Controlled Gene Delivery Research Articles

Temperature/GSH Dual Responsive Fluorinated Mesoporous Silica‐Coated Au NRs for Serum‐Resistant Gene Delivery**

AbstractMesoporous silica nanoparticles (MSN) are emerging as one of the most appealing candidates for gene carriers. Nevertheless, one major obstacle for their clinical application is the lack of efficient controlled release and gene delivery. In this study, a mesoporous silica‐modified gold nanorods (Au NRs) controlled release platform was synthesized. Then a thermosensitive copolymer was grafted to the surface of release platform, to impart the switching response of carrier under the photothermal of Au NRs. Fluorination were modified on the surface of MSN through a disulfide bond to achieve glutathione (GSH) response release and improve serum‐resistant ability. This gene carrier, with notable DNA condensation ability and negligible cytotoxicity, could be easily internalized into cells, enhanced green fluorescent protein expression in the presence of serum under laser irradiation. This work inspired us to solve the problem of traditional controllable sustained release and realize the performance of intracellular switch response to gene sustained release.

High-throughput cell optoporation system based on Au nanoparticle layers mediated by resonant irradiation for precise and controllable gene delivery

The development of approaches based on genetically modified cells is accompanied by a constant intensive search for new effective and safe delivery systems and the study of existing ones. Recently, we developed a new plasmonic nanoparticle layers-mediated optoporation system that can be proposed for precisely controlled, high-performance laser transfection compatible with broad types of cells and delivered objects of interest. The main goal of the present study is to demonstrate the broad possibilities and advantages of our system for optoporation of several mammalian cells, classified as "easy-to-transfect" cells, namely HeLa and CHO lines, and "hard-to-transfect" cells, namely A431 and RAW 264.7 cells. We show the efficient delivery of various sized cargo molecules: from small molecular dyes propidium iodide (PI) with molecular mass 700 Da, control plasmids (3–10 kb) to fluorophore-labeled dextranes with masses ranging from 10 kDa up to 100 kDa. The performance of optoporation was investigated for two types of laser sources, 800-nm continuous-wave laser, and 1064-nm ns pulsed laser. We provided a comparative study between our system and commercial agent Lipofectamine for transient transfection and stable transfection of HeLa cells with plasmids encoding fluorescent proteins. The quantitative data analysis using flow cytometry, Alamar blue viability assay, and direct fluorescence microscopy revealed higher optoporation efficacy for hard-to-transfect A431 cells and Raw 264.7 cells than lipofection efficacy. Finally, we demonstrated the optoporation performance at the single-cell level by successful delivering PI to the individual CHO cells with revealed high viability for at least 72 h post-irradiation.

Nucleic acid-based nanotherapeutics for treating sepsis and associated organ injuries.

In recent years, gene therapy has been made possible with the success of nucleic acid drugs against sepsis and its related organ dysfunction. Therapeutics based on nucleic acids such as small interfering RNAs (siRNAs), microRNAs (miRNAs), messenger RNAs (mRNAs), and plasmid DNAs (pDNAs) guarantee to treat previously undruggable diseases. The advantage of nucleic acid-based therapy against sepsis lies in the development of nanocarriers, achieving targeted and controlled gene delivery for improved efficacy with minimal adverse effects. Entrapment into nanocarriers also ameliorates the poor cellular uptake of naked nucleic acids. In this study, we discuss the current state of the art in nanoparticles for nucleic acid delivery to treat hyperinflammation and apoptosis associated with sepsis. The optimized design of the nanoparticles through physicochemical property modification and ligand conjugation can target specific organs-such as lung, heart, kidney, and liver-to mitigate multiple sepsis-associated organ injuries. This review highlights the nanomaterials designed for fabricating the anti-sepsis nanosystems, their physicochemical characterization, the mechanisms of nucleic acid-based therapy in working against sepsis, and the potential for promoting the therapeutic efficiency of the nucleic acids. The current investigations associated with nanoparticulate nucleic acid application in sepsis management are summarized in this paper. Noteworthily, the potential application of nanotherapeutic nucleic acids allows for a novel strategy to treat sepsis. Further clinical studies are required to confirm the findings in cell- and animal-based experiments. The capability of large-scale production and reproducibility of nanoparticle products are also critical for commercialization. It is expected that numerous anti-sepsis possibilities will be investigated for nucleic acid-based nanotherapeutics in the future.

