Branched co-polymers of histidine and lysine are efficient carriers of plasmids

We previously determined that a linear co-polymer of histidine and lysine (HK) in combination with liposomes enhanced the transfection efficiency of cationic liposomes. In the current study, we designed a series of HK polymers with increased branching and/or histidine/lysine ratio to determine if either variable affects transfection efficiency. In the presence of liposomes, the branched polymer with the highest number of histidines, HHK4b, was the most effective at enhancing gene expression. Furthermore, when serum was added to the medium during transfection, the combination of HHK4b and liposomes as a gene-delivery vehicle increased luciferase expression 400-fold compared to liposomes alone. In contrast to linear HK polymers, the higher branched HHK polymers were effective carriers of plasmids in the absence of liposomes. Without liposomes, the HHK4b carrier enhanced luciferase expression 15-fold in comparison with the lesser branched HHK2b carrier and increased expression by 5-logs in comparison with the HHK or HK carrier. The interplay of several parameters including increased condensation of DNA, buffering of acidic endosomes and differential binding affinities of polymer with DNA have a role in the enhancement of transfection by the HK polymers. In addition to suggesting that branched HK polymers are promising gene-delivery vehicles, this study provides a framework for the development of more efficient peptide-bond-based polymers of histidine and lysine.     

A branched histidine/lysine peptide, H2K4b, in complex with plasmids encoding antitumor proteins inhibits tumor xenografts

BACKGROUND: In this study we investigated whether a particular branched HK polymer, H2K4b, was an effective in vivo carrier of plasmids expressing the antiangiogenic kringle 1-5 or the tumor suppressor p53. METHODS: H2K4b was synthesized on a solid-phase peptide synthesizer. Distribution, optimization and time course studies were done in tumor-bearing nude mice by systemically administering H2K4b in complex with a luciferase-expressing plasmid. We examined the amount of tumor angiogenesis in C6 with MDA-MB-435 xenografts utilizing the carmine dye. The ability of H2K4b to carry luciferase plasmids to different tissues was compared with several liposomal carriers. Medium from cells transfected with mKr1-5 was tested for its capacity to inhibit angiogenesis with an in vivo Matrigel assay. We then determined if systemically delivered H2K4b in complex with plasmid encoding mKr1-5 inhibited tumor growth; we also compared the antitumor activity of HK polyplexes containing hKr1-5, mKr1-5, and p53 plasmids. RESULTS: H2K4b carried the luciferase-expressing plasmid in order of descending efficacy to these tissues: lung, spleen, tumor, and liver. Compared to DOTAP-containing liposomes, H2K4b was a more effective carrier of a luciferase-containing plasmid to extrapulmonary tissues. We then determined that mKr1-5 in complex with H2K4b reduced MDA-MB-435 tumor growth by approximately 50% compared to the control group (P < 0.01). Similarly, H2K4b/mKr1-5 polyplexes reduced the growth of C6 xenografts. In MDA-MB-435 xenografts, p53- and Kr1-5-expressing plasmids in complex with H2K4b had comparable antitumor activity. CONCLUSION: H2K4b demonstrates potential as a carrier of plasmids encoding antiangiogenic and/or tumor suppressor proteins in a tumor-bearing mouse model.     

Modified branched peptides with a histidine-rich tail enhance in vitro gene transfection

Successful gene therapy depends on the development of efficient, non-toxic gene delivery systems. To accomplish this objective, our laboratory has focused on solid-phase synthesized peptide carriers, in which the amino acid sequence can be varied precisely to augment intracellular DNA transport. We previously determined that linear and branched co-polymers of histidine and lysine in combination with liposomes enhanced the efficiency of gene transfection. In this study, we have modified two branched histidine-lysine (HK) peptides by adding a histidine-rich tail. In a variety of cell lines, this histidine-rich tail markedly improved transfection efficiency, presumably by increasing the buffering capacity of the polymer. One polymer with a histidine-rich tail, H²K4bT, compared favorably with the commonly used transfection agents. Together with modification of our transfection protocol, these improved HK peptides alone, without liposomes, are the effective carriers of plasmids into a variety of cells. We anticipate that branched HK peptides will continue to be developed as carriers of nucleic acids for in vitro and in vivo applications.     

Optimal transfection with the HK polymer depends on its degree of branching and the pH of endocytic vesicles

We have recently reported that liposomes in combination with histidine (HK)-containing polymers enhanced the expression of luciferase in transfected cells. In transformed or malignant cell lines, branched HK polymers (combined with liposome carriers) were significantly more effective than the linear HK polymer in stimulating gene expression. In the current study, we found that the linear HK polymer enhanced gene expression in primary cell lines more effectively than the branched polymers. The differences in the optimal carrier (linear versus branched) were not due to initial cellular uptake, size of the complexes or level of gene expression. There was, however, a strong association between the optimal type of HK polymer and the pH of endocytic vesicles (P = 0.0058). By altering the percentage of histidines carrying a positive charge, the endosomal pH of a cell may determine the amount of DNA released from the linear or branched HK polymer. In the two cell lines in which the linear HK was the optimal polymer, the endocytic vesicles were strongly acidic with a pH of <5.0. Conversely, in the four cell lines in which the branched polymers were optimal transfection agents, the pH of endocytic vesicles was >6.0. Furthermore, binding data support the relationship between DNA release from the optimal HK polymer and endosomal pH. The interplay between optimal HK polymers and the endosomal pH may lead to improved gene-delivery polymers tailored to a particular cell.     

