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Human Gene Therapy Dec 2022Lentiviral transduction of human mesenchymal stem cells (MSCs) induces long-term transgene expression and holds great promise for multiple gene therapy applications....
Lentiviral transduction of human mesenchymal stem cells (MSCs) induces long-term transgene expression and holds great promise for multiple gene therapy applications. Polybrene is the most commonly used reagent to improve viral gene transfer efficiency in laboratory research; however, it is not approved for human use and has also been shown to impair MSC proliferation and differentiation. Therefore, there is a need for optimized transduction protocols that can also be adapted to clinical settings. LentiBOOST (LB) and protamine sulfate are alternative transduction enhancers (TEs) that can be manufactured to current Good Manufacturing Practice standards, are easily applied to existing protocols, and have been previously studied for the transduction of human CD34 hematopoietic stem cells. In this study, we investigated these reagents for the enhancement of lentiviral transduction of adipose-derived MSCs. We found that the combination of LB and protamine sulfate could yield comparable or even superior transduction efficiency to polybrene, with no dose-dependent adverse effects on cell viability or stem cell characteristics. This combination of TEs represents a valuable clinically compatible alternative to polybrene with the potential to significantly improve the efficiency of lentiviral transduction of MSCs for gene therapy applications.
Topics: Humans; Lentivirus; Transduction, Genetic; Hexadimethrine Bromide; Genetic Vectors; Mesenchymal Stem Cells; Cell Differentiation; Protamines
PubMed: 35859364
DOI: 10.1089/hum.2022.117 -
Drug Delivery Dec 2020Food protein and polysaccharide complex emulsions are safe carriers of hydrophobic drugs and nutrients. To improve oral bioavailability and therapeutic/healthy efficacy...
Food protein and polysaccharide complex emulsions are safe carriers of hydrophobic drugs and nutrients. To improve oral bioavailability and therapeutic/healthy efficacy of hydrophobic drugs and nutrients, herein, protamine (PRO), a cationic cell-penetrating peptide, was introduced into protein and polysaccharide complex emulsion. The electrostatic complex of PRO and BSA-dextran conjugate (BD) produced by Maillard reaction was used as emulsifier to produce oil-in-water emulsion (@BD/PRO). The BSA molecules were crosslinked at the oil-water interface by a heat treatment and the PRO chains were simultaneously anchored in the interface. BD emulsion (@BD) without PRO was produced for comparation. Paclitaxel (PTX), a hydrophobic antineoplastic drug, was encapsulated in the emulsions with 99% loading efficiency and 6.4% loading capacity. The emulsions had long-term stability. The bioavailability and H22 tumor inhibition efficacy of PTX@BD/PRO were 40% and 70% higher than those of PTX@BD, respectively, after oral administration in the mice. More importantly, orally administrated PTX@BD/PRO had the same anti-tumor efficacy as intravenously injected commercial PTX injection. No abnormality was observed in the main organs of the mice after consecutive oral administration of PTX@BD/PRO. This study indicates that @BD/PRO is an excellent carrier of hydrophobic drugs/nutrients and is suitable for long-term oral administration.
Topics: Administration, Oral; Animals; Antineoplastic Agents, Phytogenic; Biological Availability; Dextrans; Drug Carriers; Gastrointestinal Tract; Male; Mice; Mice, Inbred ICR; Neoplasms; Paclitaxel; Protamines; Serum Albumin, Bovine; Treatment Outcome; Xenograft Model Antitumor Assays
PubMed: 32985911
DOI: 10.1080/10717544.2020.1825543 -
Effects of Modified Ultrafiltration on Thromboelastographic Profile after Pediatric Cardiac Surgery.The Journal of Extra-corporeal... Mar 2021Modified ultrafiltration (MUF) is still used after pediatric cardiopulmonary bypass (CPB) in some pediatric cardiac surgery centers to decrease transfusion requirements....
