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1.  Caveolae-mediated endocytosis of conjugated polymer nanoparticlesa 
Macromolecular bioscience  2013;13(7):913-920.
Understanding cellular interactions and entry pathways of synthetic biomaterials are highly important to improve overall labeling and delivery efficiency. Conjugated polymer nanoparticles (CPNs) are emerging, fluorescent materials that have been used for cancer cell labeling and small interfering RNA (siRNA) delivery. In this contribution, detailed biophysical properties of CPNs including entry mechanisms and subcellular localization were studied using fluorescent-based techniques. While CPNs cause no toxicity, decreased CPN uptake was observed from cancer cells pretreated with genistein, which is an inhibitor of caveolae-mediated endocytosis (CvME). CvME was further confirmed by high co-localization with caveolin-1 proteins, which are found in the caveolae and caveosomes. Excellent photophysical properties, non-toxicity, and non-destructive delivery pathways support that CPNs are promising multifunctional carriers minimizing degradation of contents during delivery.
PMCID: PMC3787714  PMID: 23629923
conjugated polymer; conjugated polyelectrolyte; conjugated polymer nanoparticle; endocytosis mechanism; small interfering RNA delivery
2.  A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts 
Plant Methods  2006;2:13.
Transient assays using protoplasts are ideal for processing large quantities of genetic data coming out of hi-throughput assays. Previously, protoplasts have routinely been prepared from dicot tissue or cell suspension cultures and yet a good system for rice protoplast isolation and manipulation is lacking.
We have established a rice seedling protoplast system designed for the rapid characterization of large numbers of genes. We report optimized methods for protoplast isolation from 7–14 day old etiolated rice seedlings. We show that the reporter genes luciferase GL2 and GUS are maximally expressed approximately 20 h after polyethylene glycol (PEG)-mediated transformation into protoplasts. In addition we found that transformation efficiency varied significantly with plasmid size. Five micrograms of a 4.5 kb plasmid resulted in 60–70% transformation efficiency. In contrast, using 50 μg of a 12 kb plasmid we obtained a maximum of 25–30% efficiency. We also show that short interfering RNAs (siRNAs) can be used to silence exogenous genes quickly and efficiently. An siRNA targeting luciferase resulted in a significant level of silencing after only 3 hours and up to an 83% decrease in expression. We have also isolated protoplasts from cells prepared from fully green tissue. These green tissue-derived protoplasts can be transformed to express high levels of luciferase activity and should be useful for assaying light sensitive cellular processes.
We report a system for isolation, transformation and gene silencing of etiolated rice leaf and stem-derived protoplasts. Additionally, we have extended the technology to protoplasts isolated from fully green tissue. The protoplast system will bridge the gap between hi-throughput assays and functional biology as it can be used to quickly study large number of genes for which the function is unknown.
PMCID: PMC1524957  PMID: 16808845
3.  A Chaperonin Subunit with Unique Structures Is Essential for Folding of a Specific Substrate 
PLoS Biology  2011;9(4):e1001040.
Type I chaperonins are large, double-ring complexes present in bacteria (GroEL), mitochondria (Hsp60), and chloroplasts (Cpn60), which are involved in mediating the folding of newly synthesized, translocated, or stress-denatured proteins. In Escherichia coli, GroEL comprises 14 identical subunits and has been exquisitely optimized to fold its broad range of substrates. However, multiple Cpn60 subunits with different expression profiles have evolved in chloroplasts. Here, we show that, in Arabidopsis thaliana, the minor subunit Cpn60β4 forms a heterooligomeric Cpn60 complex with Cpn60α1 and Cpn60β1–β3 and is specifically required for the folding of NdhH, a subunit of the chloroplast NADH dehydrogenase-like complex (NDH). Other Cpn60β subunits cannot complement the function of Cpn60β4. Furthermore, the unique C-terminus of Cpn60β4 is required for the full activity of the unique Cpn60 complex containing Cpn60β4 for folding of NdhH. Our findings suggest that this unusual kind of subunit enables the Cpn60 complex to assist the folding of some particular substrates, whereas other dominant Cpn60 subunits maintain a housekeeping chaperonin function by facilitating the folding of other obligate substrates.
Author Summary
Chaperonins assist the folding of some nascent and denatured proteins to their native, functional forms. Each chaperonin consists of a pair of protein complexes resembling two stacked toroids; folding occurs inside the toroid cavity. Chaperonins are ubiquitous in both bacteria and more complex nucleated cells, as well as in the intracellular organelles that have evolved from bacteria by endosymbiosis: mitochondria and, in plants, chloroplasts. They are indispensable for cellular function. Many different chaperonin subunits have evolved in various species of bacteria as well as in most mitochondria and chloroplasts. The physiological and functional relevance of these multiple chaperonin subunits is poorly understood, however. In this study, we have characterized the minor chaperonin subunit Cpn60β4 from Arabidopsis chloroplasts, which differs in structure from other chloroplast chaperonins. When the Cpn60β4 gene is defective, the plants fail to accumulate one protein complex in particular: the chloroplast NADH dehydrogenase-like complex (NDH). We discovered that Cpn60β4 forms a complex with other Cpn60 α and β subunits and that this complex is essential for the folding of the NDH subunit NdhH. Cpn60β4 has a unique protein “tail” that is required for the efficient folding of NdhH. Our findings suggest that Cpn60β4 has evolved with distinctive structural features that facilitate the folding of one specific substrate and that this strategy is used by plants to satisfy their conflicting requirements for chaperonins with both specialized and general functions.
PMCID: PMC3071376  PMID: 21483722
4.  Effects of the tom1 mutation of Arabidopsis thaliana on the multiplication of tobacco mosaic virus RNA in protoplasts. 
Journal of Virology  1993;67(9):5328-5338.
