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Chitosan (CS) shows in vitro and in vivo efficacy for siRNA delivery but with contradictory findings for incompletely characterized systems. For understanding which parameters produce effective delivery, a library of precisely characterized chitosans was produced at different degrees of deacetylation (DDAs) and average molecular weights (Mn). Encapsulation and transfection efficiencies were characterized in vitro. Formulations were selected to examine the influence of Mn and N:P ratio on nanoparticle uptake, metabolic activity, genotoxicity, and in vitro transfection. Hemocompatibility and in vivo biodistribution were then investigated for different Mn, N:P ratios, and doses. Nanoparticle uptake and gene silencing correlated with increased surface charge, which was obtained at high DDA and high Mn. A minimum polymer length of â¼60-70 monomers (â¼10 kDa) was required for stability and knockdown. In vitro knockdown was equivalent to lipid control with no metabolic or genotoxicity. An inhibitory effect of serum on biological performance was dependent on DDA, Mn, and N:P. In vivo biodistribution in mice show accumulation of nanoparticles in kidney with 40-50% functional knockdown.
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Aminas/metabolismo , Materiais Biocompatíveis/química , Quitosana/administração & dosagem , Inativação Gênica , Nanopartículas/química , Fosfatos/metabolismo , RNA Interferente Pequeno/administração & dosagem , Acetilação , Sangue , Linhagem Celular Tumoral , Quitosana/química , Quitosana/farmacocinética , Ensaio Cometa , Células Epiteliais/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/metabolismo , Peso Molecular , Nanopartículas/toxicidade , Reação em Cadeia da Polimerase em Tempo Real , Distribuição TecidualRESUMO
Analytical ultracentrifugation (AUC) and dynamic light scattering (DLS) were combined to characterize polyplexes formed with 10 kDa chitosan or 10 kDa PEI and oligodeoxynucleotides (ODN). Combined analysis revealed that both polyplexes were highly porous (over 80%) and that their weight average hydrodynamic diameters were of 46 and 55 nm for chitosan/ODN and PEI/ODN complexes, respectively. Transformation of the sedimentation coefficient distribution to a size and molecular weight distribution gave an average molecular weight of 19 and 29 MDa for chitosan and PEI polyplexes, respectively. Data from AUC also allowed for the calculation of the actual dn/dc and N/P ratios of each polyplex. Additional data from scanning electron microscopy and static light scattering confirmed the conclusions that were initially derived from AUC and DLS, thus validating that the combination of AUC and DLS is a powerful approach to characterize polyplexes in terms of refractive index increment, size, and molecular weight distributions, as well as porosity.
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Quitosana/química , Oligodesoxirribonucleotídeos/química , Poliaminas/química , Microscopia Eletrônica de Varredura , Peso Molecular , Polieletrólitos , Polietilenoimina/química , Porosidade , Refratometria , UltracentrifugaçãoRESUMO
Prime editing shows potential as a precision genome editing technology, as well as the potential to advance the development of next-generation nanomedicine for addressing neurological disorders. However, turning in prime editors (PEs), which are macromolecular complexes composed of CRISPR/Cas9 nickase fused with a reverse transcriptase and a prime editing guide RNA (pegRNA), to the brain remains a considerable challenge due to physiological obstacles, including the blood-brain barrier (BBB). This review article offers an up-to-date overview and perspective on the latest technologies and strategies for the precision delivery of PEs to the brain and passage through blood barriers. Furthermore, it delves into the scientific significance and possible therapeutic applications of prime editing in conditions related to neurological diseases. It is targeted at clinicians and clinical researchers working on advancing precision nanomedicine for neuropathologies.
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Copper-chlorophyllin is a water-soluble derivative of chlorophylls and shows low cytotoxicity and antimutagenic properties in cultured cells. It has multiple applications, including its use as a photosensitizer in photothermal therapy because of its green light-activated photothermal performance. In this work, it was copolymerized with a poly(ethylene glycol) methacrylic monomer to yield random copolymers by free radical polymerization, which showed dual temperature- and pH-dependent phase transitions in aqueous solutions. The cloud points of the copolymer solutions were raised by lowering the pH of the aqueous solutions due to the protonation of the carboxylic groups on the chlorophyllin moieties, which decreased the overall hydrophilicity of the polymers. At low pH values, complete protonation of the carboxylic acid groups of the chlorophyllin moieties led to an irreversible aggregation of the copolymers in water. The incorporation of chlorophyllin in the copolymer improved its stability over its single molecular form.