A flexible electrode Array for genetic transfection of different layers of the retina by electroporation.

Electroporation (in which the permeability of a cell membrane is increased transiently by exposure to an appropriate electric field) has exhibited great potential of becoming an alternative to adeno-associated virus (AAV)-based retina gene delivery. Electroporation eliminates the safety concerns of employing exogenous viruses and exceeds the limit of AAV cargo size. Unfortunately, several concerns (e.g., relatively high electroporation voltage, poor surgical operability and a lack of spatial selectivity of retina tissue) have prevented electroporation from being approved for clinical application (or even clinical trials). In this study, a flexible micro-electrode array for retina electroporation (FERE) was developed for retina electroporation. A suitably shaped flexible substrate and well-placed micro-electrodes were designed to adapt to the retina curvature and generate an evenly distributed electric field on the retina with a significantly reduced electroporation voltage of 5 V. The FERE provided (for the first time) a capability of controlled gene delivery to the different structural layers of retina tissue by precise control of the distribution of the electrical field. After ensuring the surgical operability of the FERE on rabbit eyeballs, the FERE was verified to be capable of transfecting different layers of retina tissue with satisfactory efficiency and minimum damage. Our method bridges the technical gap between laboratory validation and clinical use of retina electroporation.

Applications of Natural Polymers in Regenerative Medicine and Tissue Engineering

Natural polymers, or biopolymers, are widely utilized in regenerative medicine and tissue engineering. These polymers, derived from proteins, polysaccharides, and nucleic acids, serve as biomaterials for scaffolds, drug delivery systems, and bioactive materials that mimic the extracellular matrix. They offer advantages such as biocompatibility, biodegradability, versatility, and integration with gene therapy. Collagen, gelatin, chitosan, hyaluronic acid, fibrin, and alginate are commonly used natural polymers in regenerative medicine. They promote cell growth, tissue formation, wound healing, and tissue regeneration. Natural polymers also play a crucial role in controlled drug and gene delivery systems, providing safe and effective alternatives to synthetic polymers. Moreover, they contribute to developing bioactive and bio-functional materials, including hydrogels, which mimic natural biological processes and have applications in tissue engineering, drug delivery, and wound healing. Overall, natural polymers hold great promise for advancing regenerative medicine and tissue engineering. However, several challenges impede the widespread adoption and utilization of natural polymers in regenerative medicine. These challenges include variations in batch-to-batch composition, limited mechanical strength, rapid degradation rates, immunogenicity concerns, and difficulties achieving precise control over their properties. Overcoming these challenges necessitates a comprehensive understanding of the structure-function relationships of natural polymers and the development of innovative processing techniques to enhance their mechanical properties and stability. The future of natural polymers in regenerative medicine holds immense potential. Ongoing research efforts focus on refining their properties, tailoring their degradation rates, and integrating them with advanced technologies like 3D bioprinting and nanotechnology. By leveraging these advancements, natural polymers can be further optimized for specific tissue engineering applications, enabling the creation of patient-specific scaffolds, enhanced wound healing materials, and personalized drug delivery systems. Additionally, harnessing the innate bioactivity of natural polymers and their interactions with cells and tissues opens new avenues for the development of bioactive materials that promote tissue regeneration and healing.