For the multiplication of RNA viruses, specific host factors are considered essential, but as of yet little is known about this aspect of virus multiplication. To identify such host factors, we previously isolated PD114, a mutant of Arabidopsis thaliana, in which the accumulation of the coat protein of tobacco mosaic virus (TMV) in uninoculated leaves of an infected plant was reduced to low levels. The causal mutation, designated tom1, was single, nuclear, and recessive. Here, we demonstrate that the tom1 mutation affects the amplification of TMV-related RNAs in a single cell. When protoplasts were inoculated with TMV RNA by electroporation, the percentage of TMV-positive protoplasts (detected by indirect immunofluorescence staining with anti-TMV antibodies) was lower (about 1/5 to 1/10) among PD114 protoplasts than among wild-type protoplasts. In TMV-positive PD114 protoplasts, the amounts of the positive-strand RNAs (the genomic RNA and subgenomic mRNAs) and coat protein reached levels similar to, or slightly lower than, those reached in TMV-positive wild-type protoplasts, but the accumulation of the positive-strand RNAs and coat protein occurred more slowly than with the wild-type protoplasts. The parallel decrease in the amounts of the coat protein and its mRNA suggests that the coat protein is translated from its mRNA with normal efficiency. These observations support the idea that the TOM1 gene encodes a host factor necessary for the efficient amplification of TMV RNA in an infected cell. Furthermore, we show that TMV multiplication in PD114 protoplasts is severely affected by the coinoculation of cucumber mosaic virus (CMV) RNA. When PD114 protoplasts were inoculated with a mixture of TMV and CMV RNAs by electroporation, the accumulation of TMV-related molecules was approximately one-fifth of that in PD114 protoplasts inoculated with TMV RNA alone. No such reduction in the accumulation of TMV-related molecules was observed when wild-type protoplasts were inoculated with a mixture of TMV and CMV RNAs or when wild-type and PD114 protoplasts were inoculated with a mixture of TMV and turnip crinkle virus RNAs. These observations are compatible with a hypothetical model in which a gene(s) that is distinct from the TOM1 gene is involved in both TMV and CMV multiplication.
PMCID: PMC237932  PMID: 8350399
5.  Novel epithelial cell adhesion molecule antibody conjugated polyethyleneimine-capped gold nanoparticles for enhanced and targeted small interfering RNA delivery to retinoblastoma cells 
Molecular Vision  2013;19:1029-1038.
Several nanoconjugates have been designed to deliver nucleic acids such as small interfering RNA (siRNA) and DNA to cells to study silencing and expression efficacies. In the present study, we prepared novel epithelial cell adhesion molecule (EpCAM) monoclonal antibody conjugated polyethyleneimine (PEI) capped gold nanoparticles (AuNPs) loaded with EpCAM-specific siRNA molecules to knock-down the EpCAM gene in retinoblastoma (RB) cells. We chose EpCAM as a target moiety to deliver siRNA because this molecule is highly expressed in various epithelial cancers and is an ideal target as it is highly expressed in the apical surface of tumor cells while showing basolateral expression in normal cells.
The EpCAM antibody was conjugated to AuNP-PEI loaded with siRNA molecules to specifically deliver siRNA to EpCAM-expressing RB cells. Conjugation efficiencies were confirmed with ultraviolet-visible spectrophotometry, Fourier transform infrared spectroscopy, and agarose and SDS–polyacrylamide gel electrophoresis. The size and zeta potential were measured using a Zeta sizer analyzer. Nanoparticle internalization and uptake were studied using fluorescent microscopy and flow cytometry. Gene silencing efficacy was monitored with western blot analysis and real-time quantitative PCR.
Optimal size and neutral zeta potential properties of the AuNP-PEI- EpCAM antibody (EpAb) antibody were achieved for the transfection studies. The AuNP-PEI nanoparticles did not show any cytotoxicity to the cells, which means these nanomaterials are suitable for intracellular delivery of siRNA for therapeutic interventions. With EpCAM antibody conjugation, PEI-capped AuNPs loaded with EpCAM siRNA were significantly internalized in the Y79 cells as observed with fluorescence microscopy and flow cytometry and induced a highly significant reduction in the cell viability of the Y79 cells. Through increased binding of EpCAM antibody–conjugated AuNP-PEI nanoparticles, significant downregulation of EpCAM gene was observed in the Y79 cells when compared to the cells treated with the antibody-unconjugated AuNP-PEI nanoparticles.
Thus, a novel antibody conjugated nanocarrier designed to deliver siRNA holds promise as an effective gene therapy strategy for retinoblastoma in the near future. In addition to serving as an siRNA delivery tool for therapy, gold nanoparticles can also serve as imaging modality in diagnosis.
PMCID: PMC3654846  PMID: 23687439
6.  Quantitative Silencing of EGFP Reporter Gene by Self-Assembled siRNA Lipoplexes of LinOS and Cholesterol 
Molecular Pharmaceutics  2012;9(11):3384-3395.
