Abstract
Objective(s):
Although viral vectors are considered efficient gene transfer agents, their board application has been limited by toxicity, immunogenicity, mutagenicity and small gene carrying capacity. Non-viral vectors are safe but they suffer from low transfection efficiency. In the present study, polyallylamine (PAA) in two molecular weights (15 and 65 kDa) was modified by alkane derivatives in order to increase transfection activity and to decrease cytotoxicity.
Materials and Methods:
Modified PAA was synthesized using three alkane derivatives (1-bromobutane, 1-bromohexane and 1-bromodecane) in different grafting percentages (10, 30 and 50). The condensation ability of modified PAA was determined by ethidium bromide test. The prepared polyplexes, complexes of modified PAA and DNA, were characterized by size and zeta potential. Transfection activity of polyplexes was checked in Neuro2A cells. The cytotoxicity of vector was examined in the same cell line.
Results:
DNA condensation ability of PAA was decreased after modification but modified polymer could still condense DNA at moderate and high carrier to plasmid (C/P) ratios. Most of polyplexes composed of modified polymer had mean size less than 350 nm. They showed a positive zeta potential, but some vectors with high percentage of grafting had negative surface charge. Transfection efficiency was increased by modification of PAA by 1-bromodecane in grafting percentages of 30 and 50%. Modification of polymer reduced polymer cytotoxicity especially in C/P ratio of 2.
Conclusion:
Results of the present study indicated that modification of PAA with alkane derivatives can help to prepare gene carriers with better transfection activity and less cytotoxicity.
Abstract
Abstract
Among synthetic carriers, dendrimers with the more flexible structure have attracted a great deal of researchers’ attention in the field of gene delivery. Followed by the promising results upon hydrophobic modification on polymeric structures in our laboratory, alkylcarboxylated poly (propylenimine)-based carriers were synthesized by nucleophilic substitution of amines with alkyl moieties and were further characterized for their physicochemical and biological characteristics for plasmid DNA delivery. Although not noticeably effective gene transfer activity for hexanoate- and hexadecanoate-modified series was observed, but alkylation by decanoic acid significantly improved the transfection efficiency of the final constructs up to 60 fold in comparison with unmodified poly(propylenimine) (PPI). PPI modified by 10-bromodecanoic acid at 50% grafting, showed significantly higher gene expression at c/p ratio of 2 compared to Superfect as positive control.
Overall, modification of PPI with 50% primary amines grafting with 10-bromodecanoic acid could increase the transfection efficiency which is occurred at lower c/p ratio when compared to Superfect, i.e. less amount of modified vector is required to exhibit the same efficiency as Superfect. Therefore, the obtained constructs seem to be safer carriers for long-term gene therapy applications.
Abstract
Abstract
Among synthetic carriers, dendrimers with the more flexible structure have attracted a great deal of researchers’ attention in the field of gene delivery. Followed by the promising results upon hydrophobic modification on polymeric structures in our laboratory, alkylcarboxylated poly (propylenimine)-based carriers were synthesized by nucleophilic substitution of amines with alkyl moieties and were further characterized for their physicochemical and biological characteristics for plasmid DNA delivery. Although not noticeably effective gene transfer activity for hexanoate- and hexadecanoate-modified series was observed, but alkylation by decanoic acid significantly improved the transfection efficiency of the final constructs up to 60 fold in comparison with unmodified poly(propylenimine) (PPI). PPI modified by 10-bromodecanoic acid at 50% grafting, showed significantly higher gene expression at c/p ratio of 2 compared to Superfect as positive control.
Overall, modification of PPI with 50% primary amines grafting with 10-bromodecanoic acid could increase the transfection efficiency which is occurred at lower c/p ratio when compared to Superfect, i.e. less amount of modified vector is required to exhibit the same efficiency as Superfect. Therefore, the obtained constructs seem to be safer carriers for long-term gene therapy applications.
Effective in vitro gene delivery to murine cancerous brain cells using carbon...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
Carbon nanotube (CNT) has been widely applied at molecular and cellular levels due to its exceptional properties. Studies based on conjugation of CNTs with biological molecules indicated that biological activity is preserved. Polyethylenimine (PEI) is explored in designing novel gene delivery vectors due to its ability to condense plasmid DNA through electrostatic attraction. In this study functionalization and grafting polyethylenimine onto the surface of carbon nanotube was used to improve the solubility and biocompatibility.
Materials and Methods:
The effect of molecular weight of polymer on final efficacy of vectors has been investigated using three different molecular weights of polymer. In this study no linker was used and both segments (PEI and CNT) were directly attached resulted in the synthesis of three different vectors. Synthesized vectors were tested for their ability to condense plasmid DNA and cellular toxicity using ethidium bromide and MTT assays. Size and Zeta potential of nanoparticles was determined using Malvern zeta sizer. Evaluation of transfection efficiency of vectors was carried out on N2A cell line by different methods including qualitative fluorescence imaging, flow cytometry and luciferase assay.
Results:
All three synthesized vectors bear positive surface charges with sizes in the range of 85-190 nm. More than 80 percent of treated cells were viable and in the case of V25 significant improvement in reducing cytotoxicity compared to unmodified polymer was observed. Obtained results indicated that vector containing PEI 1.8 kDa has the greatest improvement in terms of its transfection efficiency compared to unmodified polymer.
Conclusion:
Conjugation of PEI with carbon nanotube les to new vectors with lowered cytotoxicity and higher transfection efficiency. The highest transfection efficiency was obtained with the lowest molecular weight PEI.
Effective in vitro gene delivery to murine cancerous brain cells using carbon...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
Carbon nanotube (CNT) has been widely applied at molecular and cellular levels due to its exceptional properties. Studies based on conjugation of CNTs with biological molecules indicated that biological activity is preserved. Polyethylenimine (PEI) is explored in designing novel gene delivery vectors due to its ability to condense plasmid DNA through electrostatic attraction. In this study functionalization and grafting polyethylenimine onto the surface of carbon nanotube was used to improve the solubility and biocompatibility.
Materials and Methods:
The effect of molecular weight of polymer on final efficacy of vectors has been investigated using three different molecular weights of polymer. In this study no linker was used and both segments (PEI and CNT) were directly attached resulted in the synthesis of three different vectors. Synthesized vectors were tested for their ability to condense plasmid DNA and cellular toxicity using ethidium bromide and MTT assays. Size and Zeta potential of nanoparticles was determined using Malvern zeta sizer. Evaluation of transfection efficiency of vectors was carried out on N2A cell line by different methods including qualitative fluorescence imaging, flow cytometry and luciferase assay.
Results:
All three synthesized vectors bear positive surface charges with sizes in the range of 85-190 nm. More than 80 percent of treated cells were viable and in the case of V25 significant improvement in reducing cytotoxicity compared to unmodified polymer was observed. Obtained results indicated that vector containing PEI 1.8 kDa has the greatest improvement in terms of its transfection efficiency compared to unmodified polymer.
Conclusion:
Conjugation of PEI with carbon nanotube les to new vectors with lowered cytotoxicity and higher transfection efficiency. The highest transfection efficiency was obtained with the lowest molecular weight PEI.
Analysis of Peroxisomal Lipid Metabolism in the Oleaginous Microalga Nannochloropsis and Development of Synthetic Biology Tools for Genetic Engineering
Abstract
Objective(s):
Lipid-based nanoparticles (NLP) are PEGylated carriers composed of lipids and encapsulated nucleic acids with a diameter less than 100 nm. The presence of PEG in the NLP formulation improves the particle pharmacokinetic behavior. The purpose of this study was to prepare and characterize NLPs containing MDR1 siRNA and evaluate their cytotoxicity and cellular uptake. MDR1 siRNA could be used in multidrug resistance reversal in cancer therapy.
