ABSTRACT
The surface properties of nanoparticles (NPs) are a major factor that influences how these nanomaterials interact with biological systems. Interactions between NPs and macrophages of the reticuloendothelial system (RES) can reduce the efficacy of NP diagnostics and therapeutics. Traditionally, to limit NP clearance by the RES system, the NP surface is neutralized with molecules like poly(ethylene glycol) (PEG) which are known to resist protein adsorption and RES clearance. Unfortunately, PEG modification is not without drawbacks including difficulties with the synthesis and associations with immune reactions. To overcome some of these obstacles, we neutralized the NP surface by acetylation and compared this modification to PEGylation for RES clearance and tumor-specific targeting. We found that acetylation was comparable to PEGylation in reducing RES clearance. Additionally, we found that dendrimer acetylation did not impact folic acid (FA)-mediated targeting of tumor cells whereas PEG surface modification reduced the targeting ability of the NP. These results clarify the impact of different NP surface modifications on RES clearance and cell-specific targeting and provide insights into the design of more effective NPs.
Subject(s)
Folic Acid/pharmacology , Macrophages/chemistry , Nanoparticles/chemistry , Animals , Cell Survival/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Folic Acid/chemistry , Humans , KB Cells , Mice , Molecular Structure , Polyethylene Glycols/chemical synthesis , Polyethylene Glycols/chemistry , RAW 264.7 Cells , Structure-Activity Relationship , Surface PropertiesABSTRACT
Despite the fact that nucleotides and adenosine help regulate vascular tone through purinergic signaling pathways, little is known regarding their contributions to the pathobiology of pulmonary arterial hypertension, a condition characterized by elevated pulmonary vascular resistance and remodeling. Even less is known about the potential role that alterations in CD39 (ENTPD1), the ectonucleotidase responsible for the conversion of the nucleotides ATP and ADP to AMP, may play in pulmonary arterial hypertension. In this study we identified decreased CD39 expression on the pulmonary endothelium of patients with idiopathic pulmonary arterial hypertension. We next determined the effects of CD39 gene deletion in mice exposed to normoxia or normobaric hypoxia (10% oxygen). Compared with controls, hypoxic CD39(-/-) mice were found to have a markedly elevated ATP-to-adenosine ratio, higher pulmonary arterial pressures, more right ventricular hypertrophy, more arterial medial hypertrophy, and a pro-thrombotic phenotype. In addition, hypoxic CD39(-/-) mice exhibited a marked increase in lung P2X1 receptors. Systemic reconstitution of ATPase and ADPase enzymatic activities through continuous administration of apyrase decreased pulmonary arterial pressures in hypoxic CD39(-/-) mice to levels found in hypoxic CD39(+/+) controls. Treatment with NF279, a potent and selective P2X1 receptor antagonist, lowered pulmonary arterial pressures even further. Our study is the first to implicate decreased CD39 and resultant alterations in circulating purinergic signaling ligands and cognate receptors in the pathobiology of pulmonary arterial hypertension. Reconstitution and receptor blocking experiments suggest that phosphohydrolysis of purinergic nucleotide tri- and diphosphates, or blocking of the P2X1 receptor could serve as treatment for pulmonary arterial hypertension.
Subject(s)
Antigens, CD/metabolism , Apyrase/metabolism , Hypertension, Pulmonary/metabolism , Lung/metabolism , Pulmonary Artery/metabolism , Receptors, Purinergic P2X1/metabolism , Adenosine/metabolism , Adenosine Triphosphate/metabolism , Animals , Antigens, CD/genetics , Antihypertensive Agents/pharmacology , Apyrase/deficiency , Apyrase/genetics , Apyrase/pharmacology , Arterial Pressure , Disease Models, Animal , Genetic Predisposition to Disease , Humans , Hydrolysis , Hypertension, Pulmonary/drug therapy , Hypertension, Pulmonary/genetics , Hypertension, Pulmonary/physiopathology , Hypertrophy, Right Ventricular/genetics , Hypertrophy, Right Ventricular/metabolism , Hypertrophy, Right Ventricular/physiopathology , Hypoxia/complications , Lung/drug effects , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Pulmonary Artery/drug effects , Pulmonary Artery/physiopathology , Purinergic P2X Receptor Antagonists/pharmacology , Receptors, Purinergic P2X1/drug effects , Severity of Illness Index , Signal Transduction , Suramin/analogs & derivatives , Suramin/pharmacology , Vascular Remodeling , Ventricular RemodelingABSTRACT
Neutral generation 3 poly(amidoamine) dendrimers were labeled with Oregon Green 488 (G3-OGn) to obtain materials with controlled fluorophore:dendrimer ratios (n = 1-2), a mixture containing mostly 3 dyes per dendrimer, a mixture containing primarily 4 or more dyes per dendrimer (n = 4+), and a stochastic mixture (n = 4avg). The UV absorbance of the dye conjugates increased linearly as n increased and the fluorescence emission decreased linearly as n increased. Cellular uptake was studied in RAW cells and HEK 293A cells as a function of the fluorophore:dendrimer ratio (n). The cellular uptake of G3-OG n (n = 3, 4+, 4avg) into RAW cells was significantly lower than G3-OG n (n = 1, 2). The uptake of G3-OG n (n = 3, 4+, 4avg) into HEK 293A cells was not significantly different from G3-OG1. Thus, the fluorophore:dendrimer ratio was observed to change the extent of uptake in the macrophage uptake mechanism but not in the HEK 293A cell. This difference in endocytosis indicates the presence of a pathway in the macrophage that is sensitive to hydrophobicity of the particle.