Nonviral siRNA vectors prepared by the direct mixing of siRNA and mixtures of an asymmetric N4,N9-diacyl spermine conjugate, N4-linoleoyl-N9-oleoyl-1,12-diamino-4,9-diazadodecane (LinOS), with either cholesterol or DOPE, at various molar ratios of the neutral lipids, are reported. The effects of varying the lipid formulation and changing the N/P charge ratio on the intracellular delivery of siRNA to HeLa cells and on the siRNA-mediated gene silencing of a stably expressed reporter gene (EGFP) were evaluated. The presence of either cholesterol or DOPE in the mixture resulted in a marked increase in the delivery of the siRNA as well as enhanced EGFP silencing as evaluated by FACS. A LinOS/Chol 1:2 mixture resulted in the highest siRNA delivery and the most efficient EGFP silencing (reduced to 20%) at N/P = 3.0. Lowering the amount of siRNA from 15 pmol to 3.75 pmol, thus increasing the N/P charge ratio to 11.9, resulted in decreasing the amount of delivered siRNA, while the efficiency of gene silencing was comparable to that obtained with 15 pmol (N/P = 3.0) of siRNA. Mixtures of symmetrical N4,N9-dioleoyl spermine (DOS) with cholesterol at 1:2 molar ratio showed less siRNA delivery than with LinOS/Chol at N/P = 3.0 (15 pmol of siRNA), and comparable delivery at N/P = 11.9 (3.75 pmol of siRNA). The EGFP silencing was comparable with LinOS and with DOS when mixed with cholesterol 1:2 (lipoplexes prepared with 15 pmol of siRNA), but LinOS mixtures showed better EGFP silencing when the siRNA was reduced to 3.75 pmol. Lipoplex particle size determination by DLS of cholesterol mixtures was 106–118 nm, compared to 194–356 nm for lipoplexes prepared with the spermine conjugates only, and to 685 nm for the LinOS/DOPE 1:1 mixture. Confocal microscopy showed successful siRNA delivery of red tagged siRNA and quantitative EGFP knockdown in HeLa EGFP cells; Z-stack photomicrographs showed that the delivered siRNA is distributed intracellularly. Cryo-TEM of siRNA LinOS/Chol 1:2 lipoplexes shows the formation of multilamellar spheres with a size of ∼100 nm, in good agreement with the particle size measured by DLS. The constant distance between lamellar repeats is ∼6 nm, with the electron-dense layers fitting a monolayer of siRNA. AlamarBlue cell viability assay showed that the lipoplexes resulted in cell viability ≥81%, with LinOS/Chol 1:2 mixtures resulting in cell viabilities of 89% and 94% at siRNA 15 nM and 3.75 nM respectively. These results show that lipoplexes of siRNA and LinOS/Chol mixtures prepared by the direct mixing of the lipid mixture and siRNA, without any preceding preformulation steps, result in enhanced siRNA delivery and EGFP knockdown, with excellent cell viability. Thus, LinOS/Chol 1:2 mixture is a promising candidate as a nontoxic nonviral siRNA vector.
PMCID: PMC3495574  PMID: 23057412
cholesterol; cryo-TEM; lipoplexes; nanoparticles; polyamine; self-assembly; siRNA; spermine; Z-stack
7.  Polymeric Nanoparticles for siRNA Delivery and Gene Silencing 
International journal of pharmaceutics  2008;367(1-2):195-203.
Gene silencing using small interfering RNA (siRNA) has several potential therapeutic applications. In the present study, we investigated nanoparticles formulated using the biodegradable polymer, poly(d,l-lactide-co-glycolide) (PLGA) for siRNA delivery. A cationic polymer, polyethylenimine (PEI), was incorporated in the PLGA matrix to improve siRNA encapsulation in PLGA nanoparticles. PLGA-PEI nanoparticles were formulated using double emulsion-solvent evaporation technique and characterized for siRNA encapsulation and in vitro release. The effectiveness of siRNA-loaded PLGA-PEI nanoparticles in silencing a model gene, fire fly luciferase, was investigated in cell culture. Presence of PEI in PLGA nanoparticle matrix increased siRNA encapsulation by about 2-fold and also improved the siRNA release profile. PLGA-PEI nanoparticles carrying luciferase-targeted siRNA enabled effective silencing of the gene in cells stably expressing luciferase as well as in cells that could be induced to overexpress the gene. Quantitative studies indicated that presence of PEI in PLGA nanoparticles resulted in 2-fold higher cellular uptake of nanoparticles while fluorescence microscopy studies showed that PLGA-PEI nanoparticles delivered the encapsulated siRNA in the cellular cytoplasm; both higher uptake and greater cytosolic delivery could have contributed to the gene silencing effectiveness of PLGA-PEI nanoparticles. Serum stability and lack of cytotoxicity further add to the potential of PLGA-PEI nanoparticles in gene silencing-based therapeutic applications.
PMCID: PMC2660441  PMID: 18940242
RNA interference; siRNA; poly(d,l-lactide-co-glycolide); luciferase; sustained release
8.  Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method 
Plant Methods  2009;5:16.
Protoplasts isolated from leaves are useful materials in plant research. One application, the transient expression of recombinant genes using Arabidopsis mesophyll protoplasts (TEAMP), is currently commonly used for studies of subcellular protein localization, promoter activity, and in vivo protein-protein interactions. This method requires cutting leaves into very thin slivers to collect mesophyll cell protoplasts, a procedure that often causes cell damage, may yield only a few good protoplasts, and is time consuming. In addition, this protoplast isolation method normally requires a large number of leaves derived from plants grown specifically under low-light conditions, which may be a concern when material availability is limited such as with mutant plants, or in large scale experiments.
In this report, we present a new procedure that we call the Tape-Arabidopsis Sandwich. This is a simple and fast mesophyll protoplast isolation method. Two kinds of tape (Time tape adhered to the upper epidermis and 3 M Magic tape to the lower epidermis) are used to make a "Tape-Arabidopsis Sandwich". The Time tape supports the top side of the leaf during manipulation, while tearing off the 3 M Magic tape allows easy removal of the lower epidermal layer and exposes mesophyll cells to cell wall digesting enzymes when the leaf is later incubated in an enzyme solution. The protoplasts released into solution are collected and washed for further use. For TEAMP, plasmids carrying a gene expression cassette for a fluorescent protein can be successfully delivered into protoplasts isolated from mature leaves grown under optimal conditions. Alternatively, these protoplasts may be used for bimolecular fluorescence complementation (BiFC) to investigate protein-protein interactions in vivo, or for Western blot analysis. A significant advantage of this protocol over the current method is that it allows the generation of protoplasts in less than 1 hr, and allows TEAMP transfection to be carried out within 2 hr.
The protoplasts generated by this new Tape-Arabidopsis Sandwich method are suitable for the same range of research applications as those that use the current method, but require less operator skill, equipment and time.
PMCID: PMC2794253  PMID: 19930690
9.  The relative brightness of PEG lipid-conjugated polymer nanoparticles as fluid-phase markers in live cells 
Analytical and bioanalytical chemistry  2012;404(10):3003-3014.