Materials and Methods:
siRNAs were encapsulated into NLPs consisted of mPEG-DSPE/DOTAP/DOPE (10:50:40 molar ratio) by the detergent dialysis method. The particle diameters of NLPs and their surface charge were measured using dynamic light scattering. siRNA encapsulation efficiency was determined by an indirect method via filtration and free siRNA concentration determination. NLPs cytotoxicity was investigated by MTT assay. The ability of NLPs for siRNA delivery checked in two human cell lines (MCF-7/ADR and EPP85-181/RDB) by fluorescence microscopy and compared with oligofectamine.
Results:
NLPs containing MDR1 siRNA were prepared with the stable size of 80-90 nm and the zeta potential near to neutral. The siRNA encapsulation efficacy was more than 80%. These properties are suitable for in vivo siRNA delivery. NLPs cytotoxicity studies demonstrated they were non-toxic at the doses used. NLPs improved siRNA localization in both cell lines.
Conclusion:
NLPs containing MDR1 siRNA can be a good candidate for in vivo siRNA delivery studies.
This is an internship report on molecular biology techniques, which was performed at PERD center under the guidance of Dr. Anshu Srivastava. This pdf contains all the basic information which is a preliminary requisite to know while approaching the molecular biology experimentally.
Genipin cross-linked electrospun chitosan-based nanofibrous mat as tissue eng...Nanomedicine Journal (NMJ)
Objective(s):
To improve water stability of electrospun chitosan/ Polyethylene oxide (PEO) nanofibers, genipin, a biocompatible and nontoxic agent, was used to crosslink chitosan based nanofibers.
Materials and Methods:
Different amounts of genipin were added to the chitosan/PEO solutions, chitosan/PEO weight ratio 90/10 in 80 % acetic acid, and the solutions were then electrospun to form nanofibers. The spun nanofibers were exposed to water vapor to complete crosslinking. The nanofibrous membranes were subjected to detailed analysis by scanning electron microscopy (SEM), Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy, swelling test, MTT cytotoxicity, and cell attachment.
Results:
SEM images of electrospun mats showed that genipin-crosslinked nanofibers retained their fibrous structure after immerging in PBS (pH=7.4) for 24 hours, while the uncrosslinked samples lost their fibrous structure, indicating the water stability of genipin-crosslinked nanofibers. The genipin-crosslinked mats also showed no significant change in swelling ratio in comparison with uncrosslinked ones. FTIR-ATR spectrum of uncrosslinked and genipin-crosslinked chitosan nanofibers revealed the reaction between genipin and amino groups of chitosan. Cytotoxicity of genipin-crosslinked nanofibers was examined by MTT assay on human fibroblast cells in the presence of nanofibers extraction media. The genipin-crosslinked nanofibers did not show any toxic effects on fibroblast cells at the lowest and moderate amount of genipin. The fibroblast cells also showed a good adhesion on genipin-crosslinked nanofibers.
Conclusion:
This electrospun matrix would be used for biomedical applications such as wound dressing and scaffold for tissue engineering without the concern of toxicity.
COMPARISON FREE ENERGY BINDING SITES NEURAMINIDASEijabjournal
Neuraminidase (NA) is the essential surface glycoprotein of the influenza virus. High- affinity neuraminidase inhibitors have been designed that interact only with the conserved active site and binding site residues. The neuraminidase (NA) of influenza virus is the target of anti – flu drug.for treatment of this
disease a thorough knowledge of neuraminidase protein is essential in order to produce potent drugs to suppress this enzyme..Drug design is by QSAR and docking methods, so we need a complete knowledge of receptor ligand, target site and binding site. This paper, using bioinformatics, Molecular Dynamics, Monte carlo and studied binding site NA enzyme in 310K temperature and different dielectrics (1, 78.39 and
32.63) for the best drug designing. We measured the potential energy of amino acids binding to the drug.Molecular Mechanics, Molecular Dynamic and Nanobiological have done a great assistance in drug designing.
DNA damage repair Neil3 gene Knockout in MOLT-4iosrjce
RNAi is superannuated cellular mechanism that protect organism against viruses that replicate
through double- stranded RNA. RNAi can be used to diminish gene expression from plasmid expressing and
inserted sequence repeat. A stable harpin would be expressed after the vector was integrated into the genome.
In this paper a shiRNA expressing vector for Neil3 was designed and developed which is capable of replication
in MOLT-4. This shiRNA vector had the ability to arose the RNAi pathway, and reduce the gene expression of
Neil3. This was assessed by using pSilence 4.1CMV as a vector, and Gapdh as positive control.
for cloning and expression of exogenous gene or gene throthrough vector it must be introduced into the host cell through transformation , ,transduction, electroporation gene gun etc.
International Journal of Pharmaceutical Science Invention (IJPSI)inventionjournals
International Journal of Pharmaceutical Science Invention (IJPSI) is an international journal intended for professionals and researchers in all fields of Pahrmaceutical Science. IJPSI publishes research articles and reviews within the whole field Pharmacy and Pharmaceutical Science, new teaching methods, assessment, validation and the impact of new technologies and it will continue to provide information on the latest trends and developments in this ever-expanding subject. The publications of papers are selected through double peer reviewed to ensure originality, relevance, and readability. The articles published in our journal can be accessed online
ShRNA-specific regulation of FMNL2 expression in P19 cellsYousefLayyous
This video encompasses all the steps and data produced for my graduation project in BSc in Biopharmaceutical science. During the course of the project we modified mammalian cells using Short Hairpin RNA to inhibit the correct function of the cytoskelleton. In this way we studied the importance of FMNL2 for the activation and regulation of actin fibers. Among the methods used are Flourescent microscopy, mamallian cell culture, cloning and flow cytometry.
Electrophoresis principle and types by Dr. Anurag YadavDr Anurag Yadav
the general principle on how the electrophoresis performs.
the different types of electrophoresis and the mechanism of separation based on different character of the medium and type of electrophoresis.
electrophoresis-
principle
types
details on paper electrophoresis
cellulose acetate electrophoresis
zone electrophoresis
SDS-PAGE
iso-electric focussing gel electrophoresis
two-dimensional gel electrophoresis
pulsed gel electrophoresis
isotachophoresis
capillary electrophoresis
microchip electrophoresis
Similar to Preparation, characterization and transfection efficiency of nanoparticles composed of alkane-modified polyallylamine (20)
Evaluation of the effect of crocetin on antitumor activity of doxorubicin enc...Nanomedicine Journal (NMJ)
Objective(s): The current study reports investigation of codelivery by PLGA nanoparticles (NPs) loaded with crocetin (Cro), a natural carotenoid dicarboxylicHYPERLINK “http://en.wikipedia.org/wiki/Carboxylic_acid” acid that is found in the crocus flower, and Doxorubicin (DOX).
Materials and Methods: Double emulsion/solvent evaporation method was used for preparation of PLGA nanoparticles containing Dox and Cro. Characterizations of prepared NPs were investigated by atomic force microscopy (AFM) and dynamic light scattering analysis. In vitro Cytotoxicity of DOX and Cro loaded PLGA NPs (PLGA-DOX-Cro) on MCF-7 cell line was evaluated using MTT test. Flow cytometry experiments were implemented to distinguish cells undergoing apoptosis from those undergoing necrosis. Furthermore the expression of caspase 3 was examined by western blot analysis.
Results: The prepared formulations had size of 150- 300 nm. Furthermore, PLGA-DOX-Cro nanoparticles inhibited MCF-7 tumor cells growth more efficiently than either DOX or Cro alone at the same concentrations, as quantified by MTT assay and flow cytometry. Studies on cellular uptake of DOX-Cro-NPs demonstrated that NPs were effectively taken up by MCF-7 tumor cells.
Conclusion: This study suggested that DOX-Cro-NPs may have promising applications in breast cancer therapy.
Effects of combination of magnesium and zinc oxide nanoparticles and heat on ...Nanomedicine Journal (NMJ)
Objective: The objective of this study was to investigate the antibacterial activities of combination of MgO and ZnO nanoparticles in the presence of heat against Escherichia coli and Staphylococcus aureus.