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Clorofilídeos , Polímeros , Polímeros/farmacologia , Polímeros/química , Polietilenoglicóis/química , ÁguaRESUMO
Polycations having a high buffering capacity in the endosomal pH range, such as polyethylenimine (PEI), are known to be efficient at delivering nucleic acids by overcoming lysosomal sequestration possibly through the proton sponge effect, although other mechanisms such as membrane disruption arising from an interaction between the polycation and the endosome/lysosome membrane, have been proposed. Chitosan is an efficient delivery vehicle for nucleic acids, yet its buffering capacity has been thought to be significantly lower than that of PEI, suggesting that the molecular mechanism responsible for endolysosomal escape was not proton sponge based. However, previous comparisons of PEI and chitosan buffering capacity were performed on a mass concentration basis instead of a charge concentration basis, the latter being the most relevant comparison basis because polycation-DNA complexes form at ratios of charge groups (amine to phosphate), rather than according to mass. We hypothesized that chitosan has a high buffering capacity when compared to PEI on a molar basis and could therefore possibly mediate endolysosomal release through the proton sponge effect. In this study, we examined the ionization behavior of chitosan and chitosan-DNA complexes and compared to that of PEI and polylysine on a charge concentration basis. A mean field theory based on the use of the Poisson-Boltzmann equation and an Ising model were also applied to model ionization behavior of chitosan and PEI, respectively. We found that chitosan has a higher buffering capacity than PEI in the endolysosomal pH range, while the formation of chitosan-DNA complexes reduces chitosan buffering capacity because of the negative electrostatic environment of nucleic acids that facilitates chitosan ionization. These data suggest that chitosans have a similar capacity as PEI to mediate endosomal escape through the proton sponge effect, possibly in a manner which depends on the presence of excess chitosan.
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Quitosana/química , DNA/química , Polietilenoimina/química , Soluções Tampão , DNA/administração & dosagem , Concentração de Íons de Hidrogênio , PrótonsRESUMO
UNLABELLED: Chitosan is a biocompatible polysaccharide composed of glucosamine and N-acetylglucosamine. The polymer has a unique behavior of fluctuating between soluble chains at pH 6 and insoluble microparticles at pH 7. The purpose of this study was to test the hypothesis that chitosan structure, solubility state, and serum influence the rate of cell uptake. Chitosans with 80% and 95% degree of deacetylation (medium and low viscosity) were tagged with rhodamine and analyzed for particle size, media solubility, and uptake by HEK293 epithelial cells using live confocal microscopy and flow cytometry. In media pH 7.4 with or without 10% serum, chitosans fully precipitated into 0.5 to 1.4 µm diameter microparticles with a slight negative charge. During 24 h of culture in serum-free medium, chitosan particles remained extracellular. In cultures with serum, particles were taken up into intracellular vesicles in a serum dose-dependent manner. Opsonization of chitosan with serum, or replacement of serum by epidermal growth factor (EGF) failed to mediate serum-free chitosan particle uptake. Serum stimulated cells to acidify the media, partly by lactate generation. Media acidified to pH 6.5 by 7 mM lactate maintained 50% of chitosan in the soluble fraction, and led to minor uniform serum-free uptake in small vesicles. CONCLUSION: Media acidification mediates minor in vitro uptake of non-biofouled soluble chitosan chains, while serum-biofouled insoluble chitosan microparticles require sustained serum exposure to generate energy required for macropinocytosis.