While conjugated polymer nanoparticles (CPNs) have been widely touted as ultra-bright labels for biological imaging, no direct comparative measurements of their intracellular brightness have been reported. Simple in vitro comparisons are not definitive since fluorophore brightness in vitro may not correspond with intracellular brightness. We have compared the fluorescence brightness of J774A.1 cells loaded with 24 nm methoxy-capped 2000 Mr polyethylene glycol lipid PFBT nanoparticles (PEG lipid-PFBT CPNs) to cells loaded with carboxy-functionalized quantum dots (Qdots) or a dextran-linked small molecule organic dye, Alexa fluor 488-dextran (AF488-dex). Under conditions likely to be used for biological imaging or flow cytometry, these CPNs are 175X brighter than Qdots and 1400X brighter than AF488-dex in cells. Evaluation of the minimum incubation concentration required for detection of nanoparticle fluorescence with a commercial flow cytometer indicated that the limit of detection for PEG lipid-PFBT CPNs was 19 pM (86 ppb), substantially lower than values obtained for Qdots (980 pM) or AF488-dex (11.2 nM). Investigation of the mechanism of cellular uptake of the three fluid-phase labels indicates that these particles are passively taken into macrophage cells via macropinocytosis without interaction with cell surface receptors, and ultimately localize in lysosomes. In addition, no cytotoxicity could be observed at any of the CPN concentrations tested. Together, these data suggest that these CPNs are appropriate and attractive candidates as fluid phase markers with significantly greater fluorescence brightness than existing dyes or nanoparticles. We expect that these CPNs will find application in both imaging and flow cytometry.
PMCID: PMC3501596  PMID: 23052882
Alexa dextran; Quantum dots; Conjugated Polymer Nanoparticles; Semiconducting Polymer Nanoparticles; LAMP-1; Cellular toxicity
10.  VEGF siRNA Delivery System Using Arginine-Grafted Bioreducible Poly(disulfide amine) 
Molecular pharmaceutics  2009;6(3):718-726.
Small interfering RNAs (siRNAs) are able to silence their target genes when they are successfully delivered intact into the cytoplasm. Delivery systems that enhance siRNA localization to the cytoplasm can facilitate gene silencing by siRNA therapeutics. We describe an arginine-conjugated poly(cystaminebisacrylamide-diaminohexane) (poly(CBA-DAH-R)), a bioreducible cationic polymer, as an siRNA carrier for therapeutic gene silencing for cancer. After intracellular uptake of the siRNA/poly(CBA-DAH-R) polyplexes, the reductive environment of the cytoplasm cleaves the disulfide linkages in the polymeric backbone, resulting in decomplexing of the siRNA/poly(CBA-DAH-R) polyplexes and release of siRNA molecules throughout the cytoplasm. The siRNA/poly(CBA-DAH-R) polyplexes, which demonstrate increased membrane permeability with arginine modification, have a similar level of cellular uptake as siRNA/bPEI polyplexes. The VEGF siRNA/poly(CBA-DAH-R) polyplexes, however, inhibit VEGF expression to a greater degree than VEGF siRNA/bPEI in various human cancer cell lines. The improved RNAi activity demonstrated by the VEGF siRNA/poly(CBA-DAH-R) polyplexes is due to enhanced intracellular delivery and effective localization to the cytoplasm of the VEGF siRNAs. These results demonstrate that the VEGF siRNA/poly(CBA-DAH-R) polyplex delivery system may useful for siRNA-based approaches for cancer therapy.
PMCID: PMC2754713  PMID: 19055368
Cytoplasmic localization; siRNA; bioreducible cationic polymer; gene silencing; vascular endothelial growth factor; cancer therapy
11.  Plastid chaperonin proteins Cpn60α and Cpn60β are required for plastid division in Arabidopsis thaliana 
BMC Plant Biology  2009;9:38.
Plastids arose from a free-living cyanobacterial endosymbiont and multiply by binary division as do cyanobacteria. Plastid division involves nucleus-encoded homologs of cyanobacterial division proteins such as FtsZ, MinD, MinE, and ARC6. However, homologs of many other cyanobacterial division genes are missing in plant genomes and proteins of host eukaryotic origin, such as a dynamin-related protein, PDV1 and PDV2 are involved in the division process. Recent identification of plastid division proteins has started to elucidate the similarities and differences between plastid division and cyanobacterial cell division. To further identify new proteins that are required for plastid division, we characterized previously and newly isolated plastid division mutants of Arabidopsis thaliana.
Leaf cells of two mutants, br04 and arc2, contain fewer, larger chloroplasts than those of wild type. We found that ARC2 and BR04 are identical to nuclear genes encoding the plastid chaperonin 60α (ptCpn60α) and chaperonin 60β (ptCpn60β) proteins, respectively. In both mutants, plastid division FtsZ ring formation was partially perturbed though the level of FtsZ2-1 protein in plastids of ptcpn60β mutants was similar to that in wild type. Phylogenetic analyses showed that both ptCpn60 proteins are derived from ancestral cyanobacterial proteins. The A. thaliana genome encodes two members of ptCpn60α family and four members of ptCpn60β family respectively. We found that a null mutation in ptCpn60α abolished greening of plastids and resulted in an albino phenotype while a weaker mutation impairs plastid division and reduced chlorophyll levels. The functions of at least two ptCpn60β proteins are redundant and the appearance of chloroplast division defects is dependent on the number of mutant alleles.
Our results suggest that both ptCpn60α and ptCpn60β are required for the formation of a normal plastid division apparatus, as the prokaryotic counterparts are required for assembly of the cell division apparatus. Since moderate reduction of ptCpn60 levels impaired normal FtsZ ring formation but not import of FtsZ into plastids, it is suggested that the proper levels of ptCpn60 are required for folding of stromal plastid division proteins and/or regulation of FtsZ polymer dynamics.
PMCID: PMC2670834  PMID: 19344532
12.  Fluorescence Activated Cell Sorting of Plant Protoplasts 
High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.