Materials and Methods:Bacteria were grown on either agar or broth media followed by the addition of ZnO and MgO nanoparticles. Then the combined effect of ZnO and MgO nanoparticles was investigated. Furthermore, the media containing nanoparticles were treated with mild heat and their synergistic antibacterial activity was investigated against E. coli and S. aureus in milk.
Results: The data showed that the nanoparticles used in this study had no effect on the bacteria in the agar medium. However, the results showed that ZnO and MgO nanoparticles resulted in a significant decrease in the number of E. coli (P<0.000) and S. aureus (Pd”0.05) in the broth medium. The combination of nanoparticles and mild heat exhibited a significant decrease in the number of E. coli and S. aureus indicating the synergistic effects of nanoparticles and heat.
Conclusion: Using a combination of mild heat, ZnO and MgO nanoparticles, E. coli and S. aureus can be controlled successfully in the milk. Mild heating plus ZnO and MgO nanoparticles has a synergistic effect which would reduce the need for high temperature and also the concentrations of ZnO and MgO nanoparticles required for pathogen control in minimally processed milk during maintaining.
Preparation and evaluation of electrospun nanofibers containing pectin and ti...Nanomedicine Journal (NMJ)
Objective(s):The aim of this study was to prepare electrospun nanofibers of celecoxib using combination of time-dependent polymers with pectin to achieve a colon-specific drug delivery system for celecoxib.
Materials and Methods:Formulations were produced based on two multilevel 22 full factorial designs. The independent variables were the ratio of drug:time-dependent polymer (X1) and the amount of pectin in formulations (X2). Electrospinning process was used for preparation of nanofibers. The spinning solutions were loaded in 5 mL syringes. The feeding rate was fixed by a syringe pump at 2.0 mL/h and a high voltage supply at range 10-18 kV was applied for electrospinning. Electrospun nanofibers were collected and evaluated by scanning electron microscopy and drug release in the acid and buffer with pH 6.8 with and without pectinase.
Results:Electrospun nanofibers of celecoxib with appropriate morphological properties were produced via electrospinning process. Drug release from electrospun nanofibers was very low in the acidic media; while, drug release in the simulated colonic media was the highest from formulations containing pectin.
Conclusion: Formulation F2 (containing drug:ERS with the ratio of 1:2 and 10% pectin) exhibited acceptable morphological characteristics and protection of drug in the upper GI tract and could be a good candidate as a colonic drug delivery system for celecoxib.
The combined effects of Aloe vera gel and silver nanoparticles on wound heali...Nanomedicine Journal (NMJ)
Objective(s): This study was aimed at investigating the synergy effects of Aloe vera gel and silver nanoparticles on the healing rate of the cutting wounds.
Materials and Methods: In order to determine the concentration of silver nanoparticles in Aloe vera gel, the MBC methods were applied on the most common bacteria infecting wounds, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa. The cutting wounds with Full-thickness skin were dorsally created on rats; then the rats were divided into 4 groups. The treatments groups included: mixture of Aloe vera gel and silver nanoparticles, Aloe vera gel alone and silver nanoparticles alone in addition to control groups. The treatment was carried out for 2 weeks and the size of the wound closures were measured by an image software analysis.
Results:There was no significant difference (p<0.05) in healing rate between the control and mixture group. However, there were significant differences between the silver nanoparticles and Aloe vera groups using Tukey’s analysis on the 6th, 8th and 10th days.
Conclusion:The Aloe vera gel increased the rate of wound healing whereas the silver nanoparticles had a delay effect; and when they were mixed, it was similar to the average effect of both Aloe vera gel and silver nanoparticles.
Simultaneous loading of 5-florouracil and SPIONs in HSA nanoparticles: Optimi...Nanomedicine Journal (NMJ)
Objective(s): Over the past two decades, considerable interest has been focused on utilizing biocompatible magnetic nanoparticles (MNPs) for biomedical applications. In this study, production of human serum albumin (HSA) nanoparticles using desolvation technique that were simultaneous loaded with high amounts of superparamagnetic iron oxide nanoparticles (SPIONs) and 5-flourouracil (5-FU) was investigated.
Materials and Methods: 5-FU loading (%) and SPIONs entrapment efficiency (%) were optimized using response surface methodology (RSM). The design expert software used to analyse the interactive effects of pH, 5-FU and SPIONs concentrations.
Results:The optimum conditions found to be pH of 8.2, drug concentration of 1.5 mg/ml and SPIONs concentration of 2.79 mg/ml. Under the mentioned optimum conditions, particles with the size of 111.8 nm, zeta potential of -37.1 mV, 5-FU loading of 15.8% and SPIONs entrapment efficiency of 41.1% were obtained. In vitro cumulative release of 5-FU from the nanoparticles was evaluated in phosphate buffer saline (pH 7.4, 37 °C). Results indicated that 85% of the 5-FU released during 95 h, which revealed a sustained release profile. In addition, Vibrating Sample Magnetometer (VSM) analyses confirmed the superparamagnetic properties of magnetic albumin nanoparticles manufactured under the optimum conditions.
Conclusion: According to the findings,SPIONs and 5-FU loaded HAS nanoparticles arepromising for use as novel targeted delivery system due to proper magnetic and drug release behaviours.
Antimicrobial and cytotoxicity effect of silver nanoparticle synthesized by C...Nanomedicine Journal (NMJ)
Objective(s): For the development of reliable, ecofriendly, less expensive process for the synthesis of silver nanoparticles and to evaluate the bactericidal, and cytotoxicity properties of silver nanoparticles synthesized from root extract of Croton bonplandianum, Baill.
Materials and Methods: The synthesis of silver nanoparticles by plant part of Croton bonplandianum was carried out. The formation of nanoparticles was confirmed by Transmission Electron Microscopy (TEM), Scanning Electron Microscopy (SEM), XRD and UV-Vis spectrophotometric analysis. The biochemical properties were assayed by antibacterial study, cytotoxicity assay using cancer cell line.
Results: The formation of silver nanoparticles was confirmed by UV-VIS spectroscopic analysis which showed absorbance peak at 425 nm. X-ray diffraction photograph indicated the face centered cubic structure of the synthesized AgNPs. TEM has displayed the different dimensional images of biogenic silver nanoparticles with particle size distribution ranging from 15-40 nm with an average size of 32 nm. Silver particles are spherical in shape, clustered. The EDX analysis was used to identify the elemental composition of synthesized AgNPs. Antibacterial activity of the synthesized AgNPs against three Gram positive and Gram negative bacteria strains like Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa carried out showed significant zones of inhibition. The cytotoxicity study by AgNPS also showed cytotoxicity on ovarian cancer cell line PA-1 and lung epithelial cancer cell line A549.
Conclusion: The present study confirms that the AgNPs have great promise as antibacterial, and anticancer agent.
Investigation of the effect of different parameters on the phase inversion te...Nanomedicine Journal (NMJ)
Objective(s): Nanoemulsions are a kind of emulsions that can be transparent, translucent (size range 50-200 nm) or “milky” (up to 500 nm). Nanoemulsions are adequatly effective for transfer of active component through skin which facilitate the entrance of the active component . The transparent nature of the system and lack of the thickener and fluidity are among advantages of nanoemulsion.
Materials and Methods: In this study, a nanoemulsion of lemon oil in water was prepared by the phase inversion temperature (PIT) emulsification method in which the tween 40 was used as surfactant. The effect of concentration of NaCl in aqueous phase, pH and weight percent of surfactant and aqueous on the PIT and droplet size were investigated. Results: The results showed that with increasing of concentration of NaCl from 0.05 M to 1 M, PIT decrease from 72 to 50. The average droplet sizes, for 0.1, 0.5 and 1 M of NaCl in 25 ºC are 497.3, 308.1 and 189.9 nm, respectively and the polydispersity indexes are 0.348, 0.334 and 0.307, respectively.
