Good Transfection Efficiency Research Articles

Saponin and fluorine-functionalized polyethyleneimine/covered metal organic framework: A novel gene delivery system for targeted delivery of green fluorescent protein plasmid

Up to now, the existence of a safe and proper nanocarrier to improve the delivery efficiency of plasmid DNA into cells is a challenge. The large surface area and adjustable architectures of metal-organic framework (MOF), have gained a lot of interest and great potential for gene delivery. Herein, the Zirconium-Fumarate MOF (MOF-801) as novel MOF-polymer hybrid nanocarrier with high ability to gene delivery, was synthesized and modified with saponin- and fluorine-functionalized polyethyleneimine through an electrostatic interaction. The size and zeta potential of polyplex (DNA/nanocarrier) was 102 nm and +40 mV, respectively. Not only these powerful nanocarrier transfect the green fluorescent protein (plasmid-non encoded CRISPR/Cas systems) expression plasmid into the cell with an efficiency of about 60 %, but also, they showed good transfection efficiency at low plasmid doses and in existence of 30 % fetal bovine serum. Also, the nanocarrier was applied to study the plasmid delivery into cancer cell lines including MDA, PC12, HCT119, NIHT3, and MCF7 cells. The novel vector displayed low toxicity compared to other vectors which could be utilized for in vivo studies. Therefore, the saponin- and fluorine-modified polyethyleneimine/functionalized MOF-801 nanocomplex with remarkable properties was recommended for efficient gene transference.

Structural and Property Characterizations of Dual-Responsive Core-Shell Tecto Dendrimers for Tumor Penetration and Gene Delivery Applications.

Core-shell tecto dendrimers (CSTDs) with excellent physicochemical properties and good tumor penetration and gene transfection efficiency have been demonstrated to have the potential to replace high-generation dendrimers in biomedical applications. However, their characterization and related biological properties of CSTDs for enhanced tumor penetration and gene delivery still lack in-depth investigation. Herein, three types of dual-responsive CSTDs are designed for thorough physicochemical characterization and investigation of their tumor penetration and gene delivery efficiency. Three types of CSTDs are prepared through phenylborate ester bonds of phenylboronic acid (PBA)-decorated generation 5 (G5) poly(amidoamine) (PAMAM) dendrimers as cores and monose (galactose, glucose, or mannose)-conjugated G3 PAMAM dendrimers as shells and thoroughly characterized via NMR and other techniques. It is shown that the produced CSTDs display strong correlation signals between the PBA and monose protons, similar hydrodynamic diameters, and dual reactive oxygen species- and pH-responsivenesses. The dual-responsive CSTDs are proven to have structure-dependent tumor penetration property and gene delivery efficiency in terms of small interference RNA for gene silencing and plasmid DNA for gene editing, thus revealing a great potential for different biomedical applications.

Recent advances on two-dimensional material-based nanosystems for gene delivery

Gene therapy has been extensively investigated and widely used in biomedical fields, such as cancer treatment. However, the most important issues for gene therapy are stability, targeting effect, transfection efficacy, and safety of gene formulation after administration, which seriously limit the further application of gene therapy in clinic. Therefore, gene delivery could be a promising strategy for overcoming these challenges. Two-dimensional (2D) materials are rising nanomaterials with excellent physical and chemical properties, including large specific surface area, easy modification, high conversion efficiency of light, and good biocompatibility, which have achieved promising applications as vehicles for gene delivery in the disease treatment. In this review, we first summarized the research progress of 2D material-based nanosystems for gene delivery to improve the therapeutic efficacy. We discussed that 2D material-based gene delivery nanosystems showed high therapeutic efficacy for many diseases treatment, especially cancer. Furthermore, we also proposed that surface modification of 2D materials might be a promising strategy to prepare multi-functional gene carriers for combination therapy with enhanced treatment efficacy. Finally, the future research progress, challenges, and prospects of 2D material-based nanosystems for gene therapy were discussed and concluded. Conclusively, we believe that 2D material-based nanosystems with good biocompatibility and high transfection efficiency would be potentially used in clinical settings to improve the therapeutic efficacy of gene therapy.