PMCID: PMC2896036  PMID: 20168296
FACS; plant protoplasts; GFP; cell type-specific; Arabidopsis thaliana; roots
13.  The preferred route for the degradation of silencing target RNAs in transgenic plants depends on pre-established silencing conditions 
Nucleic Acids Research  2004;32(11):3400-3409.
RNA silencing can be initiated upon dsRNA accumulation and results in homology-dependent degradation of target RNAs mediated by 21–23 nt small interfering RNAs (siRNAs). These small regulatory RNAs can direct RNA degradation via different routes such as the RdRP/Dicer- and the RNA-induced silencing complex (RISC)-catalysed pathways. The relative contribution of both pathways to degradation of target RNAs is not understood. To gain further insight in the process of target selection and degradation, we analysed production of siRNAs characteristic for Dicer-mediated RNA degradation during silencing of mRNAs and chimeric viral RNAs in protoplasts from plants of a transgenic tobacco silencing model line. We show that small RNA accumulation is limited to silencing target regions during steady-state mRNA silencing. For chimeric viral RNAs, siRNA production appears dependent on pre-established cellular silencing conditions. The observed siRNA accumulation profiles imply that silencing of viral target RNAs in pre-silenced protoplasts occurs mainly via a RISC-mediated pathway, guided by (pre-existing) siRNAs derived from cellular mRNAs. In cells that are not silenced at the time of infection, viral RNA degradation seems to involve Dicer action directly on the viral RNAs. This suggests that the silencing mechanism flexibly deploys different components of the RNA degradation machinery in function of the prevailing silencing status.
PMCID: PMC443538  PMID: 15220468
14.  Delivery of Quantum Dot-siRNA Nanoplexes in SK-N-SH Cells for BACE1 Gene Silencing and Intracellular Imaging 
The fluorescent quantum dots (QDs) delivered small interfering RNAs (siRNAs) targeting β-secretase (BACE1) to achieve high transfection efficiency of siRNAs and reduction of β-amyloid (Aβ) in nerve cells. The CdSe/ZnS QDs with the conjugation of amino-polyethylene glycol (PEG) were synthesized. Negatively charged siRNAs were electrostatically adsorbed to the surface of QDs to develop QD-PEG/siRNA nanoplexes. The QD-PEG/siRNAs nanoplexes significantly promote the transfection efficiency of siRNA, and the siRNAs from non-packaged nanoplexes were widely distributed in cell bodies and processes and efficiently silenced BACE1 gene, leading to the reduction of Aβ. The biodegradable PEG polymer coating could protect QDs from being exposed to the intracellular environment and restrained the release of toxic Cd2+. Therefore, the QD-PEG/siRNA nanoplexes reported here might serve as ideal carriers for siRNAs. We developed a novel method of siRNA delivery into nerve cells. We first reported that the QD-PEG/siRNA nanoplexes were generated by the electrostatic interaction and inhibited the Alzheimer's disease (AD)-associated BACE1 gene. We also first revealed the dynamics of QD-PEG/siRNAs within nerve cells via confocal microscopy and the ultrastructural evidences under transmission electron microscopy (TEM). This technology might hold promise for the treatment of neurodegenerative diseases such as AD.
PMCID: PMC3381642  PMID: 23343930
Alzheimer's disease; BACE1; quantum dot; siRNA; SK-N-SH cells
15.  Transcriptome of Protoplasts Reprogrammed into Stem Cells in Physcomitrella patens 
PLoS ONE  2012;7(4):e35961.
Differentiated plant cells can retain the capacity to be reprogrammed into pluripotent stem cells during regeneration. This capacity is associated with both cell cycle reactivation and acquisition of specific cellular characters. However, the molecular mechanisms underlying the reprogramming of protoplasts into stem cells remain largely unknown. Protoplasts of the moss Physcomitrella patens easily regenerate into protonema and therefore provide an ideal system to explore how differentiated cells can be reprogrammed to produce stem cells.
Principal findings
We obtained genome-wide digital gene expression tag profiles within the first three days of P. patens protoplast reprogramming. At four time-points during protoplast reprogramming, the transcript levels of 4827 genes changed more than four-fold and their expression correlated with the reprogramming phase. Gene ontology (GO) and pathway enrichment analysis of differentially expressed genes (DEGs) identified a set of significantly enriched GO terms and pathways, most of which were associated with photosynthesis, protein synthesis and stress responses. DEGs were grouped into six clusters that showed specific expression patterns using a K-means clustering algorithm. An investigation of function and expression patterns of genes identified a number of key candidate genes and pathways in early stages of protoplast reprogramming, which provided important clues to reveal the molecular mechanisms responsible for protoplast reprogramming.
We identified genes that show highly dynamic changes in expression during protoplast reprogramming into stem cells in P. patens. These genes are potential targets for further functional characterization and should be valuable for exploration of the mechanisms of stem cell reprogramming. In particular, our data provides evidence that protoplasts of P. patens are an ideal model system for elucidation of the molecular mechanisms underlying differentiated plant cell reprogramming.
PMCID: PMC3335808  PMID: 22545152
16.  Effective Non-Viral Delivery of siRNA to Acute Myeloid Leukemia Cells with Lipid-Substituted Polyethylenimines 
PLoS ONE  2012;7(8):e44197.