Cell-cycle dependent nuclear gene delivery enhances the effects of E-cadherin against tumor invasion and metastasis

Gene delivery is the process by which foreign DNA is transferred to host cells, released from intracellular vesicles, and transported to the nuclei for transcription. This process is frequently inefficient and difficult to control spatiotemporally. We developed a gene delivery strategy that uses ultrasound to directly deliver plasmid DNA into nuclei via gas vesicles (GVs)-based intracellular cavitation. pDNA-binding GVs can be taken up by cells and cause intracellular cavitation when exposed to acoustic irradiation and delivering their pDNA payloads into nuclei. Importantly, GVs can remain stable in the cytoplasm in the absence of acoustic irradiation, allowing for temporally controlled nuclear gene delivery. We were able to achieve spatiotemporal control of E-cadherin nuclear gene delivery in this manner, demonstrating its efficacy in tumor invasion and metastasis inhibition. Interestingly, we discovered that nuclear gene delivery of E-cadherin during the G2/M phase of the cell cycle in C6 tumor cells inhibited tumor invasion and metastasis more effectively than during the G1 and S phases. The gene delivery of E-cadherin at the G2/M phase resulted in significantly lower expression of Fam50a, which reduced Fam50a/Runx2 interaction and led to reduced transactivation of MMP13, an important factor for epithelial-mesenchymal transition, as observed in a molecular mechanism assay. Thus, using remote acoustic control of intracellular cavitation of pDNA-GVs, we developed a high spatiotemporally controllable gene delivery strategy and achieved stronger tumor invasion and metastasis inhibition effects by delivering the E-cadherin gene at the G2/M phase.

3D bioprinting of gene delivery scaffolds with controlled release

Localized gene delivery via engineered scaffolds offers spatiotemporal control of the gene vector release. Here, we explored the capability of digital light processing based bioprinting to fabricate 3D scaffolds in hydrogels for controlled gene delivery. We demonstrated the compatibility of the method with three representative hydrogel biomaterials for gene delivery. We further investigated the highly tunable release profile with these scaffolds by creating and combining distinct release mechanisms of diffusion and ion exchange. The efficacy of gene delivery of these scaffolds was validated in vitro using 293T cells. Results from this work could potentially facilitate the development of synergistic and personalized gene therapies.

Multi-sensitive functionalized niosomal nanocarriers for controllable gene delivery in vitro and in vivo

MicroRNAs, which can contribute to numerous cellular functions through post-transcriptional silencing, have become well-documented candidates for cancer treatment applications, particularly in chemo-resistant cancers. Herein, several formulations were examined to optimize the essential parameters, and the niosomal formulation consisting of cholesterol:tween-80:DOTAP:PEG with 9:69:15:7 ratio had the best physicochemical parameters including spherical shape, high entrapment efficiency, small diameter (81 ± 0.65 nm), and appropriate positive charge (23 ± 0.64 mV). Here, we aimed to design a system with increased delivery efficiency which was tested by the encapsulation of miR-34a within niosome NPs and assessed the nano-niosomal delivery of miR-34a as a tumor suppressor in MCF-7 human adenocarcinoma cells. The results showed that our novel niosome systems with non-ionic surfactants can successfully eliminate cancer cells by increasing the expression of p53 and reducing the expression of NF-κB. In comparison with the free dispersion of miR-34a, the lysis of a nano-sized delivery system demonstrated a better cytotoxicity effect against cancer cells. Similar results were obtained by performing in vivo test on the 4T1 xenografted Balb/C mouse tumor model and the miR-34a-loaded niosomes displayed a better reduction in tumor size by improving approximately + 13% in tumor inhabitation rate while maintaining the bodyweight close to the first day. Therefore, it is concluded that miR-34a delivery via niosomes has high potential as a tumor suppressor and a reliable procedure for breast cancer treatment.Graphical

Enzyme-responsive polysaccharide supramolecular nanoassembly for enhanced DNA encapsulation and controlled release

An enzyme-responsive polysaccharide supramolecular targeted nanoassembly was successfully constructed by the host-guest complexation of positively charged mono-(6-(tetraethylenepentamine)-6-deoxy)-β-cyclodextrin (TEPA-CD) with adamantane-grafted hyaluronic acid (HA-ADA). Possessing a series of positively charged polyamine chains, the obtained polysaccharide nanoassembly could serve as a biocompatible plasmid DNA (pDNA) container. More interestingly, the pDNA could be released from the nanoassembly through the enzymatic degradation of HA skeleton, which realized the controlled pDNA binding and release. Besides, the polysaccharide nanoassembly exhibited lower cytotoxicity than the commercial transfection reagents 25 kDa bPEI (PEI25k), accompanied by similar gene delivery effect. We believe that this work might present a convenient method for targeted, controlled gene delivery.

