mode of action of WRAP5:siRNA by RNA interference was first confirmed in vitro and then investigated in vivo using a combination of bioluminescent reporter genes. Finally, histological analyses were performed to elucidate the cell specificity of this PBN in the context of brain tumors. In vitro and in vivo results showed efficient knock-down of Fluc expression with no toxicity. WRAP5:siFluc remained in the tumor for at least 10 days in vivo. Messenger RNA (mRNA) analyses indicated a specific decrease in Fluc mRNA without affecting tumor growth. Histological studies identified PBN accumulation in the cytoplasm of tumor cells but also in glial and neuronal cells. Through in vivo molecular imaging, our findings established the proof of concept for specific gene silencing in solid tumors. The evidence generated could be translated into therapy for any specific gene in different types of tumors without cell type specificity but with high molecular specificity.

Keywords:  cancer therapy; gene silencing; optical imaging; peptide-based nanoparticles; siRNA

DOI:  https://doi.org/10.3390/pharmaceutics13050749
Biomedicines. 2021 May 20. pii: 583. [Epub ahead of print]9(5):

Peptide-Based Nanoparticles for Therapeutic Nucleic Acid Delivery.

Prisca Boisguérin, Karidia Konate, Emilie Josse, Eric Vivès, Sébastien Deshayes.

  Gene therapy offers the possibility to skip, repair, or silence faulty genes or to stimulate the immune system to fight against disease by delivering therapeutic nucleic acids (NAs) to a patient. Compared to other drugs or protein treatments, NA-based therapies have the advantage of being a more universal approach to designing therapies because of the versatility of NA design. NAs (siRNA, pDNA, or mRNA) have great potential for therapeutic applications for an immense number of indications. However, the delivery of these exogenous NAs is still challenging and requires a specific delivery system. In this context, beside other non-viral vectors, cell-penetrating peptides (CPPs) gain more and more interest as delivery systems by forming a variety of nanocomplexes depending on the formulation conditions and the properties of the used CPPs/NAs. In this review, we attempt to cover the most important biophysical and biological aspects of non-viral peptide-based nanoparticles (PBNs) for therapeutic nucleic acid formulations as a delivery system. The most relevant peptides or peptide families forming PBNs in the presence of NAs described since 2015 will be presented. All these PBNs able to deliver NAs in vitro and in vivo have common features, which are characterized by defined formulation conditions in order to obtain PBNs from 60 nm to 150 nm with a homogeneous dispersity (PdI lower than 0.3) and a positive charge between +10 mV and +40 mV.

Keywords:  cell-penetrating peptide; delivery; nanoparticle; nucleic acid; self-assembly

DOI:  https://doi.org/10.3390/biomedicines9050583
Polymers (Basel). 2021 May 31. pii: 1823. [Epub ahead of print]13(11):

Development of a Rapid-Onset, Acid-Labile Linkage Polyplex-Mixed Micellar System for Anticancer Therapy.

Shiou-Fen Hung, Yu-Han Wen, Lu-Yi Yu, Hsin-Cheng Chiu, Yi-Ting Chiang, Chun-Liang Lo.

  In the treatment of cancers, small interfering ribonucleic acids (siRNAs) are delivered into cells to inhibit the oncogenic protein's expression; however, polyanions, hydrophilicity, and rapid degradations in blood, endosomal or secondary lysosomal degradation hamper clinal applications. In this study, we first synthesized and characterized two copolymers: methoxy poly(ethylene glycol)-b-poly(2-hydroxy methacrylate-ketal-pyridoxal) and methoxy poly(ethylene glycol)-b-poly(methacrylic acid-co-histidine). Afterwards, we assembled two polymers with the focal adhesion kinase (FAK) siRNA, forming polyplex-mixed micelles for the treatment of the human colon cancer cell line HCT116. In terms of the physiological condition, the cationic pyridoxal molecules that were conjugated on the copolymer with ketal bonds could electrostatically attract the siRNA. Additionally, the pyridoxal could form a hydrophobic core together with the hydrophobic deprotonated histidine molecules in the other copolymer and the hydrophilic polyethylene glycol (PEG) shell to protect the siRNA. In an acidic condition, the pyridoxal would be cleaved from the polymers due to the breakage of the ketal bonds and the histidine molecules can simultaneously be protonated, resulting in the endosome/lysosome escape effect. On the basis of our results, the two copolymers were successfully prepared and the pyridoxal derivatives were identified to be able to carry the siRNA and be cleavable by the copolymers in an acidic solution. Polyplex-mixed micelles were prepared, and the micellar structures were identified. The endosome escape behavior was observed using a confocal laser scanning microscopy (CLSM). The FAK expression was therefore reduced, and the cytotoxicity of siRNA toward human colon cancer cells was exhibited, rapidly in 24 h. This exceptional anticancer efficiency suggests the potential of the pH-sensitive polyplex-mixed micellar system in siRNA delivery.