Nontoxic, Biodegradable Hyperbranched Poly(β-amino ester)s for Efficient siRNA Delivery and Gene Silencing.

RNA interference (RNAi)-mediated gene silencing is a promising therapeutic approach to treat various diseases, but safe and efficient delivery remains a major challenge to its clinical application. Non-viral gene vectors, such as poly(β-amino esters) (pBAEs), have emerged as a potential candidate due to their biodegradability, low toxicity profile, ease of synthesis, and high gene transfection efficiency for both DNA and siRNA delivery. However, achieving significant gene silencing using pBAEs often requires a large amount of polymer carrier (with polymer/siRNA weight ratio >100) or high siRNA dose (>100 nM), which might potentially exacerbate toxicity concerns during delivery. To overcome these barriers, we designed and optimized a series of hyperbranched pBAEs capable of efficiently condensing siRNA and achieving excellent silencing efficiency at a lower polymer/siRNA weight ratio (w/w) and siRNA dose. Through modulation of monomer combinations and branching density, we identified the top-performing hyperbranched pBAEs, named as h(A2B3)-1, which possess good siRNA condensation ability, low cytotoxicity, and high cellular uptake efficiency. Compared with Lipofectamine 2000, h(A2B3)-1 achieved lower cytotoxicity and higher siRNA silencing efficiency in HeLa cells at a polymer/siRNA weight ratio of 30 and 30 nM siRNA dose. Notably, h(A2B3)-1 enhanced the gene uptake in primary neural cells and effectively silenced the target gene in hard-to-transfect primary cortical neurons and oligodendrocyte progenitor cells, with gene knockdown efficiencies of 34.8 and 53.4% respectively. By incorporating a bioreducible disulfide compartment into the polymer backbone, the cytocompatibility of the h(A2B3)-1 was greatly enhanced while maintaining their good transfection efficiency. Together, the low cytotoxicity and high siRNA transfection efficiency of hyperbranched h(A2B3)-1 in this study demonstrated their great potential as a non-viral gene vector for efficient siRNA delivery and RNAi-mediated gene silencing. This provides valuable insight into the future development of safe and efficient non-viral siRNA delivery systems as well as their translation into clinical applications.

Osteophilic and Dual-Regulated Alendronate-Gene Lipoplexes for Reversing Bone Loss.

The pathogenesis of postmenopausal osteoporosis (PMOP) is mainly determined by the adhesion of osteoclasts to the bone matrix and the involvement of various molecules in bone resorption. The dual regulation strategy of the physical barriers of bone matrix and intracellular gene regulation generated by advanced biomaterials is a decent alternative for the treatment of PMOP. Herein, for the first time, it is identified that hsa-miR-378i/mmu-miR-378a-3p are closely associated with PMOP. Then, an osteophilic and dual-regulated alendronate-gene lipoplex (antagomir@Aln-Lipo), composed of medicative alendronate-functionalized liposomal vehicle and encapsulated specific microRNAs is engineered, for bone-targeting delivery of genes to achieve combined mitigation of bone loss. Alendronate targets hydroxyapatite in the bone matrix and occupies the adhesion site of osteoclasts, thus providing the "physical barriers". Antagomir is coupled precisely to specific endogenous microRNAs, thus providing the "genetic signals". These functionalized lipoplexes exhibited long-term stability and good transfection efficiency. It is proven that antagomir@Aln-Lipo could synergistically regulate osteoclastogenesis and bone resorption in vitro and in vivo. Furthermore, intravenous injection of antagomir@Aln-Lipo efficiently reverses bone loss through a dual mechanism driven by alendronate and antagomir-378a-3p. In conclusion, the osteophilic and dual-regulated antagomir@Aln-Lipo offers a brand-new bifunctional strategy for the precise treatment of PMOP.

Hyper-Branched Cationic Cyclodextrin Polymers for Improving Plasmid Transfection in 2D and 3D Spheroid Cells.