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Quitosana/metabolismo , Ácido Láctico/metabolismo , Soro/fisiologia , Configuração de Carboidratos , Quitosana/química , Meios de Cultura , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Luz , Microscopia Confocal , Microscopia de Fluorescência , Tamanho da Partícula , Rodaminas/química , Rodaminas/metabolismo , Espalhamento de Radiação , Solubilidade , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Freeze-dried chitosan formulations solubilized in platelet-rich plasma (PRP) are currently evaluated as injectable implants with the potential for augmenting the standard of care for tissue repair in different orthopedic conditions. The present study aimed to shorten the solidification time of such implants, leading to an easier application and a facilitated solidification in a wet environment, which were direct demands from orthopedic surgeons. The addition of thrombin to the formulation before lyophilization was explored. The challenge was to find a formulation that coagulated fast enough to be applied in a wet environment but not too fast, which would make handling/injection difficult. Four thrombin concentrations were analyzed (0.0, 0.25, 0.5, and 1.0 NIH/mL) in vitro (using thromboelastography, rheology, indentation, syringe injectability, and thrombin activity tests) as well as ex vivo (by assessing the implant's adherence to tendon tissue in a wet environment). The biomaterial containing 0.5 NIH/mL of thrombin significantly increased the coagulation speed while being easy to handle up to 6 min after solubilization. Furthermore, the adherence of the biomaterial to tendon tissues was impacted by the biomaterial-tendon contact duration and increased faster when thrombin was present. These results suggest that our biomaterial has great potential for use in regenerative medicine applications.
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Lipid nanoparticle (LNP)-formulated messenger RNA (mRNA) vaccineare a promising platform to prevent infectious diseases as demonstrated by the recent success of SARS-CoV-2 mRNA vaccines. To avoid immune recognition and uncontrolled inflammation, nucleoside-modified mRNA is used. However, such modification largely abrogates the innate immune responses that are critical to orchestrating robust adaptive immunity. Here we develop an LNP component-an adjuvant lipidoid-that can enhance the adjuvanticity of mRNA-LNP vaccines. Our results show that partial substitution of ionizable lipidoid with adjuvant lipidoid not only enhanced mRNA delivery, but also endowed LNPs with Toll-like receptor 7/8-agonistic activity, which significantly increased the innate immunity of the SARS-CoV-2 mRNA-LNP vaccine with good tolerability in mice. Our optimized vaccine elicits potent neutralizing antibodies against multiple SARS-CoV-2 pseudovirus variants, strong Th1-biased cellular immunity, and robust B cell and long-lived plasma cell responses. Importantly, this adjuvant lipidoid substitution strategy works successfully in a clinically relevant mRNA-LNP vaccine, demonstrating its translational potential.
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COVID-19 , Nanopartículas , Animais , Humanos , Camundongos , Vacinas contra COVID-19 , SARS-CoV-2/genética , COVID-19/prevenção & controle , Adjuvantes Imunológicos/farmacologia , RNA Mensageiro/genética , Vacinas de mRNARESUMO
Freeze-dried formulations of chitosan can be solubilized in platelet-rich plasma (PRP) to form injectable implants that are used as an adjunct treatment during surgical repair of the rotator cuff. The purpose of the current study was to assess chitosan-PRP implant residency, test safety, and assess efficacy over standard-of-care controls in a sheep model of rotator cuff repair. The infraspinatus tendon was transected unilaterally and immediately repaired with suture anchors in 22 skeletally mature ewes. In treatment groups, formulations containing chitosan, trehalose, and calcium chloride were solubilized with autologous leukocyte-rich PRP and injected at the tendon-bone interface and on top of the repaired site (1 mL or 2 mL doses). Implant residency was assessed histologically at 1 day. Outcome measures included MRI assessment at baseline, 6 weeks, and 12 weeks, histopathology and clinical pathology. Chitosan-PRP implants were resident at the injection site at 1 day and induced recruitment of polymorphonuclear cells. The tendon gap, which corresponds to the length of abnormally hyperintense tissue attached to the humeral head, was decreased by treatment with the 2 mL dose when compared to controls at 12 weeks on MRI images. Some histological features were improved by the 2 mL dose treatment compared to controls at 12 weeks. There was no treatment-specific effect on all standard safety outcome measures, which suggests high safety. This study provides preliminary evidence on the safety and efficacy of chitosan-PRP implants in a large animal model that could potentially be translated to a clinical setting.
