siRNA Delivery with Chitosan: Influence of Chitosan Molecular Weight, Degree of Deacetylation, and Amine to Phosphate Ratio on in Vitro Silencing Efficiency, Hemocompatibility, Biodistribution, and in Vivo Efficacy

A major hurdle for exploring RNA interference (RNAi) in a therapeutic setting is still the issue of in vivo delivery of small RNA molecules (siRNAs). The chemical modification of polyethylenimines (PEIs) offers a particularly attractive avenue towards the development of more efficient non-viral delivery systems. Here, we explore tyrosine-modified polyethylenimines with low or very low molecular weight (P2Y, P5Y, P10Y) for siRNA delivery. In comparison to their respective parent PEI, they reveal considerably increased knockdown efficacies and very low cytotoxicity upon tyrosine modification, as determined in different reporter and wildtype cell lines. The delivery of siRNAs targeting the anti-apoptotic oncogene survivin or the serine/threonine-protein kinase PLK1 (polo-like kinase 1; PLK-1) oncogene reveals strong inhibitory effects in vitro. In a therapeutic in vivo setting, profound anti-tumor effects in a prostate carcinoma xenograft mouse model are observed upon systemic application of complexes for survivin or PLK1 knockdown, in the absence of in vivo toxicity. We thus demonstrate the tyrosine-modification of (very) low molecular weight PEIs for generating efficient nanocarriers for siRNA delivery in vitro and in vivo, present data on their physicochemical and biological properties, and show their efficacy as siRNA therapeutic in vivo, in the absence of adverse effects.

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Determination of indicators allowing to evaluating the hemostatic activity of chitosan without a biological experiment

Journal of Bioactive and Compatible Polymers ◽

10.1177/08839115211030607 ◽

2021 ◽

pp. 088391152110306

Author(s):

Oksana Vladimirovna Kadyseva ◽

Vladimir Nikolaevich Bykov ◽

Olga Yurievna Strelova ◽

Alexander Nikolaevich Grebenyuk

Keyword(s):

Molecular Weight ◽

Physicochemical Parameters ◽

Light Transmission ◽

Resonance Spectroscopy ◽

Biological Experiment ◽

Degree Of Deacetylation ◽

Hemostatic Activity ◽

Rigid Rod

The biopolymer chitosan is widely used for the development of local hemostatic agents. However, the physicochemical parameters of chitosan that determine its hemostatic properties have not yet been determined. Standard quality control of chitosan-containing raw materials and medical products on its basis do not allow us to make a conclusion about the effectiveness of their use for stopping bleeding. The most reliable method for assessing hemostatic activity remains in vivo experiment on large animals. The aim of this study was to determine additional physicochemical parameters of chitosan, which would make it possible to predict its hemostatic activity without conducting a biological experiment. In this work, using the methods of nuclear magnetic resonance spectroscopy, spectrophotometry and viscometry, it has been shown that the ability to initiate hemostasis is depending of the molecular weight and degree of deacetylation of chitosan, but not enough linearly. The hemostatic properties in vitro increases in a series of samples with a relatively constant molecular weight with an increase in the degree of deacetylation. As well as in a series with the same degree of deacetylation with an increase in molecular weight. However, at molecular weight values more than 300 kDa, the viscosity of the polymer causes the opposite effect: with an increase in the degree of deacetylation, the hemostatic activity decreases. The best ability to initiate hemostasis have chitosan samples with a degree of deacetylation of 90.0%–97.4% and molecular weight 145.7–284.7 kDa, in which at pH of solution close to physiological, a significant part of the molecules transitioned from conformation state rigid rod to state globule. It was accompanied by an abrupt change in light transmission of the solution. It was concluded, that it is possible to study conformational states by spectrophotometry to assess the hemostatic activity of chitosan samples without performing biological experiment.

