ABSTRACT
To alleviate concerns in health security, emergency flu vaccine stockpiles are required for ensuring rapid availability of vaccines when needed. Cold chain preservation, at high cost and risk, is necessary to maintain vaccine efficacy. This study aimed to develop a dry, easily storable formula for influenza vaccine preparation. The formulation with mucoadhesive properties is expected to facilitate rapid delivery via nasal administration. Chitosan, a cationic polymer, was used as cryo-protectant and to promote mucoadhesion. Optimal concentrations and molecular weights of chitosan polymers were screened, with short chain chitosan (10 kDa) being most suitable. H1N1 dry powder, in different formulations, was prepared via freeze-drying. A series of cryo-protectants, trehalose (T), chitosan (C), fetal bovine serum (FBS; F), or a combination of these (TCF), were screened for their effects on prolonging vaccine shelf life. Physicochemical monitoring (particle size and zeta potential) of powders complexed with mucin revealed that the order of cryo-protectant mixing during preparation was of critical importance. Results indicated that the TCF formula retains its activity up to 1 year as indicated by TCID50 analysis. This approach was also successful at prolonging the shelf life of H3N2 vaccine, and has the potential for large-scale implementation, especially in developed countries where long-term storage of vaccines is problematic.
Subject(s)
Cell Adhesion/drug effects , Freeze Drying/standards , Influenza Vaccines/chemistry , Refrigeration/standards , Administration, Intranasal , Animals , Cell Adhesion/physiology , Cell Survival/drug effects , Cell Survival/physiology , Chick Embryo , Dogs , Dose-Response Relationship, Drug , Drug Compounding , Drug Storage/methods , Drug Storage/standards , Freeze Drying/methods , Influenza A Virus, H1N1 Subtype/drug effects , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H3N2 Subtype/drug effects , Influenza A Virus, H3N2 Subtype/immunology , Influenza Vaccines/administration & dosage , Madin Darby Canine Kidney Cells , Particle Size , Powders , Refrigeration/methodsABSTRACT
Sericin protein (SP) is widely used as a nutrient biomaterial for biomedical and cosmeceutical applications although it shows low stability to heat and light. To overcome these problems and add value to wastewater from the silk industry, sericin protein was recovered as sericin-loaded copolymer-liposomes (SP-PVA-LP), prepared through thin film hydration. The size and morphology of the liposomes were investigated using dynamic light scattering (DLS), and electron microscopy (SEM and TEM). The particle size, liposome surface morphology and encapsulation efficiency of SP were dependent on PVA concentration. The hydrodynamic size of the nanoparticles was between 200 and 400nm, with the degree of negative charge contingent on sericin loading. SEM and TEM images confirmed the mono-dispersity, and spherical nature of the particles, with FTIR measurements confirming the presence of surface bound PVA. Exposure of liposomes to 500ppm sericin highlighted a dependence of encapsulation efficiency on PVA content; 2% surface PVA proved the optimal level for sericin loading. Cytotoxicity and viability assays revealed that SP-loaded surface modified liposomes promote cellular attachment and proliferation of human skin fibroblasts without adverse toxic effects. Surface modified copolymer liposomes show high performance in maintaining structural stability, and promoting enhancements in the solubility and bio-viability of sericin. Taken together, these biocompatible constructs allow for effective controlled release, augmenting sericin activity and resulting in effective drug delivery systems.