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Quitosana , Plasma Rico em Plaquetas , Lesões do Manguito Rotador , Animais , Feminino , Projetos Piloto , Manguito Rotador/cirurgia , Lesões do Manguito Rotador/cirurgia , Ovinos , CicatrizaçãoRESUMO
Chitosan (CS)-based polyplexes are produced by spontaneous electrostatic association with nucleic acids using CS in excess. Interactions of positively charged polyplexes, and the unbound CS, with negatively charged blood components limit the applicable dosage of such polymeric nanoparticles (NPs) and development of formulations with improved hemocompatibility and transfection efficiency is needed. Here, we introduce a strategy based on Tangential Flow Filtration (TFF) to remove unbound CS, concentrate polyplexes and subsequently coat with hyaluronic acid (HA) to improve hemocompatibility and bioactivity. Optimal TFF conditions were established. A library of HA with different molecular weights and degrees of sulfation was used at different carboxyl + sulfate to phosphate ratios for polyplex coating, bioactivity and hemocompatibility assessment. A systematic optimization of TFF conditions allowed for purification of polylpexes from excess unbound CS and subsequent coating with HA. Except for high molecular weight HA, for which macroscopic aggregation was observed, both sulfated and non-sulfated HAs resulted in small sized and homogenous coated complexes. However, sulfated HAs displayed higher stability during the second filtration process indicating their stronger binding affinity to polyplexes. Finally, we found that low molecular weight HA-coated polyplexes have equivalent silencing efficiency in vitro and improved hemocompatibility compared to uncoated polyplexes.
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Quitosana , Nanopartículas , Ácidos Nucleicos , Quitosana/química , Ácido Hialurônico/química , Nanopartículas/química , Fosfatos , TransfecçãoRESUMO
The transfection efficiency (TE) of chitosan-plasmid DNA (pDNA) polyplexes can be critically modulated by the polymer degree of deacetylation (DDA) and molecular weight (MW). This study was performed to test the hypothesis that the TE dependence on chitosan MW and DDA is related to the polyplex stability, hence their intracellular decondensation/unpacking kinetics. Major barriers to nonviral gene transfer were studied by image-based quantification. Although uptake increased with increased DDA, it did not appear to be a structure-dependent process affecting TE, nor was nuclear entry. Colocalization analysis showed that all chitosans trafficked through lysosomes with similar kinetics. Fluorescent resonant energy transfer (FRET) analysis revealed a distinct relationship between TE and polyplex dissociation rate. The most efficient chitosans showed an intermediate stability and a kinetics of dissociation, which occurred in synchrony with lysosomal escape. In contrast, a rapid dissociation before lysosomal escape was found for the inefficient low DDA chitosan whereas the highly stable and inefficient complex formed by a high MW and high DDA chitosan did not dissociate even after 24 hours. This study identified that the kinetics of decondensation in relation to lysosomal escape was a most critical structure-dependent process affecting the TE of chitosan polyplexes.
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Quitosana/química , DNA/química , DNA/metabolismo , Plasmídeos/química , Plasmídeos/genética , Transfecção/métodos , Linhagem Celular , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência , Humanos , Cinética , Lisossomos/metabolismo , Peso MolecularRESUMO
The purpose of this study was to assess the safety and efficacy of chitosan-platelet-rich plasma (PRP) hybrid implants used as an adjunct to surgical rotator cuff repair in a pivotal Good Laboratory Practice (GLP)-compliant study. The infraspinatus tendon was transected in 48 skeletally mature ewes and repaired with a transosseous-equivalent (TOE) technique. In the two treatment groups, a chitosan-PRP solution was injected at the footprint between the tendon and the bone and on top of the repaired site (2 mL or 3 mL doses, n = 12 per group). To further assess chitosan safety, a chitosan-water solution was injected at the same sites (3 mL, n = 12). Outcome measures included Magnetic Resonance Imaging (MRI) assessment and clinical pathology at 3 months and 6 months and histopathology at 6 months. The tendon gap was decreased at 3 months on MRI images and certain histopathological features were improved at 6 months by chitosan-PRP treatment compared to controls. The group treated with chitosan-water was not different from controls. Chitosan-PRP treatment induced no negative effects in the sheep, which suggests high safety. This study provides further evidence on the safety and efficacy of chitosan-PRP for rotator cuff repair augmentation, which could eventually be used in a clinical setting.