Electronic Blood Vessel

Summary Advances in bioelectronics have great potential to address unsolved biomedical problems in the cardiovascular system. By using poly(L-lactide-co-ϵ-caprolactone), which encapsulates liquid metal to make flexible and biodegradable electrical circuitry, we develop an electronic blood vessel that can integrate flexible electronics with three layers of blood-vessel cells, to mimic and go beyond the natural blood vessel. It can improve the endothelialization process through electrical stimulation and can enable controlled gene delivery into specific parts of the blood vessel via electroporation. The electronic blood vessel has excellent biocompatibility in the vascular system and shows great patency 3 months post-implantation in a rabbit model. The electronic blood vessel would be an ideal platform to enable diagnostics and treatments in the cardiovascular system and can greatly empower personalized medicine by creating a direct link of the vascular tissue-machine interface.

Externally-Controlled Systems for Immunotherapy: From Bench to Bedside.

Immunotherapy is a very promising therapeutic approach against cancer that is particularly effective when combined with gene therapy. Immuno-gene therapy approaches have led to the approval of four advanced therapy medicinal products (ATMPs) for the treatment of p53-deficient tumors (Gendicine and Imlygic), refractory acute lymphoblastic leukemia (Kymriah) and large B-cell lymphomas (Yescarta). In spite of these remarkable successes, immunotherapy is still associated with severe side effects for CD19+ malignancies and is inefficient for solid tumors. Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of immunotherapy. The aim is to develop smart immunogene therapy-based-ATMPs, which can be controlled by the addition of innocuous drugs or agents, allowing the clinicians to manage the intensity and durability of the therapy. In the present manuscript, we will review the different inducible, versatile and externally controlled gene delivery systems that have been developed and their applications to the field of immunotherapy. We will highlight the advantages and disadvantages of each system and their potential applications in clinics.

In Vitro Synthesis of Modified RNA for Cardiac Gene Therapy.

Modified mRNA (modRNA) is a promising new gene therapy approach that has safely and effectively delivered genes into different tissues, including the heart. Current efforts to use DNA-based or viral gene therapy to induce cardiac regeneration postmyocardial infarction (MI) or in heart failure (HF) have encountered key challenges, e.g., genome integration and delayed and noncontrolled expression. By contrast, modRNA is a transient, safe, non-immunogenic, and controlled gene delivery method that is not integrated into the genome. For most therapeutic applications, especially in regenerative medicine, the ability to deliver genes to the heart transiently and with control is vital for achieving therapeutic effect. Additionally, modRNA synthesis is comparatively simple and inexpensive compared to other gene delivery methods (e.g., protein), though a simple, clear in vitro transcription (IVT) protocol for synthesizing modRNA is needed for it to be more widely used. Here, we describe a simple and improved step-by-step IVT protocol to synthesize modRNA for in vitro or in vivo applications.

Dual-Location Dual-Acid/Glutathione-Degradable Cationic Micelleplexes through Hydrophobic Modification for Enhanced Gene Silencing.

Gene therapy holds great promise for the treatment of acquired genetic disorders such as cancer with reduced side effects compared to chemotherapy. For gene therapy to be successful, it is crucial to develop efficient and nontoxic gene carriers to overcome the poor in vivo stability and low cellular uptake of nucleic acid-based therapeutic agents. Here, we report a new and versatile approach exploring a combination of hydrophobic modifications and dual-stimuli-responsive degradation (SRD) for controlled gene delivery with amphiphilic block copolymer-based nanocarriers. The block copolymer, synthesized by atom transfer radical polymerization, is designed with an acid-labile acetal linkage at the block junction and a pendant disulfide group in the hydrophobic block. The incorporation of labile linkages enables both disulfide-core-cross-linking and dual-location dual-acid/reduction-responsive degradation (DL-DSRD). Furthermore, the disulfide linkages integrated as hydrophobic moieties facilitate the nucleic acids to condense into nanometer-sized micelleplexes through electrostatic interactions of pendant dimethylamino groups with the anionic phosphate groups of the nucleic acids. Our preliminary results demonstrate that the DL-DSRD approach through hydrophobic modification is a robust platform in the development of gene delivery systems with enhanced colloidal stability, reduced cytotoxicity, and improved gene transfection efficiency.