In this article, we used monolayer two dimensional (2D) and 3D multicellular spheroid models to improve our understanding of the gene delivery process of a new modified cationic hyper-branched cyclodextrin-based polymer (Ppoly)-loaded plasmid encoding Enhanced Green Fluorescent Protein (EGFP). A comparison between the cytotoxicity effect and transfection efficiency of the plasmid DNA (pDNA)-loaded Ppoly system in 2D and 3D spheroid cells determined that the transfection efficiency and cytotoxicity of Ppoly-pDNA nanocomplexes were lower in 3D spheroids than in 2D monolayer cells. Furthermore, histopathology visualization of Ppoly-pDNA complex cellular uptake in 3D spheroids demonstrated that Ppoly penetrated into the inner layers. This study indicated that the Ppoly, as a non-viral gene delivery system in complex with pDNA, is hemocompatible, non-toxic, high in encapsulation efficiency, and has good transfection efficiency in both 2D and 3D cell cultures compared to free pDNA and lipofectamine (as the control).

Sodium Alginate-Doping Cationic Nanoparticle As Dual Gene Delivery System for Genetically Bimodal Therapy.

Photodynamic therapy occupies an important position in cancer therapy because of its minimal invasiveness and high spatiotemporal precision, and photodynamic/gene combined therapy is a promising strategy for additive therapeutic effects. However, the asynchronism and heterogeneity between traditional chemical photosensitizers and nucleic acid would restrict the feasibility of this strategy. KillerRed protein, as an endogenous photosensitizer, could be directly expressed and take effect in situ by transfecting KillerRed reporter genes into cells. Herein, a simple and easily prepared sodium alginate (SA)-doping cationic nanoparticle SA@GP/DNA was developed for dual gene delivery. The nanoparticles could be formed through electrostatic interaction among sodium alginate, polycation, and plasmid DNA. The title complex SA@GP/DNA showed good biocompatibility and gene transfection efficiency. Mechanism studies revealed that SA doping could facilitate the cellular uptake and DNA release. Furthermore, SA@GP/DNA was applied to the codelivery of p53 and KillerRed reporter genes for the synergistic effect combining p53-mediated apoptosis therapy and KillerRed-mediated photodynamic therapy. The ROS generation, tumor cell growth inhibition, and apoptosis assays proved that the dual-gene transfection could mediate the better effect compared with single therapy. This rationally designed dual gene codelivery nanoparticle provides an effective and promising platform for genetically bimodal therapy.

Development of a novel vector for siRNA delivery based on arginine‐modified polyvinylamine

AbstractAn ideal carrier that delivers small interfering RNA (siRNA) should be designed based on two criteria: high gene transfection efficiency and low cytotoxicity. In this work, arginine was used to modify polyvinylamine (PVAm) to form a novel PVAm derivative (PVAm‐Arg) for siRNA delivery and the derivative was characterized using Fourier transform infrared spectroscopy. PVAm‐Arg/siRNA complexes were formed by the self‐assembly method. The zeta potential and size of the complexes were measured using electrophoretic light scattering and dynamic light scattering, respectively. The siRNA binding capacity of PVAm‐Arg was assessed using gel retardation assay. The results showed that PVAm‐Arg derivatives can bind siRNA effectively and form complexes with sizes of about 72 ± 2 nm. What is more, the ability of PVAm‐Arg to condense siRNA was better than that of PVAm. Cytotoxicity assay with RSC96 cells also showed that PVAm‐Arg had lower cytotoxicity compared with PVAm. In in vitro cellular uptake assay, PVAm‐Arg showed good transfection efficiency. Therefore, we concluded that PVAm‐Arg would be a promising candidate as a gene delivery vector. © 2022 Society of Industrial Chemistry.

Evaluation of pentaerythritol-based and trimethylolpropane-based cationic lipidic materials for gene delivery

The chemical and physical structure of cationic liposomes pays an important effect on their gene transfection efficiency. Investigation on the structure-function relationship of cationic liposomes will guide the design of novel cationic liposomes with high transfection efficiency and biosafety. In this paper, two novel series of lipids based on the backbone of pentaerythritol and trimethylolpropane were discovered, and their gene transfection efficiencies were assayed in vitro. The four lipids 8c, 9c, 14b, and 15b, exhibited much better transfection efficiency in the HEK293 cell lines compared with Lipo2000, lipid 9c also showed good transfection efficiency in the SW480 cell lines. And the structure-efficiency relationship revealed that a hydroxyethyl polar head group boosted transfer potency in trimethylolpropane-type lipids, but reduced in pentaerythritol-type lipids.