Use of small interfering RNA (siRNA) is a promising approach for AML treatment as the siRNA molecule can be designed to specifically target proteins that contribute to aberrant cell proliferation in this disease. However, a clinical-relevant means of delivering siRNA molecules must be developed, as the cellular delivery of siRNA is problematic. Here, we report amphiphilic carriers combining a cationic polymer (2 kDa polyethyleneimine, PEI2) with lipophilic moieties to facilitate intracellular delivery of siRNA to AML cell lines. Complete binding of siRNA by the designed carriers was achieved at a polymer:siRNA ratio of ∼0.5 and led to siRNA/polymer complexes of ∼100 nm size. While the native PEI2 did not display cytotoxicity on AML cell lines THP-1, KG-1 and HL-60, lipid-modification on PEI2 slightly increased the cytotoxicity, which was consistent with increased interaction of polymers with cell membranes. Cellular delivery of siRNA was dependent on the nature of lipid substituent and the extent of lipid substitution, and varied among the three AML cell lines used. Linoleic acid-substituted polymers performed best among the prepared polymers and gave a siRNA delivery equivalent to better performing commercial reagents. Using THP-1 cells and a reporter (GFP) and an endogenous (CXCR4) target, effective silencing of the chosen targets was achieved with 25 to 50 nM of siRNA concentrations, and without adversely affecting subsequent cell growth. We conclude that lipid-substituted PEI2 can serve as an effective delivery of siRNA to leukemic cells and could be employed in molecular therapy of leukemia.
PMCID: PMC3432090  PMID: 22952927
17.  Molecular Diagnostic Tools for Detection and Differentiation of Phytoplasmas Based on Chaperonin-60 Reveal Differences in Host Plant Infection Patterns 
PLoS ONE  2014;9(12):e116039.
Phytoplasmas (‘Candidatus Phytoplasma’ spp.) are insect-vectored bacteria that infect a wide variety of plants, including many agriculturally important species. The infections can cause devastating yield losses by inducing morphological changes that dramatically alter inflorescence development. Detection of phytoplasma infection typically utilizes sequences located within the 16S–23S rRNA-encoding locus, and these sequences are necessary for strain identification by currently accepted standards for phytoplasma classification. However, these methods can generate PCR products >1400 bp that are less divergent in sequence than protein-encoding genes, limiting strain resolution in certain cases. We describe a method for accessing the chaperonin-60 (cpn60) gene sequence from a diverse array of ‘Ca.Phytoplasma’ spp. Two degenerate primer sets were designed based on the known sequence diversity of cpn60 from ‘Ca.Phytoplasma’ spp. and used to amplify cpn60 gene fragments from various reference samples and infected plant tissues. Forty three cpn60 sequences were thereby determined. The cpn60 PCR-gel electrophoresis method was highly sensitive compared to 16S-23S-targeted PCR-gel electrophoresis. The topology of a phylogenetic tree generated using cpn60 sequences was congruent with that reported for 16S rRNA-encoding genes. The cpn60 sequences were used to design a hybridization array using oligonucleotide-coupled fluorescent microspheres, providing rapid diagnosis and typing of phytoplasma infections. The oligonucleotide-coupled fluorescent microsphere assay revealed samples that were infected simultaneously with two subtypes of phytoplasma. These tools were applied to show that two host plants, Brassica napus and Camelina sativa, displayed different phytoplasma infection patterns.
PMCID: PMC4281212  PMID: 25551224
18.  Optimal Electroporation Condition for Small Interfering RNA Transfection into MDA-MB-468 Cell Line 
Background: Electroporation is a valuable tool for small interfering RNA (siRNA) delivery into cells because it efficiently transforms a wide variety of cell types. Since electroporation condition for each cell type must be determined experimentally, this study presents an optimal electroporation strategy to reproducibly and efficiently transfect MDA-MB 468 human breast cancer cell with siRNA.
Methods: To identify the best condition, the cells were firstly electroporated without siRNA and cell viability was determined by trypan blue and MTT assays. Then siRNA transfection in the best condition was performed. Western blot analysis was used for monitoring successful siRNA transfection.
Results: The best condition for electroporation of this cell line was 220 volt and 975 µF in exponential decay using the Gene Pulser X cell electroporation system. Our data demonstrated that by using proper electroporation condition, DNA methyl transferase mRNA was silenced by 10 nmol DNMT1 siRNA in MDA-MB 468 cells when compared with negative control siRNA electroporation. Analysis of cell viability demonstrated that optimal electroporation condition resulted in 74% and 78% cell viability by trypan blue staining and MTT assay, respectively.
Conclusion: Transfection of the MDA-MB-468 breast cancer cell line with siRNA in the obtained electroporation condition was successful and resulted in effective gene silencing and high cellular viability.
PMCID: PMC3470088  PMID: 23115451
Small interfering RNA; electroporation; breast cancer
19.  Low-weight polyethylenimine cross-linked 2-hydroxypopyl-β-cyclodextrin and folic acid as an efficient and nontoxic siRNA carrier for gene silencing and tumor inhibition by VEGF siRNA 
Targeted delivery of small interfering RNA (siRNA) has been regarded as one of the most important technologies for the development of siRNA therapeutics. However, the need for safe and efficient delivery systems is a barrier to further development of RNA interference therapeutics. In this work, a nontoxic and efficient siRNA carrier delivery system of low molecular weight polyethyleneimine (PEI-600 Da) cross-linked with 2-hydroxypopyl-β-cyclodextrin (HP-β-CD) and folic acid (FA) was synthesized for biomedical application.
The siRNA carrier was prepared using a simple method and characterized by nuclear magnetic resonance and Fourier transform infrared spectroscopy. The siRNA carrier nanoparticles were characterized in terms of morphology, size and zeta potential, stability, efficiency of delivery, and gene silencing efficiency in vitro and in vivo.
The siRNA carrier was synthesized successfully. It showed good siRNA binding capacity and ability to protect siRNA. Further, the toxicity of the carrier measured in vitro and in vivo appeared to be negligible, probably because of degradation of the low molecular weight PEI and HP-β-CD in the cytosol. Flow cytometry and confocal microscopy confirmed that the FA receptor-mediated endocytosis of the FA-HP-β-CD-PEI/siRNA complexes was greater than that of the HP-β-CD-PEI/siRNA complexes in FA receptor-enriched HeLa cells. The FA-HP-β-CD-PEI/siRNA complexes also demonstrated excellent gene silencing efficiency in vitro (in the range of 90%), and reduced vascular endothelial growth factor (VEGF) protein expression in the presence of 20% serum. FA-HP-β-CD-PEI/siRNA complexes administered via tail vein injection resulted in marked inhibition of tumor growth and reduced VEGF protein expression in the tumors.