Electronic Blood Vessel

Advances in bioelectronics have great potential to address unsolvable biomedical problems in the cardiovascular system. By using poly(L-lactide-co-e-caprolactone) (PLC) that encapsulates the liquid metal to make flexible and bio-degradable electrical circuitry, we develop an electronic blood vessel that can integrate flexible electronics with three layers of blood vessel cells, to mimic and go beyond the natural blood vessel. It can improve the endothelization process through electrical stimulation and can enable controlled gene delivery into specific part of the blood vessel via electroporation. The electronic blood vessel has excellent biocompatibility in the vascular system and shows great patency three months post-implantation in a rabbit model. The electronic blood vessel would be an ideal platform to enable diagnostics and treatments in the cardiovascular system and can greatly empower personalized medicine by creating a direct link of vascular tissue-machine interface.

Controlled Gene Delivery Systems: Nanomaterials and Chemical Approaches.

Successful gene therapy depends on the design of effective gene delivery systems. A gene delivery system is considered a powerful tool for the release of genetic material within cells resulting in a change in cell functions and protein production. The release of genes in a controlled manner by using appropriate carriers facilitates their release without side effects and increases the expression of genes at the released site. It is expected that significant changes in the combination of several genes and drugs can be provided by developing treatment systems sensitive to different stimuli such as redox potential, pH variations, temperature gradients, light irradiation, and enzyme activity. The most important advantages for the release of genes and stimuli-responsive therapeutics include delivering vectors locally, reducing side effects and causing no toxicity to distant tissues while at the same time reducing the immune response to the vectors. In this review, we aim to discuss different types of gene carriers involved in the controlled transfer of nucleic acids (non-viral inorganic and organic nanoparticles (NPs) and virus-like particles (VLPs)) as well as the simultaneous transfer of several genes and/or drugs into cells or different tissues, providing for an efficient and safe treatment of numerous diseases.

Cover Image, Volume 69, Issue 2

The cover image is based on the Research Article Redox‐responsive PEI‐coated magnetic iron oxide nanoparticles for controllable gene delivery and magnetic resonance imaging by Si Peng et al., https://doi.org/10.1002/pi.5943. imageCover image © Qinghan Zhou Images.

Polymeric nano-carriers for on-demand delivery of genes via specific responses to stimuli.

Polymeric nano-carriers have been developed as a most capable and feasible technology platform for gene therapy. As vehicles, polymeric nano-carriers are obliged to possess high gene loading capability, low immunogenicity, safety, and the ability to transfer various genetic materials into specific sites of target cells to express therapeutic proteins or block a process of gene expression. To this end, various types of polymeric nano-carriers have been prepared to release genes in response to stimuli such as pH, redox, enzymes, light and temperature. These stimulus-responsive nano-carriers exhibit high gene transfection efficiency and low cytotoxicity. In particular, dual- and multi-stimulus-responsive polymeric nano-carriers can respond to a combination of signals. Markedly, these combined responses take place either simultaneously or in a sequential manner. These dual-stimulus-responsive polymeric nano-carriers can control gene delivery with high gene transfection both in vitro and in vivo. In this review paper, we highlight the recent exciting developments in stimulus-responsive polymeric nano-carriers for gene delivery applications.

