Plasmid Loading Research Articles

Enzyme-like nanoparticle-engineered mesenchymal stem cell secreting HGF promotes visualized therapy for idiopathic pulmonary fibrosis in vivo.

Stem cell therapy is being explored as a potential treatment for idiopathic pulmonary fibrosis (IPF), but its effectiveness is hindered by factors like reactive oxygen species (ROS) and inflammation in fibrotic lungs. Moreover, the distribution, migration, and survival of transplanted stem cells are still unclear, impeding the clinical advancement of stem cell therapy. To tackle these challenges, we fabricate AuPtCoPS trimetallic-based nanocarriers (TBNCs), with enzyme-like activity and plasmid loading capabilities, aiming to efficiently eradicate ROS, facilitate delivery of therapeutic genes, and ultimately improve the therapeutic efficacy. TBNCs also function as a computed tomography contrast agent for tracking mesenchymal stem cells (MSCs) during therapy. Accordingly, we enhanced the antioxidant stress and anti-inflammatory capabilities of engineered MSCs and successfully visualized their biological behavior in IPF mice in vivo. Overall, this study provides an efficient and forward-looking treatment approach for IPF and establishes a framework for a stem cell-based therapeutic system aimed at addressing lung disease.

Optimized plasmid loading of human erythrocytes for Plasmodium falciparum DNA transfections

In vitro modification of Plasmodium falciparum genes is the cornerstone of basic and translational malaria research. Achieved through DNA transfection, these modifications may entail altering protein sequence or abundance. Such experiments are critical for defining the molecular mechanisms of key parasite phenotypes and for validation of drug and vaccine targets. Despite its importance, successful transfection remains difficult and is a resource-intensive, rate-limiting step in P. falciparum research. Here, we report that inefficient loading of plasmid into erythrocytes limits transfection efficacy with commonly used electroporation methods. As these methods also require expensive instrumentation and consumables that are not broadly available, we explored a simpler method based on plasmid loading through hypotonic lysis and resealing of erythrocytes. We used parasite expression of a sensitive NanoLuc reporter for rapid evaluation and optimization of each step. Hypotonic buffer composition, resealing buffer volume and composition, and subsequent incubation affected plasmid retention and successful transfection. While ATP was critical for erythrocyte resealing, addition of Ca++ or glutathione did not improve transfection efficiency, with increasing Ca++ concentrations proving detrimental to outcomes. Compared with either the standard electroporation method or a previously reported hypotonic loading protocol, the optimized method yields greater plasmid loading and higher expression of the NanoLuc reporter 48 h after transfection. It also produced significantly faster outgrowth of parasites in transfections utilizing either episomal expression or CRISPR-Cas9 mediated integration. This new method produces higher P. falciparum transfection efficiency, reduces resource requirements and should accelerate molecular studies of malaria drug and vaccine targets.

Biomimetic gene editing system for precise tumor cell reprogramming and augmented tumor therapy.

The abnormal level of hypoxia-inducible factor-1 alpha (HIF-1α) is closely related to cancer metastasis and treatment resistance. CRISPR-Cas9-based gene editing technology has sparked profound hope to solve this issue by precise gene disruption, although the in vivo application remains hindered by the lack of a safe and efficient delivery strategy. Herein, we developed a cell membrane biomimetic core-shell system for light-controllable, precise gene editing. The inner core of the system comprises protamine for CRISPR-Cas9/sgRNA plasmid (pCas9) loading and calcium ions for efficient pCas9 transfection. The shell of the system is camouflaged by a cell membrane and modified with AS1411 aptamers for tumor targeting and photosensitizers to induce lysosomal escape and pCas9 release through reactive oxygen species production, thereby producing light-controllable enhanced gene editing. Neoplastic H1299 cells were reprogrammed using the biomimetic gene editing system upon laser irradiation with reduced VEGF and Vimentin expression, leading to enhanced antimetastatic effects. Genetic disruption of HIF-1α augmented the in vivo chemotherapeutic efficacy of paclitaxel. Our approach of using a membrane-camouflaged system combined with light augmentation provides a potential solution for the in vivo delivery of CRISPR-Cas9 as well as a feasible strategy for cancer therapy.

