This document summarizes a study that prepared and characterized nanolipoparticles (NLPs) containing MDR1 siRNA for delivery to cancer cells. NLPs were produced using the detergent dialysis method with PEGylated lipids. The resulting NLPs were 80-90 nm in size with a neutral surface charge and over 80% siRNA encapsulation efficiency. In vitro studies showed the NLPs had low cytotoxicity and improved cellular uptake of siRNA compared to a commercial transfection agent. The document concludes that NLPs containing MDR1 siRNA may be a good candidate for further in vivo siRNA delivery studies aimed at reversing multidrug resistance in cancer therapy.
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.
Preparation, characterization and transfection efficiency of nanoparticles co...Nanomedicine Journal (NMJ)
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.
WDR7 up-regulation upon knocking down of neighboring noncoding RNA using siRN...Vahid Erfani-Moghadam
Objective(s): Breast cancer is the second leading cause of cancer death in females. Understanding molecular mechanisms in cancer cells compared with normal cells is crucial for diagnostic and therapeutic strategies. Long intergenic non-protein coding RNA, a regulator of reprogramming (lincRNA-RoR) is a noncoding RNA which initially was detected in induced pluripotent stem cells, and it has an important role in cell reprogramming and highly expressed in breast cancer cells. A key point in successful gene silencing is the usage of siRNA delivery system that is safe and efficient. Materials and Methods: In this study, the fifth-generation of PAMAM dendrimer is used as a nanocarrier for entering siRNA molecules for gene silencing of lincRNA-RoR. WDR7 is the gene encoding adjacent of lincRNA-RoR, which has an important role in apoptosis and cell cycle. Gel retardation assay was used to find the best Negative/Positive (N/P) molar charge ratio of siRNA- PAMAM transfected into MDA-MB 231 cells. MTT assay was performed 24 hr after transfection revealed the IC50 value (half maximal inhibitory concentrations) about 100 nanomolar for lincRNA-ROR siRNA. Results: The lincRNA-RoR and WDR7 gene expression changes were evaluated by real-time PCR after siRNA treatment and showed an increase in the gene expression of WDR7. Conclusion: This study showed that PAMAM dendrimer G5/ siRNA could be a useful system delivery for future gene therapy approaches.
Polymerase chain reaction (PCR)
Polymerase chain reaction (PCR) is a common laboratory technique used to make many copies (millions or billions) of a particular region of DNA.
Assessment of Genetic Diversity of Ficus Species Using Rapd Markers as a Meas...iosrjce
Random Amplified Polymorphic DNA analysis is an excellent tool as a biomarker in determining
genetic purity and estimating genetic diversity in plant species. Seven species of Ficus genus that are locally
available in Hyderabad such as Ficus benjamina, Ficus microcarpa, Ficus elastica, Ficus binnebdjkii, Ficus
benghalensis, Ficus religiosa and Ficus carica. Those ficus cultivars were then analysed by using three
oligonucletide primer Z13 (5’GACTAAGCCC 3’), Z17 (5’ CCTTCCCACT 3’) and Z18 (5’AGGGTCTGTG 3’).
A total of 340 amplified fragments, in which 212 (62.4%) were polymorphic fragments. The number of
polymorphic bands scored per primer ranged from 9 (primer Z13) to 25 (primer Z18). Sixty out of 340 RAPDPCR
fragments were found to be useful as cultivar specific markers. Genetic similarities among the seven Ficus
cultivars were estimated according to RAPD and cultivar distribution on the concensus tree according to
banding pattern of RAPD. Results of combined data exhibited that Ficus benghalensis and Ficus elastica are
related cultivars with highest similarity index. On the other hand Ficus benghalensis and Ficus benjamina are
two distant related species with low similarity index.
A TaqMan-based Quantitative RT-PCR Method for Detection of Apple Chlorotic Le...Agriculture Journal IJOEAR
Abstract—ACLSV is one of the major fruit viruses and can cause severe diseases in species of family Rosaceae. Previous RT-PCR methods are available to detect ACLSV in hawthorn samples, but not to evaluate the infected level of ACLSV. In this study, a TaqMan-based quantitative RT-PCR detection method targeting CP gene of ACLSV was first established and the sensitivity and reproducibility were investigated. The results indicated that this standard curve between log of plasmid DNA concentration versus the cycle threshold (Ct) value generated a linear fit with a linear correlation (R2) of 0.99 and the PCR efficiency was more than 90%. The quantitative RT-PCR method was high sensitive and able to detect 6.9 × 102 copies•μL-1 of ACLSV RNA. Compared with the conventional RT-PCR method, it was 100-fold sensitive in detection of ACLSV. In addition, different organs of hawthorn samples were examined using the quantitative RT-PCR repeatedly and the result revealed that the quantitative RT-PCR is not only an effective detection method, and can obtain an absolute quantitation for ACLSV.
