text:
View all Nature Research journals
ZnAl nano layered double hydroxides for dual functional CRISPR/Cas9 delivery and enhanced green fluorescence protein biosensor
Navid Rabiee1,
Mojtaba Bagherzadeh1,
Amir Mohammad Ghadiri1,
Ghazal Salehi1,
Yousef Fatahi2,3 &
Rassoul Dinarvand2,3
Scientific Reports volume 10, Article number: 20672 (2020) Cite this article
Nanoscience and technology
Evaluation of the effect of different parameters for designing a non-viral vector in gene delivery systems has great importance. In this manner, 2D crystals, precisely layered double hydroxides, have attracted the attention of scientists due to their significant adjustability and low-toxicity and low-cost preparation procedure. In this work, the relationship between different physicochemical properties of LDH, including pH, size, zeta potential, and synthesis procedure, was investigated and optimized for CRISPR/Cas9 delivery and reverse fluorescence response to the EGFP. In this manner, ZnAl LDH and ZnAl HMTA LDH were synthesized and characterized and applied in the HEK-293 cell line to deliver CRISPR/Cas9. The results were optimized by different characterizations as well as Gel Electrophoresis and showed acceptable binding ability with the DNA that could be considered as a promising and also new gold-standard for the delivery of CRISPR/Cas9. Also, the relationship of the presence of tertiary amines (in this case, hexamethylenetetramine (HMTA) as the templates) in the structure of the ZnAl LDH, as well as the gene delivery application, was evaluated. The results showed more than 79% of relative cell viability in most of the weight ratios of LDH to CRISPR/Cas9; fully quenching the fluorescence intensity of the EGFP/LDH in the presence of 15 µg mL−1 of the protoporphyrins along with the detection limit of below 2.1 µg mL−1, the transfection efficiency of around 33% of the GFP positive cell for ZnAl LDH and more than 38% for the ZnAl LDH in the presence of its tertiary amine template.
In the last decade, countless researches have been done on different synthesis methods and modifications of different nanomaterials for various applications. However, very little of this research focuses on finding the cause of various phenomena. Therefore, focusing on the cause of each chemical, physical, and biological phenomenon is significant. In the meantime, investigating the involved parameters in the studies related to specific nanomaterials' biological properties is very difficult because each of the parameters can affect each other. Therefore, it is very important to design experiments that lead to studying each of this basic parameters1,2,3.
As known CRISPR/Cas [clustered regularly interspaced short palindromic repeat/CRISPR-associated protein (Cas)], the technology can make some critical and permanent changes in the genome in an exact sequence of that. This technology is considered a reasonable and achievable goal for one-time cures of inherited diseases4,5,6,7. Generally, gene delivery systems are based on different vectors, and for nanotechnology, viral and non-viral vectors were considered8,9. Till now, there are several reports in designing polymer-based and also carbon-based nanomaterials as non-viral CRISPR delivery vector, but these carbon-based nanomaterials suffer from several drawbacks, including required multiple optimizations, high dependence on molecular weights, difficult adjustment of homogeneity on the surface, and also the need of using a surface or a carrier. However, inorganic-based nanomaterials don't have these problems10,11,12,13,14,15. Also, there is an approach regarding the design and synthesis of 2D nano layered inorganic systems to increase the accessible surface of the nanomaterial and the potential of physical interaction with the genetic domain. In this regard, the use of a highly accessible 2D surface with the specific spaces between them is of great importance.
2D nanocrystals, especially layered double hydroxide nanoparticles (LDH-NPs), due to their sandwich form and cationic layers, have been considered a new generation of drug/gene delivery carriers non-viral vectors. These nanoparticles are usually biocompatible, with significant loading capacity, which leads to considerable cellular uptake and could modify with a specific linker that is sensitive to different stimuli, including light, pH, redox, etc. Engineering of LDH-NPs with application in gene delivery systems have been introduced by Choy et al.16,17,18, and after that, several scientists, including Lu19,20, Giannelis21,22, Xu23,24,25, O'Hare26,27,28, Andrea29, Yazdani30, etc. In a recent study, LDH conjugated with folic acid reported in a mouse model of xenograft that has bearing with nasopharyngeal carcinoma of humans, which represented the significant gene silencing31. These layered NPs can practically capture the genetic domain of any genetic material, including CRISPR/Cas9, and improve the stable interaction between the nanocarrier and the pCRISPR. However, there is still no report regarding the use of these layers as adsorbent of the genetic domain.
In this study, we aimed to investigate the ZnAl LDH effect on the transfection efficiency of pCRISPR as well as cytotoxicity of these nanoparticles in HEK-293 cell line besides generating a simple idea for increasing the transfection efficiency as well as sensing the enhanced green fluorescence protein in a biological matrix (Fig. 1). Besides, the role of the addition of the template to the synthesis procedure and preserving the template in the structural chemistry of ZnAl LDH was investigated. In this regard, the trends of pDNA/nanoparticles and pDNA/nanomaterials size distribution and their zeta potential to design a low cost, simple, and highly efficient non-viral gene delivery vector were evaluated. Furthermore, the relationship between the structural chemistry of ZnAl LDH and the synergic effect of the cationic metal nanoparticles and layered double hydroxide along with the effect of the tertiary amines as template in the structure of these ZnAl LDH were considered in this phase of the study. A full investigation in terms of considering this nanomaterial in the biosensor assays of enhanced green fluorescence protein was conducted as well. A schematic illustration of the synthesis of LDH and transfection studies shows in Fig. 2. In this figure, the brief illustration of LDH synthesis corresponded to the addition of ZnCl2·6H2O and Al(NO3)3·9H2O to a beaker and followed by that, the mixture transferred to an autoclave for the reaction completion; a full description of the synthesis is presented in the Materials and methods section.
Schematic illustration of the application of the synthesized LDH's in this work.
A schematic illustration of the synthesis of LDH, and using them in transfection of pCRISPR.
Synthesis of the LDH
In the present study, two types of ZnAl LDH were synthesized and investigated comprehensively for the CRISPR delivery application. Both of them were synthesized with a safe and highly trusted procedure, hydrothermal method32,33,34. Briefly, 4.8 mmol of ZnCl2·6H2O and 2.4 mmol of Al(NO3)3·9H2O and water as the solvent stirred for about 30 min, and then poured into the autoclave (Teflon-lined stainless-steel autoclave) and heated to 120 °C for 48 h. For the synthesis of ZnAl LDH with the template, the hexamethylene tetraamine (HMT) was added with the metal salts. Then the autoclave was held at room temperature to cool down, and the ZnAl LDH product was washed several times with ethanol and deionized water and drying in a vacuum oven at 80 °C overnight. It should be noted that no attention was paid to control the chemical and physical characteristics in this synthesis method.
Loading CRISPR and tagged DNA on the surface of modified materials
The different procedures of the loading of CRISPR and tagged DNA on the surface of modified materials were reported previously35,36,37. Initially, the modified material, LDH, was dissolved with the concentration of 2.5 mg mL−1 in the ultrapure water, and different weight ratios of the modified material and CRISPR plasmid formulated through the addition of sterilized modified material with different concentration to an exact volume of the solution of CRISPR and the homogenous solutions were prepared. The final solutions were incubated for about 45 min at room temperature to form the ultimate nanocomplexes.
Size and surface charge analysis
The size of the Material (M)/CRISPR (C) nanocomplex was investigated using dynamic light scattering on a Zetasizer Nano ZS (Malvern Instruments, UK). The investigations were performed at the standard angle of 25° and 173°, and each measurement was verified three times. In this technique, the size of the nanocomplex is reported as the mean diameter achieved by the refractive index and viscosity of water correlation functions cumulants analysis. For aggregation kinetics measurements, the final nanocomplexes were diluted with a ratio of 1:2 with double HBSS at the neutral pH, which obtained isotonic formulations that are identified as the technique used in the transfection experiments. For zeta potential investigation, Doppler velocimetry was applied on the same instrument.
Adsorption and buffering capacity studies
To investigate the buffering capacity of the synthesized LDH, the method based on the adsorption studies of the transition metals was performed. In this regard, a stock nitrate salts solution of Cd2+, Cu2+, and Pb2+ was prepared, and 0.05 g of the ZnAl LDH (in the first study) and ZnAl HMTA (hexamethylenetetramine) LDH (in the second study) added to the solution (initial concentrations C0 = 1 mM). The initial concentrations of the metals were ranged from 0 to 2.5 mM in the experiments for Cd2+ and Pb2+, but this concentration increased up to 5.0 mM for Cu2+ to reach the plateau of the sorption isotherm. In the next step, the final suspensions were vigorously shaken, and after an exact time, they were centrifuged for 20 min with 10,000 rpm, and the supernatants were filtered and analyzed by atomic absorption spectrometry (AAS). The sorbed concentrations of the metals (mmol g−1) was investigated based on the below equation:
$$ {\text{C}}_{{\text{s}}} = \, \left( {{\text{C}}_{0} {-}{\text{ C}}_{{\text{e}}} } \right){\text{V}}/{\text{W}}{.} $$
In which the Cs is the sorbed concentration of the metal per sorbent, Ce and C0 are the equilibrium and initial solution concentrations (mmol L−1), and V is the volume of the metal solution (L), and W is the weight of the solid (g).
In order to investigate the buffering capacity of the synthesized LDH, the effect of temperature, time, media, and different adsorption models were skipped, and in this study, the effect of pH was investigated.
The possible interaction between the material and pCRISPR was evaluated by using gel electrophoresis. In this manner, the ratio between plasmid solution and loading buffer was 4:1; to be exact, 1 µL of the loading buffer was added to 4 µL of the plasmid solution, and several weight ratio of them were prepared. In the following, 5 µL of the prepared solution was poured into each wells, which have agarose gel (1%), including ethidium bromide (0.5 mL mL−1). The electrophoresis procedure was proceeded in TBE buffer for a period time of about 45 min by using the voltage of 80 V.
The human embryonic kidney cell line, HEK-293, was applied between 40 and 60 passage numbers in all of the following experiments. HEK-293 cells were grown in DMEM (Gibco, Invitrogen, Norway), which have been supplemented with 1 mM amino acids (non-essential ones) and fetal bovine serum (10%) (FBS) and 5% CO2 at 37 °C.