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Vaccination is a key component of public health policy with demonstrated cost-effective benefits in protecting both human and animal populations. Vaccines can be manufactured under multiple forms including, inactivated (killed), toxoid, live attenuated, Virus-like Particles, synthetic peptide, polysaccharide, polysaccharide conjugate (glycoconjugate), viral vectored (vector-based), nucleic acids (DNA and mRNA) and bacterial vector/synthetic antigen presenting cells. Several processes are used in the manufacturing of vaccines and recent developments in medical/biomedical engineering, biology, immunology, and vaccinology have led to the emergence of innovative nucleic acid vaccines, a novel category added to conventional and subunit vaccines. In this review, we have summarized recent advances in vaccine technologies and platforms focusing on their mechanisms of action, advantages, and possible drawbacks.
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OBJECTIVE: Homogeneity is a notion used to describe images in various fields and is often linked to critical aspects of those fields. However, this term is rarely defined in the literature and no gold standard exists for its quantification. A few quantification algorithms have been proposed, but they lack both simplicity and robustness. As a result, the scientific community uses the notion of homogeneity in subjective analysis, preventing objective comparison of a large number of data or of different studies. The main objectives of this manuscript are to propose a definition of homogeneity and an algorithm for its quantification. METHOD: This algorithm, called MASQH, rely on a multi-scale, statistical and segmentation-free approach and outputs a single homogeneity index, which makes it robust and easy to use. RESULTS: The performance and reliability of the method are demonstrated through three case studies: Firstly, on synthetic images to study the behavior and assess the relevance of the algorithm in diverse situations and hence, in various potential fields. Secondly, on histological images derived from experimental chitosan-platelet-rich-plasma hybrid biomaterial, where the quantitative results are compared to a qualitative classification provided by an expert in the field. Thirdly, on experimental nanocomposites images for which results are compared to two other homogeneity quantification algorithms from the field of nanocomposites. CONCLUSION AND SIGNIFICANCE: By quantifying homogeneity, the MASQH method may help to compare disparate studies in the literature and quantitatively demonstrate the impact of homogeneity in various fields. The MASQH method is freely available online.
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Chitosan-based nanoparticles have been extensively studied for the delivery of nucleic acids. Previous results suggest that these nanoparticles have limited ability to escape the endosome, one of the main cellular barriers hindering nucleic acid delivery. Escape can be improved by the addition of endosomolytic agents during the formulation process or by developing delivery systems with intrinsic properties to disrupt endosomal membranes. In this study, Poly(2-Propylacrylic Acid) (PPAA), an anionic synthetic polymer with known membrane lytic activity was added to the binary chitosan/mRNA nanoparticles to improve bioactivity. The ionization behavior of PPAA was characterized to identify conditions in which PPAA is sufficiently charged to interact electrostatically with chitosan and thus form nanoparticles. The physicochemical characteristics (hydrodynamic diameter, polydispersity index, ζ-potential) and the in vitro transfection efficiency (bioactivity) of this new family of CS/mRNA/PPAA ternary nanoparticles were evaluated. The addition of PPAA to CS/mRNA nanoparticles was shown to be an efficient strategy to augment in vitro bioactivity. The optimal formulation reached an expression level ~86% of the commercial lipid control at pH 6.5 without any signs of metabolic toxicity. In this paper, we report the effect of salt and pH on the ionization behavior of PPAA and demonstrate 1) successful incorporation of PPAA into/onto nanoparticles, 2) improved bioactivity with PPAA, and 3) that the kosmotropic effects of trehalose play a minimal role in the apparent increase in bioactivity in presence of trehalose.
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Quitosana , Nanopartículas , Resinas Acrílicas , RNA Mensageiro/genéticaRESUMO
Chitosan (CS) and Platelet-Rich Plasma (PRP) both display interesting properties for wound healing applications. A hybrid CS-PRP biomaterial was previously developped, consisting of a freeze dried CS formulation solubilized in PRP that promotes tissue repair and regeneration. The purpose of the current study was to investigate the ability of the CS-PRP biomaterial to stimulate cell migrationin vitro. Scratch assays revealed that CS-PRP significantly stimulates the migration rate of cells compared to cells in culture medium but not differently than PRP alone. The increase in the migration rate is dose-dependent at low dose and reaches a plateau corresponding with maximum cell motility. Cell migration rate as a function of the number of platelets that have degranulated in culture medium (to which total concentration of growth factors contributing to cell response is proportionnal), follows a modified Hill model. To analyze photographs taken during the assay and follow cell migration, an open source image analysis algorithm was developed: SAMScratch (Systematic Area Measurement of Scratch - available here:https://github.com/Biomaterials-and-Cartilage-Laboratory/SAM-Scratch). Compared with other existing analysis tools, the algorithm is precise in the determination of the scratch area and performs equally well with usual and challenging images. This study resulted in the creation of a freely available application for scratch assay analysis and provided evidence that CS-PRP implants hold promise for treatment of wounds.