Placental Extracellular Vesicles Can Be Loaded with Plasmid DNA.

Recently, extracellular vesicles (EVs) have garnered considerable interest as potential vehicles for drug delivery, including gene therapy. Although EVs from diverse sources have been investigated, current techniques used in the field for EV generation limit large-scale EV production. The placenta is essentially a tissue transplant and has unique properties that allow it to avoid the maternal immune system making it likely that placental EVs will not generate inflammatory responses and will avoid clearance by the immune system. We propose that placental EVs produced from explant cultures are an efficient method to produce considerable quantities of EVs that would be safe to administer, and we hypothesize that placental EVs can be loaded with large exogenous plasmids. To this end, we trialed three strategies to load plasmid DNA into placental EVs, including loading via electroporation of placental tissue prior to EV isolation and loading directly into placental EVs via electroporation or direct incubation of the EVs in plasmid solution. We report that the placenta releases vast quantities of EVs compared to placental cells in monolayer cultures. We show successful loading of plasmid DNA into both large- and small-EVs following both exogenous loading strategies with more plasmid encapsulated in large-EVs. Importantly, direct incubation did not alter EV size nor quantity. Further, we showed that the loading efficiency into EVs was dependent on the exogenous plasmid DNA dose and the DNA size. These results provide realistic estimates of plasmid loading capacity into placental EVs using current technologies and showcase the potential of placental EVs as DNA delivery vehicles.

TOP-EVs: Technology of Protein delivery through Extracellular Vesicles is a versatile platform for intracellular protein delivery.

Extracellular vesicles (EVs) have emerged as biocompatible drug delivery vehicles due to their native ability to deliver bioactive cargo to recipient cells. However, the application of EVs as a therapeutic delivery vehicle is hampered by effective methods for endogenously loading target proteins inside EVs and unloading proteins after delivery to recipient cells. Most EV-based engineered loading methods have a limited delivery efficiency owing to their inefficient endosomal escape or cargo release from the intraluminal attachment from the EV membrane. Here, we describe the 'Technology Of Protein delivery through Extracellular Vesicles' (TOP-EVs) as a tool for efficient intracellular delivery of target proteins mediated via EVs. The vesicular stomatitis virus glycoprotein and the rapamycin-heterodimerization of the FKBP12/T82L mutant FRB proteins were both important for the effective protein delivery through TOP-EVs. We showed that TOP-EVs could efficiently deliver Cre recombinase and CRISPR/Cas9 ribonucleoprotein complex in vitro. Moreover, our results demonstrated that the capacity of TOP-EVs to deliver intracellular proteins in recipient cells was not an artifact of plasmid contamination or direct plasmid loading into EVs. Finally, we showed that TOP-EVs could successfully mediate intracellular protein delivery in the liver in vivo. Taken together, TOP-EVs are a versatile platform for efficient intracellular protein delivery in vitro and in vivo, which can be applied to advance the development of protein-based therapeutics.

Deagglomeration of DNA nanomedicine carriers using controlled ultrasonication

Control over the agglomeration state of manufactured particle systems for drug and oligonucleotide intracellular delivery is paramount to ensure reproducible and scalable therapeutic efficacy. Ultrasonication is a well-established mechanism for the deagglomeration of bulk powders in dispersion. Its use in manufacturing requires strict control of the uniformity and reproducibility of the cavitation field within the sample volume to minimise within-batch and batch-to-batch variability. In this work, we demonstrate the use of a reference cavitating vessel which provides stable and reproducible cavitation fields over litre-scale volumes to assist the controlled deagglomeration of a novel non-viral particle-based plasmid delivery system. The system is the Nuvec delivery platform, comprising polyethylenimine-coated spiky silica particles with diameters of ∼ 200 nm. We evaluated the use of controlled cavitation at different input powers and stages of preparation, for example before and after plasmid loading. Plasmid loading was confirmed by X-ray photoelectron spectroscopy and gel electrophoresis. The latter was also used to assess plasmid integrity and the ability of the particles to protect plasmid from potential degradation caused by the deagglomeration process. We show the utility of laser diffraction and differential centrifugal sedimentation in quantifying the efficacy of product de-agglomeration in the microscale and nanoscale size range respectively. Transmission electron microscopy was used to assess potential damages to the silica particle structure due to the sonication process.