Subject(s)
Liposomes/chemistry , Polymers/chemistry , Polyvinyl Alcohol/chemistry , Sericins/chemistry , Biocompatible Materials/chemistry , Biocompatible Materials/pharmacokinetics , Biocompatible Materials/pharmacology , Cell Line , Cell Survival/drug effects , Chemical Phenomena , Drug Liberation , Drug Stability , Fibroblasts/cytology , Fibroblasts/drug effects , Humans , Liposomes/pharmacokinetics , Liposomes/ultrastructure , Microscopy, Confocal , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Nanoparticles/chemistry , Nanoparticles/ultrastructure , Particle Size , Silk , Spectroscopy, Fourier Transform Infrared , Textile Industry , Wastewater/chemistryABSTRACT
This study emphasizes the development of a novel surface modified liposome as an anticancer drug nanocarrier. Quaternized N,O-oleoyl chitosan (QCS) was synthesized and incorporated into liposome vesicles, generating QCS-liposomes (Lip-QCS). The Lip-QCS liposomes were spherical in shape (average size diameter 171.5±0.8nm), with a narrow size distribution (PDI 0.1±0.0) and zeta potential of 11.7±0.7mV. In vitro mucoadhesive tests indicated that Lip-QCS possesses a mucoadhesive property. Moreover, the presence of QCS was able to induce the cationic charge on the surface of liposome. Cellular internalization of Lip-QCS was monitored over time, with the results revealing that the cell entry level of Lip-QCS was elevated at 24h. Following this, Lip-QCS were then employed to load cisplatin, a common platinum-containing anti-cancer drug, with a loading efficiency of 27.45±0.78% being obtained. The therapeutic potency of the loaded Lip-QCS was investigated using a 3D spheroid cervical cancer model (SiHa) which highlighted their cytotoxicity and apoptosis effect, and suitability as a controllable system for sustained drug release. This approach has the potential to assist in development of an effective drug delivery system against cervical cancer.
Subject(s)
Antineoplastic Agents/administration & dosage , Chitosan/chemistry , Cisplatin/administration & dosage , Drug Delivery Systems , Nanostructures/chemistry , Phospholipids/chemistry , Uterine Cervical Neoplasms/drug therapy , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Cell Proliferation/drug effects , Cisplatin/pharmacology , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Female , Humans , Liposomes/chemistry , Molecular Structure , Structure-Activity Relationship , Uterine Cervical Neoplasms/pathologyABSTRACT
Biomimetic nanofibrous scaffolds derived from natural biopolymers for bone tissue engineering applications require good mechanical and biological performances including biomineralization. The present work proposes the utility of chitin whisker (CTWK) to enhance mechanical properties of chitosan/poly(vinyl alcohol) (CS/PVA) nanofibers and to offer osteoblast cell growth with hydroxyapatite (HA) mineralization. By using diacid as a solvent, electrospun CS/PVA nanofibrous membranes containing CTWK can be easily obtained. The dimension stability of nanofibrous CS/PVA/CTWK bionanocomposite is further controlled by exposing to glutaraldehyde vapor. The nanofibrous membranes obtained allow mineralization of HA in concentrated simulated body fluid resulting in an improvement of Young's modulus and tensile strength. The CTWK combined with HA in bionanocomposite is a key to promote osteoblast cell adhesion and proliferation. The present work, for the first time, demonstrates the use of CTWKs for bionanocomposite fibers of chitosan and its hydroxyapatite biomineralization with the function in osteoblast cell culture. These hydroxyapatite-hybridized CS/PVA/CTWK bionanocomposite fibers (CS/PVA/CTWK-HA) offer a great potential for bone tissue engineering applications.
Subject(s)
Bone and Bones/cytology , Chitin/chemistry , Chitosan/chemistry , Durapatite/chemistry , Nanocomposites/chemistry , Nanofibers/chemistry , Tissue Engineering/methods , 3T3 Cells , Animals , Biocompatible Materials/chemistry , Biocompatible Materials/pharmacology , Cell Proliferation/drug effects , Cell Survival/drug effects , Mechanical Phenomena , Mice , Osteoblasts/cytology , Osteoblasts/drug effects , Polyvinyl Alcohol/chemistryABSTRACT
Monocarboxylic acids are common solvents for chitosan to fabricate nanofibers however the unpleasant odor and the additional step of fiber stabilization using crosslinkers, which might cause toxicity, are always the points to be aware of. The present work demonstrates the potential use of multicarboxylic acids as environment-friendly solvents and in situ crosslinking agents for chitosan electrospinning. The use of these solvents leads to the tunable physicochemical properties, cellular compatibility, and cost effective production. By changing di-, to tri-, and tetracarboxylic acids combining with the simple thermal treatment, the stability and mechanical properties of the nanofibrous mats, especially the elastic modulus and elongation at break, can be altered. The resulting nanofibers exhibit biocompatibility favorable for proliferation and adhesion of the osteoblast cells. The multicarboxylic acids allow us lab-scale reproducibility and possibility to semi-production of nanofibrous chitosan using Nanospider™.