Redox‐responsive polyethyleneimine‐coated magnetic iron oxide nanoparticles for controllable gene delivery and magnetic resonance imaging

AbstractRecently, theranostic candidates that provide a combination of gene delivery and image diagnosis have attracted much interest in medical research. However, there are still many challenges for their clinical applications, such as uncontrollable gene delivery, high cytotoxicity, low transfection efficiency and reduced image contrast. Herein, redox‐responsive polyethyleneimine‐coated magnetic iron oxide nanoparticles (IONs@rPEI) were prepared for both efficient gene delivery and magnetic resonance (MR) imaging. Firstly, crosslinked rPEI was synthesized by Michael addition reaction with N,N‐bis(acryloyl)cystamine, dopamine and low‐molecular‐weight branched PEI. The rPEI was then coated onto IONs by ligand exchange reaction forming IONs@rPEI. The physicochemical properties of the IONs@rPEI, such as chemical structure, size, zeta potential and DNA condensation ability, were investigated. In addition, a rapid degradation of the as‐prepared nanoparticles was observed, which was triggered by reducing glutathione via destruction of disulfide linkages suggesting a potential controllable DNA release in tumor cells. In MR imaging detection, the IONs@rPEI had a high T2 relaxivity of 81 L mmol−1 s−1 indicating a potential usage as MR imaging contrast reagent. In cell assay, the IONs@rPEI exhibited low cytotoxicity and good transfection efficiency. In conclusion, the as‐prepared crosslinked IONs@rPEI can be used as a promising technology platform for gene therapy and MR imaging in theranostics. © 2019 Society of Chemical Industry

Supramolecular Cyclodextrin-Based Hydrogels for Controlled Gene Delivery.

Controlled delivery of gene transfer vectors is a powerful strategy to enhance the temporal and spatial presentation of therapeutic agents in a defined target. Hydrogels are adapted biomaterials for gene delivery capable of acting as a localized depot of genes while maintaining the long term local availability of DNA vectors at a specific location. Supramolecular hydrogels based on cyclodextrins (CDs) have attracted considerable attention as potential biomaterials in a broad range of drug delivery applications. Their unique characteristics of thixotropicity and low cytotoxicity due to their production under mild conditions make them potential candidates to form injectable delivery systems. This work aims to provide an overview of the use of CD-based polypseudorotaxane hydrogels as controlled gene delivery systems for different applications in regenerative medicine.

Two-Photon-Induced Charge-Variable Conjugated Polyelectrolyte Brushes for Effective Gene Silencing.

Cationic conjugated polyelectrolytes can absorb negatively charged small interfering RNA (siRNA) and also visualize the cellular internalization of siRNA, which thus have been extensively explored as siRNA carriers. However, their low charge density cannot afford a high carrying capability, severely impeding gene transfection efficiency. Moreover, the intracellular controlled release of siRNA is another factor that limits the widespread use of siRNA therapeutics. Herein, we present a novel two-photon-induced charge-variable conjugated polyelectrolyte brush as an efficient siRNA carrier. This cationic conjugated polyelectrolyte brush (PPENBr-ONB) with densely cationic charges produces remarkable carrying capability with siRNA. In addition, PPENBr-ONB with large two-photon absorption (TPA) cross-section represents effective fluorescence resonance energy transfer (FRET) to photoresponsive side chain with 720 nm illumination for two-photon-induced photolysis. Hence, the charge transformation of the photoresponsive side chain from cations to zwitterions would remarkably elevate siRNA release. The obtained PPENBr-ONB shows considerable fluorescence quantum yields (0.16) in aqueous solution, sufficient to serve as a reporter for cellular imaging. Agarose gel electrophoresis experiments indicate that PPENBr-ONB exhibit excellent siRNA-loading capacity (1 mol PPENBr-ONB to more than 20 mol siRNA). Furthermore, PPENBr-ONB with large TPA cross-section (1.47 × 105 GM) exhibits promoted siRNA release (78%) under 720 nm illumination. In vitro experiment shows that PPENBr-ONB/siRNA complex could efficaciously knock out of targeted Plk1 mRNA to 24.7% under 720 nm illumination for 1 h. This two-photon excitation siRNA carrier offers an efficacious strategy for the exploitation of photo controlled gene delivery system.