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The Journal of Biological Chemistry Apr 1996We have developed a lipidic gene transfer vector, LPDII, where DNA was first complexed to polylysine at a ratio of 1:0.75 (w/w) and then entrapped into folate-targeted...
We have developed a lipidic gene transfer vector, LPDII, where DNA was first complexed to polylysine at a ratio of 1:0.75 (w/w) and then entrapped into folate-targeted pH-sensitive anionic liposomes composed of dioleoyl phosphatidylethanolamine (DOPE)/cholesteryl hemisuccinate/folate-polyethlene glycol-DOPE (6:4:0.01 mol/mol) via charge interaction. LPDII transfection of KB cells, a cell line overexpressing the tumor marker folate receptor, was affected by both the lipid to DNA ratio and the lipid composition. At low lipid to DNA ratios (e.g. 4 and 6), LPDII particles were positively charged; transfection and cellular uptake levels were independent of the folate receptor and did not require a pH-sensitive lipid composition. Meanwhile, transfection and uptake of negatively charged LPDII particles, i.e. those with high lipid to DNA ratios (e.g. 10 and 12), were folate receptor-dependent and required a pH-sensitive lipid composition. The transfection activity of LPDII was lost when the inverted cone-shaped DOPE was replaced by dioleoyl phosphatidylcholine. LPDII particles with lipid to DNA ratios of 4, 6, 10, and 12 were approximately 20-30 times more active than DNA.3-beta-[N-(N',N'-dimethylethane)carbamoyl]cholesterol cationic liposome complexes in KB cells and were much less cytotoxic. On the sucrose gradient, LPDII particles had a migration rate in between those of the free DNA and the DNA.polylysine complex. An electron micrograph of LPDII showed a structure of spherical particles with a positively stained core enclosed in a lipidic envelope with a mean diameter of 74 +/- 14 nm. This novel gene transfer vector may potentially be useful in gene therapy for tumor-specific delivery.
Topics: Anions; Biomarkers, Tumor; DNA; Folic Acid; Gene Transfer Techniques; Genetic Vectors; Humans; Liposomes; Macromolecular Substances; Polylysine; Structure-Activity Relationship; Transfection; Tumor Cells, Cultured
PubMed: 8626549
DOI: 10.1074/jbc.271.14.8481 -
PloS One 2011Ras protein, as one of intracellular signal switches, plays various roles in several cell activities such as differentiation and proliferation. There is considerable...
BACKGROUND
Ras protein, as one of intracellular signal switches, plays various roles in several cell activities such as differentiation and proliferation. There is considerable evidence showing that calmodulin (CaM) binds to K-RasB and dissociates K-RasB from membrane and that the inactivation of CaM is able to induce K-RasB activation. However, the mechanism for the interaction of CaM with K-RasB is not well understood.
METHODOLOGY/PRINCIPAL FINDINGS
Here, by applying fluorescence spectroscopy and isothermal titration calorimetry, we have obtained thermodynamic parameters for the interaction between these two proteins and identified the important elements of K-RasB for its interaction with Ca(2+)/CaM. One K-RasB molecule interacts with one CaM molecule in a GTP dependent manner with moderate, micromolar affinity at physiological pH and physiologic ionic strength. Mutation in the polybasic domain of K-Ras decreases the binding affinity. By using a chimera in which the C-terminal polylysine region of K-RasB has been replaced with that of H-Ras and vice versa, we find that at physiological pH, H-Ras-(KKKKKK) and Ca(2+)/CaM formed a 1:1 complex with an equilibrium association constant around 10(5) M(-1), whereas no binding reaction of K-RasB-(DESGPC) with Ca(2+)/CaM is detected. Furthermore, the interaction of K-RasB with Ca(2+)/CaM is found to be enhanced by the farnesylation of K-RasB.
CONCLUSIONS/SIGNIFICANCE
We demonstrate that the polylysine region of K-RasB not only contributes importantly to the interaction of K-RasB with Ca(2+)/CaM, but also defines its isoform specific interaction with Ca(2+)/CaM. The farnesylation of K-RasB is also important for its specific interaction with Ca(2+)/CaM. Information obtained here can enhance our understanding of how CaM interacts with K-RasB in physiological environments.