ABSTRACT
BACKGROUND: Distinct monocyte subsets predict cardiovascular risk and contribute to heart failure progression in murine models, but they have not been examined in clinical acute decompensated heart failure (ADHF). METHODS AND RESULTS: Blood samples were obtained from 11 healthy control subjects (HCs) and at admission and discharge from 19 ADHF patients. Serologic markers of inflammation were assessed at admission and discharge. Monocyte populations were defined with the use of flow cytometry for cell-surface expression of CD14 and CD16: CD14++CD16- (classic), CD14++CD16+ (intermediate), and CD14+CD16++ (nonclassic). In ADHF patients, C-reactive protein (CRP) and interleukin-6 (IL-6) were higher compared with HCs (both P < .001) and decreased from admission to discharge (CRP: 12.1 ± 10.1 to 8.6 ± 8.4 mg/L [P = .005]; IL-6: 19.8 ± 34.5 to 7.1 ± 4.7 pg/mL [P = .08]). In ADHF patients, the admission proportion of CD14++CD16- monocytes was lower (68% vs 85%; P < .001) and that of CD14++CD16+ (15% vs 8%; P = .002) and CD14+CD16++ (17% vs 7%, P = .07) monocytes higher compared with HCs. Additionally, the proportion of CD14++CD16- monocytes increased (68% to 79%, P = .04) and the CD14+CD16++ monocytes decreased (17% to 7%, P = .049) between admission and discharge. CONCLUSIONS: Following standard treatment of ADHF, the monocyte profile and circulating inflammatory markers shifts to more closely resemble those of HC, suggesting a resolution of the acute inflammatory state. Functional studies are warranted to understand how specific monocyte subsets and systemic inflammation may contribute to ADHF pathophysiology.
Subject(s)
Cytokines/blood , Heart Failure/blood , Heart Failure/immunology , Monocytes/immunology , Acute Disease , Aged , Biomarkers/blood , Cytokines/immunology , Female , Humans , Inflammation/blood , Inflammation/immunology , Male , Middle AgedABSTRACT
Histone deacetylase inhibitors (HDACi) are promising therapeutics for cancer. HDACi alter the epigenetic state of tumors and provide a unique approach to treat cancer. Although studies with HDACi have shown promise in some cancers, variable efficacy and off-target effects have limited their use. To overcome some of the challenges of traditional HDACi, we sought to use a tumor-specific dendrimer scaffold to deliver HDACi directly to cancer cells. Here we report the design and evaluation of tumor-specific dendrimer-HDACi conjugates. The HDACi was conjugated to the dendrimer using an ester linkage through its hydroxamic acid group, inactivating the HDACi until it is released from the dendrimer. Using a cancer cell model, we demonstrate the functionality of the tumor-specific dendrimer-HDACi conjugates. Furthermore, we demonstrate that unlike traditional HDACi, dendrimer-HDACi conjugates do not affect tumor-associated macrophages, a recently recognized mechanism through which drug resistance emerges. We anticipate that this new class of cell-specific epigenetic therapeutics will have tremendous potential in the treatment of cancer.
ABSTRACT
Generation 5 poly(amidoamine) (G5 PAMAM) methotrexate (MTX) conjugates employing two small molecular linkers, G5-(COG-MTX)n, G5-(MFCO-MTX)n were prepared along with the conjugates of the G5-G5 (D) dimer, D-(COG-MTX)n, D-(MFCO-MTX)n. The monomer G5-(COG-MTX)n conjugates exhibited only a weak, rapidly reversible binding to folate binding protein (FBP) consistent with monovalent MTX binding. The D-(COG-MTX)n conjugates exhibited a slow onset, tight-binding mechanism in which the MTX first binds to the FBP, inducing protein structural rearrangement, followed by polymer-protein van der Waals interactions leading to tight-binding. The extent of irreversible binding is dependent on total MTX concentration and no evidence of multivalent MTX binding was observed.