Conclusion: Considering the characteristics of nanoemulsions such as being transparent, endurance of solution and droplet size can provide suitable reaction environment for polymerization process used in making hygienic and medical materials.
Mechanism of oxidative stress involved in the toxicity of ZnO nanoparticles a...Nanomedicine Journal (NMJ)
ZnO NPs (zinc oxide nanoparticles) has generated significant scientific interest as a novel antibacterial and anticancer agent. Since oxidative stress is a critical determinant of ZnO NPs-induced damage, it is necessary to characterize their underlying mode of action. Different structural and physicochemical properties of ZnO NPs such as particle surface, size, shape, crystal structure, chemical position, and presence of metals can lead to changes in biological activities including ROS (reactive oxygen species) production. However, there are some inconsistencies in the literature on the relation between the physicochemical features of ZnO NPs and their plausible oxidative stress mechanism. Herein, the possible oxidative stress mechanism of ZnO NPs was reviewed. This is worthy of further detailed evaluations in order to improve our understanding of vital NPs characteristics governing their toxicity. Therefore, this study focuses on the different reported oxidative stress paradigms induced by ZnO NPs including ROS generated by NPs, oxidative stress due to the NPs-cell interaction, and role of the particle dissolution in the oxidative damage. Also, this study tries to characterize and understand the multiple pathways involved in oxidative stress induced by ZnO NPs. Knowledge about different cellular signaling cascades stimulated by ZnO NPs lead to the better interpretation of the toxic influences induced by the cellular and acellular parameters. Regarding the potential benefits of toxic effects of ZnO NPs, in-depth evaluation of their toxicity mechanism and various effects of these nanoparticles would facilitate their implementation for biomedical applications.
Combined effects of PEGylation and particle size on uptake of PLGA particles ...Nanomedicine Journal (NMJ)
Abstract
Objective:
At the present study, relationship between phagocytosis of PLGA particles and combined effects of particle size and surface PEGylation was investigated.
Materials and Methods:
Microspheres and nanospheres (3500 nm and 700 nm) were prepared from three types of PLGA polymers (non-PEGylated and PEGylation percents of 9% and 15%). These particles were prepared by solvent evaporation method. All particles were labeled with FITC-Albumin. Interaction of particles with J744.A.1 mouse macrophage cells, was evaluated in the absence or presence of 7% of the serum by flowcytometry method.
Results:
The study revealed more phagocytosis of nanospheres. In the presence of the serum, PEGylated particles were phagocytosed less than non-PEGylated particles. For nanospheres, this difference was significant (P<0/05) and their uptake was affected by PEGylation degree. In the case of microsphere formulation, PEGylation did not affect the cell uptake. In the serum-free medium, the bigger particles had more cell uptake rate than smaller ones but the cell uptake rate was not influenced by PEGylation.
Conclusion:
The results indicated that in nanosized particles both size and PEgylation degree could affect the phagocytosis, but in micron sized particles just size, and not the PEGylation degree, could affect this.
Synthesis of silver nanoparticles and its synergistic effects in combination ...Nanomedicine Journal (NMJ)
Abstract
Objectives:
Biofilms are communities of bacteria attached to surfaces through an external polymeric substances matrix. In the meantime, Acinetobacterbaumannii is the predominant species related to nosocomial infections. In the present study, the effect of silver nanoparticles alone and in combination with biocides and imipenem against planktonic and biofilms of A. baumannii was assessed.
Materials and Methods:
Minimum inhibitory concentrations (MICs) of 75 planktonic isolates of A. baumannii were determined by using the microdilution method as described via clinical and laboratory standards institute (CLSI). Among all strains, 10 isolates which formed strong biofilms were selected and exposed to silver nanoparticles alone and in combination with imipenem, bismuth ethandithiol (BisEDT) and bismuth propanedithiol (BisPDT) to determine minimum biofilm inhibitory concentrations (MBIC). Subsequently, minimum biofilm eradication concentrations (MBECs) of silver nanoparticles alone and in combination with imipenem against mature biofilm of the isolates were evaluated.
Results:
Results showed that 29.3% of isolates were susceptible to silver nanoparticles and could inhibit the growth and eradicate biofilms produced by the isolates. For this reason, ∑FIC, ∑FBIC and ∑FBEC ≤ 0.05 were reported which shows synergism between silver nanoparticles and imipenem against not only planktonic cells but also inhibition and eradication of biofilms. The results of ∑FBIC >2 indicated to antagonistic impacts between silver nanoparticles and BisEDT/BisPDT against biofilms.
Conclusion:
It can be concluded that silver nanoparticles alone can inhibit biofilm formation but in combination with imipenem are more effective against A. baumannii in planktonic and biofilm forms.
Abstract
Objective(s):
Zinc oxide nanoparticles (ZNP) are increasingly used in sunscreens, biosensors, food additives and pigments. In this study the effects of ZNP on liver of rats was investigated.
Materials and Methods:
Experimental groups received 5, 50 and 300 mg/kg ZNP respectively for 14 days. Control group received only distilled water. ALT, AST and ALP were considered as biomarkers to indicate hepatotoxicity. Lipid peroxidation (MDA), SOD and GPx were detected for assessment of oxidative stress in liver tissue. Histological studies and TUNEL assay were also done.
Results:
Plasma concentration of zinc (Zn) was significantly increased in 5 mg/kg ZNP-treated rats. Liver concentration of Zn was significantly increased in the 300 mg/kg ZNP-treated animals. Weight of liver was markedly increased in both 5 and 300 mg/kg doses of ZNP. ZNP at the doses of 5 mg/kg induced a significant increase in oxidative stress through the increase in MDA content and a significant decrease in SOD and GPx enzymes activity in the liver tissue. Administration of ZNP at 5 mg/kg induced a significant elevation in plasma AST, ALT and ALP. Histological studies showed that treatment with 5 mg/kg of ZNP caused hepatocytes swelling, which was accompanied by congestion of RBC and accumulation of inflammatory cells. Apoptotic index was also significantly increased in this group. ZNP at the dose of 300 mg/kg had poor hepatotoxicity effect.
Conclusion:
It is concluded that lower doses of ZNP has more hepatotoxic effects on rats, and recommended to use it with caution if there is a hepatological problem.
Synthesis of graphene oxide-TiO2 nanocomposite as an adsorbent for the enrich...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
In our study, graphene oxide-TiO2 nanocomposite (GO/TiO2) was prepared and used for the enrichment of rutin from real samples for the first time.
Materials and Methods:
The synthesized GO/TiO2 was characterized by X-ray diffraction, scanning electron microscopy, and FT-IR spectra. The enrichment process is fast and highly efficient. The factors including contact time, pH, and amount of GO/TiO2 affecting the adsorption process were studied.
Results:
The maximum adsorption capacity for ciprofloxacin was calculated to be 59.5 mg/g according to the Langmuir adsorption isotherm. The method yielded a linear calibration curve in the concentration ranges from 15 to 200 μg/L for the rutin with regression coefficients (r2) of 0.9990. The limits of detection (LODs, S/N=3) and limits of quantification (LOQs, S/N=10) were found to be 8 μg/Land 28 μg/L, respectively. Both the intra-day and inter-day precisions (RSDs) were < 10% .
Conclusion:
The developed approach offered wide linear range, and good reproducibility. Owing to the diverse structures and unique characteristic, GO/TiO2 possesses great potential in the enrichment and analysis of trace rutin in real aqueous samples.
Preparation and evaluation of vitamin A nanosuspension as a novel ocular drug...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
The aim of this study was to prepare a nanosuspension formulation as a new vehicle for the improvement of the ocular delivery of vitamin A.