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Fibroblastos , Algoritmos , Materiais Biocompatíveis/farmacologia , Movimento Celular , Quitosana , Meios de Cultura , Plasma Rico em PlaquetasRESUMO
Messenger RNA (mRNA)-containing nanoparticles were produced by electrostatic complexation with a library of pharmaceutical grade chitosans with different degrees of deacetylation and hyaluronic acids (HAs) (native vs. sulfated). Polymer length (Mn), HA degree of sulfation (DS), and amine to phosphate to carboxyl + sulfate (from HA) ratio (N:P:C) were controlled. In vitro transfections were performed in the presence/absence of trehalose and at different pH. Particle size and ζ-potential were correlated with transfection efficiency. Polymer length and charge densities (degree of deacetylation, degree of sulfation) of both HA and chitosan had a direct influence on transfection efficiency through modulation of avidity to mRNA. N:P:C ratio, trehalose, mixing concentration, and nucleic acid dose influenced transfection efficiency with optimized formulations reaching â¼60%-65% transfection efficiency relative to commercially available lipid control with no apparent toxicity for transfection at slightly acidic pH 6.5.
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Quitosana , Nanopartículas , Ácido Hialurônico , Peso Molecular , Tamanho da Partícula , RNA Mensageiro/genética , TransfecçãoRESUMO
The interaction of chitosan with plasmid DNA was investigated as a function of pH, buffer composition, degree of deacetylation (DDA), and molecular weight (M(n)) of chitosan, using isothermal titration microcalorimetry (ITC). The Single Set of Identical Sites model was used to obtain the enthalpy of interaction, the binding constant, and the stoichiometry of binding. The chitosan-DNA interaction was shown to be coupled with proton transfer from the buffer to chitosan, as revealed by the dependence of the measured heat release on the ionization enthalpy of the buffer. The measured enthalpy of binding was almost entirely due to proton transfer, because it was accounted for by the enthalpy of ionization of the buffer and of chitosan once the number of protons transferred was calculated. This proton transfer during binding resulted in the protonation of an additional 17, 37, and 58% of total glucosamine units at pH 5.5, 6.5, and 7.4, respectively. The strong polyanionic nature of DNA facilitates the ionization of glucosamines of chitosan upon complexation and is responsible for proton transfer. Interestingly, using the chitosan-DNA stoichiometry provided by ITC and the calculated degree of ionization of chitosan in the complex, the charge ratio of protonated amines to negative phosphate groups in the complex was nearly constant at 0.50-0.75 after saturation and was independent of the pH, buffer type and chitosan molecular characteristics. The chitosan-DNA binding constant was in the range of 10(9)-10(10) M(-1). The binding constant was pH-dependent and was greater at lower pH due to increased electrostatic attraction to DNA when chitosan is highly charged. Furthermore, the DDA and molecular weight of chitosan exerted a great influence on binding affinity which increased by almost an order of magnitude with an increase of the latter from 7 to 153 kDa. The binding affinity did not change significantly with DDA from 72 to 80% when the M(n) was kept constant near 80 kDa, but it increased substantially with DDA from 80 to 93% to reach a value similar to that obtained with chitosan of M(n) = 153 kDa and 80% DDA. These results provide insight into the previously reported dependence of the transfection efficiency of DNA/chitosan complexes on chitosan DDA and molecular weight, where complex stability and chitosan-DNA binding strength play a critical role.