PCR is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested.
Cell-free amplification for synthesizing multiple identical copies (billions) of any DNA of interest.
Basic tool for the molecular biologist.
The purpose of a PCR is to make a huge number of copies of a gene. As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.
Like Xerox machine for gene copying.
TaqMan®Advanced miRNA cDNA synthesis kit to simultaneously study expression o...Thermo Fisher Scientific
MicroRNAs (miRNA) are a class of small non-coding RNAs (approximately 21 nt long) that bind complementary sequences in target mRNAs to specifically regulate gene expression. Aberrant regulation of miRNAs and their targets has been associated with several diseases including cancer. The relationship between miRNA and mRNA has been found to be important in cancer development and progression. Simultaneous expression studies of miRNA and mRNA and detection of mutations in mRNA transcripts can be valuable in understanding molecular mechanisms that
have an underlying role in various diseases. We demonstrate the technical verification of a novel method to reverse-transcribe and pre-amplify miRNA and mRNA from sample-limiting serum research samples using the TaqMan® Advanced miRNA cDNA Synthesis Kit. Based on results from previous studies, a signature of 49 mRNA and 37 miRNA targets has been identified that may help distinguish between benign and malignant pancreatic tissues. In this study, these targets and an additional set of transcript mutations were analyzed in serum from normal and test samples. TaqMan assays for miRNA and mRNA targets and custom TaqMan Mutation Detection Assays (TMDAs) were placed on TaqMan Array Cards to facilitate investigation of several samples in a single experiment. Results demonstrate that transcript mutations can be detected and miRNA and mRNA targets can be reliably quantified from a single reverse transcription reaction. For research use only. Not for use in diagnostic purposes.
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.
Preparation, characterization and transfection efficiency of nanoparticles co...Nanomedicine Journal (NMJ)
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.
WDR7 up-regulation upon knocking down of neighboring noncoding RNA using siRN...Vahid Erfani-Moghadam
Objective(s): Breast cancer is the second leading cause of cancer death in females. Understanding molecular mechanisms in cancer cells compared with normal cells is crucial for diagnostic and therapeutic strategies. Long intergenic non-protein coding RNA, a regulator of reprogramming (lincRNA-RoR) is a noncoding RNA which initially was detected in induced pluripotent stem cells, and it has an important role in cell reprogramming and highly expressed in breast cancer cells. A key point in successful gene silencing is the usage of siRNA delivery system that is safe and efficient. Materials and Methods: In this study, the fifth-generation of PAMAM dendrimer is used as a nanocarrier for entering siRNA molecules for gene silencing of lincRNA-RoR. WDR7 is the gene encoding adjacent of lincRNA-RoR, which has an important role in apoptosis and cell cycle. Gel retardation assay was used to find the best Negative/Positive (N/P) molar charge ratio of siRNA- PAMAM transfected into MDA-MB 231 cells. MTT assay was performed 24 hr after transfection revealed the IC50 value (half maximal inhibitory concentrations) about 100 nanomolar for lincRNA-ROR siRNA. Results: The lincRNA-RoR and WDR7 gene expression changes were evaluated by real-time PCR after siRNA treatment and showed an increase in the gene expression of WDR7. Conclusion: This study showed that PAMAM dendrimer G5/ siRNA could be a useful system delivery for future gene therapy approaches.
Polymerase chain reaction (PCR)
Polymerase chain reaction (PCR) is a common laboratory technique used to make many copies (millions or billions) of a particular region of DNA.
Assessment of Genetic Diversity of Ficus Species Using Rapd Markers as a Meas...iosrjce
Random Amplified Polymorphic DNA analysis is an excellent tool as a biomarker in determining
genetic purity and estimating genetic diversity in plant species. Seven species of Ficus genus that are locally
available in Hyderabad such as Ficus benjamina, Ficus microcarpa, Ficus elastica, Ficus binnebdjkii, Ficus
benghalensis, Ficus religiosa and Ficus carica. Those ficus cultivars were then analysed by using three
oligonucletide primer Z13 (5’GACTAAGCCC 3’), Z17 (5’ CCTTCCCACT 3’) and Z18 (5’AGGGTCTGTG 3’).
A total of 340 amplified fragments, in which 212 (62.4%) were polymorphic fragments. The number of
polymorphic bands scored per primer ranged from 9 (primer Z13) to 25 (primer Z18). Sixty out of 340 RAPDPCR
fragments were found to be useful as cultivar specific markers. Genetic similarities among the seven Ficus
cultivars were estimated according to RAPD and cultivar distribution on the concensus tree according to
banding pattern of RAPD. Results of combined data exhibited that Ficus benghalensis and Ficus elastica are
related cultivars with highest similarity index. On the other hand Ficus benghalensis and Ficus benjamina are
two distant related species with low similarity index.