Keywords:  acid-labile; anticancer therapy; mixed micelle; siRNA delivery

DOI:  https://doi.org/10.3390/polym13111823
Pharmaceutics. 2021 May 08. pii: 676. [Epub ahead of print]13(5):

Targeted RNAi of BIRC5/Survivin Using Antibody-Conjugated Poly(Propylene Imine)-Based Polyplexes Inhibits Growth of PSCA-Positive Tumors.

Willi Jugel, Achim Aigner, Susanne Michen, Alexander Hagstotz, Alexander Ewe, Dietmar Appelhans, Gabriele Schackert, Achim Temme, Stefanie Tietze.

  Delivery of siRNAs for the treatment of tumors critically depends on the development of efficient nucleic acid carrier systems. The complexation of dendritic polymers (dendrimers) results in nanoparticles, called dendriplexes, that protect siRNA from degradation and mediate non-specific cellular uptake of siRNA. However, large siRNA doses are required for in vivo use due to accumulation of the nanoparticles in sinks such as the lung, liver, and spleen. This suggests the exploration of targeted nanoparticles for enhancing tumor cell specificity and achieving higher siRNA levels in tumors. In this work, we report on the targeted delivery of a therapeutic siRNA specific for BIRC5/Survivin in vitro and in vivo to tumor cells expressing the surface marker prostate stem cell antigen (PSCA). For this, polyplexes consisting of single-chain antibody fragments specific for PSCA conjugated to siRNA/maltose-modified poly(propylene imine) dendriplexes were used. These polyplexes were endocytosed by PSCA-positive 293TPSCA/ffLuc and PC3PSCA cells and caused knockdown of reporter gene firefly luciferase and Survivin expression, respectively. In a therapeutic study in PC3PSCA xenograft-bearing mice, significant anti-tumor effects were observed upon systemic administration of the targeted polyplexes. This indicates superior anti-tumor efficacy when employing targeted delivery of Survivin-specific siRNA, based on the additive effects of siRNA-mediated Survivin knockdown in combination with scFv-mediated PSCA inhibition.

Keywords:  Survivin; maltose-modified poly(propylene imine); prostate stem cell antigen; targeted siRNA delivery

DOI:  https://doi.org/10.3390/pharmaceutics13050676
Molecules. 2021 May 27. pii: 3238. [Epub ahead of print]26(11):

Size- and Surface- Dual Engineered Small Polyplexes for Efficiently Targeting Delivery of siRNA.

Shuang Liu, Shaohui Deng, Xiaoxia Li, Du Cheng.

  Though siRNA-based therapy has achieved great progress, efficient siRNA delivery remains a challenge. Here, we synthesized a copolymer PAsp(-N=C-PEG)-PCys-PAsp(DETA) consisting of a poly(aspartate) block grafted with comb-like PEG side chains via a pH-sensitive imine bond (PAsp(-N=C-PEG) block), a poly(l-cysteine) block with a thiol group (PCys block), and a cationic poly(aspartate) block grafted with diethylenetriamine (PAsp(DETA) block). The cationic polymers efficiently complexed siRNA into polyplexes, showing a sandwich-like structure with a PAsp(-N=C-PEG) out-layer, a crosslinked PCys interlayer, and a complexing core of siRNA and PAsp(DETA). Low pH-triggered breakage of pH-sensitive imine bonds caused PEG shedding. The disulfide bond-crosslinking and pH-triggered PEG shedding synergistically decreased the polyplexes' size from 75 nm to 26 nm. To neutralize excessive positive charges and introduce the targeting ligand, the polyplexes without a PEG layer were coated with an anionic copolymer modified with the targeting ligand lauric acid. The resulting polyplexes exhibited high transfection efficiency and lysosomal escape capacity. This study provides a promising strategy to engineer the size and surface of polyplexes, allowing long blood circulation and targeted delivery of siRNA.