Material and Methods:
Formulations were designed based on full factorial design. A high pressure homogenization technique was used to produce nanosuspensions. Fifteen formulations were prepared by the use of different combinations of surfactants Tween 80, benzalkonium chloride and Pluronic and evaluated for pH, particle size, entrapment efficiency, differential scanning calorimetry (DSC), stability and drug release. Also, Draize test was used to evaluate the irritation of rabbit eye by formulations.
Results:
All formulations showed a small mean size that is well suited for ocular application. Also it was observed that the particle size decreased with increase in the amount of surfactant. Drug entrapment increased with increasing amount of surfactant. It was shown that initial and final drug release can be controlled by the ratio and the total amount of surfactants, respectively.
Conclusion:
It was concluded that the use of Tween 80 and Pluronic in the formualtions with a proper ratio does not show eye irritation and could be useful to achieve a suitable nanosuspension of vitamin A as a novel ocular delivery system.
A comparative study about toxicity of CdSe quantum dots on reproductive syste...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
Medicinal benefits of quantum dots have been proved in recent years but there is little known about their toxicity especially in vivo toxicity. In order to use quantum dots in medical applications, studies ontheir in vivo toxicity is important.
Materials and Methods:
CdSe:ZnS quantum dots were injected in 10, 20, and 40 mg/kg doses to male mice10 days later, mice were sacrificed and five micron slides were prepared structural and optical properties of quantum dots were evaluated using XRD.
Results:
Histological studies of testis tissue showed high toxic effect of CdSe:ZnS in 40 mg/kg group. Histological studies of epididymis did not show any effect of quantum dots in terms of morphology and tube structure. Mean concentration of LH and testosterone and testis weight showed considerable changes in mice injected with 40 mg/kg dose of CdSe:ZnS compared to control group. However, FSH and body weight did not show any difference with control group.
Conclusion:
Although it has been reported that CdSe is highly protected from the environment by its shell, but this study showed high toxicity for CdSe:ZnS when it is used in vivo which could be suggested that shell could contribute to increased toxicity of quantum dots. Considering lack of any previous study on this subject, our study could potentially be used as an basis for further extensive studies investigating the effects of quantum dots toxicity on development of male sexual system.
Functionalization of carbon nanotubes and its application in nanomedicine: A ...Nanomedicine Journal (NMJ)
Abstract
This review focuses on the latest developments in applications of carbon nanotubes (CNTs) in medicine. A brief history of CNTs and a general introduction to the field are presented.
Then, surface modification of CNTs that makes them ideal for use in medical applications is highlighted. Examples of common applications, including cell penetration, drug delivery, gene delivery and imaging, are given. At the same time, there are concerns about their possible adverse effects on human health, since there is evidence that exposure to CNTs induces toxic effects in experimental models. However, CNTs are not a single substance but a growing family of different materials possibly eliciting different biological responses. As a consequence, the hazards associated with the exposure of humans to the different forms of CNTs may be different. Understanding the structure–toxicity relationships would help towards the assessment of the risk related to these materials. Finally, toxicity of CNTs, are discussed. This review article overviews the most recent applications of CNTs in Nanomedicine, covering the period from 1991 to early 2015.
The role of surface charge of ISCOMATRIX nanoparticles on the type of immune ...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
ISCOMATRIX vaccines have now been shown to induce strong antigen-specific cellular or humoral immune responses to a broad range of antigens of viral, bacterial, parasite or tumor. In the present study, we investigated the role of ISCOMATRIX charge in induction of a Th1 type of immune response and protection against Leishmania major infection in BALB/c mice.
Materials and Methods:
Positively and negatively charged ISCOMATRIX were prepared. BALB/C mice were immunized subcutaneously, three times with 2-week intervals, with different ISCOMATRIX formulations. Soluble Leishmania antigens (SLA) were mixed with ISCOMATRIX right before injection. The extent of protection and type of immune response were studied in different groups of mice.
Results:
The group of mice immunized with negatively charged ISCOMATRIX showed smaller footpad swelling upon challenge with L. major and the highest IgG2a production compared with positively charged one. The mice immunized with positively charged ISCOMATRIX showed the lowest splenic parasite burden compared to the other groups. Cytokine assay results indicated that the highest level of IFN- γ and IL-4 secretion was observed in the splenocytes of mice immunized with negatively charged ISCOMATRIX as compared to other groups.
Conclusion:
The results indicated that ISCOMATRIX formulations generate an immune response with mixed Th1/Th2 response that was not protective against challenge against L. major.
Abstract
In the last decade, developments in nanotechnology have provided a new field in medicine called “Nanomedicine”. Nanomedicine has provided new tools for photodynamic therapy. Quantum dots (QDs) are approximately spherical nanoparticles that have attracted broad attention and have been used in nanomedicine applications. QDs have high molar extinction coefficients and photoluminescence quantum yield, narrow emission spectra, broad absorption, large effective stokes shifts. QDs are more photostable and resistant to metabolic degradation. These photosensitizing properties can be used as photosensitizers for Photodynamic Therapy (PDT). PDT has been recommended for its unique characteristic, such as low side effect and more efficiency. Therefore, nanomedicine leads a promising future for targeted therapy in cancer tumor. Furthermore, QDs have recently been applied in PDT, which will be addressed in this review letter. Also this review letter evaluates key aspects of nano-particulate design and engineering, including the advantage of the nanometer scale size range, biological behavior, and safety profile.
Preparation of protein-loaded PLGA-PVP blend nanoparticles by nanoprecipitati...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
Despite of wide range applications of polymeric nanoparticles in protein delivery, there are some problems for the field of protein entrapment, initial burst and controlled release profile.
Materials and Methods:
In this study, we investigated the influence of some changes in PLGA nanoparticles formulation to improve the initial and controlled release profile. Selected parameters were: pluronic F127, polysorbate 80 as surfactant, pH of inner aqueous phase, L/G ratio of PLGA polymer, volume of inner aqueous phase and addition of polyvinylpyrrolidone as an excipient. FITC-HSA was used as a model hydrophilic drug. The nanoparticles were prepared by nanoprecipitation.
Results:
Initial release of FITC-HSA from PLGA-tween 80 nanoparticles (opt-4, 61%) was faster than control (PLGA-pluronic) after 2.30 h of incubation. Results showed that decrease in pH of inner aqueous phase to pI of protein can decrease IBR but the release profile of protein is the same as control. Release profile with three phases including a) initial burst b) plateau and c) final release phase was observed when we changed volume of inner aqueous phase and L/G ratio in formulation. Co-entrapment of HSA with PVP and pluronic reduced the IBR and controlled release profile in opt-19. Encapsulation efficiency was more than 97% and nanoparticles size and zeta potentials were mono-modal and -18.99 mV, respectively.
Conclusion:
In this research, we optimized a process for preparation of PLGA-PVP-pluronic nanoparticles of diameter less than 300 nm using nanoprecipitation method. This formulation showed a decreased initial burst and long lasting controlled release profile for FITC-HSA as a model drug for proteins.
2024.06.01 Introducing a competency framework for languag learning materials ...Sandy Millin
http://sandymillin.wordpress.com/iateflwebinar2024
Published classroom materials form the basis of syllabuses, drive teacher professional development, and have a potentially huge influence on learners, teachers and education systems. All teachers also create their own materials, whether a few sentences on a blackboard, a highly-structured fully-realised online course, or anything in between. Despite this, the knowledge and skills needed to create effective language learning materials are rarely part of teacher training, and are mostly learnt by trial and error.
Knowledge and skills frameworks, generally called competency frameworks, for ELT teachers, trainers and managers have existed for a few years now. However, until I created one for my MA dissertation, there wasn’t one drawing together what we need to know and do to be able to effectively produce language learning materials.
This webinar will introduce you to my framework, highlighting the key competencies I identified from my research. It will also show how anybody involved in language teaching (any language, not just English!), teacher training, managing schools or developing language learning materials can benefit from using the framework.