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay
To investigate the cytotoxicity of the prepared material, a HEK-293 cell line was applied based on the protocol was mentioned in the literature38,39. Briefly, the mentioned cells were seeded in a 96-well plate tissue culture at a standard density (105 cells per well) and incubated in 100 µL of DMEM/F12, which have been supplemented with 10% FBS for a day. Afterward, the culture media were replaced with the fresh one containing several dilutions of the synthesized material, and the prepared cells were incubated for 5 h. In the following, the resulted media were replaced with 100 µL of the fresh one for additional 24 h. Finally, the medium was replaced with 100 µL of the fresh one, including MTT, and was also incubated again at 37 °C for 4 h. After successful incubation for 4 h, the resulted medium was aspirated, and the MTT formazan, which has been generated in this step, was dissolved in the next 100 µL of DMSO, and the absorbance of each well was recorded utilizing a microplate reader (570 nm). The resulted data are presented as average ± SD (n = 3).
In vitro gene expression
Same as the above section, the transfection experiments were performed in HEK-293 cell lines using the pCRISPR, which have been expressed the GFP gene as a receptor gene. Transfections were proceeded by utilizing final nanocomplexes that formed at different weight ratios of the synthesized materials to pCRISPR. In the following, the cells were seeded in well plates at a standard density of 2 × 105 cells per well in 600 µL of complete medium, allowed to accomplish the procedure of attachment at 37 °C in an atmosphere containing CO2 (5%) for a day. Briefly, about 65% confluence of the cells were rinsed with PBS, and in the following, were incubated with 80 µL of the dispersion of nanocomplexes and 350 µL of serum-free culture medium for 3 h at 37 °C. In this step, the amount of pCRISPR should be kept at one µg in each well. The nanocomplex, which has been contained medium, was replaced with fresh DMEM/F12 with FBS (10%), and then cells were kept at that temperature for an additional 48 h before the transfection efficacy analysis. Afterward, the transfection efficacy was evaluated via green fluorescent protein (GFP) expression under a fluorescence microscope. Live cell imaging was performed using an Olympus FV-1000 inverted confocal fluorescence microscope, and GFP-positive cells surface area was investigated by applying ImageJ 1.45 software (Under the license of National Institutes of Health, USA). All of the experiments were done in triplicates.
Colloidal stability and reactivity in the biological fluids
The synthesized LDH was investigated in terms of colloidal stability in the biological fluids (based on the literature40,41,42), human-like plasma, by using dynamic light scattering as well as determining the zeta potential. For this purpose, the synthesized LDH was dispersed in an aqueous solution with a concentration of 280 mg L−1, and the NaCl solution (5 mM) was used for equilibration for 16 h. Furthermore, to analyze the protein corona formation effect along with the colloidal stability, typical adsorption of albumin to the nanostructure was conducted. In this case, the albumin concentrations were selected from 0.01 to 1.40 g L−1, and the pH was adjusted between 8 to 9.5 for the final product. The albumin adsorption ratio was represented as the ratio of the adsorbent amount to the maximum capacity of the adsorbent amount. In addition, the synthesized LDH reactivity in the biological media was investigated by the kinetics of dissolution in the acidic media. The initial pH was adjusted to 2.5, and by dispersing the synthesized LDH (with the same concentration as mentioned above), the pH was measured continuously.
Single-layer EGFP/LDH preparation
In this step, a substrate of quartz glass was carefully washed and cleaned with concentrated sulfuric acid (30%) and hydrogen peroxide (v/v 7:3) for 40 min and washed with deionized water two times. The colloidal LDH suspension was used for immersion of the prepared quartz glass for 20 min. The quartz glasses were removed from the suspension and dried at room temperature under nitrogen gas glow. In the next step, the modified quartz glasses with LDH were immersed in the EGFP solution at a pH of 8 with a concentration of 15 mg L−1, for another 20 min. The prepared quartz glasses were removed from the suspension again and dried at room temperature under nitrogen gas flow.
All of the statistical analyses related to MTT assays and transfection experiments were performed by one-way analysis of variance (ANOVA) followed by OriginPro 9.1 software compatible tests of Bonferroni post-hoc. In addition, all data represent means of ± SD of at least n = 3 independent sets of experiments.
Ethical concern
There are no human or animal experiments in this work; and all methods were carried out in accordance with the guidelines and statements. Human-like plasma were created in the laboratory and does not have any human or animal parts.
Characterizations
Both of the synthesized ZnAl LDH were investigated in terms of their crystallinity and solid-state chemistry via the powder XRD (PXRD) technique (Fig. 3a). As observed in Fig. 3, diffraction peaks at around 2θ values of 31°, 35°, 39°, 48°, 57°, 64° and 66° correspond to the crystallographic planes of (220), (012), (311), (102), (110), (103) and (112) which is in good agreement with the literature and JCPDS database (JCPDS 48-1023)43,44,45. It should be noted that the aim of this study was to investigate the potential biomedical application of the facile and without purified nanomaterial. Therefore, the impurities in the structures are not important. However, based on the literature46,47,48, these impurities are belonging to the metal salts as well as their byproducts. Based on Fig. 3, b, a strong and broad peak at around 350 cm−1 represents both stretching frequencies of hydroxide groups of the LDH structure and also the hydrogen bonds between them and water molecules in the interlayer structures. The absorption peak at around 1640 cm−1 is another sign of the bonding vibrations of these water molecules in the interlayer structures. Two characteristic peaks at around 1370 cm−1 and 1510 cm−1 corresponds to the symmetrical and asymmetrical stretching vibrations of the carboxylate functional groups, respectively. It should be noted that both of the ZnAl LDH and ZnAl HMTA LDH shows very similar FTIR spectra. However, the broad peak assigned for hydrogen bonds at around 3500 cm−1 is very narrower than for the structure in the presence of HMTA, which is completely based on the literature and acceptable43,49,50.
(a) PXRD and (b) FTIR spectra of the ZnAl LDH and ZnAl HMTA LDH.
Based on the results that appeared in Fig. 4, the shape and morphology of the synthesized ZnAl LDH showed layered nanostructure, with the metal nanoparticles on the surface. It should be noted that considerable aggregations were observed in both ZnAl LDH and ZnAl HMTA LDH FESEM results due to the non-controlled synthesis method. In this study, the aim was to synthesis cost-effective and simple LDH to investigate the role of tertiary amines on the structure for gene delivery applications. Therefore no attention was concentrated on the controlled synthesis method as well as filtrations; however, the aggregations do not seem to be important in CRISPR/Cas9 delivery due to the ability to confine the gene in this form. Furthermore, by the addition of HMTA in the synthesis procedure, more aggregations were observed, and the nanoparticles do not seem to be isolated from the structure.
FESEM results of the ZnAl LDH (a–d) and HMTA ZnAl LDH (e–h) with different magnifications.
In addition, the hydrodynamic size distribution of the synthesized ZnAl LDH and ZnAl HMTA LDH were investigated via the DLS technique (Supplementary Fig. S1). Based on the DLS's, the size of the ZnAl LDH is in the range of 90–500 nm, and the size of the ZnAl HMTA LDH is in the range of 70–300 nm. By the addition of HMTA to the synthesis procedure, some spaces would be created due to the presence of HMTA, and those spaces lead to a smaller size of the final product. In addition, the size range of the synthesized LDH in the presence of HMTA is narrower than a template free LDH due to the homogeneity that the tertiary amines created.
Formation of the material/pCRISPR nanoplatforms
The stability of a synthetic chemical is very important because of its ability to be used in the industry. In biomedical engineering, this is very crucial to make a balance between the stability of the nanoplatform outside the organism and the release of the cargo in the cytoplasm51,52,53. The materials with the positive charge can effectively interact with the negatively charged phosphate backbone of pCRISPR, and this mechanism is considered in the physiological pH and also above the isoelectric pH of their functional groups. In addition, some transition metal ions, including Zn2+, have the ability of DNA condensation, and for this reason, an LDH based on zinc could be considered as a promising pCRISPR carrier due to both the positive zeta potential as well as the condensation ability of the metal ions. Till now, there is no report about the investigation of these synergic effects, and also there is no report about the delivery of pCRISPR via ZnAl LDH. However, there is a report of ZnAl LDH for pCEP4/Cdk9 gene, which is not comprehensive as well30. In the following, Simplicity in the production of this type of non-viral vector can reduce costs, reduce preparation time, as well as simplify optimization for different cell lines and continue to be used in various diseases. In the synthesis of this group of compounds, LDH, the importance of using molecular molds is important, but the role of these molecular molds and templates in the application of gene delivery and especially CRISPR has not been studied so far. Therefore, we investigate the effect of the HMTA template in the pCRISPR delivery in terms of particle size, eta potential, relative cell viability (Fig. 5d), and also gel electrophoresis. In this manner, the ability of the synthesized nanomaterial to pCRISPR condensation was investigated by gel electrophoresis technique (Fig. 5a,b). As a result, the different binding strength between the synthesized nanoparticles and materials and pCRISPR with increasing the ratio was shown by pCRISPR migration bonds intensity (the quantity of those bands are not considered in this study, and just the quality of the migration was the main point of this study), and it has been shown that by increasing the ratio of pCRISPR and the synthesized materials and nanoparticles, the amount of migrated pCRISPR in the agarose gel was decreased. However, the slope of this decreasing in the migration of pCRISPR in the agarose gel is different for those materials. Among ZnAl LDH and HMTA ZnAl LDH, both have shown logical interaction with pCRISPR, and both can be able to condense the pDNA effectively. However, based on the literature54,55, negatively charged materials cannot be able to condense pDNA, but there is another mechanism that would be correct for this study, the synthesized LDH due to their three-dimensional structure as well as layered chemistry cannot be able to condense the genetic material, in fact, they are located around the CRISPR and surround it. Based on this fact, our synthesized LDH is able to surround the CRISPR/Cas9 successfully and make a strong electrostatic interaction with this56,57,58. Besides these, the effect of LDH and the role of the template on the performance of gene delivery were evaluated as well (Fig. 5c). In this case, the slope of increasing the zeta potential to the positive numbers was increased, and also the condensation process improved that would be because of more positive zeta potential as well as suitable morphology (the size and zeta potential were investigated from the ratio of material to CRISPR of 10–100) (Fig. 5e). It has been proved that very small size nanomaterials and nanoparticles cannot condense the pDNA effectively. However, by increasing their size, the ability of effective interaction with pDNA, and ultimately condensation process would improve dramatically. And in our case, the ability of the synthesized nanomaterials with increasing the M/C ratio towards surrounding pCRISPR increasing dramatically. It should be noted that both the HMTA template and also the metal ions have a synergic effect on increasing the electrostatic interactions with the pCRIPSR, and by this method, we can conclude that there is no need for removing the HMTA template from the synthesis batch.
