Surface polyethylene glycol enhances substrate-mediated gene delivery by nonspecifically immobilized complexes

Substrate-mediated gene delivery describes the immobilization of gene therapy vectors to a biomaterial, which enhances gene transfer by exposing adhered cells to elevated DNA concentrations within the local microenvironment. Surface chemistry has been shown to affect transfection by nonspecifically immobilized complexes using self-assembled monolayers (SAMs) of alkanethiols on gold. In this report, SAMs were again used to provide a controlled surface to investigate whether the presence of oligo(ethylene glycol) (EG) groups in a SAM could affect complex morphology and enhance transfection. EG groups were included at percentages that did not affect cell adhesion. Nonspecific complex immobilization to SAMs containing combinations of EG- and carboxylic acid-terminated alkanethiols resulted in substantially greater transfection than surfaces containing no EG groups or SAMs composed of EG groups combined with other functional groups. Enhancement in transfection levels could not be attributed to complex binding densities or release profiles. Atomic force microscopy imaging of immobilized complexes revealed that EG groups within SAMs affected complex size and appearance and could indicate the ability of these surfaces to preserve complex morphology upon binding. The ability to control the morphology of the immobilized complexes and influence transfection levels through surface chemistry could be translated to scaffolds for gene delivery in tissue engineering and diagnostic applications.     

Synergistic effect of amino acids modified on dendrimer surface in gene delivery

Design of an efficient gene vector based on dendrimer remains a great challenge due to the presence of multiple barriers in gene delivery. Single-functionalization on dendrimer cannot overcome all the barriers. In this study, we synthesized a list of single-, dual- and triple-functionalized dendrimers with arginine, phenylalanine and histidine for gene delivery using a one-pot approach. The three amino acids play different roles in gene delivery: arginine is essential in formation of stable complexes, phenylalanine improves cellular uptake efficacy, and histidine increases pH-buffering capacity and minimizes cytotoxicity of the cationic dendrimer. A combination of these amino acids on dendrimer generates a synergistic effect in gene delivery. The dual- and triple-functionalized dendrimers show minimal cytotoxicity on the transfected NIH 3T3 cells. Using this combination strategy, we can obtain triple-functionalized dendrimers with comparable transfection efficacy to several commercial transfection reagents. Such a combination strategy should be applicable to the design of efficient and biocompatible gene vectors for gene delivery.     

Gold immuno-functionalisation via self-assembled monolayers: study of critical parameters and comparative performance for protein and bacteria detection

Surface functionalisation is of extreme importance in assay and biosensor development because it ensures the selective capture and detection of the targets of interest. In the present report, we compare the performance of several gold functionalisation strategies/chemistries, based on SAM self-assembly and Ab conjugation, for protein and bacteria detection. The first part of the work summarises the optimisation of the various protocols considered. Their efficiency was initially evaluated in terms of reduction of biomolecule non-specific adsorption and specific detection competence impairment, using as a model-target an enzyme-labelled protein. With this purpose, the effect of several parameters, such as thiomolecule length and concentration, self-assembly time and temperature, polymer incorporation, or Ab conjugation strategy was determined. The three best performing strategies consisted of antibody (Ab) conjugation to self-assembled monolayers (SAM) containing mercaptoundecanoic acid alone, or conjugated to either long-chain hydrophilic diamines or CM-dextran. In the three cases, results demonstrated that Abs had been successfully incorporated and remained functional for protein detection. Nevertheless, as showed in the second part of the work, we demonstrate for the first time that these chemistries can be inadequate for bacteria detection. The possible reasons and implications will be discussed. Ab physisorption is proposed as a cost-effective gold immuno-functionalisation strategy alternative to SAM-based Ab incorporation for bacteria detection.     