Topics: Algorithms; Amino Acid Sequence; Animals; Blotting, Western; Calcium; Calmodulin; Calorimetry; Cattle; Escherichia coli; Kinetics; Light; Models, Chemical; Polylysine; Prenylation; Protein Binding; Proto-Oncogene Proteins p21(ras); Recombinant Proteins; Spectrometry, Fluorescence; Thermodynamics
PubMed: 21750741
DOI: 10.1371/journal.pone.0021929 -
Nanomedicine (London, England) Aug 2009Treatment of sensorineural hearing loss could be advanced using novel drug carriers such as hyperbranched polylysine (HBPL) or lipid nanocapsules (LNCs). This study...
AIM
Treatment of sensorineural hearing loss could be advanced using novel drug carriers such as hyperbranched polylysine (HBPL) or lipid nanocapsules (LNCs). This study examined HBPL and LNCs for their cellular uptake and possible toxicity in vitro and in vivo as the first step in developing novel nanosized multifunctional carriers.
METHOD
Having incubated HBPL and LNCs with fibroblasts, nanoparticle uptake and cell viability were determined by confocal laser scanning microscopy, fluorescence measurements and neutral red staining. In vivo, electrophysiology, confocal laser scanning microscopy and cytocochleograms were performed for nanoparticle detection and also toxicity studies after intracochlear application.
RESULTS
Both nanoparticles were detectable in the fibroblasts' cytoplasm without causing cytotoxic effects. After in vivo application they were visualized in cochlear cells, which did not lead to a change in hearing threshold or loss of hair cells. Biocompatibility and traceability were demonstrated for HBPL and LNCs. Thus, they comply with the basic requirements for drug carriers for potential application in the inner ear.
Topics: Animals; Cochlea; Drug Carriers; Ear, Inner; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Guinea Pigs; Hair Cells, Auditory; Hearing Loss, Sensorineural; Indoles; Lipids; Nanocapsules; Phalloidine; Polylysine; Rhodamines
PubMed: 19663591
DOI: 10.2217/nnm.09.41 -
International Journal of Biological... May 2024Alginate-based dressings have been shown to promote wound healing, leveraging the unique properties of alginate. This work aimed to develop and characterize flexible...
Alginate-based dressings have been shown to promote wound healing, leveraging the unique properties of alginate. This work aimed to develop and characterize flexible individual and bilayered films to deliver bacteriophages (phages) and ε-Poly-l-lysine (ε-PLL). Films varied in different properties. The moisture content, swelling and solubility increased with higher alginate concentrations. The water vapour permeability, crucial in biomedical films to balance moisture levels for effective wound healing, reached optimal levels in bilayer films, indicating these will be able to sustain an ideal moist environment. The bilayer films showed improved ductility (lower tensile strength and increased elongation at break) compared to individual films. The incorporated phages maintained viability for 12 weeks under vacuum and refrigerated conditions, and their release was sustained and gradual. Antibacterial immersion tests showed that films with phages and ε-PLL significantly inhibited Pseudomonas aeruginosa PAO1 growth (>3.1 Log CFU/cm). Particle release was influenced by the swelling degree and diffusional processes within the polymer network, providing insights into controlled release mechanisms for particles of varying size (50 nm to 6 μm) and charge. The films developed, demonstrated modulated release capabilities for active agents, and may show potential as controlled delivery systems for phages and wound healing adjuvants.
Topics: Polylysine; Pseudomonas aeruginosa; Bacteriophages; Wound Healing; Anti-Bacterial Agents; Alginates; Bandages; Steam; Permeability; Anti-Infective Agents
PubMed: 38688343
DOI: 10.1016/j.ijbiomac.2024.131963 -
Activation of toll-like receptor signaling pathway for protection against influenza virus infection.Vaccine May 2009This study aims to evaluate the antiviral role of nucleic acid-based agonists for the activation of toll-like receptor (TLR) signaling pathways, and its protective role...