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Effects of Unfractionated and Low Molecular Weight Heparins on Platelet Thromboxane Biosynthesis “In Vivo”

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648988 ◽

1994 ◽

Vol 72 (06) ◽

pp. 942-946 ◽

Cited By ~ 20

Author(s):

Raffaele Landolfi ◽

Erica De Candia ◽

Bianca Rocca ◽

Giovanni Ciabattoni ◽

Armando Antinori ◽

...

Keyword(s):

Molecular Weight ◽

Unfractionated Heparin ◽

Low Molecular Weight Heparin ◽

Primary Source ◽

In Vivo Studies ◽

Low Molecular Weight ◽

Heparin Administration ◽

To Receive

SummarySeveral “in vitro” and “in vivo” studies indicate that heparin administration may affect platelet function. In this study we investigated the effects of prophylactic heparin on thromboxane (Tx)A2 biosynthesis “in vivo”, as assessed by the urinary excretion of major enzymatic metabolites 11-dehydro-TxB2 and 2,3-dinor-TxB2. Twenty-four patients who were candidates for cholecystectomy because of uncomplicated lithiasis were randomly assigned to receive placebo, unfractionated heparin, low molecular weight heparin or unfractionaed heparin plus 100 mg aspirin. Measurements of daily excretion of Tx metabolites were performed before and during the treatment. In the groups assigned to placebo and to low molecular weight heparin there was no statistically significant modification of Tx metabolite excretion while patients receiving unfractionated heparin had a significant increase of both metabolites (11-dehydro-TxB2: 3844 ± 1388 vs 2092 ±777, p <0.05; 2,3-dinor-TxB2: 2737 ± 808 vs 1535 ± 771 pg/mg creatinine, p <0.05). In patients randomized to receive low-dose aspirin plus unfractionated heparin the excretion of the two metabolites was largely suppressed thus suggesting that platelets are the primary source of enhanced thromboxane biosynthesis associated with heparin administration. These data indicate that unfractionated heparin causes platelet activation “in vivo” and suggest that the use of low molecular weight heparin may avoid this complication.

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Toxicity of Heparinoids, with Special Reference to the Precipitation of Fibrinogen

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655587 ◽

1964 ◽

Vol 12 (01) ◽

pp. 232-261 ◽

Cited By ~ 2

Author(s):

S Sasaki ◽

T Takemoto ◽

S Oka

Keyword(s):

Molecular Weight ◽

Dextran Sulfate ◽

Anticoagulant Activity ◽

Molecular Structures ◽

Severe Reaction ◽

Clinical Use ◽

Molecular Weights ◽

Close Relationship

SummaryTo demonstrate whether the intravascular precipitation of fibrinogen is responsible for the toxicity of heparinoid, the relation between the toxicity of heparinoid in vivo and the precipitation of fibrinogen in vitro was investigated, using dextran sulfate of various molecular weights and various heparinoids.1. There are close relationships between the molecular weight of dextran sulfate, its toxicity, and the quantity of fibrinogen precipitated.2. The close relationship between the toxicity and the precipitation of fibrinogen found for dextran sulfate holds good for other heparinoids regardless of their molecular structures.3. Histological findings suggest strongly that the pathological changes produced with dextran sulfate are caused primarily by the intravascular precipitates with occlusion of the capillaries.From these facts, it is concluded that the precipitates of fibrinogen with heparinoid may be the cause or at least the major cause of the toxicity of heparinoid.4. The most suitable molecular weight of dextran sulfate for clinical use was found to be 5,300 ~ 6,700, from the maximum value of the product (LD50 · Anticoagulant activity). This product (LD50 · Anticoagulant activity) can be employed generally to assess the comparative merits of various heparinoids.5. Clinical use of the dextran sulfate prepared on this basis gave satisfactory results. No severe reaction was observed. However, two delayed reactions, alopecia and thrombocytopenia, were observed. These two reactions seem to come from the cause other than intravascular precipitation.