A TaqMan-based Quantitative RT-PCR Method for Detection of Apple Chlorotic Le...Agriculture Journal IJOEAR
Abstract—ACLSV is one of the major fruit viruses and can cause severe diseases in species of family Rosaceae. Previous RT-PCR methods are available to detect ACLSV in hawthorn samples, but not to evaluate the infected level of ACLSV. In this study, a TaqMan-based quantitative RT-PCR detection method targeting CP gene of ACLSV was first established and the sensitivity and reproducibility were investigated. The results indicated that this standard curve between log of plasmid DNA concentration versus the cycle threshold (Ct) value generated a linear fit with a linear correlation (R2) of 0.99 and the PCR efficiency was more than 90%. The quantitative RT-PCR method was high sensitive and able to detect 6.9 × 102 copies•μL-1 of ACLSV RNA. Compared with the conventional RT-PCR method, it was 100-fold sensitive in detection of ACLSV. In addition, different organs of hawthorn samples were examined using the quantitative RT-PCR repeatedly and the result revealed that the quantitative RT-PCR is not only an effective detection method, and can obtain an absolute quantitation for ACLSV.
PCR is a technique which is used to amplify the number of copies of a specific region of DNA, in order to produce enough DNA to be adequately tested.
Cell-free amplification for synthesizing multiple identical copies (billions) of any DNA of interest.
Basic tool for the molecular biologist.
The purpose of a PCR is to make a huge number of copies of a gene. As a result, it now becomes possible to analyze and characterize DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.
Like Xerox machine for gene copying.
TaqMan®Advanced miRNA cDNA synthesis kit to simultaneously study expression o...Thermo Fisher Scientific
MicroRNAs (miRNA) are a class of small non-coding RNAs (approximately 21 nt long) that bind complementary sequences in target mRNAs to specifically regulate gene expression. Aberrant regulation of miRNAs and their targets has been associated with several diseases including cancer. The relationship between miRNA and mRNA has been found to be important in cancer development and progression. Simultaneous expression studies of miRNA and mRNA and detection of mutations in mRNA transcripts can be valuable in understanding molecular mechanisms that
have an underlying role in various diseases. We demonstrate the technical verification of a novel method to reverse-transcribe and pre-amplify miRNA and mRNA from sample-limiting serum research samples using the TaqMan® Advanced miRNA cDNA Synthesis Kit. Based on results from previous studies, a signature of 49 mRNA and 37 miRNA targets has been identified that may help distinguish between benign and malignant pancreatic tissues. In this study, these targets and an additional set of transcript mutations were analyzed in serum from normal and test samples. TaqMan assays for miRNA and mRNA targets and custom TaqMan Mutation Detection Assays (TMDAs) were placed on TaqMan Array Cards to facilitate investigation of several samples in a single experiment. Results demonstrate that transcript mutations can be detected and miRNA and mRNA targets can be reliably quantified from a single reverse transcription reaction. For research use only. Not for use in diagnostic purposes.
Cybersecurity in a Transparent World, Cyber Security Technology Symposium, Li...G. S. McNamara
Discussed privacy and security issues present in a connected world from a variety of perspectives. Moderator: Dr. Anup K. Ghosh, Chief Scientist, Center for Secure Information Systems (CSIS). Additional panelists: Gary McGraw, chief technology officer at Cigital, Michael Lefebvre, cybersecurity strategist at Northrop Grumman Corporation, and Elizabeth A. Hight, Rear Admiral (USN, ret.) vice president, U.S. Defense Command and Control Infrastructure Practice at Hewlett-Packard Company
Non-biological gene carriers designed for overcoming the major extra- and int...Nanomedicine Journal (NMJ)
Abstract
Gene therapy as a modern therapeutic approach has not yet advanced to a globally-approved therapeutic approach. Lack of adequate reliable gene delivery system seems to be one of the major reasons from the pharmaceutical biotechnology point of view. Main obstacles delaying successful application of human gene therapy are presented in this review. The unique advantages of non-biological gene carriers as compared to their biological counterparts make them ideal alternatives for overcoming extra- and intracellular barriers in a more safely manner. We, therefore, highlight the significant contributions in non-biological gene delivery and favorable characteristics of different design attitudes with focus on in vivo approaches. Bypassing the rapid extracellular enzymatic degradation of genetic materials is covered in extracellular segment of this review with emphasis on PEGylated and targeted formulations. The successful approaches to pave the rest of the way from cellular uptake to intracellular transfer and gene expression of unpacked DNA are also discussed. From these approaches, we emphasize more on optimization of cationic-based polymers and dendrimers, developing newly designed membrane-effective components, and adjusting the hydrophilic-hydrophobic balance of the synthesized vectors
Molecular markers are the DNA sequences that are used to tag or highlight specific genes or nucleotide sequence in the genome. In the past, diagnosis, and monitoring of infectious disease, there many conventional methods like Biotyping, Ribotyping and Protein analyses are used but these methods are not so reliable and exact. So by the invention of the molecular markers, the detection of diseases was started by using these molecular markers. In this review, we discussed different types of molecular markers that are used to detect diseases and many purposes as diagnostic tools. The Molecular markers include Restriction fragment length polymorphism (RFLP), Random amplification of polymorphic DNA (RAPD), Amplified fragment length polymorphism (AFLP), Simple sequence repeats (SSR), Inter-Simple Sequence Repeat Amplification (ISSR), Cleaved Amplified Polymorphic Sequences (CAPS) and Sequence Characterized Amplified Region (SCAR) are discussed in the article.