Unit 8 - Information and Communication Technology (Paper I).pdfThiyagu K
This slides describes the basic concepts of ICT, basics of Email, Emerging Technology and Digital Initiatives in Education. This presentations aligns with the UGC Paper I syllabus.
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The simplified electron and muon model, Oscillating Spacetime: The Foundation...RitikBhardwaj56
Discover the Simplified Electron and Muon Model: A New Wave-Based Approach to Understanding Particles delves into a groundbreaking theory that presents electrons and muons as rotating soliton waves within oscillating spacetime. Geared towards students, researchers, and science buffs, this book breaks down complex ideas into simple explanations. It covers topics such as electron waves, temporal dynamics, and the implications of this model on particle physics. With clear illustrations and easy-to-follow explanations, readers will gain a new outlook on the universe's fundamental nature.
Executive Directors Chat Leveraging AI for Diversity, Equity, and InclusionTechSoup
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Exploiting Artificial Intelligence for Empowering Researchers and Faculty, In...Dr. Vinod Kumar Kanvaria
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Preparation, characterization and transfection efficiency of nanoparticles composed of alkane-modified polyallylamine
1. Please cite this paper as:
Kazemi Oskuee R, Zakeri V, Gholami L, Malaekeh-Nikouei B. Preparation, characterization and
transfection efficiency of nanoparticles composed of alkane modified polyallylamine, Nanomed J, 2015;
2(2):111-120 .
Received: Sep. 5, 2014; Accepted: Dec. 23, 2014
Vol. 2, No. 2, Spring 2015, page 111-120
Received: Apr. 22, 2014; Accepted: Jul. 12, 2014
Vol. 1, No. 5, Autumn 2014, page 298-301
Online ISSN 2322-5904
http://nmj.mums.ac.ir
Original Research
Preparation, characterization and transfection efficiency of nanoparticles
composed of alkane-modified polyallylamine
Reza Kazemi Oskuee1,2
, Vahideh Zakeri3
, Leila Gholami4
, Bizhan Malaekeh-Nikouei5
*
1
Neurogenic Inflammation Research Center, School of Medicine, Mashhad University of Medical Sciences,
Mashhad, Iran
2
Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical Sciences,
Mashhad, Iran
3
School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
4
Targeted Drug Delivery Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
5
Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad,
Iran
Abstract
Objective(s): Although viral vectors are considered efficient gene transfer agents, their board
application has been limited by toxicity, immunogenicity, mutagenicity and small gene
carrying capacity. Non-viral vectors are safe but they suffer from low transfection efficiency.
In the present study, polyallylamine (PAA) in two molecular weights (15 and 65 kDa) was
modified by alkane derivatives in order to increase transfection activity and to decrease
cytotoxicity.
Materials and Methods: Modified PAA was synthesized using three alkane derivatives (1-
bromobutane, 1-bromohexane and 1-bromodecane) in different grafting percentages (10, 30
and 50). The condensation ability of modified PAA was determined by ethidium bromide
test. The prepared polyplexes, complexes of modified PAA and DNA, were characterized by
size and zeta potential. Transfection activity of polyplexes was checked in Neuro2A cells.
The cytotoxicity of vector was examined in the same cell line.
Results: DNA condensation ability of PAA was decreased after modification but modified
polymer could still condense DNA at moderate and high carrier to plasmid (C/P) ratios. Most
of polyplexes composed of modified polymer had mean size less than 350 nm. They showed
a positive zeta potential, but some vectors with high percentage of grafting had negative
surface charge. Transfection efficiency was increased by modification of PAA by 1-
bromodecane in grafting percentages of 30 and 50%. Modification of polymer reduced
polymer cytotoxicity especially in C/P ratio of 2.
Conclusion: Results of the present study indicated that modification of PAA with alkane
derivatives can help to prepare gene carriers with better transfection activity and less
cytotoxicity.
Keywords: Alkane derivatives, Cytotoxicity, Gene delivery, Polyallylamine
*Corresponding author: Bizhan Malaekeh-Nikouei, Mashhad University of Medical
Sciences, Mashhad, Iran.
Tel: 05138823255,Email: malaekehb@mums.ac.ir
2. Alkane-modified polyallylamine as gene delivery vector
112 Nanomed J, Vol. 2, No. 2, Spring 2015
Introduction
Gene transfer systems are classified into
two categories, viral and non-viral vectors.
Despite recent advances, the major
problem in the application of gene transfer
systems is lack of carrier with high
efficiency and low toxicity (1, 2). Viral
vectors exhibit high transfection efficiency
but application of these vectors has been
limited due to induction of immune
response, low gene transfer capacity, high
cost of production and insertional
mutagenesis (3, 4). Therefore, non-viral
systems have been considered more in
recent decades. These vectors are less
toxic, able to carry large DNA molecules,
producible with low-cost in large-scale.
However, their low transfection efficiency
compared to viral carriers is fundamental
problem in implementing of these vectors
(5, 6).
Among non-viral systems, cationic
polymers show special advantages as
carriers for gene transfer. Among the
cationic polymers, polyallylamine (PAA)
can be noted. PAA is a synthetic polymer
with high primary amine groups.
Interaction of this cationic polymer is
provided with the negatively charged DNA
due to its high positive charge. Toxicity
due to the high positive charge of the
polymer and low buffering capacity has
limited the administration of PAA as a
gene transfer carrier (7).
PAA structure can be modified to create
new derivatives with different structures in
order to improve transfection activity and
reduce the cytotoxicity of polymer. In the
present study, PAA was modified by
alkane derivatives with different chain
lengths to achieve a library of compound
with different physicochemical properties.
The aim of the present study was to obtain
a vector capable to enter into the cell to the
greater extent and act efficiently in
escaping from endosomes. Also, we expect
reduction in cytotoxicity of polymer due to
covering of amine groups. The mentioned
modifications have not been conducted on
PAA in previous studies. Nanocomplexes
composed of PAA-dextran-DNA with an
average size of about 150 nm were
prepared in the study of Nimesh et al (8).
The results of this study showed that
transfection efficiency of these nano-
particles was higher and cytotoxicity as
well as significantly reduced compared to
the PAA-DNA nanoparticles. Also,
conjugation of mildly basic groups such as
imidazole to PAA was also investigated to
increase the proton sponge effect (9). By
this modification, in vitro transfection
efficiency of synthesized nanoparticles
was increased up to several folds
compared to native PAA.
Materials and Methods
Materials
PAA with different molecular weights (15
and 65 kDa) were purchased from
Polyscience Inc (USA). Bromoalkanes (1-
bromobutane, 1-bromohexane and 1-
bromodecane) were ordered from Aldrich
(USA). Ethidium bromide (EtBr) was from
Cinnagen (Iran).
Cell culture
Neuro2A (murine neuroblastoma) cells
(ATCC CCL-131) were cultured in
DMEM supplemented with fetal bovine
serum (10%), streptomycin (100 μg/ml),
and penicillin (100 U/ml). The cultured
cells were incubated at 37 °C under 5%
CO2.
Modification of PAA
PAA was modified by the reaction between
PAA and a series of bromoalkanes (Figure
3. Kazemi Oskuee R, et al
Nanomed J, Vol. 2, No. 2, Spring 2015 113
1). Briefly, a solution of bromoalkane in
DMF was added dropwise to the stirring
solution of PAA (0.1 g in 5 mL DMF) over
3 hours. After 24 hour additional stirring at
room temperature, the reactions mixture was
dialyzed once against 0.25 M NaCl and
twice against water (10000 Da cut-off
dialysis tubing) to remove unreacted
compounds. Finally after freeze-drying of
final solution, the achieved powder was kept
at 2-8 °C. The modified polymers were
labeled as PAAX-YC AlkZ%, in which X is
molecular weight of PAA, Y is number of
carbons in alkyl chain of bromoalkane
derivative and Z% is the percentage of PAA
primary amines substituted with alkyl
chains. The primary amine content of
modified polymer was evaluated using
quantification of accessible primary amines
by coupling with 2, 4, 6 trinitrobenzene
sulfonic acid (TNBS). Briefly, freshly
aqueous TNBS solution (15 mg/ml) was
added to various amounts of PAA dissolved
in double distilled water. The mixture was
diluted by sodium bicarbonate buffer
solution (0.8 M, pH 8.5) and the UV
absorbance of this solution was recorded at
410 nm.