The effect of 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase gene overexpression in the kynurenine pathway on the expression levels of indoleamine 2,3-dioxygenase 1 and interferon-γ in inflammatory conditions: an in vitro study.

The kynurenine pathway (KP) can be involved in the pathogenesis of neurodegenerative diseases and excessive neurotoxic metabolite production. This study aimed to evaluate the effects of overexpression of murine 2-amino-3-carboxymuconate-6-semialdehyde decarboxylase (Acmsd) gene in inflammatory conditions in RAW 264.7 cell line to present more information about the effect of this gene on inflammatory conditions and the KP cycle. The coding sequence of the Acmsd gene was cloned into pCMV6-AC-IRES-GFP expression vector with a green fluorescent protein (GFP) marker. To simulate inflammatory conditions, RAW 264.7 macrophage cells were stimulated by Lipopolysaccharide (LPS) 24h before transfection, and transfected by Polyethyleneimine(PEI) with constructed plasmids expressing the Acmsd gene. The effect of Acmsd gene expression level on murine Interferon-gamma (Ifn-γ) and murine Indoleamine 2,3-dioxygenase 1 (Ido1) gene expression level was investigated by Real-Time PCR. According to the results of this study, good transfection efficiency was observed 72h after transfection, and Acmsd expression level increased 29-fold (P < 0.001) in transfected LPS-stimulated cells compared to the control group (LPS-stimulated cells that were not transfected). Additionally, increased Acmsd expression level significantly down-regulated Ifn-γ (P < 0.001) and Ido1 (P < 0.01) expression level in transfected LPS-stimulated cells compared to LPS-stimulated cells. Acmsd gene overexpression in inflammatory conditions can reduce the expression levels of the Ido1 gene, and its regulator, Ifn-γ. Consequently, it may be considered as a novel regulatory factor in the KP balance.

The Process of Binding and Releasing of Genetic Material from Lipoplexes Based on Trimeric Surfactants and Phospholipids.

Nonviral vectors for gene therapy such as lipoplexes are characterized by low toxicity, high biocompatibility, and good transfection efficiency. Specifically, lipoplexes based on polymeric surfactants and phospholipids have great potential as gene carriers due to the increased ability to bind genetic material (multiplied positive electric charge) while lowering undesirable effects (the presence of lipids makes the system more like natural membranes). This study aimed to test the ability to bind and release genetic material by lipoplexes based on trimeric surfactants and lipid formulations of different compositions and to characterize formed complexes by circular dichroism (CD) spectroscopy and atomic force microscopy (AFM). The cytotoxicity of studied lipoplexes was tested on HeLa cells by the MTT cell viability assay and the dye exclusion test (trypan blue). The presence of lipids in the system lowered the surfactant concentration required for complexation (higher efficiency) and reduced the cytotoxicity of lipoplexes. Surfactant/lipids/DNA complexes were more stable than surfactant/DNA complexes. Surfactant molecules induced the genetic material condensation, but the presence of lipids significantly intensified this process. Systems based on trimeric surfactants and lipid formulations, particularly TRI_N and TRI_IMI systems, could be used as delivery carrier, and have proven to be highly effective, nontoxic, and universal for DNA of various lengths.

Highly Photoluminescent Nitrogen- and Zinc-Doped Carbon Dots for Efficient Delivery of CRISPR/Cas9 and mRNA.