Subject(s)
Biocompatible Materials/chemistry , Carboxylic Acids/chemistry , Chitosan/chemistry , Nanofibers/chemistry , Polyvinyl Alcohol/chemistry , Solvents/chemistry , Animals , Biocompatible Materials/pharmacology , Cell Line , Cell Survival/drug effects , Mice , Microscopy, Electron, Scanning , Nanofibers/toxicity , Solubility , Spectroscopy, Fourier Transform Infrared , Tensile StrengthABSTRACT
Target-specific MRI contrast agent based on super-paramagnetic iron oxide-chitosan-folic acid (SPIONP-CS-FA) nanoparticles was fabricated by using an ionotropic gelation method, which involved the loading of SPIONPs at various concentrations into CS-FA nanoparticles by electrostatic interaction. The SPIONP-CS-FA nanoparticles were characterized by ATR-FTIR, XRD, TEM, and VSM techniques. This study revealed that the advantages of this system would be green fabrication, low cytotoxicity at iron concentrations ranging from 0.52 mg/L to 4.16 mg/L, and high water stability (pH 6) at 4°C over long periods. Average particle size and positive zeta-potential of the SPIONP-CS-FA nanoparticles was found to be 130 nm with narrow size distribution and 42 mV, respectively. In comparison to SPIONP-0.5-CS nanoparticles, SPIONP-0.5-CS-FA nanoparticles showed higher and specific cellular uptake levels into human cervical adenocarcinoma cells due to the presence of folate receptors, while in vivo results (Wistar rat) indicated that only liver tissue showed significant decreases in MR image intensity on T2 weighted images and T2* weighted images after post-injection, in comparison with other organs. Our results demonstrated that SPIONP-CS-FA nanoparticles can be applied as an either tumor or organ specific MRI contrast agents.
Subject(s)
Chitosan/chemistry , Ferric Compounds/chemistry , Magnetic Resonance Imaging/methods , Magnets/chemistry , Nanoparticles/chemistry , Animals , Biological Transport , Cell Survival/drug effects , Ferric Compounds/metabolism , Ferric Compounds/toxicity , HeLa Cells , Humans , Male , RatsABSTRACT
Two guest molecules (eugenol and (-)-menthol) were investigated on inclusion complex formation with water-soluble quaternized ß-CD grafted with chitosan (QCD-g-CS). The inclusion complexes were prepared at varying mole ratios between eugenol or (-)-menthol and ß-CD (substituted on QCD-g-CS) by a conventional shaking method and obtained as solid powder by freeze-drying process. The results showed that encapsulation efficiency %EE decreased with increasing of initial eugenol or (-)-menthol loading whereas %loading increased with increasing of initial eugenol or (-)-menthol loading. The results indicated that inclusion complex formation between eugenol and QCD-g-CS was more favorable than that of (-)-menthol. To clarify this mechanism, molecular dynamics simulations were performed to explore their binding energy, solvation energy and total free energy of those complexes. It was found that the total free energy (ΔG) of eugenol and (-)-menthol against QCD-g-CS (mole ratio of 1) in water-explicit system were -2108.91 kJ/mol and -344.45 kJ/mol, respectively. Moreover, molecular dynamic simulation of eugenol absorbed on surface QCD-g-CS (-205.73 kJ/mol) was shown to have a higher negative value than that of (-)-menthol on QCD-gCS (3182.31 kJ/mol). Furthermore, the release characteristics of the encapsulated powder were also investigated in simulated saliva pH 6.8 at 32 °C. The results suggested that (-)-menthol had higher release rate from the complexes than eugenol. In all cases, the release characteristics for those guest molecules could be characterized by the limited-diffusion kinetics.
Subject(s)
Chitosan/chemistry , Eugenol/chemistry , Menthol/chemistry , beta-Cyclodextrins/chemistry , Calorimetry, Differential Scanning , Eugenol/administration & dosage , Menthol/administration & dosage , Models, Molecular , Molecular Conformation , Solubility , Spectroscopy, Fourier Transform Infrared , Thermodynamics , Water/chemistryABSTRACT
The aim of this study was to synthesize pH responsive chitosan and to evaluate the influence of drug-loaded micelle methods on loading efficiency, particle size and micelle stability. N-naphthyl-N,O-succinyl chitosan (NSCS) was successfully synthesized and meloxicam (MX) was loaded into the inner core of the NSCS micelles by physical entrapment methods (dialysis, O/W emulsion, dropping and evaporation) with a regular spherical shape (particle size 84-382nm). MX-loaded micelles by evaporation method showed the highest entrapment efficiency. The stability of the drug-loaded micelles depended on not only the methods but also the initial of drug. NSCS micelles are less toxic on Caco-2 cells. In acidic medium at 0-2h, percentage cumulative release of MX from MX-loaded micelles was similar to free drug. When the pH was adjusted to pH 6.8, the MX release was increased significantly. Therefore, this NSCS micelle would be desirable to develop MX carrier for oral drug delivery.