Cationic lipid/pDNA complex formation as potential generic method to generate specific IRF pathway stimulators

Cationic liposome – CpG DNA complexes (lipoplexes) are known as stimulators of innate immunity via Toll-like receptor 9 (TLR9)-triggered activation of the nuclear factor kappa B (NF-κB) pathway. More recent reports suggest that DNA lipoplexes also engage DNA sensors in the cytosol leading to the stimulation of the interferon response factor (IRF) pathway. In this study a range of lipoplexes were formulated by using an invariable helper lipid, three different cationic lipids (DOTAP, DOTMA and DDA) and three different CpG-containing plasmids of different sizes. These lipoplexes exhibited similar hydrodynamic diameters, zeta-potentials and plasmid loading rates, despite the different lipid blends and CpG-containing plasmids. Binding and uptake of liposomal lipids by J774.A1 macrophages and JAWSII dendritic cells increased significantly (up to 4-fold) upon lipoplex formation. Cellular plasmid DNA uptake via lipoplexes compared to naked DNA was increased up to 18-fold. Analysis of signal transduction pathway activation in J774-DUAL™ reporter cells by liposomes or naked CpG plasmid DNA compared to their derived lipoplexes showed only minor activation of the NF-κB pathway, while the IRF pathway displayed massive activation factors of up to 46-fold. DOTAP- and DOTMA lipoplexes also led to massive interferon-alpha and –beta secretion of J774A.1 macrophages and JAWSII dendritic cells, which is a hallmark of IRF pathway activation. Cellular distribution studies on DOTAP lipoplexes suggest delivery of plasmid DNA via vesicular compartments into the cytosol. Taken together, the CpG plasmid DNA lipoplexes generated in this study appear to selectively stimulate DNA receptors activating the IRF pathway, while bypassing TLR9 and NF-κB activation.

Abstract 6241: PAX8-directed nanotherapeutics for high-grade serous ovarian carcinoma

Abstract Objective: High-grade serous ovarian cancer (HGSOC) is the most common epithelial ovarian cancer subtype. Targeted therapy for HGSOC is challenging because the disease is characterized by copy number variation rather than recurrent somatic mutations. HGSOC displays well-conserved epigenetic features such as expression of transcription factor PAX8. We propose employing epigenetic therapies that capitalize on interactions between PAX8 and specific DNA-binding sites to develop potential therapeutic approaches to target HGSOC. Our hypothesis is to use a biomimetic approach wherein PAX8 is induced to transcribe an ectopically transfected CRISPR-base editor (BE)-guide RNA (gRNA) plasmid containing a PAX8 binding site in its promoter. The gRNA will impart a dominant-negative allele of Polo Like Kinase 1 (PLK1) which has been shown to be synergistically lethal with deficient tp53 in cancer cells. In the presence of PAX8, the plasmid will be transcribed, while in the absence of PAX8, the plasmid is degraded, with minimal toxicity observed in healthy cells. Methods: PAX8-expressing ovarian cancer cell lines including COV362, Kuramochi, OVCAR8 and OVSAHO or PAX8-negative control cell lines including JEG3, MCF-7, HeLa and HEK293T were transfected with polyplexes bearing the plasmid of interest. Polyplex diameter, polydispersity indices, and surface zeta potentials were measured on a Malvern Zetasizer. Polyplexes were imaged for uptake and functionality using fluorescence microscopy and Amnis ImageStream. Polyplex toxicity was assessed in vitro using cell viability assays. Preliminary in vivo experiments to demonstrate luciferase plasmid-bearing polyplexes have been performed in NOD-SCID mice. To monitor in vivo transfection efficiencies, we will deliver fluorescently labeled plasmids or luciferase-encoding plasmids to test bio-distribution and pharmacokinetics. Results: We have synthesized sub-200nm (diameter) polyplexes using the polyamine PPLG-g-azidopropylamine (PPLG) and BE or gRNA plasmids. Using -/-PLK1 gRNA, we observed that the BE and the gRNA can be efficiently packaged into two distinct polymeric nanoplexes and co-delivered to ovarian cancer cells or control cells to impart a dominant-negative mutant of PLK1, which is also synthetically lethal with mutated tp53, as observed from cancer cell death compared to controls. Ongoing work includes repetitive polyplex injections in vitro and in vivo, quantifying percentage of total sequencing reads with target base pair conversions after treatment, determination of plasmid loading efficiency, and testing gRNAs against other targets for efficacy in ovarian cancer targeting. Conclusion: Well-defined lineage transcription factors have the potential to serve as novel targets for gene-editing therapies in ovarian cancer and other malignancies. Combining nanotechnology and gene editing can improve specificity toward ovarian cancer cells as well as incorporate precision therapy aimed at the underlying genetic architecture of this disease. Citation Format: Akshaya Chandrasekaran, Mariam Ahmed, Wade Wang, Paula T. Hammond, Kevin M. Elias. PAX8-directed nanotherapeutics for high-grade serous ovarian carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6241.