Our results suggest that the FA-HP-β-CD-PEI complex is a nontoxic and highly efficient gene carrier with the potential to deliver siRNA for cancer gene therapy effectively in vitro and in vivo.
PMCID: PMC3678862  PMID: 23766646
polyethyleneimine; 2-hydroxypropyl-β-cyclodextrin; folic acid; siRNA carrier; vascular endothelial growth factor; gene silencing
20.  Efficient Intracellular siRNA Delivery by Ethyleneimine-Modified Amphiphilic Macromolecules 
Macromolecular bioscience  2011;11(9):1192-1200.
New materials that can bind and deliver oligonucleotides such as short interfering RNA (siRNA) without toxicity are greatly needed to fulfill the promise of therapeutic gene silencing. Amphiphilic macromolecules (AMs) were functionalized with linear ethyleneimines to create cationic AMs capable of complexing with siRNA. Structurally, the parent AM is formed from a mucic acid backbone whose tetra-hydroxy groups are alkylated with 12-carbon aliphatic chains to form the hydrophobic component of the macromolecule. This alkylated mucic acid is then mono-functionalized with poly(ethylene glycol) (PEG) as a hydrophilic component. The resulting AM contains a free carboxylic acid within the hydrophobic domain. In this work, linear ethyleneimines were conjugated to the free carboxylic acid to produce an AM with one primary amine (1N) or one primary amine and four secondary amines (5N). Further, an AM with amine substitution both to the free carboxylic acid in the hydrophobic domain and also to the adjacent PEG was synthesized to produce a polymer with one primary amine and eight secondary amines (9N), four located on each side of the AM hydrophobic domain. All amine-functionalized AMs formed nanoscale micelles but only the 5N and 9N AMs had cationic zeta potentials, which increased with increasing number of amines. All AMs exhibited less inherent cytotoxicity than linear polyethyleneimine (L-PEI) at concentrations of 10 µM and above. By increasing the length of the cationic ethyleneimine chain and the total number of amines, successful siRNA complexation and cellular siRNA delivery was achieved in a malignant glioma cell line. In addition, siRNA-induced silencing of firefly luciferase was observed using complexes of siRNA with the 9N AM and comparable to L-PEI, yet showed better cell viability at higher concentrations (above 10 µM). This work highlights the promise of cationic AMs as safe and efficient synthetic vectors for siRNA delivery. Specifically, a novel polymer (9N) was identified for efficient siRNA delivery to cancer cells and will be further evaluated.
PMCID: PMC3549469  PMID: 21793212
amphiphiles; gene delivery; nanotechnology; siRNA
21.  Selective Modification of HK Peptides Enhances siRNA Silencing of Tumor Targets In Vivo 
Cancer gene therapy  2011;18(10):707-716.
Our research has focused on systemic delivery of small interference RNA (siRNA) by branched peptides composed of histidine and lysine, called HK peptides. After studying several HK peptides, one four-branched peptide, H3K(+H)4b, with a predominant repeating pattern of -HHHK-, was found to be an effective carrier of siRNA. Although the unmodified H3K(+H)4b carrier of siRNA targeting an oncogene was previously shown to have promise in a tumor-bearing mouse model, we sought to develop a more effective HK carrier of siRNA in the current study. Our primary goal was to determine whether different ligand (cyclic RGD)-pegylation patterns on the H3K(+H)4b peptide affect siRNA delivery in vitro and in vivo. We compared the unmodified H3K(+H)4b with two modified H3K(+H)4b peptides for their ability to deliver siRNA in a tumor-bearing mouse model; one modified HK peptide, (RGD-PEG)4-H3K(+H)4b, had four cRGD-PEG conjugated to each molecule, while the other peptide, (RGD-PEG)-H3K(+H)4b, had one cRGD-PEG per molecule. Although the modified HK peptides by themselves did not form stable nanoplexes with siRNA, combination of a highly charged unmodified HK peptide, H2K4b, with either of the modified HK peptides did form stable siRNA nanoparticles. For in vitro experiments with MDA-MB-435 cells that expressed luciferase, the H3K(+H)4b siRNA nanoplexes targeting luciferase decreased its activity by 90% compared with negligible down-regulation by the modified H3K(+H)4b nanoplexes (P<0.01). In contrast, the two modified H3K(+H)4b siRNA nanoplexes administered intravenously were more effective than the H3K(+H)4b nanoplexes in silencing luciferase in a tumor xenograft model. The luciferase activity in tumor lysates of mice administered H3K(+H)4b, (RGD-PEG)-H3K(+H)4b, and (RGD-PEG)4-H3K(+H)4b nanoplexes decreased by 18%, 35%, and 75%, respectively. Thus, the siRNA nanoplex incorporating the highly modified peptide, (RGD-PEG)4-H3K(+H)4b, was the most effective at silencing its target in vivo (P<0.01). These studies demonstrate that selectively modified HK polymers are promising candidates for targeting oncogenes with siRNA.
PMCID: PMC3177007  PMID: 21818135
non-viral; siRNA; MDA-MB-435 cells; xenograft; systemic; mouse model
22.  Dissecting RNA silencing in protoplasts uncovers novel effects of viral suppressors on the silencing pathway at the cellular level 
Nucleic Acids Research  2004;32(22):e179.