Subject(s)
Chitosan/analogs & derivatives , Chitosan/chemistry , Chitosan/chemical synthesis , Drug Carriers/chemistry , Drug Carriers/chemical synthesis , Micelles , Succinates/chemistry , Succinates/chemical synthesis , Thiazines/chemistry , Thiazoles/chemistry , Administration, Oral , Caco-2 Cells , Chemistry Techniques, Synthetic , Chitosan/toxicity , Drug Carriers/toxicity , Drug Liberation , Humans , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Meloxicam , Succinates/toxicity , Thiazines/administration & dosage , Thiazoles/administration & dosageABSTRACT
Curcumin (CM) has demonstrated safety and efficacy as a drug, but its pharmaceutical role is restricted as a result of extremely low aqueous solubility, rapid systemic elimination, inadequate tissue absorption and degradation at alkaline pH; properties that severely curtail its bioavailability. To address this issue, CM was encapsulated within pH responsive amphiphilic chitosan, resulting in the formation of 100 nm spontaneously self-assembled polymeric micelles in water. The amphiphilic chitosan, namely N-benzyl-N,O-succinyl chitosan (BSCS), was prepared by reductive N-benzylation and N,O-succinylation. The stability of micelles after being re-dispersed in water was investigated using glycine as a cryoprotectant, and the average sizes were shown to be maintained at a level lower than 200 nm for up to 4 months, at temperatures of 4°C and 25°C. In vitro drug release results showed that CM was slowly released from the micelles without any burst effect in the intestine (pH 5.5-7.4), with limited release in the stomach (pH 1.2). Cytotoxicity assays indicated that CM loaded micelles showed half maximal inhibitory concentrations (IC50) 4.7-, 3.6-, and 12.2-fold lower than that of free CM in HeLa, SiHa and C33a cervical cell lines, respectively. Cellular uptake of micelles was confirmed by confocal laser scanning microscopy and flow cytometry, with a 6-fold significant increase in the amount of CM loaded micelles compared to free CM in all cervical cancer cells. Notably, CM loaded micelles promoted an increase (30-55%) in the percentage of early apoptosis of HeLa, SiHa and C33a cells, compared to free CM. These results suggest that BSCS micelles may be a promising carrier for effective oral delivery of CM.
Subject(s)
Antineoplastic Agents/administration & dosage , Apoptosis/drug effects , Chitosan/analogs & derivatives , Curcumin/administration & dosage , Drug Carriers/chemical synthesis , Surface-Active Agents/chemical synthesis , Uterine Cervical Neoplasms/pathology , Antineoplastic Agents/pharmacology , Cell Culture Techniques , Chitosan/chemical synthesis , Chitosan/chemistry , Curcumin/pharmacology , Drug Carriers/chemistry , Drug Liberation , Drug Stability , Female , HeLa Cells , Humans , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Micelles , Microscopy, Electron, Transmission , Particle Size , Surface Properties , Surface-Active Agents/chemistry , Uterine Cervical Neoplasms/drug therapyABSTRACT
Super-paramagnetic iron oxide nanoparticles (SPIONPs) were encapsulated at various concentrations within chitosan-triphosphate (SPIONPs-CS) nanoparticles using an ionotropic gelation method. The encapsulation of SPIONPs within CS nanoparticles enhanced their dispersion ability in aqueous solution, with all particles being lower than 130 nm in size and having highly positive surface charge. The SPIONPs-CS nanoparticles exhibited crystalline structure and super-paramagnetic behavior, as seen in non-encapsulated SPIONPs. The morphology of SPIONPs-CS nanoparticles showed that they almost spherical in shape. The effect of phantom environments (culture medium and 3% agar solution) on either T1 or T2 weighted MRI was investigated using a clinical 1.5T MRI scanner. The results revealed that 3% agar solution showed relaxation values higher than the culture medium, leading to a significant decrease in the MR image intensity. Our results demonstrated that the SPIONPs-CS nanoparticles can be applied as tissue-speciï¬c MRI contrast agents.