This study aims to evaluate the antiviral role of nucleic acid-based agonists for the activation of toll-like receptor (TLR) signaling pathways, and its protective role in respiratory influenza A virus infections. TLR-3 is expressed on myeloid dendritic cells, respiratory epithelium, and macrophages, and appears to play a central role in mediating both the antiviral and inflammatory responses of the innate immunity in combating viral infections. Influenza viruses can effectively inhibit the host's ability to produce interferons, and thereby suppress the immune system's antiviral defence mechanisms. Poly ICLC is a synthetic double stranded RNA comprising of polyriboinosinic-poly ribocytidylic acid (Poly IC) stabilized with l-lysine (L) and carboxymethylcellulose (C). Poly ICLC and liposome-encapsulated Poly ICLC (LE Poly ICLC) are TLR-3 agonists and are potent inducer of interferons and natural killer cells. Intranasal pre-treatment of mice with Poly ICLC and LE Poly ICLC provided high level of protection against lethal challenge with a highly lethal avian H5N1 influenza (HPAI) strain (A/H5N1/chicken/Henan clade 2), and against lethal seasonal influenza A/PR/8/34 [H1N1] and A/Aichi/2 [H3N2] virus strains. The duration of protective antiviral immunity to multiple lethal doses of influenza virus A/PR/8/34 virus had been previously found to persist for up to 3 weeks in mice for LE Poly ICLC and 2 weeks for Poly ICLC. Similarly, pre-treatment of mice with CpG oligonucleotides (TLR-9 agonist) was also found to provide complete protection against influenza A/PR/8/34 infection in mice. RT-PCR analysis of lung tissues of mice treated with Poly ICLC and LE Poly ICLC revealed upregulation of TLR-3 mRNAs gene expression. Taken together, these results do support the potential role of TLR-3 and TLR-9 agonists such as Poly ICLC and LE Poly ICLC in protection against lethal seasonal and HPAI virus infection.
Topics: Animals; Antiviral Agents; Carboxymethylcellulose Sodium; Female; Influenza A Virus, H1N1 Subtype; Influenza A Virus, H3N2 Subtype; Influenza A Virus, H5N1 Subtype; Influenza A virus; Liposomes; Mice; Mice, Inbred BALB C; Oligodeoxyribonucleotides; Orthomyxoviridae Infections; Poly I-C; Polylysine; RNA, Messenger; Signal Transduction; Toll-Like Receptor 3
PubMed: 19200852
DOI: 10.1016/j.vaccine.2009.01.048 -
Cells, Tissues, Organs 2023Human mesenchymal stromal cells (hMSCs) are multipotent cells that have been proposed for the treatment of immune-mediated diseases. Culturing hMSCs on tissue culture...
Human mesenchymal stromal cells (hMSCs) are multipotent cells that have been proposed for the treatment of immune-mediated diseases. Culturing hMSCs on tissue culture plastic reduces their therapeutic potential in part due to the lack of extracellular matrix components. The aim of this study is to evaluate multilayers of heparin and poly(L-lysine) (HEP/PLL) as a bioactive surface for hMSCs stimulated with soluble interferon gamma (IFN-γ). Multilayers were formed, via layer-by-layer assembly, with HEP as the final layer and supplemented with IFN-γ in the culture medium. Multilayer construction and chemistry were confirmed using Azure A staining, quartz crystal microbalance, and X-ray photoelectron spectroscopy. hMSCs adhesion, viability, and differentiation, were assessed. Results showed that (HEP/PLL) multilayer coatings were poorly adhesive for hMSCs. However, performing chemical crosslinking using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide significantly enhanced hMSCs adhesion and viability. The immunosuppressive properties of hMSCs cultured on crosslinked (HEP/PLL) multilayers were confirmed by measuring indoleamine 2,3-dioxygenase activity. Lastly, hMSCs cultured on crosslinked (HEP/PLL) multilayers in the presence of soluble IFN- γ successfully differentiated towards the osteogenic and adipogenic lineages as confirmed by Alizarin red, and oil-red O staining, as well as alkaline phosphatase activity. This study suggests that crosslinked (HEP/PLL) films can modulate hMSCs response to soluble factors, which may improve hMSCs-based therapies aimed at treating several immune diseases.
Topics: Humans; Heparin; Polylysine; Interferon-gamma; Osteogenesis; Mesenchymal Stem Cells; Cell Differentiation
PubMed: 34937023
DOI: 10.1159/000521609 -
Biomacromolecules Sep 2016Endothelial cells (ECs) play a crucial role in regulating various physiological and pathological processes. The behavior of ECs is modulated by physical (e.g., substrate...