+ R’-Br + HBr
Ŕ =Butyl, Hexyl, Decyl
Figure 1. Schematic illustration of synthesis.
Preparation of polyplexes
For preparation of polyplexes, different
amounts of modified polymer were mixed
with DNA at different polycation to DNA
weight ratios (C/P ratio) ranging from 2:1
to 6:1. The mixture was incubated at room
temperature for 15 min before use.
Characterization of polyplexes
The mean size and zeta potential of
nanoparticles were measured with
Zetasizer Nano ZS (Malvern Instruments,
UK). EtBr, a DNA-intercalating dye, was
used to examine the association of DNA
with the polymer solution to evaluate the
condensation ability of modified polymer.
For this purpose, a solution of 400 ng/mL
EtBr glucose was prepared with further
addition of 10 μg/ml of DNA.
The fluorescence intensity of this mixture
was measured at 510 (excitation
wavelength) and 590 nm (emission
wavelength) using a spectrofluorimeter
(FP-6200; Jasco, Japan) and set to 100%.
Next, equal amounts of polymer solutions
were added stepwise to this solution, and
the fluorescence intensity was recorded.
In vitro transfection activity
Twenty four hours before transfection,
cells were seeded at a density of 1×104
cells per well in 96-well plates. Then
4. Alkane-modified polyallylamine as gene delivery vector
114 Nanomed J, Vol. 2, No. 2, Spring 2015
0
20
40
60
80
100
120
0 1 2 3 4 5
Relativeflourescence%
C/P
PAA15-4C Alk 10%
PAA15-4C Alk30%
PAA15-4C Alk50%
PAA15
0
20
40
60
80
100
120
0 1 2 3 4 5
Relativeflouroscence%
C/P
PAA15-6C Alk10%
PAA15-6C Alk30%
PAA15-6C Alk50%
PAA15
0
20
40
60
80
100
120
0 1 2 3 4
Relativeflouroscence%
C/P
PAA65-4C Alk 10%
PAA65-4C Alk30%
PAA65-4C Alk50%
PAA65
0
20
40
60
80
100
120
0 1 2 3 4 5
Relativeflouroscence%
C/P
PAA65-6C Alk10%
PAA65-6C Alk30%
PAA65-6C Alk50%
PAA65
polyplexes were added to each well in
different C/P ratios (2, 4 and 6) and
incubated with cell for 4 hours, followed
by replacement of the medium with fresh
medium. After another 24 h, the medium
was removed and lysis buffer were added
to each well. The percentage of transfected
cells was determined by measuring the
fluorescence intensity of green fluorescent
protein (GFP) at 498 nm (excitation
wavelength) and 535 nm (emission
wavelength) using fluorescent plate reader
(Victor X5, Perkin-Elmer, USA).
Cytotoxicity of polyplexes
The metabolic activity of Neuro2A cells in
the presence of polyplexes was determined
using a thiazolyl blue tetrazolium bromide
(MTT) assay.
Cells (1×104
) were seeded and treated with
the same amounts of polyplexes used for
transfection experiment. After 4 hours
incubation, the medium was replaced by
fresh culture medium. After 18 hours, 10
μl of MTT solution was added to each
well. Then the medium was removed after
incubation for 2 hours at 37°C, 100 μl of
dimethyl sulfoxide added, and the samples
further incubated at 37 °C for 30 minutes
under constant shaking. Optical
absorbance was measured at 590 nm
(reference wavelength 630 nm) using a
microplate reader (M200, Tecan,
Switzerland), and cell viability was
expressed as a percent relative to untreated
control cells. Values of metabolic
activity are presented as mean±SD for
three samples.
Statistical analysis
One-way ANOVA test was used to
analyze our data. Differences between
means were statistically significant if
the p-value was less than 0.05.
5. Kazemi Oskuee R, et al
Nanomed J, Vol. 2, No. 2, Spring 2015 115
0
20
40
60
80
100
120
0 1 2 3 4 5
Relativeflouroscence%
C/P
PAA15-10C Alk 10%
PAA15-10C Alk 30%
PAA15-10C Alk 50%
PAA15
0
20
40
60
80
100
120
0 1 2 3 4 5
Relativeflouroscence%
C/P
PAA65-10C Alk 10%
PAA65-10C Alk 30%
PAA65-10C Alk 50%
Figure 2. The DNA condensation ability of polyallylamines (15 and 65 KDa) and modified- polyallylamines by
bromoalkane derivatives. DNA condensation measured as a decrease in fluorescence of EtBr.
Results
The extent of the modification of
primary amines in the structure of PAA
after conjugation by different
boromoalkane derivatives was estimated
by the TNBS method. As it was
presented in Table 1, the degree of
conjugation was in the range of 8–30
mol% depending on the molar ratios in
the feed.
Table 2 shows the mean size and zeta
potential of the prepared polyplexes.
Size of polyplexes ranged from 117 nm
to several microns.
For both molecular weights of PAA,
nanoparticles composed of 1-
bromodecane had the highest particle
size. Type of bromoalkane derivative
used and grafting percent had effect on
mean size and zeta potential of final
vectors. Zeta potential of nanoparticles
changed from positive to negative in
some cases.
PAA in both molecular weights
condensed the plasmid at C/P ratio of 0.5
(Figure 2). The complete condensation
was occurred at higher C/P ratio after
modification of PAA. In case of
modified PAA 65 KDa with 1-
bromodecane, complete condensation
was not occurred even in high C/P ratio.
For PAA 15 KDa, the transfection
activity of polyplexes was increased by
increasing in C/P ratio (Figure 3).
Modification of PAA 15 KDa with 1-
bromohexane and 1-bromodecane at 30
and 50% in selected C/P ratios increased
the transfection efficiency significantly
(P<0.05). 15 KDa modified with 1-
bromodecane (grafting of 50%) at C/P
ratio of 4 showed the highest gene
transfer ability. In case of vectors
prepared with PAA 65 KDa,
modification of polymer with
bromoalkane derivatives did not affect
the transfection activity of vector
(P>0.05).
The cytotoxicity of PAA decreased
remarkably by covering of the amine
groups of polymer especially in the
grafting percent of 10% (Figure 4).
The cytotoxicity of the modified polymer
was increased by increasing in C/P ratio
in both molecular weight of PAA. The
vectors with the highest transfection
activity had reasonable cytotoxicity.
7. Kazemi Oskuee R, et al
Nanomed J, Vol. 2, No. 2, Spring 2015 117
Figure 3. Transfection activity of polyplexes prepared by polyallylamine (15 and 65 kDa) or modified
polyallylamine in Neuro2A cells. Complexes were prepared at three different C/P ratios (mean±SD, n=3).
0
500
1000
1500
2000
2500
3000
%GFPintensity
C/P 2
C/P 4
C/P 6
0
500
1000
1500
2000
2500
3000
%GFPintensity
C/P 2
C/P 4
C/P 6
8. Alkane-modified polyallylamine as gene delivery vector
118 Nanomed J, Vol. 2, No. 2, Spring 2015
0
10
20
30
40
50
60
70
80
90
100
%Metabolicactivity
C/P2
C/P4
C/P6
0
10
20
30
40
50
60
70
80
90
100
%Metabolicactivity
C/P2
C/P4
C/P6
Figure 4. Cytotoxicity of polyplexes prepared by polyallylamine (15 and 65 kDa) or modified polyallylamine at
different C/P ratios (mean±SD, n=3).