Towards construction of viral vectors based on avian coronavirus infectious bronchitis virus for gene delivery and vaccine development

Manipulation of the coronavirus genome to accommodate and express foreign genes is an attractive approach for gene delivery and vaccine development. By using an infectious cloning system developed recently for the avian coronavirus infectious bronchitis virus (IBV), the enhanced green fluorescent protein (EGFP) gene, the firefly luciferase gene and several host and viral genes (eIF3f, SARS ORF6, Dengue virus 1 core protein gene) were inserted into various positions of the IBV genome, and the effects on gene expression, virus recovery, and stability in cell culture were studied. Selected viruses were also inoculated into chicken embryos for studies of foreign gene expression at different tissue level. The results demonstrated the stability of recombinant viruses depends on the intrinsic properties of the foreign gene itself as well as the position at which the foreign genes were inserted. For unstable viruses, the loss of expression of the inserted genes was found to result from a large deletion of the inserted gene and even IBV backbone sequences. This represents a promising system for development of coronavirus-based gene delivery vectors and vaccines against coronavirus and other viral infections in chicken.     

Adsorption of a therapeutic enzyme to self-assembled monolayers: effect of surface chemistry and solution pH on the amount and activity of adsorbed enzyme

The adsorption of a therapeutic enzyme to self-assembled monolayers (SAMs) of different functionalities (X = CH(3)-, OH- and COOH-) was evaluated as a function of solution pH. Radiolabelling studies showed that the enzyme has higher affinity for hydrophobic surfaces than for hydrophilic surfaces, and that the highest adsorption was obtained at the more acidic pH values (4.5 and 5.5), despite the type of surface. IRAS and XPS measurements confirmed this tendency. Dye-binding studies and fluorescence quenching were used to investigate if a pH variation induces any conformational changes on the enzyme. Both methods suggest that lowering the pH from physiological to acidic values triggers an increased exposure of non-polar sites in the enzyme, which may modulate its adsorption behaviour to the more hydrophobic surfaces. At pH 4.5, the enzyme carries a substantial positive net charge and therefore relatively low native-state stability. As a consequence, surface binding may be favoured, irrespective of the type of surface, by providing increased conformational entropy to the enzyme. The specific activity (SA) of the adsorbed enzyme was strongly dependent on the conditions used. A decrease in SA (ca. 30% of control) was observed after adsorption on CH(3)-SAMs for all the pH tested. Adsorption on gold and on the more hydrophilic SAMs (OH- and COOH-) resulted in different degrees of inactivation at the more acidic pH (4.5), and in enzyme activation (up to ca. 230% of control) at higher pH (7-8), near the isoelectric point of the enzyme.     

人脐带内皮祖细胞的分离、鉴定及转染EGFP质粒的实验研究

目的 从脐带中分离内皮祖细胞(EPCs),考察其体外增殖、基因转染绿色荧光蛋白质粒的行为.方法 以酶消化法从脐带中分离出内皮祖细胞,并通过流式细胞仪和共聚焦显微镜对内皮祖细胞进行鉴定,以Lipofectamine 2000为转染试剂考察了内皮祖细胞转染绿色荧光蛋白质粒的行为.结果 从脐带中分离培养的内皮祖细胞在第9天形成了典型的内皮细胞集落,流式细胞仪分析结果显示CD133和激酶插入区受体(KDR)的含量均有所提高,并具有内皮祖细胞结合异硫氰酸荧光素(FITC)标记的荆豆凝聚素1(FITC-UEA-1)和吞噬DiI标记的低密度脂蛋白(DiI-ac-LDL)的功能,能较好地表达绿色荧光蛋白.结论 从脐带中分离的内皮祖细胞体外在适当的培养条件下,可增殖、诱导分化为内皮细胞,并能较好地表达外源基因,是基因与细胞治疗理想的载体.     