New progress in lipid nanoparticles technologyDoriaFang
Researchers have developed LNPs that can precisely deliver mRNA to lung tissue. The therapy of mRNA delivered by LNPs significantly reduced the disease burden of lymphangioleiomyomatosis in animal models.
ST8 micellar/niosomal vesicular nanoformulation for delivery of naproxen in c...Vahid Erfani-Moghadam
Naproxen (NPX) is a non-steroidal anti-inflammatory drug (NSAID) used against a variety of diseases, including autoimmune disorders and chronic inflammations. However, low water solubility limits its therapeutic efficacy and novel nanoformulations are required to bypass its poor bioavailability to reach its therapeutic effect. The purpose of the study was to investigate the role of the nanoformulation of biocompatible molecules; Squalene (S) and Tween 80 (T8) Micellar/Niosomal Vesicles (ST8MNV) prepared, by thin-film hydration method and their potential as a drug delivery system for NPX. The percentage of encapsulation efficiency was calculated to be 99.5 ± 0.2% for 5% of NPX weight in total ingredients of micellar/niosomal vesicles (w/w). The ST8MNV nanoformulation exhibited a slower rate of NPX release from the drug encapsulated over seven days, suggesting a stable complex of NPX. Finally, cell toxicity assay demonstrated that the half-maximal inhibitory concentrations (IC50) of NPX were drastically reduced by ST8MNV nanoformulation in MCF-7, A549, HeLa, and MDA-MB-231 cancer cell lines. Our data show this micellar/niosomal naproxen nanoformulation is a great candidate for the future in vitro and in vivo studies for potential clinical anti-inflammatory and anticancer applications.
ABSTRACT- Multiple Drug resistance (MDR) tuberculosis timely diagnose is of utmost clinical relevance and needs to be diagnose at initial stages for the proper treatment. The current study was done to detect the several genes for MDR tuberculosis (TB) in clinical isolates by molecular tools. 60 clinical isolates were collected and subjected for AFB smear preparation, Nested PCR (IS6110) for mycobacterium tuberculosis complex detection and MDR TB PCR targeting rpoB, kat G, mab A promoter. 12 came positive for AFB smears, out of which 08 were pulmonary and 04 were extra pulmonary. Nested PCR targeting IS6110 gene was amplified at 123 base pairs with 340 base pairs as IC (internal control) was seen in 25 cases which include 19 pulmonary and 6 extra pulmonary. The Positive TB PCR specimens were subjected for MDRTB PCR Only 06 cases yielded, an amplicon of 315 bp confirming the rpoB gene resistance for resistance for rifampcin drug. In any of the 06 positives none of the other resistance gene other than rpoB was amplified. Targeting multiple genes at once, additional information will be gained from a single test run that otherwise would require several times the reagents and more time to perform. Current study signifies the usage of quick, cost effective, DNA sequences based method for MDR TB detection where disease will be diagnosed earlier and hence treatment would be started at an early stage.
Keywords: Multiple drug resistance, amplicon, Polymerase chain reaction, Nested PCR, Rifampicin.
This is a presentation slide about cellular RNA interference process and RNA interference technology. Contains basic information about biology of cellular RNA interference processes and its discovery, and RNA interference technology. Also gives you the history and development of in-vitro and in-vivo technologies for applicability of RNA interference technology.
siRNA synthesis, siRNA libraries, siRNA delivering techniques, Electroporation, viral transfection methods, Advantages and disadvantages of RNA interference technology.
details about the preliminary and pre-clinical experiments of RNA interference as well as clinical trials of RNA interference.
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.
Preparation of protein-loaded PLGA-PVP blend nanoparticles by nanoprecipitati...
4 nmj 2-1
1. Please cite this paper as:
Nourbakhsh M, Behravan J, Lage H, Abnos Kh, mosaffa F, Badiee A, Jaafari Mr. Nanolipoparticles-mediated
MDR1 siRNA delivery: preparation, characterization and cellular uptake, Nanomed J, 2015; 2(1): 39-45.