Carbon nanotube-mediated DNA delivery without transgene integration in intact plants.

Exogenous biomolecule delivery into plants is difficult because the plant cell wall poses a dominant transport barrier, thereby limiting the efficiency of plant genetic engineering. Traditional DNA delivery methods for plants suffer from host-species limitations, low transformation efficiencies, tissue damage, or unavoidable and uncontrolled DNA integration into the host genome. We have demonstrated efficient plasmid DNA delivery into intact plants of several species with functionalized high-aspect-ratio carbon nanotube (CNT) nanoparticles (NPs), enabling efficient DNA delivery into a variety of non-model plant species (arugula, wheat, and cotton) and resulting in high protein expression levels without transgene integration. Herein, we provide a protocol that can be implemented by plant biologists and adapted to produce functionalized single-walled CNTs (SWNTs) with surface chemistries optimized for delivery of plasmid DNA in a plant species-independent manner. This protocol describes how to prepare, construct, and optimize polyethylenimine (PEI)-functionalized SWNTs and perform plasmid DNA loading. The authors also provide guidance on material characterization, gene expression evaluation, and storage conditions. The entire protocol, from the covalent functionalization of SWNTs to expression quantification, can be completed in 5 d.

Facile Gene Delivery Derived from Branched Low Molecular Weight Polyethylenimine by High Efficient Chemistry.

Convenient methods for the preparation of gene delivery platforms based on branched low molecular weight polyethylenimine (PEI) were described. Firstly, PEI lipids, with a low molecular weight PEI headgroup and hexadecyl chain tail group, were prepared through a highly efficient ring-opening reaction of glycidyl hexadecyl ether (EpoxyC16) by amine from PEI. Then, the PEI lipids were used as a component of cationic liposomes and as a surfactant for the preparation of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticle (NP) via solvent extraction/evaporation method. As potential effective gene delivery platforms, their preparation, size, size distribution, toxicities, plasmid DNA loading, in vitro transfection and intracellular trafficking were studied. Both facile platforms showed less toxicity and higher transfection efficacy when compared to high molecular weight PEI in vitro, and may have further versatile applications in the gene delivery field.

Starch-Chitosan Polyplexes: A Versatile Carrier System for Anti-Infectives and Gene Delivery.

Despite the enormous potential of nanomedicine, the search for materials from renewable resources that balance bio-medical requirements and engineering aspects is still challenging. This study proposes an easy method to make nanoparticles composed of oxidized starch and chitosan, both isolated from natural biopolymers. The careful adjustment of C/N ratio, polymer concentration and molecular weight allowed for tuning of particle characteristics. The system’s carrier capability was assessed both for anti-infectives and for nucleic acid. Higher starch content polyplexes were found to be suitable for high encapsulation efficiency of cationic anti-infectives and preserving their bactericidal function. A cationic carrier was obtained by coating the anionic polyplex with chitosan. Coating allowed for a minimal amount of cationic polymer to be employed and facilitated plasmid DNA loading both within the particle core and on the surface. Transfection studies showed encouraging result, approximately 5% of A549 cells with reporter gene expression. In summary, starch-chitosan complexes are suitable carriers with promising perspectives for pharmaceutical use.