Modified ultrafiltration (MUF) is still used after pediatric cardiopulmonary bypass (CPB) in some pediatric cardiac surgery centers to decrease transfusion requirements. Other potential benefits of MUF include clearance of inflammatory markers and improvement in myocardial function. Our hypothesis is that MUF will hemoconcentrate coagulation factors and improve thromboelastography (TEG) parameters after pediatric CPB. Patients younger than 6 months were prospectively enrolled over a year. TEG was carried out before MUF, after MUF, and after protamine administration. Paired t tests were conducted to compare values pre-MUF and post-MUF as well as post-MUF and post-protamine administration. Thirty patients were enrolled in the study, with 20 (67%) neonates in the cohort. Seven arterial switch operations and nine Norwood procedures were found to be performed among the cohort. Reaction time (), angle (α), and maximum amplitude (MA) were significantly worse post-MUF compared with pre-MUF ( < .001). They improved significantly after protamine administration compared with post-MUF ( < .001). The amount of fluid removal was significantly associated with a worse post-MUF R, angle, and MA and worse post-protamine administration, angle, and MA but with no effect on post-protamine R. MUF caused worsening of TEG parameters that is reversed by protamine administration.
Topics: Cardiac Surgical Procedures; Cardiopulmonary Bypass; Child; Humans; Infant, Newborn; Prospective Studies; Thrombelastography; Ultrafiltration
PubMed: 33814606
DOI: 10.1182/ject-2000051 -
Angewandte Chemie (International Ed. in... Jan 2022Ibrutinib is an inhibitor of Bruton's tyrosine kinase that has been approved for the treatment of patients with chronic lymphocytic leukemia, mantle cell lymphoma and...
Ibrutinib is an inhibitor of Bruton's tyrosine kinase that has been approved for the treatment of patients with chronic lymphocytic leukemia, mantle cell lymphoma and Waldenstrom's macroglobulinemia and is connected with toxicities. To minimize its toxicities, we linked ibrutinib to a cell-targeted, internalizing antibody. To this end, we synthesized a poly-anionic derivate, ibrutinib-Cy3.5, that retains full functionality. This anionic inhibitor is complexed by our anti-CD20-protamine targeting conjugate and free protamine, and thereby spontaneously assembles into an electrostatically stabilized vesicular nanocarrier. The complexation led to an accumulation of the drug driven by the CD20 antigen internalization to the intended cells and an amplification of its pharmacological effectivity. In vivo, we observed a significant enrichment of the drug in xenograft lymphoma tumors in immune-compromised mice and a significantly better response to lower doses compared to the original drug.
Topics: Adenine; Animals; Antibodies, Monoclonal; Antineoplastic Agents; Carbocyanines; Cell Proliferation; Drug Screening Assays, Antitumor; Humans; Lymphoma, Large B-Cell, Diffuse; Mice; Neoplasms, Experimental; Piperidines; Protein Engineering; Protein Kinase Inhibitors; Static Electricity
PubMed: 34725904
DOI: 10.1002/anie.202109769 -
Journal of Food and Drug Analysis Dec 2021Oversulfated chondroitin sulfate (OSCS), a non-natural sulfated glycosaminoglycan, recognizes as a significant containment in the pharmaceutical heparin, and it could... (Review)
Review
Oversulfated chondroitin sulfate (OSCS), a non-natural sulfated glycosaminoglycan, recognizes as a significant containment in the pharmaceutical heparin, and it could trigger adverse reactions. Chromatography-, electrophoresis-, electrochemistry-, and spectroscopy-related techniques are currently available for accurate and precise analysis of a trace amount of OSCS in heparin. Recently, emerging studies focus on developing colorimetric and fluorescent probes to monitor OSCS containments in heparin. Therefore, this current review aims to describe the sensing principle and procedure of the reported probes that are sensitive and selective toward OSCS in heparin without the interferences of other sulfated glycosaminoglycans. The reported OSCS-specific probes are comprehensively discussed according to the recognition elements of OSCS, including coralyne, AG73 peptides, positively charged tetraphenylethene derivatives, polythiophene polymer, and poly-L-lysine, protamine, superpositively charged green fluorescent proteins, and poly (diallyldimethylammonium chloride). The sensing of OSCS in heparin is generally achieved using, (i) the specific affinity of the recognition element with OSCS and heparin, (ii) heparinase-mediated hydrolysis of heparin, and (iii) OSCS-induced inhibition of heparinase activity. Additionally, coralyne-based DNA probes can detect OSCS in heparin in the presence of Ca ions without the assistance of heparinase. This review will pave the way to design another sensing probe towards other sulfated contaminants, like dermatan sulfate.