Endothelial cells (ECs) play a crucial role in regulating various physiological and pathological processes. The behavior of ECs is modulated by physical (e.g., substrate stiffness) and biochemical cues (e.g., growth factors). However, the synergistic influence of these cues on EC behavior has rarely been investigated. In this study, we constructed poly(l-lysine)/hyaluronan (PLL/HA) multilayer films with different stiffness and exposed ECs to these substrates with and without hepatocyte growth factor (HGF)-supplemented culture medium. We demonstrated that EC adhesion, migration, and proliferation were positively correlated with substrate stiffness and that these behaviors were further promoted by HGF. Interestingly, ECs on the lower stiffness substrates showed stronger responses to HGF in terms of migration and proliferation, suggesting that HGF can profoundly influence stiffness-dependent EC behavior correlated with EC growth. After the formation of an EC monolayer, EC behaviors correlated with endothelial function were evaluated by characterizing monolayer integrity, nitric oxide production, and gene expression of endothelial nitric oxide synthase. For the first time, we demonstrated that endothelial function displayed a negative correlation with substrate stiffness. Although HGF improved endothelial function, HGF was not able to change the stiffness-dependent manner of endothelial functions. Taken together, this study provides insights into the synergetic influence of physical and biochemical cues on EC behavior and offers great potential in the development of optimized biomaterials for EC-based regenerative medicine.
Topics: Cell Adhesion; Cell Proliferation; Cells, Cultured; Endothelium, Vascular; Hardness; Hepatocyte Growth Factor; Humans; Hyaluronic Acid; Polylysine; Surface Properties
PubMed: 27428305
DOI: 10.1021/acs.biomac.6b00318 -
Journal of Nanobiotechnology May 2018The delivery of plasmonic particles, such as gold nanorods, to the tumor microenvironment has attracted much interest in biomedical optics for topical applications as...
BACKGROUND
The delivery of plasmonic particles, such as gold nanorods, to the tumor microenvironment has attracted much interest in biomedical optics for topical applications as the photoacoustic imaging and photothermal ablation of cancer. However, the systemic injection of free particles still crashes into a complexity of biological barriers, such as the reticuloendothelial system, that prevent their efficient biodistribution. In this context, the notion to exploit the inherent features of tumor-tropic cells for the creation of a Trojan horse is emerging as a plausible alternative.
RESULTS
We report on a convenient approach to load cationic gold nanorods into murine macrophages that exhibit chemotactic sensitivity to track gradients of inflammatory stimuli. In particular, we compare a new model of poly-L-lysine-coated particles against two alternatives of cationic moieties that we have presented elsewhere, i.e. a small quaternary ammonium compound and an arginine-rich cell-penetrating peptide. Murine macrophages that are exposed to poly-L-lysine-coated gold nanorods at a dosage of 400 µM Au for 24 h undertake efficient uptake, i.e. around 3 pg Au per cell, retain the majority of their cargo until 24 h post-treatment and maintain around 90% of their pristine viability, chemotactic and pro-inflammatory functions.
CONCLUSIONS
With respect to previous models of cationic coatings, poly-L-lysine is a competitive solution for the preparation of biological vehicles of gold nanorods, especially for applications that may require longer life span of the Trojan horse, say in the order of 24 h. This biopolymer combines the cost-effectiveness of small molecules and biocompatibility and efficiency of natural peptides and thus holds potential for translational developments.
Topics: Animals; Cell Movement; Cell Survival; Cytokines; Gold; Macrophages; Mice; Nanotubes; Polylysine
PubMed: 29855304
DOI: 10.1186/s12951-018-0377-7 -
International Journal of Nanomedicine 2012It has been proposed in the literature that Fe(3)O(4) magnetic nanoparticles (MNPs) could be exploited to enhance or accelerate nerve regeneration and to provide...
PURPOSE
It has been proposed in the literature that Fe(3)O(4) magnetic nanoparticles (MNPs) could be exploited to enhance or accelerate nerve regeneration and to provide guidance for regenerating axons. MNPs could create mechanical tension that stimulates the growth and elongation of axons. Particles suitable for this purpose should possess (1) high saturation magnetization, (2) a negligible cytotoxic profile, and (3) a high capacity to magnetize mammalian cells. Unfortunately, the materials currently available on the market do not satisfy these criteria; therefore, this work attempts to overcome these deficiencies.