The effect of quantum dot size and poly(ethylenimine) coating on the efficiency of gene delivery into human mesenchymal stem cells

Quantum dot (QDs) have been employed as bioimaging agents and delivery vehicles for gene therapeutics in several types of cells. In this study, we fabricated multiple QD bundled nanoparticles (NPs) to investigate the effect of QD size and poly(ethylenimine) (PEI) coating on the efficiency of gene delivery into human mesenchymal stem cells (hMSCs). Several types of QDs, which exhibit different ranges of particle size and fluorescence when employed, were coated with PEI to alter their negative charges and to enable them to be bundled into larger particles. Using specific wavelengths of QDs for bioimaging, gene-complexed QD bundled NPs were easily detected in the hMSCs using several different methods such as fluorescence-activated cell sorter, confocal laser scanning microscopy, and in vivo optical imaging. These PEI-coated, bundled QD NPs exhibited significantly higher gene transfection efficacy than single-type QDs. Particularly, the largest QD bundled NPs examined, QD655, had a much higher uptake capability and greater gene expression ability than the other QD NPs (QD525, QD565, and QD605). We believe that our findings help to enrich knowledge of design considerations that will aid in the engineering of QD NPs for stem cell application in the future.     

Protein interactions with oligo(ethylene glycol) (OEG) self-assembled monolayers: OEG stability,surface packing density and protein adsorption

We present a study of protein adsorption on oligo(ethylene glycol) (OEG) self-assembled monolayers (SAMs) at a range of OEG surface densities. OEG SAMs were formed in mixed ethanol and water solutions at different assembly temperatures to adjust the packing density of EG₄-SAMs. These SAMs were characterized using X-ray photoelectron spectroscopy (XPS). Fibrinogen adsorption on these surfaces was measured by a surface plasmon resonance (SPR) sensor at different temperatures. This work is aimed at addressing three important issues for protein–OEG interactions, i.e., (i) OEG stability, (ii) the correlation between OEG surface densities and surface non-fouling properties, and (iii) protein adsorption on OEG surfaces at different temperatures.     

在真核细胞中表达的幽门螺杆菌UreC,OipA抗原性的检测及其对细胞的作用

目的：研究幽门螺杆菌（Hp）UreC、OipA基因重组子转染胃上皮细胞后表达产物的抗原性及其对胃上皮细胞的作用，为两种毒力因子DNA疫苗的研制提供可行性依据及安全性指标。方法：用RT-PCR方法从国际标准株NCTC11637中获取UreC、OipA全长基因，克隆人pGEM-T Easy载体并测序，以重组T载体为模板，将两种基因的开放读码框架分别定向克隆入pcDNA3．1载体；获得的重组子转染SGC-7901细胞，筛选耐潮霉素的细胞克隆，用RT-PCR及Immuno-blot方法检测细胞内UreC、OipA蛋白的表达和抗原性；用荧光染色技术、MTT、流式细胞术分别检测UreC、OipA对细胞表型、增殖及凋亡的影响。结果：SoipA、SureC细胞（分别转染OipA、UreC重组子）表达相应的产物且具有抗原性。荧光显微镜下观察SoipA、SureC细胞未见明显形态学改变；用MTT法检测细胞增殖：SoipA、SureC细胞与SpcDNA3．1细胞（转染pcDNA3．1）比较，生长增殖无显著性差异（P＞0．05），流式细胞技术检测细胞凋亡结果：SoipA和SureC的凋亡率与SpcDNA3．1比较无显著性差异（P＞0．05）。结论：OipA、UreC在培养细胞内的表达产物具有抗原性且对细胞的功能无影响，可考虑用于制备DNA疫苗。     

Delivery of Viral Vectors to Tumor Cells: Extracellular Transport, Systemic Distribution, and Strategies for Improvement

It is a challenge to deliver therapeutic genes to tumor cells using viral vectors because (i) the size of these vectors are close to or larger than the space between fibers in extracellular matrix and (ii) viral proteins are potentially toxic in normal tissues. In general, gene delivery is hindered by various physiological barriers to virus transport from the site of injection to the nucleus of tumor cells and is limited by normal tissue tolerance of toxicity determined by local concentrations of transgene products and viral proteins. To illustrate the obstacles encountered in the delivery and yet limit the scope of discussion, this review focuses only on extracellular transport in solid tumors and distribution of viral vectors in normal organs after they are injected intravenously or intratumorally. This review also discusses current strategies for improving intratumoral transport and specificity of viral vectors.     