Subject(s)
DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , Dendrimers/chemistry , Dendrimers/metabolism , Methotrexate/chemistry , Polyamines/chemistry , Calorimetry , Humans , Methotrexate/metabolism , Nuclear Magnetic Resonance, Biomolecular , Polyamines/metabolism , RNA-Binding Proteins , Surface Plasmon ResonanceABSTRACT
BACKGROUND: Although cytosolic DNA degradation plays an important role in decreasing transgene expression, the plasmid degradation pattern remains largely unexplored. METHODS: Illumina dye sequencing was employed to provide degradation site information for S1 and cytosolic nucleases. S1 nuclease provided a positive control for a comparison between the agarose gel method and sequencing approaches. RESULTS: The poly(A) region between the ß-lactamase gene and the cytomegalovirus (CMV) promoter was identified as the most likely cut site for polyplex-treated cytosol. The second most likely site, at the 5' end of the ß-lactamase gene, was identified by gel electrophoresis and sequencing. Additional sites were detected in the OriC region, the SV40/poly(A) region, the luciferase gene and the CMV promoter. Sequence analysis of plasmid treated with cytosol from control cells showed the greatest cut activity in the OriC region, the ß-lactamase gene and the poly(A) region following the luciferase gene. Additional regions of cut activity include the SV40 promoter and the ß-lactamase poly(A) termination sequence. Both cytosolic nucleases and the S1 nuclease showed substantial activity at the bacterial origin of replication (OriC). CONCLUSIONS: High-throughput plasmid sequencing revealed regions of the luciferase plasmid DNA sequence that are sensitive to cytosolic nuclease degradation. This provides new targets for improving plasmid and/or polymer design to optimize the likelihood of protein expression.
Subject(s)
DNA/metabolism , Gene Transfer Techniques , High-Throughput Nucleotide Sequencing/methods , Plasmids/metabolism , Cytomegalovirus/genetics , Electrophoresis, Agar Gel , Endonucleases/genetics , Endonucleases/metabolism , Genetic Therapy , Genetic Vectors , Luciferases/genetics , Luciferases/metabolism , Origin Recognition Complex/genetics , Poly(A)-Binding Proteins/genetics , Poly(A)-Binding Proteins/metabolism , Promoter Regions, Genetic , Sequence Analysis, DNA , Transgenes , beta-Lactamases/geneticsABSTRACT
Cytosolic nucleases have been proposed to play an important role in limiting the effectiveness of polyplex-based gene delivery agents. In order to explore the effect of cell membrane disruption on nuclease activation, nuclease activity upon polyplex uptake and localization, and nuclease activity upon gene expression, we employed an oligonucleotide molecular beacon (MB). The MB was incorporated as an integral part of the polymer/DNA polyplex, and two-color flow cytometry experiments were performed to explore the relationship of MB cleavage with propidium iodide (PI) uptake, protein expression, and polyplex uptake. In addition, confocal fluorescence microcopy was performed to examine both polyplex and cleaved MB localization. The impact of cell membrane disruption was also probed using whole-cell patch clamp measurement of the plasma membrane's electrical conductance. Differential activation of cytosolic nuclease was observed with substantial activity for B-PEI and G5 PAMAM dendrimer (G5), less cleavage for jetPEI, and little activity for L-PEI. jetPEI and L-PEI exhibited substantially greater transgene expression, consistent with the lower amounts of MB oligonucleotide cleavage observed. Cytosolic nuclease activity, although dependent on the choice of polymer employed, was not related to the degree of cell plasma membrane disruption that occurred as measured by PI uptake or whole-cell patch clamp.
Subject(s)
Deoxyribonucleases/metabolism , Transgenes/genetics , Dendrimers/chemistry , Flow Cytometry , HeLa Cells , Humans , Microscopy, Confocal , Patch-Clamp Techniques , Porosity , Propidium/metabolism , TransfectionABSTRACT
Generation 7 (G7) poly(amidoamine) (PAMAM) dendrimers with amine, acetamide, and carboxylate end groups were prepared to investigate polymer/cell membrane interactions in vitro. G7 PAMAM dendrimers were used in this study because higher-generation of dendrimers are more effective in permeabilization of cell plasma membranes and in the formation of nanoscale holes in supported lipid bilayers than smaller, lower-generation dendrimers. Dendrimer-based conjugates were characterized by (1)H NMR, UV/vis spectroscopy, GPC, HPLC, and CE. Positively charged amine-terminated G7 dendrimers (G7-NH(2)) were observed to internalize into KB, Rat2, and C6 cells at a 200 nM concentration. By way of contrast, neither negatively charged G7 carboxylate-terminated dendrimers (G7-COOH) nor neutral acetamide-terminated G7 dendrimers (G7-Ac) associated with the cell plasma membrane or internalized under similar conditions. A series of in vitro experiments employing endocytic markers cholera toxin subunit B (CTB), transferrin, and GM(1)-pyrene were performed to further investigate mechanisms of dendrimer internalization into cells. G7-NH(2) dendrimers colocalized with CTB; however, experiments with C6 cells indicated that internalization of G7-NH(2) was not ganglioside GM(1) dependent. The G7/CTB colocalization was thus ascribed to an artifact of direct interaction between the two species. The presence of GM(1) in the membrane also had no effect upon XTT assays of cell viability or lactate dehydrogenase (LDH) assays of membrane permeability.