METHODS
Magnetite particles were synthesized by an oxidative hydrolysis method and characterized based on their external morphology and size distribution (high-resolution transmission electron microscopy [HR-TEM]) as well as their colloidal (Z potential) and magnetic properties (Superconducting QUantum Interference Devices [SQUID]). Cell viability was assessed via Trypan blue dye exclusion assay, cell doubling time, and MTT cell proliferation assay and reactive oxygen species production. Particle uptake was monitored via Prussian blue staining, intracellular iron content quantification via a ferrozine-based assay, and direct visualization by dual-beam (focused ion beam/scanning electron microscopy [FIB/SEM]) analysis. Experiments were performed on human neuroblastoma SH-SY5Y cell line and primary Schwann cell cultures of the peripheral nervous system.
RESULTS
This paper reports on the synthesis and characterization of polymer-coated magnetic Fe(3)O(4) nanoparticles with an average diameter of 73 ± 6 nm that are designed as magnetic actuators for neural guidance. The cells were able to incorporate quantities of iron up to 2 pg/cell. The intracellular distribution of MNPs obtained by optical and electronic microscopy showed large structures of MNPs crossing the cell membrane into the cytoplasm, thus rendering them suitable for magnetic manipulation by external magnetic fields. Specifically, migration experiments under external magnetic fields confirmed that these MNPs can effectively actuate the cells, thus inducing measurable migration towards predefined directions more effectively than commercial nanoparticles (fluidMAG-ARA supplied by Chemicell). There were no observable toxic effects from MNPs on cell viability for working concentrations of 10 μg/mL (EC(25) of 20.8 μg/mL, compared to 12 μg/mL in fluidMAG-ARA). Cell proliferation assays performed with primary cell cultures of the peripheral nervous system confirmed moderate cytotoxicity (EC(25) of 10.35 μg/mL).
CONCLUSION
These results indicate that loading neural cells with the proposed MNPs is likely to be an effective strategy for promoting non-invasive neural regeneration through cell magnetic actuation.
Topics: Analysis of Variance; Animals; Cell Line, Tumor; Cell Membrane; Cell Movement; Cell Survival; Cells, Cultured; Humans; Iron; Magnetite Nanoparticles; Neuroblastoma; Neurons; Particle Size; Polylysine; Rats; Schwann Cells; Static Electricity
PubMed: 22811603
DOI: 10.2147/IJN.S28460 -
Journal of Visualized Experiments : JoVE Aug 2013While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors, their clinical efficacy remains to be established. The...
While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors, their clinical efficacy remains to be established. The method described below, prepared in accordance with Good Manufacturing Process (GMP) guidelines, is an optimization of the most common ex vivo preparation method for generating large numbers of DCs for clinical studies. Our method utilizes the synthetic TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysine Carboxymethylcellulose (Poly-ICLC) to stimulate the DCs. Our previous study established that Poly-ICLC is the most potent individual maturation stimulus for human DCs as assessed by an upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukmin 1 (IL-1), and type I interferons (IFN), and minimal interleukin 10 (IL-10) production. DCs are differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis. PBMCs are isolated by Ficoll gradient centrifugation and frozen in aliquots. On Day 1, PBMCs are thawed and plated onto tissue culture flasks to select for monocytes which adhere to the plastic surface after 1-2 hr incubation at 37 °C in the tissue culture incubator. After incubation, the lymphocytes are washed off and the adherent monocytes are cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage-colony stimulating factor (GM-CSF) to differentiate to immature DCs. On Day 6, immature DCs are pulsed with the keyhole limpet hemocyanin (KLH) protein which serves as a control for the quality of the vaccine and may boost the immunogenicity of the vaccine. The DCs are stimulated to mature, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed, and frozen in 1 ml aliquots containing 4-20 x 10(6) cells using a controlled-rate freezer. Lot release testing for the batches of DCs is performed and must meet minimum specifications before they are injected into patients.
Topics: Antigens, Neoplasm; Carboxymethylcellulose Sodium; Dendritic Cells; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunotherapy, Adoptive; Interleukin-4; Leukocytes, Mononuclear; Poly I-C; Polylysine
PubMed: 23928481
DOI: 10.3791/50085