siRNA delivery from triblock copolymer micelles with spatially-ordered compartments of PEG shell,siRNA-loaded intermediate layer,and hydrophobic core

Hydrophobized block copolymers have widely been developed for construction of polymeric micelles for stable delivery of nucleic acids as well as anticancer drugs. Herein, we elaborated an A-B-C type of triblock copolymer featuring shell-forming A-segment, nucleic acid-loading B-segment, and stable core-forming C-segment, directed toward construction of a three-layered polymeric micelle as a small interfering RNA (siRNA) vehicle. The triblock copolymer was prepared with nonionic and hydrophilic poly(ethylene glycol) (PEG), cationic poly(l-lysine) (PLys), and poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} [PAsp(DET)] bearing a hydrophobic dimethoxy nitrobenzyl ester (DN) moiety in the side chain [PEG-PLys-PAsp(DET-DN)]. The resulting triblock copolymers spontaneously formed sub-100 nm-sized polymeric micelles with a hydrophobic PAsp(DET-DN) core as well as PEG shell in an aqueous solution. This micelle was able to incorporate siRNA into the intermediate PLys layer, associated with slightly reduced size and a narrow size distribution. The triblock copolymer micelles (TCMs) stably encapsulated siRNA in serum-containing medium, whereas randomly hydrophobized triblock copolymer [PEG-PLys(DN)-PAsp(DET-DN)] control micelles (RCMs) gradually released siRNA with time and non-PEGylated diblock copolymer [PLys-PAsp(DET-DN)] control micelles (DCMs) immediately formed large aggregates. The TCMs thus induced appreciably stronger sequence-specific gene silencing in cultured cancer cells, compared to those control micelles. The siRNA delivery with TCMs was further examined in terms of cellular uptake and intracellular trafficking. The flow cytometric analysis revealed that the cellular uptake of TCMs was more efficient than that of RCMs, but less efficient than that of DCMs. The intracellular trafficking study using confocal laser scanning microscopy combined with fluorescence resonance energy transfer (FRET) revealed that the TCMs could readily release the siRNA payload within cells, which was in contrast to the DCMs exhibiting much slower release profile. This result indicates that PEG shell contributed to the smooth release of siRNA from TCMs within the cells, presumably due to avoiding irreversible aggregate formation. The obtained results demonstrated that the design of separately functionalized polymer segments expanded the performance of polymeric micelles for successful siRNA delivery.     

Continuing cancer care delivery during the peak of COVID-19 in the Bronx,New York: experience from a public teaching hospital

IntroductionWe report our experience with cancer care delivery during the peak of COVID-19 pandemic in New York City.MethodsRetrospective analysis of the patients treated from the 1^st of March, 2020 to the 8^th of May, 2020.ResultsTeam huddles, infection screening and patient selection strategies were implemented. One hundred and seventy patients were treated in 576 visits. Six developed severe COVID-19 requiring hospitalization, two died. Their median Charlson Comorbidity Index was 9, higher than the rest of the cohort.ConclusionsCancer care delivery is safe and feasible using an approach focused on careful patient selection, team communication and infection control.     