Keywords:  disulfide bond-crosslinking; pH-sensitive PEG shedding; siRNA delivery; small polyplex; targeting delivery

DOI:  https://doi.org/10.3390/molecules26113238
ACS Omega. 2021 May 04. 6(17): 11669-11678

Liposomal Delivery of MicroRNA-7 Targeting EGFR to Inhibit the Growth, Invasion, and Migration of Ovarian Cancer.

Xiaojuan Cui, Keqi Song, Xiaolan Lu, Weiwei Feng, Wen Di.

Ovarian cancer is highly aggressive and has high rates of recurrence and metastasis. Due to the limited effects of current treatments, it is necessary to conduct research and develop new treatment options. The application of gene therapy in tumor therapy is gradually increasing and has exciting prospects. MicroRNA-7 (miR-7) has been reported to inhibit the growth, invasion, and metastasis of a variety of solid tumors. Cationic liposomes are safe and effective gene delivery systems for transfection in vivo and in vitro. To realize the application of miR-7 in the treatment of ovarian cancer, cationic liposomes were prepared with 1,2-dioleoyl-3-trimethylammonium-propane, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and cholesterol. The miR-7 liposomes had a suitable particle size, potential, and a high cellular uptake rate. MiR-7 encapsulated by liposomes could be effectively delivered to ovarian cancer cells and successfully targeted to the tumor site in a mouse xenograft model of ovarian cancer. In vitro and in vivo experiments revealed that the miR-7 liposomes had a significant ability to inhibit the growth, invasion, and migration of ovarian cancer, probably by inhibiting the expression of the epidermal growth factor receptor. Our studies of miR-7 liposomes demonstrated a safe and efficient microRNA delivery system for the gene therapy of ovarian cancer.

DOI: https://doi.org/10.1021/acsomega.1c00992
Drug Deliv. 2021 Dec;28(1): 1055-1066

A size-shrinkable matrix metallopeptidase-2-sensitive delivery nanosystem improves the penetration of human programmed death-ligand 1 siRNA into lung-tumor spheroids.

Jiaolin Wen, Neng Qiu, Zejiang Zhu, Peng Bai, Mengshi Hu, Wenyan Qi, Yan Liu, Ailin Wei, Lijuan Chen.

  Given the maturation of small-interfering RNA (siRNA) techniques with nanotechnology, and because overexpression of human programmed death-ligand 1 (PD-L1) is crucial for T cell inactivation and immunosuppression of the tumor microenvironment, application of siRNA-PD-L1 has demonstrated positive progress in preclinical studies; however, the limited penetration of this compound into solid tumors remains a challenge. To decrease PD-L1 expression and increase the penetration efficacy of solid tumors, we synthesized a novel tumor-microenvironment-sensitive delivery polymer by conjugating hyaluronic acid (HA) to polyethyleneimine (PEI), with a matrix metalloproteinase-2 (MMP-2)-sensitive peptide acting as the linker (HA-P-PEI), for use in delivery of PD-L1-siRNA. Concurrent synthesis of a linker-less HA-PEI compound allowed confirmation that negatively charged siRNA can be complexed onto the positively charged HA-PEI and HA-P-PEI compounds to form nanoparticles with the same particle size and uniform distribution with serum stability. We found that the size of the HA-P-PEI/siRNA nanoparticles decreased to <10 nm upon addition of MMP-2, and that H1975 cells overexpressing CD44, PD-L1, and MMP-2 aided confirmation of the delivery efficacy of the HA-P-PEI/siRNA nanocomplexes. Additionally, the use of HA-P-PEI caused less cytotoxicity than PEI alone, demonstrating its high cellular uptake. Moreover, pretreatment with MMP-2 increased nanocomplex tumor permeability, and western blot showed that HA-P-PEI/PD-L1-siRNA efficiently downregulated the PD-L1 expression in H1975 cells. These results demonstrated a novel approach for siRNA delivery and tumor penetration for future clinical applications in cancer treatment.