Comparative assessment of plasmid DNA delivery by encapsulation within or adsorbed on poly (D, L-lactide-coglycolide) nanoparticles

Purpose : To compare the gene delivery effectiveness of plasmid DNA (pDNA) encapsulated within poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles with that adsorbed on PLGA nanoparticles. Methods: PLGA nanoparticles were prepared using solvent-evaporation method. To encapsulate pDNA within the particles, it was first complexed with cetyltrimethylammonium bromide (CTAB) and then added to the oil phase during the synthesis. For the adsorption, PLGA nanoparticles were first modified with either CTAB or chitosan and then pDNA was adsorbed on the particle surface by electrostatic interaction. Results : Nanoparticles encapsulating pDNA exhibited better plasmid loading and protection with significantly lower burst release (p < 0.05) compared to that of the nanoparticles with adsorbed plasmid. Cell uptake of chitosan-modified nanoparticles by murine neuroblastoma (N2a) cells was significantly (p < 0.05) higher than that of chitosan-free nanoparticles. Nanoparticles encapsulating pDNA showed higher transfection efficiency (p < 0.05) in N2a cells. Conclusion : Encapsulation of pDNA within PLGA nanoparticles presents a potential strategy for gene delivery that is superior to pDNA adsorbed on the nanoparticle surface. In addition, encapsulation keeps the particle surface free for further modifications such as the addition of targeting ligands. Keywords : Poly (D,L-lactide-co-glycolide), Plasmid DNA, Encapsulation, Adsorption, Cellular uptake, Gene therapy, Targeting ligands

A laser-activated multifunctional targeted nanoagent for imaging and gene therapy in a mouse xenograft model with retinoblastoma Y79 cells

Retinoblastoma (RB) is the most common intraocular malignancy of childhood that urgently needs early detection and effective therapy methods. The use of nanosized gene delivery systems is appealing because of their highly adjustable structure to carry both therapeutic and imaging agents. Herein, we report a folic acid (FA)-modified phase-changeable cationic nanoparticle encapsulating liquid perfluoropentane (PFP) and indocyanine green (ICG) (FA-CN-PFP-ICG, FCNPI) with good plasmid DNA (pDNA) carrying capacity, favorable biocompatibility, excellent photoacoustic (PA) and ultrasound (US) contrast, enhanced gene transfection efficiency and therapeutic effect. The liquid-gas phase transition of the FCNPI upon laser irradiation has provided splendid contrasts for US/PA dual-modality imaging in vitro as well as in vivo. More importantly, laser-mediated gene transfection with targeted cationic FCNPI nanoparticles demonstrated the best therapeutic effect compared with untargeted cationic nanoparticle (CN-PFP-ICG, CNPI) and neutral nanoparticle (NN-PFP-ICG, NNPI), both in vitro and in vivo. Such a multifunctional nanoagent is expected to combine dual-mode guided imaging with fewer side effects and proper therapeutic efficacy. These results establish an experimental foundation for the clinical detection of and therapy for RB. Statement of significanceWe successfully constructed a multifunctional targeted cationic nanoparticle (FCNPI) and meticulously compared the variations in the plasmid loading capacity and binding to Y79 cells with NNPI, CNPI, and FCNPI. FCNPI exhibited favorable plasmid loading capability, splendid ability for targeting and only it could provide optimal US and PA contrast to background during a considerable long time. The FCNPI/pDNA + Laser system also exhibited the best therapeutic effect in vivo; this finding proposes a potential strategy for the evaluation of an efficient gene delivery nanocarrier for gene targeting therapy of RB tumor. Our study showed that there are great advantages of targeting FCNPI to provide PA/US imaging and to enlighten laser-mediated gene transfection. FCNPI is a very helpful multifunctional agent with potential.