Subject(s)
Chitosan/chemistry , Contrast Media/chemistry , Ferric Compounds/chemistry , Nanoparticles/chemistry , Cell Line , Cell Survival/drug effects , Chitosan/pharmacology , Contrast Media/pharmacology , Ferric Compounds/pharmacology , Humans , Magnetic Fields , Magnetic Resonance Imaging/methodsABSTRACT
The delivery of curcumin has been explored in the form of liposomal nanoparticles to treat various cancer cells. Since curcumin is water insoluble and an effective delivery route is through encapsulation in liposomes, which were modified with three components of DDAB, cholesterol and non-ionic surfactant. The purpose of this study was to establish a critical role of DDAB in liposomes containing curcumin at cellular response against two types of cell lines (HeLa and SiHa). Here, we demonstrate that DDAB is a potent inducer of cell uptake and cell death in both cell lines. The enhanced cell uptake was found on DDAB-containing liposome, but not on DDAB-free liposome. However, the cytotoxicity of DDAB-containing liposomes was high and needs to be optimized. The cytotoxicity of liposomal curcumin was more pronounced than free curcumin in both cells, suggesting the benefits of using nanocarrier. In addition, the anticancer efficiency and apoptosis effect of the liposomal curcumin formulations with DDAB was higher than those of DDAB-free liposomes. Therefore curcumin loaded liposomes indicate significant potential as delivery vehicles for the treatment of cervical cancers.
Subject(s)
Antineoplastic Agents/therapeutic use , Curcumin/therapeutic use , Uterine Cervical Neoplasms/drug therapy , Antineoplastic Agents/pharmacology , Cations/chemistry , Cell Death/drug effects , Cell Line, Tumor , Cell Shape/drug effects , Cell Survival/drug effects , Curcumin/pharmacology , Endocytosis/drug effects , Female , Humans , Liposomes/chemistry , Microscopy, Atomic Force , Particle Size , Static Electricity , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/ultrastructureABSTRACT
In this study two types of water-soluble ßCD grafted chitosan were synthesized and compared based on similar degree of N-substitution of ßCD moiety; QCD23-g-CS contained methylene spacer and QCDCA22-g-CS contained citric acid spacer. The QCD23-g-CS demonstrated greater eugenol (EG) encapsulation efficiency than that of QCDCA22-g-CS. The micelle-like assemblies of QCD23-g-CS led to slower release of EG while it did not observe in case of QCDCA22-g-CS. It was found that EG could absorb on chitosan backbone according to in silico modeling. Cytotoxicity of both derivatives against buccal mucosa cell is concentration-dependent. The QCDCA22-g-CS demonstrated stronger mucoadhesive response than that of QCD23-g-CS, due to hydrogen bonding according to mucin particle and SPR methods. Our results revealed that the spacer on both derivatives played an important role on binding affinity with EG, releasing profile and mucoadhesive property. These derivatives could be considered as promising carriers for mucosal delivery system.