Gel electrophoresis (a) (lane 1 stands for the positive control, lane 2 stands for treatment after WR of 10, lane 3 stands for treatment after WR of 20, lane 4 stands for treatment after WR of 50, lane 5 stands for treatment after WR of 80 and lane 6 stands for treatment after WR of 100) and (b) (lane 1 stands for the positive control, lane 2 stands for treatment after WR of 10, lane 3 stands for treatment after WR of 20, lane 4 stands for treatment after WR of 50, lane 5 stands for treatment after WR of 80, lane 6 stands for treatment after WR of 100 and lane 7 stands for treatment after WR of 150), zeta potential (c), relative cell viability assay (d) and particle size (e) of ZnAl LDH and HMTA ZnAl LDH in association with pCRISPR. WR is the weight ratio between Material (M) to CRISPR (C). The indicate data are presented as the mean (± SD) from three independent experiments.
Zeta potential of the nanocomplexes
This is very important to evaluate both the physical and chemical properties of nanomaterials for each application, and in terms of designing a non-viral gene delivery vector for pCRISPR delivery, this is an important part of investigating the zeta potential of them59,60,61. In this study, we have been measured the zeta potential of ZnAl LDH/pCRISPR and also ZnAl HMTA LDH/pCRISPR nanocomplexes with different weight ratios (Fig. 4c). The zeta potential of ZnAl LDH/pCRISPR nanocomplexes was recorded + 25 and + 30 for different weight ratios (WR) of ZnAl LDH/pCRISPR (M/C) = 10 and 100, respectively. Alongside, the zeta potential of ZnAl HMTA LDH/pCRISPR nanocomplexes was recorded about + 19 and over + 35 for WR(M/C) = 10 and 100, respectively. However, by the addition of template, HMTA, to the starting material, the zeta potential shifted negatively in a gradual and considerable slope. Furthermore, these phenomena lead us to conclude that the structure with the template has less tendency to form a stable nanocomplex with pCRISPR, but there is no significant difference (for the zeta potential and the particle size results) between the template free LDH and template-assisted LDH. Therefore, other tests were conducted to investigate the whole of the idea. Based on the literature62,63, the positive zeta potential for gene delivery applications is suitable, and it seems that the resulted zeta potential is applied to improve the nanocomplexes stability, as well as the efficiency of gene expression and cellular uptake. It should be noted that a little shift to the negative phase was expected by the addition of HMTA as the template, but as far as we know, there is no report to investigate this effect, and the present study is the first work on that64,65. However, there is a hypothesis that even less negative zeta potential nanostructure could be able to have stable nanocomplexes with pCRISPR or other genetic materials, therefore66, it would be a smart idea to modify the ZnAl HMTA LDH with some functional groups to increase the interaction with pCRISPR or genetic materials and investigate them in the future.
Cell viability assay
The stability of the nanoplatform along with low toxicity for multifunctional gene delivery is important for both outside of the cells and also instability inside the target cells as well as considerable gene transfection. In this step, the cytotoxicity of these nanomaterials' conjugates against HEK-293 cells was evaluated with different concentrations by MTT assay (Fig. 4d). It is clearly depicted that all of the synthesized nanomaterials presented very low toxicity to HEK-293 cells (viability is more than 80%) at different concentrations of the synthesizing procedure. By comparing these results with the gold standard non-viral gene delivery vector, polyethyleneimine (PEI), it could be considered that the synthesized nanomaterials have very low toxicity than any type of PEI and related conjugates, which make them a promising candidate for non-viral gene delivery vector. Based on the literature67,68,69, by increasing the amino group's deteriorations, the cytotoxicity decreases gradually, and this trend has been observed in high concentrations of HMTA ZnAl LDH, but this trend was not presented for ZnAl LDH without the template because the mentioned nanomaterial does not have any nitrogen-rich functional groups. By comparing our results with the gold-standard non-viral gene delivery vectors, especially PEI, by increasing the concentration of PEI on the surface of the carrier, the viability will be reduced in a very steeper slope70,71 than the ZnAl LDH and also HMTA ZnAl LDH, and it would be because of several factors, but the structural chemistry of LDH and the template in the association of the Zn and Al have the major role in this part, which is unknown right now. There is an important role about the addition of HMTA template to the nanostructure, as observed the cell viability decreased just in a very steeper slope, and it has not very different with the free-template LDH, but the results of gel electrophoresis showed the interactions between the synthesized nanomaterials and pCRISPR increased substantially, which means not only the nanostructure has the interaction with pCRISPR, but also the synthesis media including solvent and template have a direct effect on that, and for future experiments, the role of different solvents would certainly study.
The role of HMTA on the LDH nanosystem for pCRISPR delivery
Nearly all of the CRISPR-based studies are around using polymer-based nanostructures, including PEI, PLL, and even dendrimers. However, studying the chemistry of these non-viral gene delivery vectors are of great importance. One of the most important parts of designing a nanostructure for pCRISPR, is the ability of that in the endosomal escape. In this manner, the proton sponge effect was introduced as a standard scale for designing the concepts of the non-viral gene delivery vectors. This effect expressed that in the presence of weakly basic molecules, these basic molecules being protonated and cause an endosome to burst. For analysis of the buffering capacity of the synthesized ZnAl LDH, the adsorption studies based on the transition metal uptake were performed. The sorption isotherms of Cu2+, Cd2+, and Pb2+ of both of the synthesized LDH were presented similar to type H and L of the Giles classification72,73 (Fig. 6), which is in accordance with the literature74,75. In this study, the amount of metal ions sorption is not important. However, the effect of these metal ions on the pH of the environment and buffering capacity of the synthesized nanostructure is of great importance. The pH of the final nanostructure on the sorption process of these metal ions was recorded about 7.7–8.6, which is proof of the fact that these metals were proceeded the uptake mechanism by their metal hydroxide form76,77. However, the concentration of the precipitated metal hydroxides on the ZnAl HMTA LDH was higher than ZnAl LDH, which is because of the presence of tertiary amines HMTA.
The sorption isotherms of (A) Cu2+, (B) Cd2+, and (C) Pb2+ on the ZnAl LDH and ZnAl HMTA LDH.
In the following, the removal percentage of these metals from the ZnAl LDH and ZnAl HMTA LDH were investigated in the presence of different pH's (Fig. 7). Generally, by increasing the pH value, the releasing of the mentioned metals increased78. However, for all of the three metals, the removal percentage of the metals were screened above 90% at the pH range of 5.5 to 6.5 for ZnAl LDH and 4.5 to 6.5 for ZnAl HMTA LDH, which is proof of significant performance of both LDH in the adsorption of these metals, and the ZnAl HMTA LDH showed higher percentage in comparison with the ZnAl LDH. By reducing the pH value of the nanostructure through the dissolution of the adsorbent, the ZnAl LDH structure might collapse, and the metal ions might release from the LDH nanostructure, which is not in the scope of this study, but the final equilibrium pH values of the nanostructures are important. In this regard, the pH values of the final equilibriums were screened in the range of 6.5–7.5 for ZnAl LDH and 5.5–8.0 for ZnAl HMTA LDH, which is an indicator of considerable pH buffering capacity of both nanostructures79,80,81. However, the ZnAl HMTA LDH showed better pH buffering capacity due to the presence of tertiary amines on the nanostructure.
The effect of pH on the removal of Copper, Lead, and Cadmium (adsorbent dose = 1.5 g L−1; Temperature is 25 °C; initial metal concentration is 150 mg L−1 and the contact time is 300 min) of (A) ZnAl LDH and (B) ZnAl HMTA LDH. The indicate data are presented as the mean (± SD) from three independent experiments.
In vitro gene expression efficiency
In this study, pCRISPR expressing GFP was applied as a receptor to evaluate the capability of the synthesized nanoparticles and nanomaterials in terms of in vitro gene delivery in HEK-293 cell line. Fluorescent microscopy (Fig. 8a–d) was used to investigate the EGFP expression in HEK-293 cells with different weight ratios of the synthesized nanoparticles and nanomaterials to pCRISPR for gene transfection efficiency study (Fig. 8e). Based on the results, by increasing the weight ratio of the synthesized nanomaterials to pCRISPR, the trends of gene transfection efficiency increase dramatically. And also, the best result of gene expression of EGFP is about 38% in the HEK-293 cells for ZnAl HMTA LDH, and this could be considered as a next-generation gold standard in designing the simple, low cost and efficient non-viral gene delivery vector. Based on the literature, there are several factors that could influence transfection efficiency, including polymeric length and size of DNA82,83,84, but based on our knowledge, there is no report with a simple and low-cost nanomaterial that can achieve a significant transfection efficiency without the need for costly and difficult optimizations. In addition, in the presence of HMTA as the tertiary amine template in the structure of ZnAl LDH, a small decrease in the GFP positive cells were screened from material to CRISPR ratio of 5 to 20, but above the 20 ratios, the significant increase in the EGFP was observed.
(a–d) 2D fluorescence microscopy images of the ZnAl LDH and ZnAl HMTA LDH on the HEK-293 cell line in the presence of pCRISPR, and (e) GFP positive cells percentage of those LDH with different ratio of material to CRISPR. The Scale bar is 50 µm. The data indicate the 2D fluorescence microscopy and EGFP read are presented as the mean (± SD) from three independent experiments.
Colloidal stability and reactivity in biological fluids
Based on Fig. 9, the adsorbed albumin in a solution of NaCl (5 mM at pH 9.0) directly affected the zeta potential of the synthesized LDH. By increasing the Γ/Γ(m), the zeta potential decreased because of reaching the isoelectric point. However, the zeta potential decrease slope is much higher in the ZnAl LDH in comparison with the ZnAl HMTA LDH due to the superior buffering capacity of the template-assisted ZnAl LDH nanostructure. In addition, it should be noted that the presence of HMTA in the structure of the mentioned LDH leads to increasing the interlayer anions and cations in the nanostructure, and can be able to affect the albumin adsorption significantly40,41. Both of the synthesized LDH showed acceptable stability as well as superior and applicable protein adsorption, which is another proof for the CRISPR/Cas9 results. In addition, both of the synthesized LDH showed good stability in protein corona as well as human-like plasma serum.