Received: Aug. 15, 2014; Accepted: Sep. 25, 2014
Vol. 2, No. 1, Winter 2015, page 39-45
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
Nanolipoparticles-mediated MDR1 siRNA delivery: preparation,
characterization and cellular uptake
Mahnaz Nourbakhsh 1,2,3
, Javad Behravan 1,2*
, Hermann Lage 4
, Khalil Abnous 5
, Fatemeh
mosaffa 3
, Ali Badiee 2
, Mahmoud R. Jaafari 2*
1
Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
2
Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad,
Iran
3
Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences,
Mashhad, Iran
4
Charite´ Campus Mitte, Institute of Pathology, Charite´platz 1, D-10117 Berlin, Germany
5
Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad,
Iran
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.
Keywords: Nanolipoparticles; siRNA delivery; PEG-shielded; Gene therapy; Liposome.
*Correspondence authors: Mahmoud R. Jaafari, Nanotechnology Research Center, School of Pharmacy,
Mashhad University of Medical Sciences, Mashhad, Iran.
Tel: +98-511-8823255, Fax: +98511-8823251, E-mail: jafarimr@mums.ac.ir,
Javad Behravan, Biotechnology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
Tel: +98-511-8823255, E-mail: behravanj@mums.ac.ir
J.B. and M.R.J. equally designed research
2. Nanolipoparticles-mediated siRNA delivery
40 Nanomed J, Vol. 2, No. 1, Winter 2015
Introduction
The use of the RNA interference (RNAi)
through the exploiting of small interfering
RNA (siRNA), a 21–25 nucleotide (nt)
double-stranded RNA molecule, is a
powerful tool for post-transcriptional gene
silencing. RNAi is being exploited as a
promising approach for efficient gene
silencing for therapeutic applications like
MDR1 mediated multidrug resistance
(MDR) reversal in cancer therapy. MDR1
is the gene that encodes P-glycoprotein (P-
gp). The over expression of P-gp is a
major mechanism of drug resistance in
several cancer types such as breast and
pancreatic cancers (1).
But actually the clinical siRNA application
is barricaded by some major Problems. For
instance, rapid enzymatic degradation of
siRNA results in a short blood circulation
time and its negative charge leads to poor
cellular uptake. So, therapeutic application
of siRNA requires developing efficient
carriers.
Among the vectors developed for gene
delivery, lipid based delivery systems have
proved to be one of the most effective
carriers (2). Cationic liposomes containing
nucleic acids also known as Lipoplexes,
are the most commonly used systems for
nucleic acids delivery (3). Traditional
lipoplexes such as LipofectamineTM
2000
(4) and OligofectamineTM
(5) are
frequently used, commercially available
products for both DNA and siRNA
delivery.
Despite the widespread use of lipoplexes
for in vitro experiments, these agents are
not efficient gene delivery systems in vivo.
Excessive size and positive charge result
in strong interactions with serum proteins
and sequestrating within the
reticuloendothelial system (RES) (half-
life< 5 min) (2, 6). The liposomal
nanoparticles with the general name of NP
(2) or lipid nanoparticles (LNs) (7) are the
other group of liposomes for nucleic acid
delivery. NP with small particle size (<
100 nm) and encapsulated nucleic acids in
a polyethylene glycol (PEG) -shielded
bilayer shell are different from traditional
lipoplexes. Wheeler et al. (8) exploited a
detergent dialysis method for plasmid
DNA encapsulation in nanoparticles
named ‘stabilized plasmid-lipid particles
(SPLP). In their study, liposome
aggregation was prevented during
detergent dialysis using 10 mol % of PEG
–lipid in the formulation. SPLP with small
sizes (<100 nm) and neutral charges have
better stability in vivo (half-life: 1-10 h)
(9) and are suitable for in vitro and in vivo
DNA delivery(10). This procedure was
then improved by Li et al. (11) who
developed a low concentration detergent
dialysis method to prepare DNA
containing nanolipoparticles (NLPs).
They reduced octyl glycoside (OG)
concentration from 200 mM in SPLP
preparation procedure to 28 mM (near to
critical micelle concentration (CMC)) in
NLPs. They increased DNA encapsulation
ratio from 40-50% in SPLP to 80-100% in
NLPs and improved the transfection
activity.
As NLPs have shown high efficiency for
gene delivery in in vitro studies (2, 11) and
have a good potential for in vivo
applications, they can be a promising
candidate for more siRNA delivery studies
in different phenomenon like multidrug
resistance reversal in cancer. Based on
these facts, in current study, NLPs
containing siRNA were prepared with the
low concentration detergent dialysis
method. Physical characteristics, siRNA
encapsulation efficiency and stability of
NLPs were studied. Cytotoxicity of NLPs
was checked with MTT assay. After
characterization, the ability of NLPs for
siRNA cellular uptake facilitating was
investigated by fluorescence microscopy
in vitro.