Topics: Chondroitin Sulfates; Colorimetry; Drug Contamination; Heparin; Heparin Lyase
PubMed: 35649143
DOI: 10.38212/2224-6614.3379 -
Development & Reproduction Dec 2020Spermatogonial stem cells (SSCs) have stemness characteristics, including germ cell-specific imprints that allow them to form gametes. Spermatogenesis involves changes... (Review)
Review
Spermatogonial stem cells (SSCs) have stemness characteristics, including germ cell-specific imprints that allow them to form gametes. Spermatogenesis involves changes in gene expression such as a transition from expression of somatic to germ cell-specific genes, global repression of gene expression, meiotic sex chromosome inactivation, highly condensed packing of the nucleus with protamines, and morphogenesis. These step-by-step processes finally generate spermatozoa that are fertilization competent. Dynamic epigenetic modifications also confer totipotency to germ cells after fertilization. Primordial germ cells (PGCs) in embryos do not enter meiosis, remain in the proliferative stage, and are referred to as gonocytes, before entering quiescence. Gonocytes develop into SSCs at about 6 days after birth in rodents. Although chromatin structural modification by Polycomb is essential for gene silencing in mammals, and epigenetic changes are critical in spermatogenesis, a comprehensive understanding of transcriptional regulation is lacking. Recently, we evaluated the expression profiles of Yin Yang 1 (YY1) and CP2c in the gonads of E14.5 and 12-week-old mice. YY1 localizes at the nucleus and/or cytoplasm at specific stages of spermatogenesis, possibly by interaction with CP2c and YY1-interacting transcription factor. In the present article, we discuss the possible roles of YY1 and CP2c in spermatogenesis and stemness based on our results and a review of the relevant literature.
PubMed: 33537512
DOI: 10.12717/DR.2020.24.4.249 -
Journal of Clinical Medicine Jun 2023We aimed to evaluate the efficacy and safety of low-dose protamine in reducing access site-related complications during Transcatheter Aortic Valve Implantation (TAVI) as...
OBJECTIVES
We aimed to evaluate the efficacy and safety of low-dose protamine in reducing access site-related complications during Transcatheter Aortic Valve Implantation (TAVI) as compared to full-dose protamine.
BACKGROUND
Access site-related complications represent an independent predictor of poor outcomes of TAVI. Data regarding heparin reversal with protamine and the dosage needed to prevent bleeding complications are scarce among patients undergoing TAVI.
METHODS
A total of 897 patients were retrospectively included in the study. Patients who underwent percutaneous coronary intervention within 4 weeks before or concomitantly with TAVI ( = 191) were given 0.5 mg protamine for each 100 units of unfractionated heparin. All other patients ( = 706) were considered as a control group and 1 mg protamine for each 100 units of heparin was administered.
RESULTS
The combined intra-hospital endpoint of death, life-threatening major bleeding, and major vascular complications were significantly more frequent in patients receiving low-dose protamine [29 (15.2%) vs. 50 (7.1%), < 0.001]. After propensity matching ( = 130 for each group) for relevant clinical characteristics including anti-platelet therapy [19 (14.6%) vs. 6 (4.6%), = 0.006], low-dose protamine predicted the combined endpoint (OR 3.54, 95%-CI 1.36-9.17, = 0.009), and even in multivariable analysis, low-dose protamine continued to be a predictor of the combined endpoint in the matched model (OR 3.07, 95%-CI 1.17-8.08, = 0.023) alongside baseline hemoglobin.
CONCLUSIONS
In this propensity-matched retrospective analysis, a low-dose protamine regime is associated with a higher rate of major adverse events compared to a full-dose protamine regime following transfemoral TAVI.