Cationic Polyarginine Conjugated Mesoporous Bioactive Glass Nanoparticles with Polyglycerol Coating for Efficient DNA Delivery

Mesoporous bioactive glass (MBG) is a type of material with high biological activity and excellent biocompatibility. Because of its high specific surface area and adjustable surface morphology, MBG is usable for loading and delivering molecules. In our previous report, MBG particles were used as gene vectors and showed good transfection rate. In this paper, MBG, prepared through a sacrificial liquid template method in sol–gel process, was covered with polyglycerol (PG) and the resulting MBG-PG was further functionalized with octaarginine (Arg8. More specifically, MBG-PG-Arg8 particles were synthesized by PG functionalization of MBG through ring-opening polymerization of glycidol on the MBG surface, followed by multistep organic transformations (–OH→ –OTs (tosylate)→ –N3 in the PG layer and click conjugation of the Arg8 terminated with propargyl glycine. MBG-PG-Arg8 was successfully taken up by cells more efficiently due to the cellpenetrating property of Arg8, and thus showed higher plasmid DNA loading and cell transfection efficiency than MBG modified with amino groups. This novel arginine-functionalized MBG may be a good candidate as a vector for gene delivery with higher efficiency.

Targeted antiangiogenesis gene therapy using targeted cationic microbubbles conjugated with CD105 antibody compared with untargeted cationic and neutral microbubbles.

Objective This study aimed to develop targeted cationic microbubbles conjugated with a CD105 antibody (CMB105) for use in targeted vascular endothelial cell gene therapy and ultrasound imaging. We compared the results with untargeted cationic microbubbles (CMB) and neutral microbubbles (NMB).Methods CMB105 were prepared and compared with untargeted CMB and NMB. First, the microbubbles were characterized in terms of size, zeta-potential, antibody binding ability and plasmid DNA loading capacity. A tumor model of subcutaneous breast cancer in nude mice was used for our experiments. The ability of different types of microbubbles to target HUVECs in vitro and tumor neovascularization in vivo was measured. The endostatin gene was selected for its outstanding antiangiogenesis effect. For in vitro experiments, the transfection efficiency and cell cycle were analyzed using flow cytometry, and the transcription and expression of endostatin were measured by qPCR and Western blotting, respectively. Vascular tube cavity formation and tumor cell invasion were used to evaluate the antiangiogenesis gene therapy efficiency in vitro. Tumors were exposed to ultrasound irradiation with different types of microbubbles, and the gene therapy effects were investigated by detecting apoptosis induction and changes in tumor volume.Results CMB105 and CMB differed significantly from NMB in terms of zeta-potential, and the DNA loading capacities were 16.76±1.75 μg, 18.21±1.22 μg, and 0.48±0.04 μg per 5×108 microbubbles, respectively. The charge coupling of plasmid DNA to CMB105 was not affected by the presence of the CD105 antibody. Both CMB105 and CMB could target to HUVECs in vitro, whereas only CMB105 could target to tumor neovascularization in vivo. In in vitro experiments, the transfection efficiency of CMB105 was 24.7-fold higher than the transfection efficiency of NMB and 1.47-fold higher than the transfection efficiency of CMB (P<0.05). With ultrasound-targeted microbubble destruction (UTMD)-mediated gene therapy, the transcription and expression of endostatin were the highest in the CMB105 group (P<0.001); the antiangiogenesis effect and inhibition of tumor cells invasion was better with CMB105 than CMB or NMB in vitro (P<0.01). After gene therapy, the tumor volumes of CMB105 group were significantly smaller than that of CMB and NMB, and many tumor cells had begun apoptosis in the CMB105 group, which had the highest apoptosis index (P<0.001).Conclusions As a contrast agent and plasmid carrier, CMB105 can be used not only for targeted ultrasound imaging but also for targeted gene therapy both in vitro and in vivo. The plasmid DNA binding ability of the CMB was not affected by conjugation of the CMB with the CD105 antibody, and because of its targeting ability, the gene transfection efficiency and therapeutic effect were better compared with the untargeted CMB and NMB. The advantages of targeted gene therapy with CMB105 in vivo were more prominent than with CMB or NMB because neither can target the endothelia in vivo.