Materials and Methods
Materials
1,2-Dioleoyl-3-Trimethylammonium-
propane (DOTAP), 1,2-Dioleoyl-sn-
Glycero-3-Phosphoethanolamine (DOPE),
1,2-distearoyl-sn-glycero-3-pho-sphoethan
3. Nourbakhsh M, et al
Nanomed J, Vol. 2, No. 1, Winter 2015 41
olamine-N-[methoxy(polyethelene glycol)-
2000] (mPEG 2000-DSPE) were from
Avanti Polar Lipids (Alabaster, AL, USA).
Octyl β-D-glucopyranoside (OG) and Tris
(hydroxymethyl)aminomethane and
Diethyl pyrocarbonate (DEPC) were
purchased from Invitrogen™ (USA).
Oligofectamine™ (OFA) Transfection
Reagent was purchased from Invitrogen™
(USA). Deionised water was used in all
experiments and then treated with DEPC
for siRNA containing samples. All other
chemicals used were of molecular grade.
siRNA
Two following siRNAs were used for
liposome characterization studies: The
anti-MDR1-specific siRNA duplex (siM)
homologous to nt 88-108 (sense strand 5'-
GAA ACC AAC UGU CAG UGU
AdTdT-3’) was previously designed
against the MDR1-encoding mRNA
consensus sequence (GenBank accession
number no. NM_000927)(5) and a
biologically active siRNA 5’-CGU ACG
CGG AAU ACU UCG AdTdT-3’ directed
against the non-mammalian luciferase
(GL-2) encoding mRNA (siC). In addition,
FITC labeled anti-MDR1-specific siRNA
(siF) (The same sequence as siM) was
used for siRNA localization studies. All
siRNAs were commercially obtained from
Dharmacon (Lafayette, CO, USA) in
annealed form.
Preparation of NLPs containing siRNA
siRNA was encapsulated into NLPs
containing mPEG-DSPE/DOTAP/DOPE
(10:50:40) by the detergent dialysis
method (11) with some modifications. The
charge ratio of siRNA and DOTAP was
1:2. Briefly, mPEG-DSPE/DOTAP/DOPE
(2.5 µmol total lipid) were mixed at a
molar ratio 10/50/40 in chloroform (35 µL
mPEG-DSPE (20 mg/ml) / 34.92 µL
DOTAP (25 mg/ml) / 37.2 µL DOPE (20
mg/ml)), dried by rotary evaporation,
placed under a high vacuum for 2 h. Then,
the dried lipid film was hydrated in 1.47
ml of 28 mM OG in 5 mM Tris buffer (pH
8.0) for 30 min. 14881pmol
siRNA was
separately solubilized in 1.47 ml of 28 mM
OG in 5 mM Tris buffer (pH 8.0).The
latter solution was gently added into the
lipid solution and vortexed for 30 s.
Following 10 min incubation at room
temperature for spontaneous particle
formation, the mixture was transferred to a
Slide-A-LyzerTM
dialysis cassette (MWCO
10 K; Pierce, Rockford, IL, USA) and
dialyzed against 1 L of 5 mM Tris buffer
(pH 8.0) for 2 days at 4°C. For the in vitro
study, the buffer was changed to150 mM
NaCl, 5 mM Tris buffer (pH 8.0) by
dialysis.
siRNA encapsulation efficiency assay
siRNA encapsulation efficiency was
determined in triplicate by filtration via
Amicon Ultra-4 centrifugal filter
device,100 KD. First total formulation
filtrated and free siRNA concentration
determined in the filtrate by measuring
absorption at 260 nm using a NanoDrop
1000 spectrophotometer (Thermo Fisher
Scientific, Wilmington, DE). Next total
formulation lysed by 2% OG (12) and total
siRNA concentration was determined as
described. The siRNA encapsulation
efficiency was calculated using Equation
1.
Equation 1:
Encapsulation (%) = (1-Free siRNA/Total
siRNA) × 100.
Particle size and zeta-potential
measurements
The particle diameters of empty and
loaded NLPs were measured in triplicate
using dynamic light scattering (ZetaSizer
Nano-ZS; Malvern Instruments Ltd.,
Worcestershire, United Kingdom). The
zeta-potential (ζ) was measured with the
same instrument using the zeta potential
mode as the average of 20 measurements
by diluting the particles into a low salt
buffer (1 mM NaCl and 1mM HEPES at
pH 7.5) (11, 13).
4. Nanolipoparticles-mediated siRNA delivery
42 Nanomed J, Vol. 2, No. 1, Winter 2015
Cell lines and cell culture
MCF-7/ADR and EPP85-181/RDB,
established classical multidrug-resistant
derivatives of human breast and pancreatic
carcinoma cell lines respectively, (from
Lage's laboratory stocks) were grown in
Leibovitz L-15 medium (Biowhittaker,
Walkersville, MD) supplemented by 10%
fetal calf serum (FCS) (GIBCO/BRL,
Grand Island, NY), 1 mM L-glutamine,
6.25 mg/l fetuin, 80 IE/l insulin,2.5 mg/ml
transferrin, 0.5 g/l glucose, 1.1 g/l
NaHCO3, 1% minimal essential vitamins
and 2x104
kIE/l trasylol in a humidified
atmosphere of 5% CO2 at 37 o
C as
described previously (14).