Subject(s)
Chitosan/chemistry , Cyclodextrins/chemistry , Eugenol/chemistry , Cell Line/drug effects , Chitosan/chemical synthesis , Chitosan/pharmacology , Cyclodextrins/chemical synthesis , Cyclodextrins/pharmacology , Drug Delivery Systems , Eugenol/chemical synthesis , Humans , Hydrogen Bonding , Micelles , Mouth Mucosa/cytology , Mouth Mucosa/drug effects , Mucins/chemistry , Particle Size , Water/chemistryABSTRACT
In this study, we examined the potential of cationic nanoparticle - polyethyleneimine-introduced chitosan shell/poly (methyl methacrylate) core nanoparticles (CS-PEI) for siRNA delivery. Initially, DNA delivery was performed to validate the capability of CS-PEI for gene delivery in the human cervical cancer cell line, SiHa. siRNA delivery were subsequently carried out to evaluate the silencing effect on targeted E6 and E7 oncogenes. Physicochemical properties including size, zeta potential and morphology of CS-PEI/DNA and CS-PEI/siRNA complexes, were analyzed. The surface charges and sizes of the complexes were observed at different N/P ratios. The hydrodynamic sizes of the CS-PEI/DNA and CS-PEI/siRNA were approximately 300-400 and 400-500nm, respectively. Complexes were positively charged depending on the amount of added CS-PEI. AFM images revealed the mono-dispersed and spherical shapes of the complexes. Gel retardation assay confirmed that CS-PEI nanoparticles completely formed complexes with DNA and siRNA at a N/P ratio of 1.6. For DNA transfection, CS-PEI provided the highest transfection result. Localization of siRNA delivered through CS-PEI was confirmed by differential interference contrast (DIC) confocal imaging. The silencing effect of siRNA specific to HPV 16 E6/E7 oncogene was examined at 18 and 24h post-transfection. The results demonstrated the capacity of CS-PEI to suppress the expression of HVP oncogenes.
Subject(s)
Gene Silencing , Human papillomavirus 16/metabolism , Methylmethacrylate/chemistry , Nanoparticles/chemistry , Oncogene Proteins, Viral/biosynthesis , Papillomavirus E7 Proteins/biosynthesis , Papillomavirus Infections/metabolism , Polyethyleneimine/chemistry , RNA, Small Interfering/chemistry , Repressor Proteins/biosynthesis , Uterine Cervical Neoplasms/metabolism , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic/drug effects , Gene Expression Regulation, Neoplastic/genetics , Gene Expression Regulation, Viral/drug effects , Gene Expression Regulation, Viral/genetics , Human papillomavirus 16/genetics , Humans , Methylmethacrylate/pharmacology , Oncogene Proteins, Viral/genetics , Papillomavirus E7 Proteins/genetics , Papillomavirus Infections/genetics , Papillomavirus Infections/pathology , Papillomavirus Infections/therapy , Papillomavirus Infections/virology , Particle Size , Polyethyleneimine/pharmacology , RNA, Small Interfering/pharmacology , Repressor Proteins/genetics , Uterine Cervical Neoplasms/genetics , Uterine Cervical Neoplasms/pathology , Uterine Cervical Neoplasms/therapy , Uterine Cervical Neoplasms/virologyABSTRACT
BACKGROUND: The purpose of this study was to demonstrate the potential of magnetic poly(methyl methacrylate) (PMMA) core/polyethyleneimine (PEI) shell (mag-PEI) nanoparticles, which possess high saturation magnetization for gene delivery. By using mag-PEI nanoparticles as a gene carrier, this study focused on evaluation of transfection efficiency under magnetic induction. The potential role of this newly synthesized nanosphere for therapeutic delivery of the tryptophan hydroxylase-2 (TPH-2) gene was also investigated in cultured neuronal LAN-5 cells. METHODS: The mag-PEI nanoparticles were prepared by one-step emulsifier-free emulsion polymerization, generating highly loaded and monodispersed magnetic polymeric nanoparticles bearing an amine group. The physicochemical properties of the mag-PEI nanoparticles and DNA-bound mag-PEI nanoparticles were investigated using the gel retardation assay, atomic force microscopy, and zeta size measurements. The gene transfection efficiencies of mag-PEI nanoparticles were evaluated at different transfection times. Confocal laser scanning microscopy confirmed intracellular uptake of the magnetoplex. The optimal conditions for transfection of TPH-2 were selected for therapeutic gene transfection. We isolated the TPH-2 gene from the total RNA of the human medulla oblongata and cloned it into an expression vector. The plasmid containing TPH-2 was subsequently bound onto the surfaces of the mag-PEI nanoparticles via electrostatic interaction. Finally, the mag-PEI nanoparticle magnetoplex was delivered into LAN-5 cells. Reverse-transcriptase polymerase chain reaction was performed to evaluate TPH-2 expression in a quantitative manner. RESULTS: The study demonstrated the role of newly synthesized high-magnetization mag-PEI nanoparticles for gene transfection in vitro. The expression signals of a model gene, luciferase, and a therapeutic gene, TPH-2, were enhanced under magnetic-assisted transfection. An in vitro study in neuronal cells confirmed that using mag-PEI nanoparticles as a DNA carrier for gene delivery provided high transfection efficiency with low cytotoxicity. CONCLUSION: The mag-PEI nanoparticle is a promising alternative gene transfection reagent due to its ease of use, effectiveness, and low cellular toxicity. The mag-PEI nanoparticle is not only practical for gene transfection in cultured neuronal cells but may also be suitable for transfection in other cells as well.