APPLICATION OF DENDRIMER/PLASMID hBMP-2 COMPLEXES LOADED INTO β-TCP/COLLAGEN SCAFFOLD IN THE TREATMENT OF FEMORAL DEFECTS IN RATS

Purpose: Plasmid loading into scaffolds to enhance sustained release of growth factors is an important focus of regenerative medicine. The aim of this study was to build gene-activated matrices (GAMs) and examine the bone augmentation properties. Methods: Generation 5 polyamidoamine dendrimers (G5 dPAMAM)/plasmid recombinant human bone morphogenetic protein-2 (rhBMP-2) complexes were immobilized into beta-tricalcium phosphate (β-TCP)/type I collagen porous scaffolds. After cultured with rat mesenchymal stem cells (rMSCs), transfection efficiencies were examined. The secretion of rhBMP-2 and alkaline phosphatase (ALP) were detected to evaluate the osteogenic properties. Scanning electron microscopy (SEM) was used to observe attachment and proliferation. Moreover, we applied these GAMs directly into freshly created segmental bone defects in rat femurs, and their osteogenic efficiencies were evaluated. Results: Released plasmid complexes were transfected into stem cells and were expressed, which caused osteogenic differentiations of rat mesenchymal stem cells (rMSCs). SEM analysis showed excellent cell attachment. Bioactivity of plasmid rhBMP-2 was maintained in vivo, and the X-ray observation, histological analysis and immunohistochemistry (IHC) of bone tissue demonstrated that the bone healing in segmental femoral defects was enhanced by implantation of GAMs. Conclusions: Such biomaterials offer therapeutic opportunities in critical-sized bone defects.

A universal gene carrier platform for treatment of human prostatic carcinoma by p53 transfection

Our previous work showed that a charge-reversal layer-by-layer nanosystem, PEI/PAH-Cit/AuNP-CS, effectively facilitates cellular uptake of siRNA and enhances the silencing efficacy of MDR1 siRNA. Here, the plasmid loading capacity of this vehicle was examined using EGFP-N1, and the plasmid release profile was determined in response to pH changes. The cytotoxicity of the EGFP-N1/PEI/PAH-Cit/AuNP-CS complex against HeLa and 293T cells was almost negligible. PEI/PAH-Cit/AuNP-CS efficaciously delivered the plasmids EGFP-N1 (encoding green fluorescent protein) and pGL3.0 (encoding luciferase) into 293T and HeLa cells, thus verifying the universality of PEI/PAH-Cit/AuNP-CS as a gene carrier. The results of an inverted fluorescence microscopy, flow cytometry (FCM) and western blotting methods demonstrated that PC-3 prostate cancer cells treated with EGFP-p53/PEI/PAH-Cit/AuNP-CS expressed higher levels of GFP than cells treated with EGFP-p53/PEI. Furthermore, PC-3 cells treated with EGFP-p53/PEI/PAH-Cit/AuNP-CS showed reduced cellular viability and increased nuclear fragmentation, consistent with elevated p53 expression. Propidium iodide (PI) flow cytometric assays were conducted to demonstrate that EGFP-p53/PEI/PAH-Cit/AuNP-CS elevated the level of apoptosis in PC-3 cells. Western blotting and caspase activation studies revealed that EGFP-p53/PEI/PAH-Cit/AuNP-CS complexes may induce PC-3 apoptosis via the mitochondria-mediated signaling pathway by up-regulation of Bax, down-regulation of Bcl-2, and activation of caspase-3.

Design of a Microchannel‐Nanochannel‐Microchannel Array Based Nanoelectroporation System for Precise Gene Transfection