In order to ensure maintenance of drug-
resistant phenotype, MCF-7/ADR and
EPP85-181/RDB mediums were respect-
ively supplemented with 0.05 μg/ml
doxorubicin or 2.5 μg/ml daunorubicin
(Farmitalia Carlo Erba, Freiburg,
Germany) and were regularly replaced
twice a week.
NLPs cytotoxicity analysis
MCF-7/ADR Cells were seeded in clear
96-well plates at a density of 1 x 104
cells/well. After 24 h, the cells were
transfected at 30–40% confluence.
Transfection was performed in OPTI-
MEM1 (Invitrogen™, USA) without
serum or antibiotics(5). NLPs containing
control siRNA (NLP siC) and NLPs
containing MDR1 siRNA (NLP siM) were
added at final concentrations of 25, 50,
100 or 200 nM siRNA. Final
concentrations of siRNA were calculated
based on siRNA concentration in the
formulation (5 µM). Empty NLP was
added at lipid concentrations equivalent to
loaded NLPs. Nontransfected cells were
considered as negative controls.
Cells were incubated at 37°C for 3 days in
a 5% CO2 atmosphere. Cell numbers were
evaluated using the 3-(4, 5-dimethylthiazo
l-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay (15). Assays were carried out
in triplicate and were repeated three times.
Translocation studies
MCF-7/ADR and EPP85-181/RDB Cells
were seeded on cover slips (Sigma, USA)
in 12 well plates at a density of 1.5 x 105
cells per well. After 24 h, cells were
transfected with NLPs and OFA
containing 25, 50 or 100 nM FITC-labeled
siRNA (siF) as described. Untreated cells,
cells incubated with naked siF 100 nM and
empty liposomes were used as controls. In
the case of NLP siF 100 nM, transfection
was performed in serum free and complete
media for comparison. After 4 h, cells
were directly fixed in 4% formaldehyde in
PBS for 10 min for fluorescence
microscopy, using a Keyence BZ-8100E
with excitation-mode for green dye (FITC)
(16).
Results and Discussion
Liposome characterization
The biophysical properties of these
particles including the z-average particle
diameter, the extent of siRNA
encapsulation and the zeta potential were
determined as described. The mean
particle size for empty NLPs, NLPs
containing siRNA (NLP siM) were 100.4
± 36.4, 87.6 ± 6.7 nm (n=3) as calculated
by particle size analyzer. To determine the
level of positive charge on the particle
surface, we measured the zeta-potential by
particle size analyzer. The surface charge
of NLPs was close to neutral. Zeta
potentials for empty NLPs, NLPs
containing siRNA were +9.2 ± 4.3, +3.3 ±
10.9 mV respectively. NLPs were stable
regarding their size for 10 days at 4°C.The
encapsulation efficiency (EE) for NLPs
containing siRNA was calculated to be
82.2 ± 4.4%. The results of NLPs
containing siRNA characterization are
almost similar to ones achieved for NLPs
containing DNA by Li et al. (11). They
prepared neutral NLPs with diameters
ranging from 80–150 nm depending on the
lipid composition. The percentages of
DNA encapsulation for NLPs were about
80-100%.
5. Nourbakhsh M, et al
Nanomed J, Vol. 2, No. 1, Winter 2015 43
The small size of NLPs as the neutral
charge of the surface reduces their
interactions with blood components and
enables them to escape from RES uptake.
So NLPs have the potential for long
circulation in animals. In the other hand
these NLPs characters help to exploit the
EPR effect for passive targeting and make
them good candidates for siRNA delivery
to tumors(2).
NLPs cytotoxicity
All formulation including empty NLPs,
NLPs containing siM (NLP siM) and
NLPs containing siC (NLP siC) at the
doses used for transfection were well
tolerated by MCF7/ADR cells. There was
no significant cytotoxicity (P > 0.05) even
at the highest dose (200 nM) (Figure 1).
These are while Li et al. (11) also showed
all NLPs tested were well tolerated by
monkey kidney fibroblast CV-1 cells at the
dose used for transfection and the NLPs
containing DNA were better tolerated than
traditional lipoplexes. Traditional
lipoplexes have high cytotoxicity because
of their large size and high positive surface
charge. Small size and neutral surface
charge of NLPs accompanied with PEG-
shielding can reduce blood components
interactions and accordingly reduce NLPs
cytotoxicity compared with lipoplexes.