Discussion
Gene delivery approaches have been
developed for the treatment of many
diseases (10). Transfection efficiency of
non-viral systems such as cationic polymer
is lower than with viral vectors but
because of several advantages including
ease of synthesis, low cost and safety,
these gene delivery systems have been
considered more (11).
PAA is a synthetic polymer with a high
density of primary amines. This polymer
has been used as non-viral gene transfer
system (12) but application of this polymer
has been limited because of cell toxicity.
Also, one of drawbacks of this polymer is
9. Kazemi Oskuee R, et al
Nanomed J, Vol. 2, No. 2, Spring 2015 119
low buffering capacity (8, 9). In this study,
in order to increase the hydrophobicity and
to reduce toxicity of polymer, PAA was
modified with bromoalkane derivatives in
three grafting percentages (10, 30 and
50%). Hydrophobic groups in the structure
of polymer can increase the interactions of
carrier with both cell and endosome
membranes. This modification may
increase the possibility of vector crossing
from cell membrane and improve the
efficiency of the polyplex escape from
endosomes (13). Consequently, variation
in transfection activity among different
modified PAAs can be related to favorable
hydrophobic-hydrophilic balance of
polymer (14).
Covering the amine groups of PAA
decreased the ability of polymer to
condense DNA. This speculated result is
due to the substitution of positively-
charged groups by hydrophobic groups
(15). Since the condensation is resulting
from the interaction of positively charged
groups of polymer and negatively charged
phosphate groups of DNA, full condens-
ation occurred for modified polymer in
higher C/P ratio. On the other hand, the
ability to condense DNA is an advantage
for a designed vector but DNA release
from vector after cell uptake should be
considered. Consequently, considering the
optimal balance between condensation and
separation of DNA from vector is
necessary (14). Size and surface charge are
two important parameters in controlling
uptake by cells (16). Size of the carriers
has an important role in carrier entrance
into the cell via endocytosis. Having an
appropriate surface charge is necessary for
the interaction of vector with cell surface
(17). Previous studies related to this field
have shown that the hydrophobic
modification of polymer can increase size
of resultant polyplexes compared to
unmodified polymer. This increase in size
may be cut back due to poor interactions
of modified polymer and DNA. In this
study, almost with increasing grafting
percent and also increase the number of
carbon substituent In some vectors, the
zeta potential was unusual and final
polyplexes showed negative surface
charge. As it was modeled in the study of
Kuhn et al., the charge inversion in case of
polyplexes or lipoplexes may happen with
even small concentration of cationic
amphiphile (polymer or lipid), if it is
sufficiently hydrophobic (18).
Different studies show gene carriers enter
cells according to their size. For example,
particles with a diameter less than 200 nm
are endocytosed by clathrin-mediated
pathway. Polyplexes formed from
modified polymer with 10% grafting had
the mean size around 200 nm. Probably
these carriers are entering into the cells by
clathrin mediated- endocytosis. Better
transfection achieved with this modified
polymer may be related to the difference in
uptake pathway. Although the transfection
ability of PAA after modification was
improved, it may not consider being a
great achievement but from our point of
view the importance of it will be clarified
when the results of toxicity are considered
as well. In the other word, modification of
PAA has resulted to some vectors with
better transfection ability with reduced
toxicity. Several studies have reported the
reduction of toxicity after conjugation of
hydrophobic moieties to polycations (19,
20).
Conclusion
The results of study showed that
10. Alkane-modified polyallylamine as gene delivery vector
120 Nanomed J, Vol. 2, No. 2, Spring 2015
modification of PAA with bromoalkane
derivatives increased transfection activity
and reduced the vector cytotoxicity.
Acknowledgments
This work was supported financially by a
research grant from the Vice Chancellor
for Research of Mashhad University of
Medical Sciences, Mashhad, Iran. Authors
declare that there is no conflict of interests
in this study. The results described in this
paper were part of a Pharm.D. student
thesis.
References
1. Thomas M, Klibanov AM. Non-viral gene
therapy: polycation-mediated DNA
delivery. Appl Microbiol Biotechnol.
2003; 62: 27-34.
2. Verma IM, Somia N. Gene therapy -
promises, problems and prospects. Nature.
1997; 389: 239-242.
3. Kullberg M, McCarthy R, Anchordoquy
TJ. Systemic tumor-specific gene delivery.
J Control Release. 2013; 172: 730-736.
4. Nayerossadat N, Maedeh T, Ali PA. Viral
and nonviral delivery systems for gene
delivery. Adv Biomed Res. 2012; 1:27.
5. Wang T, Upponi JR, Torchilin VP. Design
of multifunctional non-viral gene vectors
to overcome physiological barriers:
Dilemmas and strategies. Int J Pharm.
2012; 427: 3-20.
6. Yin H, Kanasty RL, Eltoukhy AA, Vegas
AJ, Dorkin JR, Anderson DG. Non-viral
vectors for gene-based therapy. Nat Rev
Genet. 2014; 15: 541-555.
7. Oskuee RK, Mohtashami E, Golami L,
Malaekeh-Nikouei B, Cationic liposomes-
polyallylamine-plasmid nanocomplexes
for gene delivery. J Exp Nanosci. 2014; 9:
1026-1034.
8. Nimesh S, Kumar R, Chandra R. Novel
polyallylamine-dextran sulfate-DNA
nanoplexes: highly efficient non-viral
vector for gene delivery. Int J Pharm.
2006; 320: 143-149.
9. Pathak A, Aggarwal A, Kurupati RK,
Patnaik S, Swami A, Singh Y, et al.
Engineered polyallylamine nanoparticles
for efficient in vitro transfection. Pharm
Res. 2007; 24: 1427-1440.
10. Pisskin E, Dincer S, Turk M. Gene
delivery: intelligent but just at the
beginning. J Biomater Sci Polym Ed.
2004; 15: 1181-1202.
11. Somia N, Verma IM. Gen therapy: trials
and tribulations. Nat. Rev. Genet. 2000; 1:
91-99.
12. Boussifi O, Delair T, Brua C, Veron L,
Pavirani A, Kolbe HV. Synthesis of
polyallylamine derivatives and their use as
gene transfer vectors in vitro. Bioconjug
Chem. 1999; 10: 877-883.
13. Neu M, Fischer D, Kissel T. Recent
advances in rational gene transfer vector
design based on poly(ethylene imine) and
its derivatives. J Gene Med. 2005; 7: 992-
1009.
14. Gabrielson NP, Pack DW. Acetylation of
polyethylenimine enhances gene delivery
via weakened polymer/DNA interactions.
Biomacromolecules. 2006; 7: 2427-2435.
15. Mahato M, Rana G, Kumar P, Sharma
AK. Tetramethy lguanidiniumolyallyl-
amine (Tmg‐PA): A new class of nonviral
vector for efficient gene transfection. J
Polym Sci. 2012; 50: 2344-2355.
16. Zanta MA, Boussifi O, Adib A, Behr J.-P.
In vitro gene delivery to hepatocytes with
galactosylated polyethylenimine. Biocon-
jug. Chem. 1997; 8: 839-844.
17. Tros de Ilarduya C., Sun Y., Duzgunes N.,
Gene delivery by lipoplexes and
polyplexes. Eur J Pharm Sci. 2010; 40:
159-170.
18. Kuhn PS, Levin Y, Barbosa MC. Charge
inversion in DNA–amphiphile complexes:
possible application to gene therapy.
Physica A. 1999; 274: 8–18.
19. Dehshahri A, Oskuee RK, Shier WT,
Hatefi A, Ramezani M. Gene transfer
efficiency of high primary amine content,
hydrophobic, alkyl-oligoamine derivatives
of polyethylenimine. Biomaterials. 2009;
30: 4187-4194.
20. Oskuee RK, Dehshahri A, Shier WT,
Ramezani M. Modified polyethylene-
imine: self-assembled nanoparticle
forming ploymer for pDNA delivery. Iran
J Basic Med Sci. 2008; 11: 33-40.