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Neutralization of a Low Molecular Weight Heparin (LHN-1) and Conventional Heparin by Protamine Sulfate in Rats

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661675 ◽

1986 ◽

Vol 56 (03) ◽

pp. 318-322 ◽

Cited By ~ 18

Author(s):

V Diness ◽

P B Østergaard

Keyword(s):

Molecular Weight ◽

Low Molecular Weight Heparin ◽

Thrombus Formation ◽

Experimental Models ◽

Protamine Sulfate ◽

Low Molecular Weight ◽

Antithrombotic Effect ◽

Higher Doses

SummaryThe neutralization of a low molecular weight heparin (LHN-1) and conventional heparin (CH) by protamine sulfate has been studied in vitro and in vivo. In vitro, the APTT activity of CH was completely neutralized in parallel with the anti-Xa activity. The APTT activity of LHN-1 was almost completely neutralized in a way similar to the APTT activity of CH, whereas the anti-Xa activity of LHN-1 was only partially neutralized.In vivo, CH 3 mg/kg and LHN-1 7.2 mg/kg was given intravenously in rats. The APTT and anti-Xa activities, after neutralization by protamine sulfate in vivo, were similar to the results in vitro. In CH treated rats no haemorrhagic effect in the rat tail bleeding test and no antithrombotic effect in the rat stasis model was found at a protamine sulfate to heparin ratio of about 1, which neutralized APTT and anti-Xa activities. In LHN-1 treated rats the haemorrhagic effect was neutralized when APTT was close to normal whereas higher doses of protamine sulfate were required for neutralization of the antithrombotic effect. This probably reflects the fact that in most experimental models higher doses of heparin are needed to induce bleeding than to prevent thrombus formation. Our results demonstrate that even if complete neutralization of APTT and anti-Xa activities were not seen in LHN-1 treated rats, the in vivo effects of LHN-1 could be neutralized as efficiently as those of conventional heparin. The large fall in blood pressure caused by high doses of protamine sulfate alone was prevented by the prior injection of LHN-1.

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Studies on Metabolism of Urokinase and Mechanism of Thrombolysis by Urokinase

Thrombosis and Haemostasis ◽

10.1055/s-0038-1666937 ◽

1979 ◽

Vol 42 (03) ◽

pp. 885-894 ◽

Cited By ~ 13

Author(s):

Tatsuo Ueno ◽

Norio Kobayashi ◽

Tadashi Maekawa

Keyword(s):

Molecular Weight ◽

Plasma Clearance ◽

Radioactive Material ◽

Optimal Dosage ◽

Plasma Clot ◽

Optimal Dosage Regimen ◽

Arterial Thrombus ◽

Contact Experiment

SummaryPharmacokinetics of intravenously injected 125I-labeled urokinase (125I-UK) of a molecular weight of 33,000 daltons in normal rabbits and patients with various diseases were investigated. The plasma clearance of 125I-UK in rabbits was described by a biexponential curve within six hours with a half-life of 8 minutes, 2.3 hours, respectively. The radioactivity in the liver and kidneys 15 minutes after iv injection with 125I-UK was 9.6% and 14.0% of the radioactivity injected, respectively. Approximately 80% of the total radioactive material injected was excreted in the urine in 18 hours. No increase in activator activity in the urine was observed after a large amount of UK injection. Activity uptake of 125I-UK by experimentally induced arterial thrombus was little. Lysis of the stasis thrombus was produced by injecting 7.5 × 104 IU of UK in only one out of 8 rabbits. In vitro contact experiment revealed that transfer of 125I-UK to plasma clot is slow (24 hours for 10% of 125I-UK by plasma clot). In 4 patients plasma clearance of 125I-UK was essentially similar to that in rabbits. From the results obtained optimal dosage regimen of UK administration for complete thrombolysis in vivo was discussed.

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Faculty Opinions recommendation of The in vitro mechanisms and in vivo efficacy of intravenous lidocaine on the neuroinflammatory response in acute and chronic pain.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726025441.793523751 ◽

2016 ◽

Author(s):

Jan Jakobsson

Keyword(s):

Chronic Pain ◽

Intravenous Lidocaine ◽

Neuroinflammatory Response ◽

In Vivo Efficacy

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