Pentablock copolymers of poly(ethylene glycol), poly((2-dimethyl amino)ethyl methacrylate) and poly(2-hydroxyethyl methacrylate) from consecutive atom transfer radical polymerizations for non-viral gene delivery

Well-defined pentablock copolymers (PBPs) of P(HEMA)-b-P(DMAEMA)-b-PEG-b-P(DMAEMA)-b-P(HEMA) (in which PEG = poly(ethylene glycol), P(DMAEMA) = poly((2-dimethyl amino)ethyl methacrylate), and P(HEMA) = poly(2-hydroxyethyl methacrylate)), with different block lengths of P(DMAEMA), for non-viral gene delivery were prepared via consecutive atom transfer radical polymerizations (ATRPs) from the same di-2-bromoisobutyryl-terminated PEG (Br–PEG–Br) center block. The PBPs demonstrate good ability to condense plasmid DNA (pDNA) into 100–160 nm size nanoparticles with positive zeta potentials of 25–35 mV at PBPs/pDNA weight ratios of 5–25. The PBPs exhibit very low in vitro cytotoxicity and excellent gene transfection efficiency in HEK293 and COS7 cells. In particular, the transfection efficiencies of all the PBPs in HEK293 cells are comparable to, or higher than those of polyethylenimine (PEI, 25 kDa) at most weight ratios. The ability of the copolymers to condense plasmid DNA and the transfection efficiency of the resulting complexes are dependent on the chain length of P(DMAEMA) blocks. In addition to reducing the cytotoxicity and increasing the stability of the plasmid complexes, the PEG center block and the short P(HEMA) end blocks also help to enhance the gene transfection efficiency. Thus, the approach to well-defined block copolymers via ATRP provides a versatile means for tailoring the structure of non-viral gene vectors to meet the requirements of low cytotoxicity, good stability and high transfection capability for gene therapy applications.     

On the suitability of nanocrystalline ferrites as a magnetic carrier for drug delivery: functionalization, conjugation and drug release kinetics

Superparamagnetic nickel ferrite nanoparticles functionalized with polyvinyl alcohol, polyethylene oxide and polymethacrylic acid (PMAA) polymers and subsequently conjugated with doxorubicin anti-cancer drug are studied for their use as a magnetic carrier for drug delivery. Fourier transform infrared spectroscopy enabled examination of the ability of the nanoparticles to be functionalized with polymers and conjugated with doxorubicin drug. The functionalized polymer-coated nanocrystalline nickel ferrites retain the magnetic characteristics of non-functionalized nanocrystalline nickel ferrites (superparamagnetism, absence of hysteresis, remanence and coercivity at room temperature), encouraging their application as a magnetic carrier for drug delivery. The PMAA-coated nanoferrites are demonstrated as being a potentially superior magnetically targeted drug carrier based on FTIR results and drug release kinetics in the absence and presence of an external magnetic field.     

The past,present and future of service delivery in genetic counseling: Keeping up in the era of precision medicine

Precision medicine aims to approach disease treatment and prevention with consideration of the variability in genes, environment, and lifestyle for each person. This focus on the individual is also key to the practice of genetic counseling, whereby foundational professional values prioritize informed and autonomous patient decisions regarding their genetic health. Genetic counselors are ideally suited to help realize the goals of the precision medicine. However, a limited genetic counseling workforce at a time in which there is a rapidly growing need for services is challenging the balance of supply and demand. This article provides historical context to better understand what has informed traditional models of genetic counseling and considers some of the current forces that require genetic counselors to adapt their practice. New service delivery models can improve access to genetic healthcare by overcoming geographical barriers, allowing genetic counselors to see a higher volume of patients and supporting other healthcare providers to better provide genetic services to meet the needs of their patients. Approaches to genetic counseling service delivery are considered with a forward focus to the challenges and opportunities that lie ahead for genetic counselors in this age of precision health.     