Short interfering RNA (siRNA)-mediated RNA silencing plays an important role in cellular defence against viral infection and abnormal gene expression in multiple organisms. Many viruses have evolved silencing suppressors for counter-defence. We have developed an RNA silencing system in the protoplasts of Nicotiana benthamiana to investigate the functions of viral suppressors at the cellular level. We showed that RNA silencing against a green fluorescent protein (GFP) reporter gene in the protoplasts could be induced rapidly and specifically by co-transfection with the reporter gene and various silencing inducers [i.e. siRNA, double-stranded RNA (dsRNA) or plasmid encoding dsRNA]. Using this system, we uncovered novel roles of some viral suppressors. Notably, the Cucumber mosaic virus 2b protein, shown previously to function predominantly by preventing the long-distance transmission of systemic silencing signals, was a very strong silencing suppressor in the protoplasts. Some suppressors thought to interfere with upstream steps of siRNA production appeared to also act downstream. Therefore, a viral suppressor can affect multiple steps of the RNA silencing pathway. Our analyses suggest that protoplast-based transient RNA silencing is a useful experimental system to investigate the functions of viral suppressors and further dissect the mechanistic details of the RNA silencing pathway in single cells.
PMCID: PMC545478  PMID: 15601991
23.  Co-delivery of doxorubicin and siRNA using octreotide-conjugated gold nanorods for targeted neuroendocrine cancer therapy† 
Nanoscale  2012;4(22):10.1039/c2nr31853a.
A multifunctional gold (Au) nanorod (NR)-based nanocarrier capable of co-delivering small interfering RNA (siRNA) against achaete-scute complex-like 1 (ASCL1) and an anticancer drug (doxorubicin (DOX)) specifically to neuroendocrine (NE) cancer cells was developed and characterized for combined chemotherapy and siRNA-mediated gene silencing. The Au NR was conjugated with (1) DOX, an anticancer drug, via a pH-labile hydrazone linkage to enable pH-controlled drug release, (2) polyarginine, a cationic polymer for complexing siRNA, and (3) octreotide (OCT), a tumor-targeting ligand, to specifically target NE cancer cells with overexpressed somatostatin receptors. The Au NR-based nanocarriers exhibited a uniform size distribution as well as pH-sensitive drug release. The OCT-conjugated Au NR-based nanocarriers (Au-DOX-OCT, targeted) exhibited a much higher cellular uptake in a human carcinoid cell line (BON cells) than non-targeted Au NR-based nanocarriers (Au-DOX) as measured by both flow cytometry and confocal laser scanning microscopy (CLSM). Moreover, Au-DOX-OCT-ASCL1 siRNA (Au-DOX-OCT complexed with ASCL1 siRNA) resulted in significantly higher gene silencing in NE cancer cells than Au-DOX-ASCL1 siRNA (non-targeted Au-DOX complexed with ASCL1 siRNA) as measured by an immunoblot analysis. Additionally, Au-DOX-OCT-ASCL1 siRNA was the most efficient nanocarrier at altering the NE phenotype of NE cancer cells and showed the strongest anti-proliferative effect. Thus, combined chemotherapy and RNA silencing using NE tumor-targeting Au NR-based nanocarriers could potentially enhance the therapeutic outcomes in treating NE cancers.
PMCID: PMC3495135  PMID: 23070403
24.  Confocal microscopy for the analysis of siRNA delivery by polymeric nanoparticles 
Microscopy research and technique  2010;73(9):878-885.
Clinical applications of genetic therapies, including delivery of short, interfering RNAs (siRNAs) for RNA interference (RNAi), are limited due to the difficulty of delivering nucleic acids to specific cells of interest while at the same time minimizing toxicity and immunogenicity. The use of cationic polymers to deliver nucleic acid therapeutics has the potential to address these complex issues but is currently limited by low delivery efficiencies. While cell culture studies have shown that some polymers can be used to deliver siRNAs and achieve silencing, it is still not clear what physical or chemical properties are needed to ensure that the polymers form active polymer-siRNA complexes. In this study, we used multicolor fluorescence confocal microscopy to analyze the cellular uptake of siRNAs delivered by novel propargyl glycolide polymeric nanoparticles (NPs). Delivery by these vehicles was compared to delivery by linear polyethyleneimine (LPEI) and Lipofectamine 2000 (LF2K), which are both known as effective delivery vehicles for siRNAs. Our results showed that when LF2K and LPEI were used, large quantities of siRNA were delivered rapidly, presumably overwhelming the basal levels of mRNA to initiate silencing. In contrast, our novel polymeric NPs showed delivery of siRNAs but at concentrations that were initially too low to achieve silencing. Nonetheless, the exceptionally low cytotoxicity of our NPs, and the simplicity with which they can be modified, makes them good candidates for further study to optimize their delivery profiles and, in turn, achieve efficient silencing.
PMCID: PMC2931363  PMID: 20803695
25.  Surface-Engineered Targeted PPI Dendrimer for Efficient Intracellular and Intratumoral siRNA Delivery 
Low penetration ability of Small Interfering RNA (siRNA) through the cellular plasma membrane combined with its limited stability in blood, limits the effectiveness of the systemic delivery of siRNA. In order to overcome such difficulties, we constructed a nanocarrier-based delivery system by taking advantage of the lessons learned from the problems in the delivery of DNA. In the present study, siRNA nanoparticles were first formulated with Poly(Propyleneimine) (PPI) dendrimers. To provide lateral and steric stability to withstand the aggressive environment in the blood stream, the formed siRNA nanoparticles were caged with a dithiol containing cross-linker molecules followed by coating them with Poly(Ethylene Glycol) (PEG) polymer. A synthetic analog of Luteinizing Hormone-Releasing Hormone (LHRH) peptide was conjugated to the distal end of PEG polymer to direct the siRNA nanoparticles specifically to the cancer cells. Our results demonstrated that this layer-by-layer modification and targeting approach confers the siRNA nanoparticles stability in plasma and intracellular bioavailability, provides for their specific uptake by tumor cells, accumulation of siRNA in the cytoplasm of cancer cells, and efficient gene silencing. In addition, in vivo body distribution data confirmed high specificity of the proposed targeting delivery approach which created the basis for the prevention of adverse side effects of the treatment on healthy organs.
PMCID: PMC2787988  PMID: 19567257
Poly(propyleneimine) dendrimer; siRNA targeted to BCL2 mRNA; LHRH peptide; tumor targeting; cytotoxicity; in vivo imaging

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