Safe and efficient delivery of CRISPR/Cas9 systems is still a challenge. Here we report the development of fluorescent nitrogen- and zinc-doped carbon dots (N-Zn-doped CDs) using one-step microwave-aided pyrolysis based on citric acid, branched PEI25k, and different zinc salts. These versatile nanovectors with a quantum yield of around 60% could not only transfect large CRISPR plasmids (∼9 kb) with higher efficiency (80%) compared to PEI25k and lipofectamine 2000 (Lipo 2K), but they also delivered mRNA into HEK 293T cells with the efficiency 20 times greater than and equal to that of PEI25k and Lipo 2K, respectively. Unlike PEI25k, N-Zn-doped CDs exhibited good transfection efficiency even at low plasmid doses and in the presence of 10% fetal bovine serum (FBS). Moreover, these nanovectors demonstrated excellent efficiency in GFP gene disruption by transferring plasmid encoding Cas9 and sgRNA targeting GFP as well as Cas9/sgRNA ribonucleoproteins into HEK 293T-GFP cells. Hence, N-Zn-doped CDs with remarkable photoluminescence properties and high transfection efficiency in the delivery of both CRISPR complexes and mRNA provide a promising platform for developing safe, efficient, and traceable delivery systems for biological research.

Hydrophobic and hydrophilic modifications of polyethylenimine towards gene delivery applications

AbstractNon‐viral gene delivery has emerged as a promising approach for therapy in cancer treatment. Polyethylenimine (PEI) is a prominent transfecting agent, but due to toxicity and poor hemocompatibility, its usage for in vivo applications is limited. However, modification of PEI with different chemical groups can lead to conjugates with good transfection efficiency and better cytocompatibility. In this study, the efficiency of PEI derivatives, namely, PEI succinate (PEIS), PEI lauryl succinate (PEILS), PEI laurate (PEIL) was analyzed for gene delivery applications. Apart from biophysical characterization such as size, zeta potential, nanoplex stability and buffering capacity, cellular internalization, polymer trafficking and p53 transgene expression in C6 cell lines were also investigated. The results indicate that PEI conjugated with lauryl succinate act as better transfecting agent with high efficiency compared to other derivatives, and such balanced hydrophilic‐hydrophobic modification of PEI can render it to be a cytocompatible and effective nucleic acid delivery system.

Spontaneous Formation of Cationic Vesicles in Aqueous DDAB-Lecithin Mixtures for Efficient Plasmid DNA Complexation and Gene Transfection.

Cationic liposomes have become an attractive tool to deliver genes and interfering RNA into cells. Herein, we report the application of spontaneously formed cationic vesicles in mixtures of lecithin and cationic amphiphiles for efficient transfection of plasmid DNA and siRNA into cells. The average hydrodynamic diameter of the phospholipid vesicles was modulated by changing the ratio of dihexadecyldimethylammonium bromide (DDAB) to phospholipid in the vesicles. The vesicles were characterized by dynamic light scattering, ζ potential, and small-angle X-ray scattering. Depending on the ratio of DDAB to phospholipid, the average size of the vesicles can be varied in the range of 150-300 nm with a ζ potential of +40 mV. The ability of these cationic vesicles to form lipoplexes upon binding with pDNA is demonstrated by ζ potential, isothermal titration calorimetry, gel retardation, and DNase I digestion assay. The enthalpy of binding between pDNA and cationic liposome was found to be -5.7 (±0.8) kJ/mol. The cellular uptake studies of lipoplexes observed by fluorescence microscopy confirmed good transfection efficiency of DDAB liposomes in MCF-7 and HeLa cells. The fluorescent imaging analysis showed effective gene delivery and expression of green fluorescent protein. In addition, the formulation has demonstrated an ability to deliver small interfering RNA (siBRD4) for efficient gene silencing as seen by a significant decrease in BRD4 protein level in siBRD4-treated cells. Comparison of the transfection efficiency of different formulations suggests that DDAB-rich mixed phospholipid vesicles with size <200 nm are better than large size vesicles for improved endocytosis and gene expression.

Cationic nanoparticles self-assembled from amphiphilic chitosan derivatives containing poly(amidoamine) dendrons and deoxycholic acid as a vector for co-delivery of doxorubicin and gene

Combination treatment through the co-delivery of drugs and genes by nanoformulations may achieve a synergistic effect. In our previous study, poly(amidoamine) dendronized chitosan derivative (PAMAM-Cs) showed good gene transfection efficiency and low cytotoxicity. Here, we incorporated hydrophobic deoxycholic acid (DCA) onto the chitosan backbone of PAMAM-Cs to obtain an amphiphilic derivative—PAMAM-Cs-DCA, which could self-assemble into cationic nanoparticles (NPs). The resulting NPs with diameters of 140–220 nm can encapsulate the hydrophobic anticancer drug doxorubicin (DOX) in the core while bind pDNA via the positively charged PAMAM shell. PAMAM-Cs-DCA NPs could completely complex with pDNA at a ratio of nitrogen to phosphorous (N/P) low as 1 and the complexes achieved a transfection efficiency up to 74 % at N/P 20. Moreover, low-dose co-delivered DOX could enhance the transgene expression, showing a synergistic effect. These results suggest that PAMAM-Cs-DCA NPs hold great promise to co-deliver chemotherapeutics and nucleic acid drugs.