A micro/nano-fabrication process of a nanochannel electroporation (NEP) array and its application for precise delivery of plasmid for non-viral gene transfection is described. A dip-combing device is optimized to produce DNA nanowires across a microridge array patterned on the polydimethylsiloxane (PDMS) surface with a yield up to 95%. Molecular imprinting based on a low viscosity resin, 1,4-butanediol diacrylate (1,4-BDDA), adopted to convert the microridge-nanowire-microridge array into a microchannel-nanochannel-microchannel (MNM) array. Secondary machining by femtosecond laser ablation is applied to shorten one side of microchannels from 3000 to 50 μm to facilitate cell loading and unloading. The biochip is then sealed in a packaging case with reservoirs and microfluidic channels to enable cell and plasmid loading, and to protect the biochip from leakage and contamination. The package case can be opened for cell unloading after NEP to allow for the follow-up cell culture and analysis. These NEP cases can be placed in a spinning disc and up to ten discs can be piled together for spinning. The resulting centrifugal force can simultaneously manipulate hundreds or thousands of cells into microchannels of NEP arrays within 3 minutes. To demonstrate its application, a 13 kbp OSKM plasmid of induced pluripotent stem cell (iPSC) is injected into mouse embryonic fibroblasts cells (MEFCs). Fluorescence detection of transfected cells within the NEP biochips shows that the delivered dosage is high and much more uniform compared with similar gene transfection carried out by the conventional bulk electroporation (BEP) method.

Tumor targeting and microenvironment-responsive nanoparticles for gene delivery

A tumor targeting nanoparticle system has been successfully developed to response to the lowered tumor extracellular pH (pHe) and upregulated matrix metalloproteinase 2 (MMP2) in the tumor microenvironment. The nanoparticles are modified with activatable cell-penetrating peptide (designated as dtACPP) that's dual-triggered by the lowered pHe and MMP2. In dtACPP, the internalization function of cell-penetrating peptide (CPP) is quenched by a pH-sensitive masking peptide, linking by a MMP2 substrate. The masking peptide is negatively charged to quench the cationic CPP well after systemic administration. Hence, dtACPP-modified nanoparticles possesses passive tumor targetability via the enhanced permeability and retention (EPR) effect. Once reaching the tumor microenvironment, the pre-existing attraction would be eliminated due to the lowered pHe, accompanying the linker cleaved by MMP2, dtACPP would be activated to expose CPP to drive the nanoparticles' internalization into the intratumoral cells. The studies of plasmid DNA loading, toxicity assessment, cellular uptake, tumor targeting delivery, and gene transfection demonstrate that dtACPP-modified nanoparticle system is a potential candidate for tumor targeting gene delivery.

Multiple release of polyplexes of plasmids VEGF and bFGF from electrospun fibrous scaffolds towards regeneration of mature blood vessels

Key challenges associated with the outcomes of vascular grafting (for example, to fully vascularize engineered tissues and promptly regenerate blood vessel substitutes) remain unsolved. The local availability of angiogenic growth factors is highly desirable for tissue regeneration, and plasmid loading in scaffolds represents a powerful alternative for local production of tissue-inductive factors. No attempt has been made so far to clarify the efficacy of electrospun fibers with the loading of multiple plasmids to promote tissue regeneration. In the present study, core–sheath electrospun fibers with the encapsulation of polyplexes of basic fibroblast growth factor-encoding plasmid (pbFGF) and vascular endothelial growth factor-encoding plasmid (pVEGF) were evaluated to promote the generation of mature blood vessels. In vitro release indicated a sustained release of pDNA for ∼4weeks with as low as ∼10% initial burst release, and the release patterns from the single and twofold plasmid-loaded systems coincided. In vitro investigations on human umbilical vein endothelial cells showed that the sustained release of pDNA from fibrous mats promoted cell attachment and viability, cell transfection and protein expression, and extracellular secretion of collagen IV and laminin. The acceleration of angiogenesis was assessed in vivo after subcutaneous implantation of fibrous scaffolds, and the explants were evaluated after 1, 2 and 4weeks’ treatment by histological and immunohistochemical staining. Compared with pDNA polyplex infiltrated fibrous mats, the pDNA polyplex encapsulated fibers alleviated the inflammation reaction and enhanced the generation of microvessels. Although there was no significant difference in the total number of microvessels, the density of mature vessels was significantly enhanced by the combined treatment with both pbFGF and pVEGF compared with those incorporating individual pDNA. The integration of the core–sheath structure, DNA condensation and multiple delivery strategies provided a potential platform for scaffold fabrication to regenerate functional tissues.