Keywords:  Programmed death-ligand 1; hyaluronic acid; matrix metalloproteinase-2; polyethyleneimine; small-interfering RNA

DOI:  https://doi.org/10.1080/10717544.2021.1931560
Pharmaceutics. 2021 May 27. pii: 800. [Epub ahead of print]13(6):

PEGylation of mRNA by Hybridization of Complementary PEG-RNA Oligonucleotides Stabilizes mRNA without Using Cationic Materials.

Naoto Yoshinaga, Mitsuru Naito, Yoshihiro Tachihara, Eger Boonstra, Kensuke Osada, Horacio Cabral, Satoshi Uchida.

  Messenger RNA (mRNA) delivery strategies are required to protect biologically fragile mRNA from ribonuclease (RNase) attacks to achieve efficient therapeutic protein expression. To tackle this issue, most mRNA delivery systems have used cationic components, which form electrostatically driven complexes with mRNA and shield encapsulated mRNA strands. However, cationic materials interact with anionic biomacromolecules in physiological environments, which leads to unspecific reactions and toxicities. To circumvent this issue of cation-based approaches, herein, we propose a cation-free delivery strategy by hybridization of PEGylated RNA oligonucleotides with mRNA. The PEG strands on the mRNA sterically and electrostatically shielded the mRNA, improving mRNA nuclease stability 15-fold after serum incubation compared with unhybridized mRNA. Eventually, the PEGylated mRNA induced nearly 20-fold higher efficiency of reporter protein expression than unhybridized mRNA in cultured cells. This study provides a platform to establish a safe and efficient cation-free mRNA delivery system.

Keywords:  RNA engineering; mRNA delivery; mRNA therapeutics

DOI:  https://doi.org/10.3390/pharmaceutics13060800
Mater Sci Eng C Mater Biol Appl. 2021 Jul;pii: S0928-4931(21)00306-4. [Epub ahead of print]126 112167

Delivery of antisense oligonucleotides using multi-layer coated gold nanoparticles to methicillin-resistant S. aureus for combinatorial treatment.

Marcel Janis Beha, Jea Sung Ryu, Yang Soo Kim, Hyun Jung Chung.

  The spread of multidrug-resistant (MDR) bacterial infections has become a serious global threat. We introduce multi-layer coated gold nanoparticles (MLGNPs) delivering antisense oligonucleotides (ASOs) targeting the resistance gene of methicillin-resistant Staphylococcus aureus (MRSA), as a selective antimicrobial by restoring susceptibility. MLGNPs were prepared by multi-step surface immobilization of gold nanoparticles (GNPs) with polyethylenimine (PEI) and loaded with ASO targeting the mecA gene. The MLGNPs were shown to be efficiently internalized into various types of Gram-positive bacteria, including MRSA, Staphylococcus epidermidis, and Bacillus subtilis, which was superior to single-layer coated GNPs and free PEI polymer. The delivery of MLGNPs into MRSA resulted in up to 74% silencing of the mecA gene with high selectivity, in a dose-dependent manner. The treatment of MLGNPs to MRSA in the presence of oxacillin, a beta-lactam antibiotic, showed major suppression (~71%) of bacterial growth, due to the recovery of antibacterial sensitivity. Furthermore, the treatment of MLGNPs in a complex system showed preferential uptake into bacteria over mammalian cells, demonstrating the suitable characteristics of MLGNPs for selective delivery into bacteria. The current approach can be potentially applied for targeting various types of MDR bacterial infections by specific silencing of a resistance gene, as a combinatorial therapeutic used with conventional antibiotics.

Keywords:  Antibiotic resistance; Antisense oligonucleotide; Bacteria; Multi-layer coating; Nanoparticle delivery

DOI:  https://doi.org/10.1016/j.msec.2021.112167