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Calorimetria/métodos , Quitosana/química , DNA/química , Concentração de Íons de Hidrogênio , Concentração Osmolar , TermodinâmicaRESUMO
BACKGROUND: Platelet-rich plasma (PRP) has been used to solubilize freeze-dried chitosan (CS) formulations to form injectable implants for tissue repair. OBJECTIVE: To determine whether the in vitro performance of the formulations depends on the type of PRP preparation used to solubilize CS. METHODS: Formulations containing 1% (w/v) CS with varying degrees of deacetylation (DDA 80.5-84.8%) and number average molar mass (Mn 32-55 kDa), 1% (w/v) trehalose and 42.2 mM calcium chloride were freeze-dried. Seven different PRP preparations were used to solubilize the formulations. Controls were recalcified PRP. RESULTS: CS solubilization was achieved with all PRP preparations. CS-PRP formulations were less runny than their corresponding PRP controls. All CS-PRP formulations had a clotting time below 9 minutes, assessed by thromboelastography, while the leukocyte-rich PRP controls took longer to coagulate (>32 min), and the leukocyte-reduced PRP controls did not coagulate in this dynamic assay. In glass culture tubes, all PRP controls clotted, expressed serum and retracted (43-82% clot mass lost) significantly more than CS-PRP clots (no mass lost). CS dispersion was homogenous within CS-PRP clots. CONCLUSIONS: In vitro performance of the CS-PRP formulations was comparable for all types of PRPs assessed.
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Materiais Biocompatíveis/química , Quitosana/química , Plasma Rico em Plaquetas/química , Materiais Biocompatíveis/administração & dosagem , Coagulação Sanguínea , Quitosana/administração & dosagem , Liofilização , Humanos , Injeções , Masculino , Próteses e Implantes , Solubilidade , CicatrizaçãoRESUMO
Recently, chitosan dissolved in solutions containing glycerol phosphate (GP) were found to undergo a sol-gel transition when heated and the proposed gelling mechanism was based on increasing hydrophobic interactions with temperature. Subsequently, an investigation of ionization and precipitation behavior of chitosan, including dependencies on temperature, added salt, and fraction of deacetylated monomers (fD) was performed. This latter study revealed important differences in the temperature dependence of pKa of chitosan versus GP and led us to propose an alternative hypothesis for the mechanism of gelation in chitosan-GP systems whereby heat induces transfer of protons from chitosan to glycerol phosphate thereby neutralizing chitosan and allowing attractive interchain forces to form a physical gel. To investigate this specific molecular thermogelling mechanism, temperature ramp experiments on dilute chitosan-GP solutions were performed. Chitosans with fD of 0.72 and 0.98 were used to prepare solutions with a range of molar ratios of GP to chitosan glucosamine monomer of 1.25 to 10 and with 0 or 150 mM added monovalent salt. Light transmittance measurements were performed simultaneously to indicate precipitation in these dilute systems as a surrogate for gelation in concentrated systems. Measured temperatures of precipitation ranged from 15 to 85 degrees C, where solutions with less GP (used in a disodium salt form) had lower precipitation temperatures. A theoretical model using acid-base equilibria with temperature dependent pKa's, including the electrostatic contribution from the polyelectrolyte nature of chitosan, was used to calculate the degree ionization of chitosan (alpha, the fraction of protonated glucosamine monomer) as a function of temperature and showed a significant decrease in alpha with increased temperature due to proton transfer from chitosan to GP. This heat-induced proton transfer from chitosan to GP was experimentally confirmed by 31P NMR measurements during temperature ramp experiments since the chemical shift of 31P of GP is an indicator of its level of protonation. By assuming average temperature independent values of alpha p that were calculated from measured T(p), the model was able to accurately predict measured temperatures of precipitation (T(p)) of all chitosan-GP mixtures. The resulting alpha(p) were temperature independent but increased with increased chitosan fD and with increased salt. Measurements and theory revealed that T(p) can be adjusted in a predictable manner by changing the chitosan-GP molar ratio and thereby systematically tailored to obtain a large range of precipitation temperatures. Finally, similar temperature ramp experiments using inorganic phosphate and MES in place of GP demonstrated that the temperature-induced precipitation of chitosan also occurs with these buffers, confirming that the key feature of the buffer used with chitosan is its ability to absorb heat-stimulated release of chitosan protons and facilitate chitosan neutralization. A theoretical expression for the variation of chitosan ionization degree with temperature in a system composed of two titratable species (chitosan and buffer) was derived and allowed us to establish the required characteristics of the buffer for efficient heat-stimulated proton transfer between a chitosan and the buffer. These results provide a useful explanation for the mechanism of heat-induced gelation of chitosan-based systems that could be exploited for numerous practical applications.