(a) zeta potential dependency with Γ/Γ(m) ratio of the synthesized LDH, (b) Albumin adsorbed amount versus equilibrium concentration, (c) The spectra of fluorescence intensity of the EGFP/LDH in the presence of different pH environments (λex = 480 nm), (d) The spectra of fluorescence intensity of the EGFP/LDH in the presence of different concentrations of the protoporphyrins (λex = 480 nm; pH = 7; 0–15 µg mL−1). The indicate data are presented as the mean (± SD) from three independent experiments.
Reversible fluorescence response of the EGFP/LDH
The active chromophore compartment of EGFP is based on the imidazole ring, which has been linked together by a pi-bonds of the glycine-tyrosine dipeptide that represents in the neutral or anionic states85,86,87. So, the pH of the environment can be able to affect the fluorescence intensity of the EGFP/LDH directly, and this nanomaterial can be able to use as a suitable pH sensor in the range of pH of 4–10. Based on the results (Fig. 9c), EGFP/LDH in this study showed the strongest fluorescence intensity at pH = 8, and by decreasing the pH value, the fluorescence intensity decays over time. In addition, a reversible behavior has been observed for the fluorescence intensity of the EGFP/LDH in the range of pH 4–8, and this behavior was consistent even after eight cycles of treatment (Supplementary Fig. S2), which is an indicator of using this nanosystem for a pH sensory application. It should be noted that, by increasing the cycles, a slight decline in the fluorescence intensity has been observed that is because of the EGFP protonation process in the acidic pH88,89, specifically under 6.
These types of chromophores have several types of interactions, including stacking and charge interactions, as well as hydrogen bonding, which all of them come from the amino acids that have been surrounded in the nano-crystalline phase. The EGFP/LDH showed different fluorescence intensity in different environments of drying, including water and in air, and also in vacuum drying protocols. Based on the literature, the EGFP/LDH displayed higher fluorescence intensity in air and water90,91,92,93. Therefore, in this study, all of the experiments were conducted in air and water. Also, from a molecular chemistry perspective, a large volume of water has been located on the surface of LDH, which is extraordinarily stable. Therefore, these molecules will not be removed or ejected from the surface of the LDH through low-vacuum conditions and high-temperatures. This intrinsic phenomenon provides a significant hydrophilic condition in the EGFP/LDH interlayers for the EGFP molecules, which is by itself, can protect the fluorescence intensity of those molecules. In this regard, a series of experiments were conducted to prove this hypothesis and displayed that the solution of EGFP quenched its fluorescence intensity at 80 °C almost completely. However, the EGFP/LDH showed over 25% of its fluorescence intensity at the mentioned temperature, and the intensity decays completely at above of the 92 °C (Supplementary Fig. S3). This observation is because of the presence of water molecules on the interlayers' spaces of the LDH, which resulted in considerable thermal stability of the EGFP/LDH.
In order to examine the sensory capability of this EGFP/LDH, the sensitivity of this nanosystem against protoporphyrins was investigated. Protoporphyrins are derived from hemoglobin and have an important role in different types of inflammatory lesions, as well as the metabolism of energy and transportation of oxygen in different tissues and organs94,95. These protoporphyrins have extended π-conjugates nano-structures, which plays an important and critical role in the photophysical and biophysical fields96,97,98. By screening the fluorescence spectra of the EGFP/LDH in the presence of different concentrations of the protoporphyrins (0–15 µg mL−1), it has been depicted that the fluorescence intensity decays in a semi-linear slope upon addition of protoporphyrins (Fig. 9d). Further experiments were conducted to examine the reversibility of these sensory observations (Supplementary Fig. S4), and the same as the pH conditions, the EGFP/LDH showed significant reversibility after eight cycles of treatment with the protoporphyrins. The mechanism of this fluorescence decay in the presence of protoporphyrins are associated with the inner-filter effect (IFE), which has been made emphasis on the quenching of the fluorescence intensity in the presence of protoporphyrins due to the fluorophore's emission and/or excitation agents' absorptions on the surface of the LDH. Interestingly, the IFE does not need any chemical or even strong physical interactions and/or bonds between the fluorophore and the absorber; however, the routine and usual fluorescence resonance energy transfer-based methods need a chemical linkage or strong physical interactions between them.
In this work, the aim was to investigate the role of layered double hydroxide and different metal cations as well as the role of tertiary amines and templates in smart gene delivery systems. In this case, ZnAl LDH and ZnAl HMTA LDH were selected and optimized based on different parameters, including pH, size, zeta potential, and the ratio of material to CRISPR. The chemical characterizations were proved successful synthesis of these nanomaterials, and biological characterization showed interestingly considerable binding ability with the DNA as well as low cellular toxicity, which is a promising result with the low cost and simple nanosystem in the delivery of pCRISPR. Furthermore, both of the synthesized LDH showed acceptable stability in protein corona and the human-like plasma serum. Based on the literature, this study would be considered as a pioneer of designing and preparing a new gold standard class of 2D crystalline platforms for pCRISPR delivery, with low cost and simple preparation procedure. It should be mentioned that, based on the chemical hypothesis, which has been mentioned and discussed earlier, a new mechanism for reducing the size of the nano platform was introduced in this study that is based on charge separation and leads to reducing the size of the final carrier.
Rabiee, N. et al. High-gravity-assisted green synthesis of NiO-NPs anchored on the surface of biodegradable nanobeads with potential biomedical applications. J. Biomed. Nanotechnol. 16, 520–530 (2020).
Rabiee, N. et al. Green synthesis of ZnO NPs via Salvia hispanica: Evaluation of potential antioxidant, antibacterial, mammalian cell viability, H1N1 influenza virus inhibition and photocatalytic activities. J. Biomed. Nanotechnol. 16, 456–466 (2020).
Ahmadi, S. et al. Controlled gene delivery systems: Nanomaterials and chemical approaches. J. Biomed. Nanotechnol. 16, 553–582 (2020).
Liu, C., Zhang, L., Liu, H. & Cheng, K. Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications. J. Control. Release 266, 17–26 (2017).
Mout, R. et al. Direct cytosolic delivery of CRISPR/Cas9-ribonucleoprotein for efficient gene editing. ACS Nano 11, 2452–2458 (2017).
Su, T. et al. A CRISPR-Cas9 assisted non-homologous end-joining strategy for one-step engineering of bacterial genome. Sci. Rep. 6, 1–11 (2016).
Yamato, T. et al. Single crossover-mediated targeted nucleotide substitution and knock-in strategies with CRISPR/Cas9 system in the rice blast fungus. Sci. Rep. 9, 1–8 (2019).
Yin, H. et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat. Biotechnol. 34, 328 (2016).
Yin, H., Kauffman, K. J. & Anderson, D. G. Delivery technologies for genome editing. Nat. Rev. Drug Discov. 16, 387 (2017).
Zhang, Z. et al. Cationic polymer-mediated CRISPR/Cas9 plasmid delivery for genome editing. Macromol. Rapid Commun. 40, 1800068 (2019).
Glass, Z., Lee, M., Li, Y. & Xu, Q. Engineering the delivery system for CRISPR-based genome editing. Trends Biotechnol. 36, 173–185 (2018).
Miller, J. B. et al. Non-viral CRISPR/Cas gene editing in vitro and in vivo enabled by synthetic nanoparticle co-delivery of Cas9 mRNA and sgRNA. Angew. Chem. Int. Ed. 56, 1059–1063 (2017).
Kam, N. W. S., Liu, Z. & Dai, H. Functionalization of carbon nanotubes via cleavable disulfide bonds for efficient intracellular delivery of siRNA and potent gene silencing. J. Am. Chem. Soc. 127, 12492–12493 (2005).
Wang, H.-X. et al. CRISPR/Cas9-based genome editing for disease modeling and therapy: Challenges and opportunities for nonviral delivery. Chem. Rev. 117, 9874–9906 (2017).
Scaletti, F. et al. Protein delivery into cells using inorganic nanoparticle—protein supramolecular assemblies. Chem. Soc. Rev. 47, 3421–3432 (2018).
Choy, J.-H., Kwak, S.-Y., Park, J.-S., Jeong, Y.-J. & Portier, J. Intercalative nanohybrids of nucleoside monophosphates and DNA in layered metal hydroxide. J. Am. Chem. Soc. 121, 1399–1400 (1999).
Choy, J. H., Kwak, S. Y., Jeong, Y. J. & Park, J. S. Inorganic layered double hydroxides as nonviral vectors. Angew. Chem. Int. Ed. 39, 4041–4045 (2000).
Park, D.-H., Kim, J.-E., Oh, J.-M., Shul, Y.-G. & Choy, J.-H. DNA core@ inorganic shell. J. Am. Chem. Soc. 132, 16735–16736 (2010).
Ladewig, K., Niebert, M., Xu, Z. P., Gray, P. P. & Lu, G. Q. M. Controlled preparation of layered double hydroxide nanoparticles and their application as gene delivery vehicles. Appl. Clay Sci. 48, 280–289 (2010).
Liu, J. et al. Synthesis of nanorattles with layered double hydroxide core and mesoporous silica shell as delivery vehicles. J. Mater. Chem. 21, 10641–10644 (2011).
Tyner, K. M. et al. Intercalation, delivery, and expression of the gene encoding green fluorescence protein utilizing nanobiohybrids. J. Control. Release 100, 399–409 (2004).
Tyner, K. M., Schiffman, S. R. & Giannelis, E. P. Nanobiohybrids as delivery vehicles for camptothecin. J. Control. Release 95, 501–514 (2004).
Xu, Z. P. et al. Layered double hydroxide nanoparticles as cellular delivery vectors of supercoiled plasmid DNA. Int. J. Nanomed. 2, 163 (2007).
Li, L., Gu, W., Liu, J., Yan, S. & Xu, Z. P. Amine-functionalized SiO2 nanodot-coated layered double hydroxide nanocomposites for enhanced gene delivery. Nano Res. 8, 682–694 (2015).
ADS Article CAS Google Scholar
Ladewig, K., Xu, Z. P. & Lu, G. Q. Layered double hydroxide nanoparticles in gene and drug delivery. Expert Opin. Drug Deliv. 6, 907–922 (2009).
Wang, Q. & O'Hare, D. Recent advances in the synthesis and application of layered double hydroxide (LDH) nanosheets. Chem. Rev. 112, 4124–4155 (2012).