Design of a New Gemini Lipoaminoacid with Immobilized Lipases Based on an Eco-Friendly Biosynthetic Process

Lipoaminoacids (LAA) are an important group of biosurfactants, formed by a polar hydrophilic part (amino acid) and a hydrophobic tail (lipid). The gemini LAA structures allow the formation of a supramolecular complex with bioactive molecules, like DNA, which provides them with good transfection efficiency. Since lipases are naturally involved in lipid and protein metabolism, they are an alternative to the chemical production of LAA, offering an eco-friendly biosynthetic process option. This work aimed to design the production of novel cystine derived gemini through a bioconversion system using immobilized lipases. Three lipases were used: porcine pancreatic lipase (PPL); lipase from Thermomyces lanuginosus (TLL); and lipase from Rizhomucor miehei (RML). PPL was immobilized in sol-gel lenses. L-cystine dihydrochloride and dodecylamine were used as substrates for the bioreaction. The production of LAA was evaluated by thin layer chromatography (TLC), and colorimetric reaction with eosin. The identification and quantification was carried out by High Performance Liquid Chromatographer-Mass Spectrometry (HPLC-MS/MS). The optimization of media design included co-solvent (methanol, dimethylsulfoxide), biphasic (n-hexane and 2-propanol) or solvent-free media, in order to improve the biocatalytic reaction rates and yields. Moreover, a new medium was tested where dodecylamine was melted and added to the cystine and to the biocatalyst, building a system of mainly undissolved substrates, leading to 5 mg/mL of LAA. Most of the volume turned into foam, which indicated the production of the biosurfactant. For the first time, the gemini derived cystine lipoaminoacid was produced, identified, and quantified in both co-solvent and solvent-free media, with the lipases PPL, RML, and TLL.

SIRT1 alleviates hepatic ischemia-reperfusion injury via the miR-182-mediated XBP1/NLRP3 pathway

The hepatoprotection of histone deacetylase sirtuin 1 (SIRT1) has been identified to attenuate ischemia-reperfusion (IR)-triggered inflammation and liver damage. This study was performed to characterize the function of SIRT1 in hepatic IR injury. In in vivo assays on liver-specific knockout mice of SIRT1, we first validated the effect of SIRT1 knockout on liver damage and XBP1/NLRP3 inflammasome activation. Next, we examined whether knockdown of XBP1/NLRP3 or miR-182 agomir could reverse the effect of SIRT1 knockout. In in vitro assays, NCTC1469 cells subjected to hypoxia/reoxygenation (H/R) were transduced with small interfering RNA (siRNA)/activator of SIRT1 or miR-182 agomir to confirm the effect of SIRT1 on NCTC1469 cell behaviors as well as the regulation of miR-182 and the XBP1/NLRP3 signaling pathway. Hepatic IR injury was appreciably aggravated in SIRT1 knockout mice, and SIRT1 knockdown abolished the inhibition of XBP1/NLRP3 inflammasome activation, which was reversed by NLRP3 knockdown, XBP1 knockdown, or miR-182 agomir. Mechanistically, miR-182 expression was positively regulated by SIRT1 in hepatic IR injury in mice, and miR-182 inhibited the expression of XBP1 by binding to the 3′ untranslated region (UTR) of XBP1. The histone deacetylase SIRT1 inhibits the downstream XBP1/NLRP3 inflammatory pathway by activating miR-182, thus alleviating hepatic IR injury in mice.

Purification, Culture, and CD19-CAR Lentiviral Transduction of Adult and Umbilical Cord Blood NK Cells.