Self-aggregated nanoparticles of linoleic acid-modified glycol chitosan conjugate as delivery vehicles for paclitaxel: preparation,characterization and evaluation

A series of linoleic acid-modified glycol chitosan (LAGC) conjugates were synthesized and characterized by FTIR and ¹H NMR. The effect of the amount of linoleic acid (LA) on the physicochemical properties of LAGC conjugates was investigated. The mean diameters of three LAGC nanoparticles determined by dynamic light scattering ranged from 204 to 289 nm. The critical aggregation concentration values of LAGC conjugates in aqueous solution were 0.0148, 0.0348, and 0.0807 mg/ml, respectively. Paclitaxel (PTX) was physically loaded into the LAGC nanoparticles by a dialysis method. The drug loading content and encapsulation efficiency of PTX-loaded LAGC (PTX-LAGC) nanoparticles increased with an increasing ratio of the hydrophobic LA to hydrophilic glycol chitosan in the conjugates. PTX-LAGC nanoparticles were almost spherical in shape observed by transmission electron microscopy. In vitro release revealed that PTX release from the nanoparticles was reduced as the LA substitution degree of LAGC conjugates increased. Compared with the commercial formulation Taxol, PTX-LAGC-1 nanoparticles exhibited comparable cellular uptake and cytotoxicity against HepG2 cells in vitro. Importantly, PTX-LAGC-1 nanoparticles demonstrated the stronger antitumor efficacy against hepatic H22 tumor-bearing mice than Taxol (p < 0.05). Therefore, glycolipid-like LAGC nanoparticles had a potential as delivery vehicles for tumor therapy.     

Chloroplast DNA from lettuce and Barnadesia (Asteraceae): structure,gene localization,and characterization of a large inversion

Summary We have cloned into plasmids 17 of 18 lettuce chloroplast DNA SacI fragments covering 96% of the genome. The cloned fragments were used to construct cleavage maps for 10 restriction enzymes for the chloroplast genomes of lettuce (Lactuca sativa) and Barnadesia caryophylla, two distantly related species in the sunflower family (Asteraceae). Both genomes are approximately 151 kb in size and contain a 25 kb inverted repeat. We also mapped the position and orientation of 37 chloroplast DNA genes. The mapping studies reveal that chloroplast DNAs of lettuce and Barnadesia differ by a 22 kb inversion in the large single copy region. Barnadesia has retained the primitive land plant genome arrangement, while the inversion has occurred in a lettuce lineage. The endpoints of the derived lettuce inversion were located by comparison to the well-characterized spinach and tobacco genomes. Both endpoints are located in intergenic spacers within tRNA gene clusters; one cluster being located downstream from the atpA gene and the other upstream from the psbD gene. The endpoint near the atpA gene is very close to one endpoint of a 20 kb inversion in wheat (Howe et al. 1983; Quigley and Weil 1985). Comparison of the restriction site maps gives an estimated sequence divergence of 3.7% for the lettuce and Barnadesia genomes. This value is relatively low compared to previous estimates for other angiosperm groups, suggesting a high degree of sequence conservation in the Asteraceae.     

Adaptational phenomena and mechanical responses during running: effect of surface, aging and task experience

The goals of the study were to identify adaptational phenomena in running mechanics over a variety of surfaces due to age related changes in the muscle-tendon units (MTUs) capacities, to examine whether running experience is associated with adaptational effects on running mechanics over a variety of surfaces even at old age, and to investigate whether surface condition affects running mechanics. The investigation was executed on 30 old and 19 young including 29 runners and 20 non-active subjects. In a previous study we documented that the older had lower MTUs capacities. In the present study running mechanics were analysed as the same subjects ran at 2.7 m/s over three surfaces having different compliance. Surface condition did not affect centre of mass trajectory, duty factor or joint kinetics (P > 0.01). Older react to the reduced MTUs capacity by increasing duty factor and benefiting from a mechanical advantage for the triceps surae MTU and a lower rate of force generation on all surfaces (P < 0.01). Runners displayed lower average horizontal forces and a higher mechanical advantage for the quadriceps femoris MTU for all surfaces (P < 0.01). The results provided strong evidence on that running strategy remained essentially unchanged over a variety of surfaces. Adaptive improvements in running mechanics due to task experience were present for all surfaces and did not depend on age. We further concluded that older adults were able to recalibrate their running strategy to adjust the task effort to the reduced MTUs capacities in a feedforward control manner for a variety of mechanical environments.