Khan, A. I., Lei, L., Norquist, A. J. & O'Hare, D. Intercalation and controlled release of pharmaceutically active compounds from a layered double hydroxideElectronic supplementary information (ESI) available: Fig. S1: X-ray diffraction patterns of (a)[LiAl2 (OH) 6] Cl· H2O and (b) LDH/Ibuprofen intercalate. See http://www.rsc.org/suppdata/cc/b1/b106465g. Chemical Communications, 2342–2343 (2001).
Bao, W., Wang, J., Wang, Q., O'Hare, D. & Wan, Y. Layered double hydroxide nanotransporter for molecule delivery to intact plant cells. Sci. Rep. 6, 1–9 (2016).
Andrea, K. A. et al. Adsorption of oligo-DNA on magnesium aluminum-layered double-hydroxide nanoparticle surfaces: Mechanistic implication in gene delivery. Langmuir 33, 3926–3933 (2017).
Yazdani, P. et al. Layered double hydroxide nanoparticles as an appealing nanoparticle in gene/plasmid and drug delivery system in C2C12 myoblast cells. Artif. Cells Nanomed. Biotechnol. 47, 436–442 (2019).
Park, D. H., Cho, J., Kwon, O. J., Yun, C. O. & Choy, J. H. Biodegradable inorganic nanovector: Passive versus active tumor targeting in siRNA transportation. Angew. Chem. Int. Ed. 55, 4582–4586 (2016).
Zhu, K., Gao, Y., Tan, X. & Chen, C. Polyaniline-modified Mg/Al layered double hydroxide composites and their application in efficient removal of Cr (VI). ACS Sustain. Chem. Eng. 4, 4361–4369 (2016).
Li, M. et al. Potent and durable antibacterial activity of ZnO-dotted nanohybrids hydrothermally derived from ZnAl-layered double hydroxides. Colloids Surf. B 181, 585–592 (2019).
Li, C. et al. Enhanced visible-light-induced photocatalytic performance of Bi2O3/ZnAl-LDH–C for dyes removal in water. Mater. Lett. 244, 215–218 (2019).
Carboni, V., Maaliki, C., Alyami, M., Alsaiari, S. & Khashab, N. Synthetic vehicles for encapsulation and delivery of CRISPR/Cas9 gene editing machinery. Adv. Ther. 2, 1800085 (2019).
Radmanesh, F. et al. Photoluminescent Functionalized Carbon Dots for CRISPR delivery: synthesis, optimization and cellular investigation. Nanotechnology 30, 135101 (2019).
ADS PubMed Article CAS Google Scholar
He, X. et al. Simple and efficient targeted intracellular protein delivery with self-assembled nanovehicles for effective cancer therapy. Adv. Func. Mater. 29, 1906187 (2019).
Fan, X., Jiang, S., Li, Z. & Loh, X. J. Conjugation of poly (ethylene glycol) to poly (lactide)-based polyelectrolytes: An effective method to modulate cytotoxicity in gene delivery. Mater. Sci. Eng. C 73, 275–284 (2017).
Liu, C., Shao, N., Wang, Y. & Cheng, Y. Clustering small dendrimers into nanoaggregates for efficient DNA and siRNA delivery with minimal toxicity. Adv. Healthc. Mater. 5, 584–592 (2016).
Vasti, C., Bedoya, D. A., Rojas, R. & Giacomelli, C. E. Effect of the protein corona on the colloidal stability and reactivity of LDH-based nanocarriers. J. Mater. Chem. B 4, 2008–2016 (2016).
Gu, Z., Zuo, H., Li, L., Wu, A. & Xu, Z. P. Pre-coating layered double hydroxide nanoparticles with albumin to improve colloidal stability and cellular uptake. J. Mater. Chem. B 3, 3331–3339 (2015).
Zuo, H., Gu, Z., Cooper, H. & Xu, Z. P. Crosslinking to enhance colloidal stability and redispersity of layered double hydroxide nanoparticles. J. Colloid Interface Sci. 459, 10–16 (2015).
ADS CAS PubMed Article Google Scholar
Liu, L., Cheng, M. & Yang, Z. Improved performance of flower-like ZnAl LDH growing on carbon nanotubes used in zinc–nickel secondary battery. Electrochim. Acta 277, 67–76 (2018).
ADS CAS Article Google Scholar
Chen, W. & Qu, B. LLDPE/ZnAl LDH-exfoliated nanocomposites: effects of nanolayers on thermal and mechanical properties. J. Mater. Chem. 14, 1705–1710 (2004).
Chakraborty, M. et al. Methotrexate intercalated ZnAl-layered double hydroxide. J. Solid State Chem. 184, 2439–2445 (2011).
Inayat, A., Klumpp, M. & Schwieger, W. The urea method for the direct synthesis of ZnAl layered double hydroxides with nitrate as the interlayer anion. Appl. Clay Sci. 51, 452–459 (2011).
Liu, Y. & Yang, Z. Intercalation of sulfate anions into a Zn–Al layered double hydroxide: their synthesis and application in Zn–Ni secondary batteries. Rsc Advances 6, 68584–68591 (2016).
Li, Z., Zhang, S. & Lee, W. E. Molten salt synthesis of zinc aluminate powder. J. Eur. Ceram. Soc. 27, 3407–3412 (2007).
Hu, J. et al. Preparation and enhanced properties of polyaniline/grafted intercalated ZnAl-LDH nanocomposites. Appl. Surf. Sci. 328, 325–334 (2015).
Guo, X., Yin, P. & Yang, H. Superb adsorption of organic dyes from aqueous solution on hierarchically porous composites constructed by ZnAl-LDH/Al (OH) 3 nanosheets. Microporous Mesoporous Mater. 259, 123–133 (2018).
Bila, H., Kurisinkal, E. E. & Bastings, M. M. Engineering a stable future for DNA-origami as a biomaterial. Biomater. Sci. 7, 532–541 (2019).
Austin, M. J. & Rosales, A. M. Tunable biomaterials from synthetic, sequence-controlled polymers. Biomater. Sci. 7, 490–505 (2019).
Bank, R. A. Limiting biomaterial fibrosis. Nat. Mater. 18, 781 (2019).
Alyami, M. Z. et al. Cell-type-specific CRISPR/Cas9 delivery by biomimetic metal organic frameworks. J. Am. Chem. Soc. 142, 1715–1720 (2020).
Rohiwal, S. et al. Polyethylenimine based magnetic nanoparticles mediated non-viral CRISPR/Cas9 system for genome editing. Sci. Rep. 10, 1–12 (2020).
Nishitani, C. et al. Efficient genome editing in apple using a CRISPR/Cas9 system. Sci. Rep. 6, 1–8 (2016).
Zhang, P. et al. Recent advances in siRNA delivery for cancer therapy using smart nanocarriers. Drug Discov. Today 23, 900–911 (2018).
Sun, W. & Gu, Z. Tailoring non-viral delivery vehicles for transporting genome-editing tools. Sci. China Mater. 60, 511–515 (2017).
Bonengel, S., Prüfert, F., Jelkmann, M. & Bernkop-Schnürch, A. Zeta potential changing phosphorylated nanocomplexes for pDNA delivery. Int. J. Pharm. 504, 117–124 (2016).
Tang, J. et al. Preparation of optimized lipid-coated calcium phosphate nanoparticles for enhanced in vitro gene delivery to breast cancer cells. J. Mater. Chem. B 3, 6805–6812 (2015).
Cheng, X. & Lee, R. J. The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery. Adv. Drug Deliv. Rev. 99, 129–137 (2016).
Imani, R., Prakash, S., Vali, H. & Faghihi, S. Polyethylene glycol and octa-arginine dual-functionalized nanographene oxide: An optimization for efficient nucleic acid delivery. Biomater. Sci. 6, 1636–1650 (2018).
Kudsiova, L. et al. Trichain cationic lipids: The potential of their lipoplexes for gene delivery. Biomater Sci. 7, 149–158 (2019).
Di Mauro, V. et al. Bioinspired negatively charged calcium phosphate nanocarriers for cardiac delivery of MicroRNAs. Nanomedicine 11, 891–906 (2016).
Tang, J. et al. Enhanced delivery of siRNA to triple negative breast cancer cells in vitro and in vivo through functionalizing lipid-coated calcium phosphate nanoparticles with dual target ligands. Nanoscale 10, 4258–4266 (2018).
Liu, S. et al. Zinc coordination substitute amine: A noncationic platform for efficient and safe gene delivery. ACS Macro Lett. 7, 868–874 (2018).
Ahn, C.-H., Chae, S. Y., Bae, Y. H. & Kim, S. W. Biodegradable poly (ethylenimine) for plasmid DNA delivery. J. Control. Release 80, 273–282 (2002).
Bishop, C. J., Tzeng, S. Y. & Green, J. J. Degradable polymer-coated gold nanoparticles for co-delivery of DNA and siRNA. Acta Biomater. 11, 393–403 (2015).
Keles, E., Song, Y., Du, D., Dong, W.-J. & Lin, Y. Recent progress in nanomaterials for gene delivery applications. Biomater. Sci. 4, 1291–1309 (2016).
Kim, J., Kim, H. & Kim, W. J. Single-layered MoS2–PEI–PEG nanocomposite-mediated gene delivery controlled by photo and redox stimuli. Small 12, 1184–1192 (2016).
Loh, X. J., Lee, T.-C., Dou, Q. & Deen, G. R. Utilising inorganic nanocarriers for gene delivery. Biomater. Sci. 4, 70–86 (2016).
Giles, C., MacEwan, T., Nakhwa, S. & Smith, D. 786. Studies in adsorption. Part XI. A system of classification of solution adsorption isotherms, and its use in diagnosis of adsorption mechanisms and in measurement of specific surface areas of solids. J. Chem. Soc. (Resumed) 23, 3973–3993 (1960).
Giles, C. H., Smith, D. & Huitson, A. A general treatment and classification of the solute adsorption isotherm. I. Theoretical. J. Colloid Interface Sci. 47, 755–765 (1974).
He, H., Kang, H., Ma, S., Bai, Y. & Yang, X. High adsorption selectivity of ZnAl layered double hydroxides and the calcined materials toward phosphate. J. Colloid Interface Sci. 343, 225–231 (2010).
Yu, J., Lu, L., Li, J. & Song, P. Biotemplated hierarchical porous-structure of ZnAl-LDH/ZnCo 2 O 4 composites with enhanced adsorption and photocatalytic performance. RSC advances 6, 12797–12808 (2016).