Natural killer cells, or NK cells, are a type of cytotoxic lymphocyte critical to the innate immune system. The role that NK cells play is analogous to that of cytotoxic T cells in that they provide rapid responses to virus-infected cells and responses to tumor formation. Unmodified NK cells have long been used in various immunotherapies to treat different tumors, with only marginal success. However, in the last few years, NK cells modified to express chimeric antigen receptors (CAR-NK cells) have emerged as particularly ideal cellular platforms for antigen-specific antitumor agents. Unlike CAR-T cells, they do not elicit allogeneic responses or graft-versus-host disease and therefore can be administered to recipients with differing MHC expression. This article outlines protocols to obtain CD19-CAR-NK cells, focusing on the importance of obtaining and culturing a purified NK cell population and how to attain good transfection efficiency. © 2020 Wiley Periodicals LLC Basic Protocol 1: Purification and culture of adult peripheral blood and umbilical cord blood NK cells Basic Protocol 2: CD19-CAR lentiviral transduction of adult peripheral blood or umbilical cord blood NK cells Support Protocol: Production of lentiviral supernatant.

Quantitating Endosomal Escape of a Library of Polymers for mRNA Delivery.

Endosomal escape is a key step for intracellular drug delivery of nucleic acids, but reliable and sensitive methods for its quantitation remain an unmet need. In order to rationally optimize the mRNA transfection efficiency of a library of polymeric materials, we designed a deactivated Renilla luciferase-derived molecular probe whose activity can be restored only in the cytosol. This probe can be coencapsulated with mRNA in the same delivery vehicle, thereby accurately measuring its endosomal escape efficiency. We examined a library of poly(amine-co-ester) (PACE) polymers with different end groups using this probe and observed a strong correlation between endosomal escape and transfection efficiency (R2 = 0.9334). In addition, we found that mRNA encapsulation efficiency and endosomal escape, but not uptake, were determinant factors for transfection efficiency. The polymers with high endosomal escape/transfection efficiency in vitro also showed good transfection efficiency in vivo, and mRNA expression was primarily observed in spleens after intravenous delivery. Together, our study suggests that the luciferase probe can be used as an effective tool to quantitate endosomal escape, which is essential for rational optimization of intracellular drug delivery systems.

Study of near-infrared light-induced excitation of upconversion nanoparticles as a vector for non-viral DNA delivery.

Clinical requirements have necessitated the development of biomedical nanomaterials that can be implanted into tissues or bodies. Physiological regulation can be achieved in these nanomaterials through external light. The combination of nanomaterials with infrared optics can be termed optogenetics. The low autofluorescence of upconversion nanoparticles (UCNPs) has several applications in the biological field. For optogenetics applications, UCNPs with high fluorescence performance and photostability can solve the penetration depth problem. NaYF4:Yb,Tm nanocrystals with controllable sizes, shapes, and compositions were synthesized using a rapid coprecipitation method in organic solvent. UCNPs using single crystal nanoparticles provide higher chemical stability than those using amorphous phase. However, because UCNPs are usually capped with hydrophobic ligands, it is particularly important to prepare biocompatible UCNPs with specific molecular recognition capabilities. Surface modification and subsequent functionalization are essential for the application of inorganic nanomaterials in the biological environment and are arousing increasing research interest. Due to the high biocompatibility and high loading of materials, mesoporous silica and amine groups were selected as the best candidates. Expression of plasmid DNA in vivo and transfection efficiency were determined by fluorescence microscopy and flow cytometry. The MTT assay was used to evaluate the particle biocompatibility; the results showed that UCNP@mSiO2 has great biocompatibility. Additionally, at neutral pH, the cell surface is negatively charged. Therefore, the surface is functionalized with amino groups and can be electrostatically bound to DNA. Finally, UCNP@mSiO2-NH2 as a vector was applied in live cells by loading DNA; according to the results, DNA-UCNPs were successfully transfected in the primary cells, and NaYF4:Yb,Tm@mSiO2-NH2-DNA were observed to have good transfection efficiency by flow cytometry. It is expected that this work will provide a different method from the traditional adenovirus method and improve the immune response and side effects caused by adenovirus.