Cellular uptake of FITC labeled siRNA
In order to easily assess the siRNA
delivery efficiency of liposomes, MCF-
7/ADR (Figure 2) and EPP85-181/RDB
(Figure 3) cells were incubated with naked
or liposome conjugated FITC labeled
siRNA (siF). Fluorescence microscopy
studies showed that cellular uptake of siF
in both NLPs (Figure 2A and 3A), and
oligofectamineTM (OFA) (Figure 2B and
3B) transfected cells was dose-dependent.
In addition cellular uptake of OFA siF was
more than NLP siF in both cell lines. No
uptake was observed in control groups
(Figure 2C and 3C). We observed no
considerable differences between NLP
mediated siRNA cellular uptake in serum
Figure 1. Cytotoxicity of NLPs containing siC and
siM at different concentrations (25, 50, 100 and
200 nM) and empty NLP at lipid concentrations
equivalent to loaded liposomes in human
multidrug-resistant breast (MCF-7/ADR) cancer
cells. The cells were incubated for 72 h with
different formulations. Cell number was evaluated
using the MTT assay. Means ± SD are shown (n =
3; P > 0.05). Cell viability in untreated cells was
taken as control (100%).
NLP: Nanolipoparticle; siC: Control siRNA; siM:
MDR1 siRNA
These results can be approved by the
results obtained for cell transfection by
NLPs containing DNA (11). Li et al
observed NLPs can deliver DNA to CV-1
cells effectively in the presence of 10%
serum. They observed NLPs in
comparison to PEI polyplex had lower
transfection activity.
The lower transfection of NLPs compared
to traditional lipoplexes or polyplexes can
be explained with the presence of PEG in
the formulation. PEG-lipids reduce the
interaction with cell membranes and can
decrease the in vitro uptake of DNA or
siRNA particles into the cells.
Nevertheless, PEG-lipid may substantially
improve systemic gene transfer by
extending the circulation time and
reducing the toxicity in vivo(2).
Conclusion
PEGylated nanolipoparticles (NLPs)
containing MDR1 siRNA were prepared
with a simple method, based on low
concentration detergent dialysis. NLPs
with the small size of 80-90 nm and the
surface charge close to neutral kept their
stability for 10 days.
6. Nanolipoparticles-mediated siRNA delivery
44 Nanomed J, Vol. 2, No. 1, Winter 2015
Figure 2. Translocation of the siF into human multidrug-resistant breast (MCF-7/ADR) cancer cells using
different NLP or OFA formulations. The cells seeded on coverslips and were incubated with different
formulation at 37 °C without FBS for 4 h. Then cells fixed in 4% formaldehyde and slides were prepared for
fluorescence microscopy evaluation. Images were photographed by Keyence BZ-8100E fluorescence
microscope. (A) Empty NLP, NLP siF in 25, 50 or 100 nM concentrations. (B) Empty OFA, OFA siF in 25, 50
or 100 nM concentrations. (C) Untreated cells, naked siF100 nM treated cells used as controls. Serum effects on
NLP mediated translocation was evaluated by NLP siF 100 nM tranfection in 10% FBS medium.
NLP: Nanolipoparticle; OFA: Oligofectamine; siF: FITC labeled siRNA.
Figure 3. Translocation of the siF into human multidrug-resistant pancreatic (EPP85-181/RDB) cancer cells
using different NLP or OFA formulations. The cells seeded on coverslips and were incubated with different
formulation at 37 °C without FBS for 4 h. Then cells fixed in 4% formaldehyde and slides were prepared for
fluorescence microscopy evaluation. Images were photographed by Keyence BZ-8100E fluorescence
microscope. (A) Empty NLP, NLP siF in 25, 50 or 100 nM concentrations. (B) Empty OFA, OFA siF in 25, 50
or 100 nM concentrations. (C) Untreated cells, naked siF100 nM treated cells used as controls. Serum effects on
NLP mediated translocation was evaluated by NLP siF 100 nM tranfection in 10% FBS medium.
NLP: Nanolipoparticle; OFA: Oligofectamine; siF: FITC labeled siRNA.
7. Nourbakhsh M, et al
Nanomed J, Vol. 2, No. 1, Winter 2015 45
The efficacy of siRNA encapsulating in
NLPs was more than 80%. The NLPs
formulations didn’t have any negative
effect on cell viability, as tested by MTT
assay. These characters make them a good
candidate for in vivo studies. The results
of fluorescent imaging by microscopic
studies showed the cellular uptake of
siRNA in resistant cancer cells is suitable
and this formulation merits further in vivo
studies for multidrug resistance reversal in
cancer therapy.
Acknowledgements
This project was supported by a grant from
Vice Chancellor for Research, Mashhad
University of Medical Sciences, Mashhad,
Iran. This report has been extracted from
the Ph.D. thesis of Mahnaz Nourbakhsh.
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