Komarneni, S., Kozai, N. & Roy, R. Novel function for anionic clays: Selective transition metal cation uptake by diadochy. J. Mater. Chem. 8, 1329–1331 (1998).
Chen, S. et al. Enhanced photocatalytic activity of molybdenum disulfide by compositing ZnAl–LDH. Colloids Surf. A 586, 124140 (2020).
Ferreira, O. P., De Moraes, S. G., Duran, N., Cornejo, L. & Alves, O. L. Evaluation of boron removal from water by hydrotalcite-like compounds. Chemosphere 62, 80–88 (2006).
González, M., Pavlovic, I., Rojas-Delgado, R. & Barriga, C. Removal of Cu2+, Pb2+ and Cd2+ by layered double hydroxide–humate hybrid. Sorbate and sorbent comparative studies. Chem. Eng. J. 254, 605–611 (2014).
Seida, Y. & Nakano, Y. Removal of humic substances by layered double hydroxide containing iron. Water Res. 34, 1487–1494 (2000).
Zhou, H., Jiang, Z. & Wei, S. A new hydrotalcite-like absorbent FeMnMg-LDH and its adsorption capacity for Pb2+ ions in water. Appl. Clay Sci. 153, 29–37 (2018).
Taranejoo, S., Liu, J., Verma, P. & Hourigan, K. A review of the developments of characteristics of PEI derivatives for gene delivery applications. J. Appl. Polym. Sci. 132, 42096 (2015).
Wu, D. et al. RGD/TAT-functionalized chitosan-graft-PEI-PEG gene nanovector for sustained delivery of NT-3 for potential application in neural regeneration. Acta Biomater. 72, 266–277 (2018).
Schulze, J. et al. Spray-dried nanoparticle-in-microparticle delivery systems (NiMDS) for gene delivery, comprising polyethylenimine (PEI)-based nanoparticles in a poly (vinyl alcohol) matrix. Small 14, 1701810 (2018).
Schwinges, P. et al. A bifunctional dermaseptin–thanatin dipeptide functionalizes the crop surface for sustainable pest management. Green Chem. 21, 2316–2325 (2019).
Bae, Y. et al. Dipeptide-functionalized polyamidoamine dendrimer-mediated apoptin gene delivery facilitates apoptosis of human primary glioma cells. Int. J. Pharm. 515, 186–200 (2016).
Brabham, R. L. et al. Rapid sodium periodate cleavage of an unnatural amino acid enables unmasking of a highly reactive α-oxo aldehyde for protein bioconjugation. Org. Biomol. Chem. 18, 4000–4003 (2020).
Zhang, P., Hu, Y., Ma, R., Li, L. & Lu, J. Enhanced green fluorescence protein/layered double hydroxide composite ultrathin films: Bio-hybrid assembly and potential application as a fluorescent biosensor. J. Mater. Chem. B 5, 160–166 (2017).
Oscar, B. G. et al. Excited-state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging. Proc. Natl. Acad. Sci. 111, 10191–10196 (2014).
Lee, W., Kim, H., Kang, Y., Lee, Y. & Yoon, Y. A biosensor platform for metal detection based on enhanced green fluorescent protein. Sensors 19, 1846 (2019).
Mishra, A. et al. A new arene–Ru based supramolecular coordination complex for efficient binding and selective sensing of green fluorescent protein. Dalton Trans. 43, 6032–6040 (2014).
Dai, G., Hu, J., Zhao, X. & Wang, P. A colorimetric paper sensor for lactate assay using a cellulose-Binding recombinant enzyme. Sens. Actuators B Chem. 238, 138–144 (2017).
Stanziale, S. F. et al. Oncolytic herpes simplex virus-1 mutant expressing green fluorescent protein can detect and treat peritoneal cancer. Hum. Gene Ther. 15, 609–618 (2004).
Smith, K. M., Parish, D. W. & Inouye, W. S. Methyl deuteration reactions in vinylporphyrins: Protoporphyrins IX, III, and XIII. J. Org. Chem. 51, 666–671 (1986).
Tripathy, B. C. & Rebeiz, C. A. Chloroplast biogenesis: Quantitative determination of monovinyl and divinyl Mg-protoporphyrins and protochlorophyll (ides) by spectrofluorometry. Anal. Biochem. 149, 43–61 (1985).
Zhang, Z. & Imae, T. Hydrogen-bonding stabilized self-assembled monolayer film of a functionalized diacid, protoporphyrin IX zinc (II), onto a gold surface. Nano Lett. 1, 241–243 (2001).
Heleg-Shabtai, V., Katz, E. & Willner, I. Assembly of microperoxidase-11 and Co (II)-protoporphyrin IX reconstituted myoglobin monolayers on Au-electrodes: integrated bioelectrocatalytic interfaces. J. Am. Chem. Soc. 119, 8121–8122 (1997).
Komatsu, T., Wang, R.-M., Zunszain, P. A., Curry, S. & Tsuchida, E. Photosensitized reduction of water to hydrogen using human serum albumin complexed with zinc–protoporphyrin IX. J. Am. Chem. Soc. 128, 16297–16301 (2006).
The authors acknowledge the Sharif University of Technology Research Council for the support.
Department of Chemistry, Sharif University of Technology, Tehran, Iran
Navid Rabiee, Mojtaba Bagherzadeh, Amir Mohammad Ghadiri & Ghazal Salehi
Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Yousef Fatahi & Rassoul Dinarvand
Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
Navid Rabiee
Mojtaba Bagherzadeh
Amir Mohammad Ghadiri
Ghazal Salehi
Yousef Fatahi
Rassoul Dinarvand
N.R. and M.B. conceived the original idea and with the contributions of A.M.G. and G.S. and Y.F. developed the study. N.R. performed the experiments with the contribution of A.M.G. and G.S, and analyzed the results with the contribution of R.D. The manuscript were written by N.R., Y.F. and R.D. and were finalized with the assistance of all authors. M.B. provided advice, expertise, reagents and materials. All authors read and edited the manuscript.
Correspondence to Mojtaba Bagherzadeh.
The authors declare no competing interests.
Supplementary Information.
Rabiee, N., Bagherzadeh, M., Ghadiri, A.M. et al. ZnAl nano layered double hydroxides for dual functional CRISPR/Cas9 delivery and enhanced green fluorescence protein biosensor. Sci Rep 10, 20672 (2020). https://doi.org/10.1038/s41598-020-77809-1
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
About Scientific Reports
Guest Edited Collections
Scientific Reports Top 100 2017
Scientific Reports Top 10 2018
Editorial Board Highlights
Author Highlights
Scientific Reports ISSN 2045-2322 (online)
Close banner Close
Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.
I agree my information will be processed in accordance with the Nature and Springer Nature Limited Privacy Policy.
Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing


text:
THE FARE STRIKE IS ON!
PAY MORE TO WAIT LONGER?
NO MUNI FARE HIKE!
NO MUNI SERVICE CUTS!
The fare strike continues, and goes on until Muni reverses its fare hike and service cuts. Basta!
Please contact us if you want flyers. You can also download flyers from our website. Click here.
* Call for Organizers
* Another Report from Day One
* Update on Muni Security Thuggery
* Muni Fare Strike Legal Team
CALL FOR ORGANIZERS
As can be seen from the report below, a small team of organizers can have a big impact if we adapt our organizing tactics to the concrete situation. We are now actively recruiting new organizers to join our teams, and help spread the fare strike throughout the city. Want to join us? Give us a call at (415) 648-2535, or email us at MuniFareStrike@yahoo.com. Solidarity in action is the key to victory.
ANOTHER REPORT from DAY ONE
The following is a report from one of the Muni Fare Strike organizing teams on Day One of the fare strike. Their experiences are very instructive, and show the evolution over the course of the day from a team working at one fixed location, to a mobile organizing team spreading the word about the fare strike by example throughout the city.
"On the first day of the fare strike, our team of six (including one Chinese, one Korean and one Spanish speaker) went to our post at Divisadero and Geary at 7:30 AM, where we had a steady flow of 38-Geary buses, the busiest line in the City, as well as a less frequent cross flow of the 24-Divisadero. We had one large banner that simply said FARE STRIKE Starting Sept. 1, and lots of smaller posters that we either carried as picket signs or taped up to the bus shelters in all four directions (click here).
"We started handing out leaflets to riders and drivers. There was no Muni security around at this point. Two of us flyered the fairly frequent 38-Geary buses going outbound and even put English, Chinese and Spanish fare strike stickers over the Muni stickers on the glass of the back doors warning riders not to enter using the back door. One driver stopped, got out of the bus motioned me forward and asked me to put stickers on the front door. I was surprised and elated. (In the late afternoon I saw the same bus downtown, and the English and Chinese fare strike stickers were still on the front door.)
"Muni light duty employees -- the first guy said he was a driver on disability -- started arriving after 8:00 AM. They did not interfere with us, but were handing out photocopied sample citations and a Muni brochure about the fare increase. We had initially concentrated all our efforts on the southwest corner to the 38-Geary buses inbound to downtown. When more Muni employees arrived, we spread out to all four bus stops.
"Many riders noticed our large FARE STRIKE banner and could see us with our picket signs. We got a lot of thumbs-up. One man slid the narrow window open and motioned with his hand for me to give him some flyers. I handed him a bunch and they instantly got distributed to everyone sitting near him. Obviously others wanted to see what the flyer said, so I gave him another larger pile and they quickly went both ways up and down the long, articulated bus. When the bus drove off I could see people eagerly reading the flyer at both the front and the rear of the bus. We got several thumbs-up from that bus.
"Around 9:00 AM more Muni fare enforcement folks showed up, one even appeared to be some kind of a bureaucrat, and it was crowding us out of the narrow sidewalk of the southwest corner stop for the 38-Geary. We were nearly out of flyers anyway, so we decided to see if we could re-supply ourselves at 16th & Mission and start getting on the buses.
"Mission and 16th had a huge cop presence when we got there just after 10:00 AM. So, after checking in with the organizers there, a couple of us hit the buses by walking down Mission a block to the next bus stop. The first time was pretty easy. Just a word or two about being on 'fare strike' and not paying and whoosh, we were on -- with the people at the front of the bus having seen what we had just done and we started flyering them. Then to the end of the bus and off, having flyered everyone, and then across the street, on again in the opposite direction and we did it again... and again... and again.