Subject(s)
Imines/chemistry , Magnetite Nanoparticles/chemistry , Neuroblastoma/genetics , Neuroblastoma/therapy , Polyethylenes/chemistry , Polymethyl Methacrylate/chemistry , Transfection/methods , Cell Line, Tumor , Cell Survival , Electrophoretic Mobility Shift Assay , Humans , Medulla Oblongata/chemistry , Medulla Oblongata/enzymology , Neuroblastoma/chemistry , Particle Size , Reverse Transcriptase Polymerase Chain Reaction , Tryptophan Hydroxylase/genetics , Tryptophan Hydroxylase/metabolismABSTRACT
RNA interference (RNAi) is a promising strategy to combat shrimp viral pathogens at lab-scale experiments. Development of effective orally delivered agents for double-stranded (ds)RNA is necessary for RNAi application at farm level. Since continuous shrimp cell lines have not been established, we are developing a dsRNA-delivery system in Spodoptera frugiperda (Sf9) cells for studying in vitro RNAi-mediated gene silencing of shrimp virus. Sf9 cells challenged with yellow head virus (YHV) were used for validating nanoparticles as effective dsRNA carriers. Inexpensive and biodegradable polymers, chitosan and its quarternized derivative (QCH4), were formulated with long dsRNA (>100 bp) targeting YHV. Their morphology and physicochemical properties were examined. When treated with chitosan- and QCH4-dsRNA complexes, at least 50% reduction in YHV infection in Sf9 cells relative to the untreated control was evident at 24h post infection with low cytoxicity. Inhibitory effects of chitosan- and QCH4-dsRNA complexes were comparable to that of dsRNA formulated with Cellfectin(®), a commercial lipid-based transfection reagent. The natural and quaternized chitosan prepared in this study can be used for shrimp virus-specific dsRNA delivery in insect cultures, and have potential for future development of dsRNA carriers in shrimp feed.
Subject(s)
Chitosan/chemistry , Gene Silencing , Nanocapsules/chemistry , RNA, Double-Stranded/administration & dosage , RNA, Double-Stranded/genetics , Roniviridae/genetics , Transfection/methods , Animals , Nanocapsules/ultrastructure , Roniviridae/drug effects , Sf9 Cells , SpodopteraABSTRACT
Inclusion complex between water-soluble ßCD-grafted chitosan derivatives (QCD-g-CS) and eugenol (EG) was investigated as a new type of mucoadhesive drug carrier. The QCD-g-CSs were synthesized with various ßCD moieties ranging from 5 to 23%. Spontaneous inclusion complex of these derivatives and EG were found and confirmed by FTIR and simulation study. Self-aggregated formations of QCD-g-CS were found, according to fluorescence and TEM studies, where the formations were preferable for QCD11g-CS and QCD5-g-CS. EG can be included in both ßCD hydrophobic cavity and hydrophobic core of QCD-g-CS self-aggregates, resulting in varying entrapment efficiencies. Degree of QCD substitution on QCD-g-CS plays an important role on their physical properties, due to steric hindrance. The QCD11-g-CS showed excellent mucoadhesion, compared to the QCD5-g-CS and QCD23-g-CS. Moreover, the inclusion complex between QCD-g-CS and EG tend to express higher antimicrobial activities against Candida albicans, Streptococcus oralis and Streptococcus mutans, than the native QCD-g-CS.