PubMed: 37445282
DOI: 10.3390/jcm12134243 -
JBRA Assisted Reproduction Jun 2024Male infertility is a great matter of concern as out of 15% of infertile couples in the reproductive age, about 40% are contributed by male factors alone. For DNA... (Review)
Review
Male infertility is a great matter of concern as out of 15% of infertile couples in the reproductive age, about 40% are contributed by male factors alone. For DNA condensation during spermatogenesis, constrained DNA nicking is required, which if increased beyond certain level results in infertility in men. High sperm DNA Fragmentation (SDF) majorly contributes to male infertility and its association with regards to poor natural conception and assisted reproductive technology (ART) outcomes is equivocal. Apoptosis, protamination failure and the excess of reactive oxygen species (ROS) are considered to be the main causes of SDF. It's testing came into existence because of the limitations of the conventional methods in explaining infertility in normozoospermic infertile individuals. Over the past 25 years, SDF's several testing strategies have been proposed to diagnose the aetiology of infertility. Various treatments combined with sperm selection techniques are being used alone or in combination to reduce DNA fragmentation index (DFI) and obtain spermatozoa with high quality chromatin for assisted reproduction. This review summarises SDF's main causes, its impact on fertility and clinical outcomes in assisted reproduction, the need to perform test, testing procedures, and the treatment strategies.
Topics: Humans; Male; DNA Fragmentation; Infertility, Male; Spermatozoa; Reproductive Techniques, Assisted
PubMed: 38289201
DOI: 10.5935/1518-0557.20230076 -
Drug Delivery Dec 2020Protamine is a natural cationic peptide mixture used as a drug for the neutralization of heparin and in formulations of slow-release insulin. In addition, Protamine can...
Protamine is a natural cationic peptide mixture used as a drug for the neutralization of heparin and in formulations of slow-release insulin. In addition, Protamine can be used for the stabilization and delivery of nucleic acids (antisense, small interfering RNA (siRNA), immunostimulatory nucleic acids, plasmid DNA, or messenger RNA) and is therefore included in several compositions that are in clinical development. Notably, when mixed with RNA, protamine spontaneously generates particles in the size range of 20-1000 nm depending on the formulation conditions (concentration of the reagents, ratio, and presence of salts). These particles are being used for vaccination and immuno-stimulation. Several grades of protamine are available, and we compared them in the context of complex formation with messenger RNA (mRNA). We found that the different available protamine preparations largely vary in their composition and capacity to transfect mRNA. Our data point to the source of protamine as an important parameter for the production of therapeutic protamine-based complexes.
Topics: Cells, Cultured; Drug Compounding; HEK293 Cells; Humans; Leukocytes, Mononuclear; Particle Size; Protamines; RNA, Messenger; Transfection
PubMed: 32804028
DOI: 10.1080/10717544.2020.1790692 -
Chemical Science Dec 2021Optical nanosensors for the detection of polyions, including protamine and heparin, have to date relied upon ion-exchange reactions involving an analyte and an optical...
Optical nanosensors for the detection of polyions, including protamine and heparin, have to date relied upon ion-exchange reactions involving an analyte and an optical transducer. Unfortunately, due to the limited selectivity of the available ionophores for polyions, this mechanism has suffered from severe interference in complex sample matrices. To date no optical polyion nanosensors have demonstrated acceptable performance in serum, plasma or blood. Herein we describe a new type of nanosensor based on our discovery of a "hyper-polarizing lipophilic phase" in which dinonylnaphthalenesulfonate (DNNS) polarizes a solvatochromic dye much more than even an aqueous environment. We have found that the apparent polarity of the organic phase is only modulated when DNNS binds to large polyions such as protamine, unlike singly charged ions that lack the cooperative binding required to cause a significant shift in the distribution of the polarizing DNNS ions. Our new sensing mechanism allows solvatochromic signal transduction without the transducer undergoing ion exchange. The result is significantly improved sensitivity and selectivity, enabling for the first time the quantification of protamine and heparin in human plasma using optical nanosensors that correlates with the current gold standard analysis method, the anti-Xa factor assay.
PubMed: 35003589
DOI: 10.1039/d1sc04930e