"We took a break for lunch, lost a couple comrades from our early morning team of six, and four of us spent the rest of the afternoon surfing buses up and down Mission and to downtown. In that stretch, we must have ridden around fifteen buses, flyered nearly everyone on board and several times we had forums at the back of the bus with pissed off folks who were totally angry and 100% behind participating in a fare strike themselves, many still fired up and talking among themselves when we jumped off and got on another bus.
"In all that time only ONE driver said 'No, you can't enter my bus without paying the fare.' (All day I rode over twenty buses and he was the only one.)
"About 4:30 PM we went to 4th & Market. It was the biggest Muni enforcement circus we had seen all day. So our team, now down to two people, headed down the block to Market and Kearny, which did not really build up crowds like at 4th and Market. We just started surfing buses and flyering on them from there into Chinatown. We had patched two signs together to read FARE STRIKE in English, NO MUNI FARE TODAY in Spanish and RIDERS DON'T PAY -- DRIVERS DON'T COLLECT in Chinese. At the bus stops it became a powerful magnet for enthusiastically supportive Chinese people. We got on and off the bus going in the same direction several times and then walked up from Columbus to Stockton.
"It was totally amazing, wherever we went with the sign, people came up and thanked us and asked for a copy from the pile of flyers in our hands. We got stopped in front of a fruit and vegetable grocery and could not leave because so many people were coming up to us. Finally, after giving out what seemed like a hundred flyers in a matter of minutes, we got on the buses again. We went a block, got off and it happened all over again. And then did it again... and again.
"At another crowded bus stop, a pleasant middle-aged Chinese woman of moderate English ability kept saying that it sounded like a good idea to fare strike, but the driver wouldn't let us do it. We assured her that she could, but she and the crowd of others hanging on our every word didn't believe us. So, we got to the front of people piling onto the bus and got on and told the driver we were on fare strike and then just kept walking down the aisle. The middle-aged woman followed, with some high school girls in tow, and all of them were beside themselves with delight, completely giddy with satisfaction of having got on for free. We got off soon after and as we were leaving asked them to show others how to do it. It was a magical experience of seeing people empower themselves.
"By then we were going through the Stockton Tunnel and got out before Union Square. Still, people would see the sign and ask for a flyer and then thank us.
"We ended up back at 4th & Market as it was getting dark, things were winding down and soon after we called it a night. All-in-all, I would have to say that it was a very successful day, especially after we realized that the best way to foment the fare strike was riding for free -- and encouraging others to do likewise."
[More reports to come...]
UPDATE on MUNI SECURITY THUGGERY
As reported in our previous bulletin, one rider was arrested at 16th and Mission on Day One of the fare strike. The incident started when a Muni security guard grabbed this rider by the seat of his pants after he had entered a bus through the back door, and dragged him back out onto the street. The inevitable altercation ensued, the man was chased up the street by several Muni guards, roughed up and then arrested.
The rider is now in jail, charged with assault. A Muni Fare Strike Legal Team lawyer will be representing him, and we have witnesses who can testify about what really happened. If anybody is to be prosecuted, it should be the Muni security squad that inflicted corporal punishment on this rider for the dastardly crime of getting on the bus by the back door.
A San Francisco jury recently awarded $27 million to the family of a 4-year-old girl who died after being struck by a Muni truck, one of the largest personal injury awards ever against the City. How many fare increases will we have to suffer to pay for that? The Muni officials who are running the security squads combating the fare strike should think long and hard about the fact that San Francisco juries, both criminal and civil, do not seem to hold Muni officialdom in very high regard.
MUNI FARE STRIKE LEGAL TEAM
Remember, our lawyers, will defend anyone who gets a ticket or has other legal problems as a result of the fare strike. If you get a ticket, call the Muni Fare Strike Legal Team at 285-1011, and let us defend you. For more info on the legal team, click here.
PAY MORE TO WAIT LONGER? NO WAY!

redpajama_set_name: RedPajamaCommonCrawl


Below are just a collection of the testimonials for derek cheshire.
Derek and Samuel delivered a Creativity In Action workshop during our Growth Week in September 2016. The Creativity In Action workshop was very well received by the team and voted one of the best sessions of the week.
Derek was engaged to provide training and consultancy to my client Espartec of San Luis Potosi, Mexico. Several months later the company has continued to move forward in difficult market conditions. I would not hesitate in recommending Derek's services. He has an in-depth knowledge of Creativity, Innovation and Strategy which he communicates easily to others.
Our company has a very good knowledge of the work and expertise of Mr. Derek Cheshire and his firm Creative4Business regarding Innovation and Creativity matters. We have been working together for the last 2 years when we started our Innovation Department in México, and we found the help, teaching and advice of Mr. Cheshire very valuable for us.
Thanks again for last night, your takeaways were top drawer and can be used easily. This is the kind of presentation our members love so thanks for delivering! If you ever need a testimonial let me know!
Thank you for this morning. It was thought provoking. The exercises were really good and engaged the audience and I am sure got them thinking about creativity and innovation being applied to their own businesses.
Thank you so much for your time today. I really enjoyed the masterclass and learned some amazing techniques.
We had positive feedback from the whole conference and your bit was very well received, thank you very much! I think we can safely say people were awake and had forgotten lunchtime by the end of it!! Thank you again for your help.
I don't think I have ever seen our members ask so many questions in a Q & A's session. You obviously got them thinking and made me want to run my business like a well-known terrorist organisation. Well done….I think!
Thank you for your interesting speech 'Creating the difference' on behalf of Inspire Success Ltd. We have had a terrific response from our delegates as we could have predicted from the reaction on the night. You clearly are an expert in your field and surprised everyone with your revelation on the night.
Thank you once more and we look forward to working again with you in the future.
Thank you and Kenny for running this workshop for the Association of MBA's in Scotland. Our members were delighted and enjoyed participating in it. Derek and Kenny have created a tremendous guide through the scary yet exhilarating landscape of Creativity and innovation. I recommend their workshop to young MBAs.
I personally wanted to thank you for your very valuable contribution to the conference at RAF Museum Cosford last Thursday. Your workshop presentation was targeted well to the audience and was an exceptional element to an excellent day.
You clearly are an expert in your field and we would not hesitate to recommend this approach to others within the public sector. It really made us think and has sown seeds for the future.
Thank you once again and I hope that we can work together in the future.


text:
College of Humanities and Sciences / Department of Mathematical Sciences
Seminars, Colloquia & Events
Statistics & Applied Math CORE
If mathematical formulas are not correctly displayed, try changing the MathJax output format.
#1: Bharath Sriraman
Handbook of the Mathematics of the Arts and Sciences
Bharath Sriraman, University of Montana- Missoula
Abstract: This Handbook aims to become a definitive source with chapters that show the origins, unification, and points of similarity between different disciplines and mathematics. The seven sections in this book explore: Mathematics and Architecture; Mathematics, Biology and Dynamical Systems; Mathematics, Humanities and the Language Arts; Mathematics, Art and Aesthetics; Mathematics, History and Philosophy; Mathematics in Society; and New Directions. Science and Art are used as umbrella terms to encompass the physical, natural and geological sciences, as well as the visual and performing arts.
Keywords: Mathematics and Architecture; Mathematics and Dynamical Systems; Mathematics and Humanities; Mathematics, Art and Aesthetics; Mathematics, History and Philosophy
AMS Subject Classification: 00
pdf files: Aims & Scope, Flyer, Table of contents
#5: Martin Schmidt, Bharath Sriraman
Nyaya Methodology and Western Mathematical Logic: Origins and Implications
Martin Schmidt
Western Nevada College
Bharath Sriraman
Abstract: In this chapter we compare the Nyaya school of logic, namely one of the six major schools of Hindu philosophy to Aristotelian logic. In particular we compare the intent, nature and structure of syllogisms in Nyaya to that in Western mathematical logic to highlight differences in premises and conclusions. In doing so, we draw on the foundational writings and commentaries on Nyaya methodology from both Hindu and Buddhist sources, as well as the major sources of Aristotelian logic. We explore the possibility of Nyaya methodology being influenced by Aristotelian logic before arguing that the Nyaya system developed on its own trajectory. Some modern implications of Nyaya methodology are given.
Keywords: Indian philosophy; Hindu philosophy; Mathematical Logic; Nyaya logic; Aristotelian logic; Syllogisms; Milinda; Law of Contrapositive; Buddhist logic
AMS Subject Classification: 03-03; 03F50
Download (228 KB pdf)
#4: Scott Chamberlin, Bharath Sriraman
Affect in Mathematical Modeling
Scott Chamberlin, University of Wyoming
Bharath Sriraman, University of Montana
Abstract: The relationship between affect and mathematical modeling must be investigated because, as educational psychologists have suggested for decades, affect directly influences achievement. Moreover, given the importance of mathematical modeling and the applications to high level mathematics, providing the field of mathematics psychology with insight regarding affect, in relation to mathematical modeling, might help determine the degree to which aspiring mathematicians facilitate understanding of mathematics and understanding affect in mathematical modeling episodes may have a direct effect on cognition. Though it existed for centuries, mathematical modeling may have first been mentioned in literature by Bell (1972). However, more than anyone else, often Lesh (1981) is credited with bringing the process of mathematical modeling to the attention mathematics educators and mathematical psychologists. In the initial version of the National Council of Teachers of Mathematics document, Principles and Standards for School Mathematics (2000), the process of mathematical modeling is embedded as a secondary task in algebra. It is not even mentioned as one of the five process standards. When the Common Core State Standards-Mathematics were released in 2010 (National Governor's Association & Council of Chief State School Officers, 2010), modeling with mathematics surfaced as the fourth mathematical practice. One may ask, "Why the sudden interest in mathematical modeling in national standards?" In fact, the models and modelling movement had been rapidly growing as a direct result of Lesh and his colleagues at PME-NA (Psychology of Mathematics-North America) and ICTMA (International Conference on the Teaching of Mathematical Modeling and Applications). Many of those colleagues are represented as authors in this book. The authors selected to contribute to this edited book were identified as a result of their work in mathematical modeling, research in affect, or both. This book will help readers develop understanding of the relationship between affect and mathematical modelling from an international perspective. In so doing, a vast array of authors will be utilized to provide insight on affective states while problem solvers create mathematical models.