Subject(s)
Anti-Infective Agents/chemistry , Chitosan/chemistry , Drug Carriers/chemistry , Epoxy Compounds/chemistry , Eugenol/chemistry , Quaternary Ammonium Compounds/chemistry , beta-Cyclodextrins/chemistry , Adhesiveness , Anti-Infective Agents/administration & dosage , Candida albicans/drug effects , Candida albicans/growth & development , Drug Carriers/administration & dosage , Eugenol/administration & dosage , Mucins/chemistry , Solubility , Streptococcus mutans/drug effects , Streptococcus mutans/growth & development , Streptococcus oralis/drug effects , Streptococcus oralis/growth & development , Water/chemistryABSTRACT
Five water-soluble chitosan derivatives were carried out by quaternizing either iodomethane or N-(3-chloro-2-hydroxypropyl) trimethylammonium chloride (Quat188) as a quaternizing agent under basic condition. The degree of quaternization (DQ) ranged between 28±2% and 90±2%. The antifungal activity was evaluated by using disc diffusion method, minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) methods against Trichophyton rubrum (T. rubrum), Trichophyton mentagrophyte (T. mentagrophyte), and Microsporum gypseum (M. gypseum) at pH 7.2. All quaternized chitosans and its derivatives showed more effective against T. rubrum than M. gypseum and T. mentagrophyte. The MIC and MFC values were found to range between 125-1000 µg/mL and 500-4000 µg/mL, respectively against all fungi. Our results indicated that the quaternized N-(4-N,N-dimethylaminocinnamyl) chitosan chloride showed highest antifungal activity against T. rubrum and M. gypseum compared to other quaternized chitosan derivatives. The antifungal activity tended to increase with an increase in molecular weight, degree of quaternization and hydrophobic moiety against T. rubrum. However, the antifungal activity was depended on type of fungal as well as chemical structure of the quaternized chitosan derivatives.
Subject(s)
Antifungal Agents/chemistry , Chitosan/chemistry , Chemistry, Pharmaceutical/methods , Drug Design , Humans , Hydrocarbons, Iodinated/chemistry , Magnetic Resonance Spectroscopy/methods , Microbial Sensitivity Tests , Microsporum/metabolism , Molecular Weight , Solubility , Spectroscopy, Fourier Transform Infrared/methods , Tinea/drug therapy , Trichophyton/metabolismABSTRACT
The complexation between two isomers of citral in lemongrass oil and varying types of cyclodextrins (CDs), α-CD, ß-CD, and HP-ß-CD, were studied by molecular modeling and physicochemical characterization. The results obtained revealed that the most favorable complex formation governing between citrals in lemongrass oil and CDs were found at a 1:2 mole ratio for all CDs. Complex formation between E-citral and CD was more favorable than between Z-citral and CD. The thermal stability of the inclusion complex was observed compared to the citral in the lemongrass oil. The release time course of citral from the inclusion complex was the diffusion control, and it correlated well with Avrami's equation. The release rate constants of the E- and Z-citral inclusion complexes at 50 °C, 50% RH were observed at 1.32×10(-2) h(-1) and 1.43×10(-2) h(-1) respectively.
Subject(s)
Chemical Phenomena , Cyclodextrins/chemistry , Models, Molecular , Monoterpenes/chemistry , Plant Oils/chemistry , Terpenes/chemistry , Acyclic Monoterpenes , Capsules , Drug Stability , Stereoisomerism , Temperature , Thermodynamics , Water/chemistryABSTRACT
Water-soluble ß-cyclodextrin grafted with chitosan (CD-g-CS) was carried out by quaternizing the CD-g-CS with glycidyltrimethyl ammonium chloride (GTMAC) under mild acidic condition, corresponding to the quaternized CD-g-CS (QCD-g-CS). The degrees of substitution (DS) and quaternization (DQ), ranging from 5% to 23% and 66% to 80%, respectively, were determined by (1)H NMR spectroscopy. Self-aggregates formation of all QCD-g-CSs were investigated in water using dynamic light scattering (DLS), atomic force microscopy (AFM), and transmission electron microscopy (TEM) techniques. The result revealed that all QCD-g-CSs are able to form self-aggregates in water. Large particle sizes ranged from 800 to 3000nm were obtained by DLS while zeta-potentials were ranging from 25 to 40mV. AFM and TEM depicted a spherical shape with particle sizes ranging from 100 to 900nm. Mucoadhesive and cytotoxic properties of all QCD-g-CSs were evaluated using a mucin particle method and MTT assay compared to quaternized chitosan (QCS). It was found that the mucoadhesive property increased with decreasing DS due to less quaternary ammonium moiety into the chitosan backbone. On the other hand, the cytotoxicity increased with increasing DS even though the DQ is decreased.