Keywords: Affect; Mathematical Modeling; Cognition; AMS Subject Classification: 97
Preprint of ToC of Affect and mathematical modeling- Book forthcoming in Advances in Mathematics Education, Springer
pdf files: Aims & Scope, Flyer, Project overview
On Measures of Measurement and Mismeasurement: A commentary on Planning and Assessment
Bharath Sriraman, University of Montana - Missoula
Abstract: In this commentary the six Canadian chapters on planning and assessment in mathematics are critiqued. Some common strands are discussed on what entails a measurable item in mathematics assessments at the secondary school level. Implications are made for teachers.
Keywords: Learning; Teaching; Mathematical Memory; Testing; Rubrics; Assessment
AMS Subject Classification: 097D60
Preprint of Commentary for "Teaching and Learning Secondary School Mathematics: Canadian Perspectives in an International Context", Springer Berlin.
#1: Bharath Sriraman, Per Haavold
Reconciling the Realist/Anti Realist Dichotomy in the Philosophy of Mathematics
Per Haavold
University of Tromso, Norway
In the philosophy of mathematics, the realist vs. anti-realist debate continues today with differing positions on the status of mathematical objects. For realists, objects sit in "Plato's heaven", immovable, objective, eternal, and we contemplate them, whereas anti-realists (or Constructionists) are the opposite, and emphasize epistemology over ontology, saying that we construct mathematical objects. There are numerous results in mathematics which can be arrived at both from a realist and an anti-realist viewpoint. In other words, they can be contemplated (proved) via methods deemed unsuitable by anti-realists- or simply arrived at it through methods (or construction) as the anti-realist would say. In this chapter, we argue that realism and anti-realism can be seen as two sides of the same coin, or different ways of knowing the same thing, and therefore the so called dichotomy between these positions is reconcilable for particular mathematical objects.
Keywords: Intuitionism - Constructionism - Realism - Platonism - Anti-realism - Brouwer - Constructive mathematics - Philosophy of mathematics
AMS Subject Classification: 03A, 03F
Uncorrected proofs of chapter to appear in "The Map and the Territory": The Frontiers Collection, Springer International Publishing
Emily Stone
Mathematics Learning Center (Tutoring)
The Lennes Collection
Contact the Math Department


Michigan Governor Gretchen Whitmer stopped by the Michigan Press Association's Annual Convention at the Amway Grand Plaza Hotel on Friday to address Michigan journalists and sign an executive directive on Freedom of Information Act requests.
During her time on stage, Whitmer signed the directive and noted that it would change how state agencies respond to such requests, making the process more accessible and user-friendly.
The directive tackles extensions to FOIA requests – limiting them and requiring agencies to notify if the request will be prolonged – assigning a liaison between state departments to fulfil requests and requesting the Department of Technology, Management and Budget develop an online system where public notices and records can easily be accessed as well as livestreaming state public meetings.
Whitmer has had a busy week tackling other issues as well like the massive winter storm passing through Michigan. On Monday she declared a state of emergency due to the severe weather conditions and pleaded with residents on Wednesday to turn down their heat in light of the storm. But the slick roads and below freezing temperatures did not keep Whitmer from attending the annual convention where she debated nearly a year ago as a candidate for her current position. After signing the executive directive, Whitmer talked about other issues plaguing Michigan residents and her plans to overcome them.
She addressed the $1.3 billion supplemental spending bill signed by then-governor Rick Snyder at the end of 2018 and stated her plans to handle those changes.
Whitmer was asked by one of the journalists in attendance if she saw an end to the struggle Republicans and Democrats face when trying to communicate with each other and how that struggle affects Michigan residents.
She also addressed Enbridge's Line 5 pipeline, which was approved by a state panel in December to build an oil pipeline tunnel under the Great Lakes, and her plans for how best to handle the project.


Awesome surfboard shaped rug, these surfboard floor rugs are thick, super soft and come in the most unique designs for adults and children. Use them next to your bed, in front of the TV or even hang it on a wall as a feature! These very versatile rugs make the perfect gift for children or someone special.
This surfboard rug are stylish, modern and bold and have been created using 100% superior Japanese acrylic pile, it has also been hand carved along the pattern for a extra design feature.


Fantasy Worm Escape is another point & click escape game developed by WOWEscape. In this fantasy escape game, you came to a Fantasy Worm forest. By finding useful objects, hints and solving the puzzlea you need to find out the Magic leaf in this forest. Good Luck and have fun!


text:
Welcome to the Official Website of Liuzhou Municipal Government
Supports IPv6
ฉบับภาษาไทย
About Liuzhou
Know Liuzhou Travel in Liuzhou
Invest in Liuzhou
Garden Zone Economic
Medical Education Employment Marriage Immigration FQA
Home > Local Service > FQA
What is Chinese currency?
Date:2009-08-18 16:19
Renminbi, the Chinese currency, is issued by the state bank, the People's Bank of China. The standard unit of the Renminbi is yuan, with jiao and fen as subsidiary units. Thus one yuan equals ten jiao and one jiao equals ten fen. Yuan, jiao and fen are issued both in paper money and coins. Renminbi features the following denominations: one, two, five, ten, twenty, fifty and a hundred yuan; one, two, and five jiao; and one, two and five fen. The abbreviation for Renminbi is RMB¥.
Foreign Currency Conversion
Conversion services are available in China for the following foreign currencies:
US Dollar, British Pound Sterling, French Franc, Deutsche Mark, Japanese Yen, Australian Dollar, Austrian Schilling, Belgian Franc, Canadian Dollar, Hong Kong Dollar, Swiss Franc, Danish Krone, Guilder (or Florin), Norwegian Krone, Swedish Krone, Singapore Dollar, Malaysian Ringgit, Italian Lira, Macao Pataca, and Finnish Markka.
Banks conduct the conversion of the above-mentioned foreign currencies into Renminbi or vice versa. This constitutes conversion services in China.
China's law governing foreign exchange bans the circulation of foreign currencies and the settling of accounts with foreign currencies in the People's Republic of China. For the convenience of foreigners and compatriots from Hong Kong, Macao and Taiwan traveling in the mainland of China, the Bank of China and other designated Chinese banks conduct the conversion into Renminbi of foreign traveler's cheques and credit cards as well as the conversion of twenty-two foreign currencies and the New Taiwan Dollar in cash. Again for the convenience of travelers in China, some hotels, restaurants and stores in China also conduct the conversion of foreign cash into Renminbi. A foreign traveler may have the remaining amount of Renminbi converted back into foreign cash and brought out of China within a grace period of six months prior to departure from China, but in doing so he has to display a foreign currency conversion receipt. Different conversion rates are applied under different circumstances. Buying prices are applied for the conversion of traveler's cheques, credit cards and remittances; selling prices are applied for the conversion of Renminbi into foreign currencies (cash included); and buying prices are used for the conversion of foreign cash into Renminbi.
Credit Cards Acceptable in China:
So far the following foreign credit cards are acceptable in China:
1) Master Card;
2) Visa Card;
3) American Express Card;
4) JCB Card;
5) Diners Card.
Foreign Exchange Regulations for "Foreign Visitors"
Article 17 of the Regulations on Foreign Exchange Control of the People's Republic of China stipulates: "Foreign exchange sent or carried in by foreign establishments and foreign nationals in China can be held at their own discretion, deposited in designated banks or sold to the designated foreign exchange banks. Such foreign exchange can also be remitted or taken abroad upon the presentation of valid documents."
The Regulations also include the following:
1) Foreign exchange sent or carried in by foreign establishments and foreign nationals in China can be held at their own discretion, deposited in designated banks or sold to the designated foreign exchange banks on a voluntary basis. Such foreign exchange can also be remitted or taken abroad upon the presentation of valid documents.
2) All the expenses incurred by foreign establishments and foreign nationals in China should be paid in Renminbi. The remaining amount of Renminbi converted from foreign exchange can be converted back into foreign currency and brought or remitted abroad upon the presentation of one's passport and the memo within a six-month grace period.
3) Foreign establishments and foreign nationals in China are not allowed to engage in unauthorized trading of foreign exchange in China.
According to China's current foreign exchange control regulations, the conversion (or transaction) between foreign exchange and Renminbi can take place only at a bank empowered to conduct foreign exchange businesses, or at the China Foreign Exchange Trade Centre and the system attached to it. All transactions between foreign exchange and Renminbi conducted through channels other than these two are unauthorized behaviors no matter at what exchange rate such transactions have been made. Such unauthorized behaviors are illegal and banned by Chinese law because they disturb the country's financial order. According to Article 45 of the Regulations on Foreign Exchange Control of the People's Republic of China, to penalize unauthorized trading, the exchange administration agencies shall serve a warning, impose the conversion of foreign exchange, confiscate the unlawful income from such transactions, and place a penalty fine in the range of more than 30 percent of and less than 3 times the amount of the foreign exchange in question.
Sponsored by Liuzhou Informational Leading Group Office (The Bureau Of The Information Industry Of Liuzhou)
Prepared by Liuzhou Network Management Centre Address:No. 66, Sanzhong Road, Liuzhou, China
Copyright (C) 2020 The People's Government of Liuzhou Municipality,All Rights Reserved ICP Certificate No.05009280


text:
Q: Jquery maphilight issue with IE9 maphilight work fine on firefox, IE7/8 but it not working on IE9. Live demo also not working with IE9. Are there any fixes?

A: SVG requires excanvas plugin to work on Explorer.

A: You can also delete the line 192 of the plugin.

redpajama_set_name: RedPajamaStackExchange


2014-06-27T08:44:31-04:00https://images.c-span.org/Files/ddf/20140627084701001_hd.jpgMichael Elliott of the Marijuana Industry Group talked by video from Denver about the new levels of regulation in Colorado following the state's legalization of recreational marijuana sales, which went into effect in January 2014. The new rules not only concern sales, but also new taxes, the tracking of product from "seed to sale," banking issues, and protections to prevent sales to minors.
The end of the segment included a taped interview with Ryan Williams, a quality control specialist for Medicine Man Marijuana Dispensary in Denver, Colorado, who talked about how marijuana was processed and cured.
The entire June 27, 2014, "Washington Journal" was devoted to Colorado allowing the legal sale of recreational marijuana to those over 21.
Michael Elliott of the Marijuana Industry Group talked by video from Denver about the new levels of regulation in Colorado following the state's legalization of recreational marijuana sales, which went into effect in January 2014. The new rules not only concern sales, but also new taxes, the tracking of product from "seed to sale," banking issues, and protections to prevent sales to minors.