Enhancement of star vector-based gene delivery to endothelial cells by addition of RGD-peptide

This study aimed to investigate the feasibility of using a cationic nonviral gene carrier in endothelial cells for enhancing gene expression by the addition of an integrin-binding RGD peptide. A 4-branched cationic polymer of poly( N,N-dimethylaminopropylacrylamide) (star vector), developed as a gene carrier, could complex with the luciferase-encoding plasmid DNA under a charge ratio of 5 (vector/pDNA) to form polymer/DNA complexes (polyplexes). The addition of the RGD-containing peptide (GRGDNP) to the polyplex solution led to a decrease in the zeta-potential from ca. +30 to +20 mV along with the reduction in the particle size from ca. 300 to 200 nm. Additionally, a marked inhibition of polyplex aggregation was observed, indicating the coating of the polyplex surface with RGD peptides. A transfection study on endothelial cells showed that the luciferase activity increased with the amount of RGD peptides added to the polyplexes and exhibited minimal cellular cytotoxicity. The transfection activity further increased when cyclic RGD peptides (RGDFV) were used; the activity with RGD peptide addition was approximately 8-fold compared to that without RGD peptide addition. Gene delivery to endothelial cells was significantly enhanced by only the addition of RGD peptides to star vector-based polyplexes.     

The impact of carboxyalkylation of branched polyethylenimine on effectiveness in small interfering RNA delivery

Background

Carboxyalkylation of branched 25 kDa polyethylenimine (PEI) was considered to reduce the positive surface charge of the polymer without reducing its ‘proton sponge’ buffering capacity, and to provide alkylene domains for hydrophobic interactions, thus generating optimized novel PEI carriers for efficient delivery of small interfering RNA (siRNA).

Methods

Substitution of PEI was evaluated in the range of 6 to > 50 mole percentage of primary amines. Additionally, variation of the carboxyalkyl chain (one to 15 methylene groups) was explored to modulate the carrier hydrophobicity. Carriers were characterized in their buffering capacity, capability of siRNA polyplex formation, and cytotoxicity. Marker gene‐silencing efficacy was evaluated using Neuro2A‐eGFPLuc neuroblastoma cells.

Results

Carboxyalkylation strongly reduced cytotoxicity of PEI and improved siRNA mediated luciferase gene knockdown. An optimum silencing activity was observed at an alkylcarboxylation degree of 6–9 mole percentage of primary amines and with a broad range of carboxyalkylene chains (containing one to 15 methylene groups). Strongly enhanced gene‐silencing efficacy also was observed when the biocompatible polymers were separately added at 1 h after transfection with tolerated doses of standard PEI25/siRNA polyplexes.

Conclusions

Carboxyalkylation of branched 25 kDa PEI resulted in polymers with strongly reduced cytotoxicity and improved silencing efficacy. Mechanistic studies demonstrated that the presence of a surplus of free carboxyalkylated polymer is responsible for the improved siRNA delivery. Copyright © 2010 John Wiley & Sons, Ltd.     

Potent gene silencing in vitro at physiological pH using chitosan polymers

Among non-viral cationic polymers, biodegradable chitosan has during the last decade become an attractive carrier for small interference RNA (siRNA) delivery. Currently, degradation of macromolecules in the lysosomes is assumed to be a major barrier for effective siRNA transfection. Hence, transfection protocols are focused toward endosomal release mechanisms. In this work, we have tested 3 novel chitosan polymers and their siRNA delivery properties in vitro. To obtain efficient gene silencing of our model gene, S100A4, various transfection parameters were investigated, such as pH, nitrogen/phosphate ratio, photochemical internalization (PCI), media for complex formation, and cell lines. Our results showed that 2 linear chitosan polymers demonstrated excellent siRNA gene silencing, better than Lipofectamine 2000. The silencing effect was achieved without PCI treatment, under physiological pH, and with no observable reduction in cell viability.     

PEI-g-PEG-RGD/small interference RNA polyplex-mediated silencing of vascular endothelial growth factor receptor and its potential as an anti-angiogenic tumor therapeutic strategy

Tumor angiogenesis appears to be achieved by the expression of vascular endothelial growth factor (VEGF) within solid tumors that stimulate host vascular endothelial cell mitogenesis and possibly chemotaxis. VEGF's angiogenic actions are mediated through its high-affinity binding to 2 endothelium-specific receptor tyrosine kinase, Flt-1 (VEGFR1), and Flk-1/KDR (VEGFR2). RNA interference-mediated knockdown of protein expression at the messenger RNA level provides a new therapeutic strategy to overcome various diseases. To achieve high efficacy in RNA interference-mediated therapy, it is critical to develop an efficient delivering system to deliver small interference RNA (siRNA) into tissues or cells site-specifically. We previously reported an angiogenic endothelial cell-targeted polymeric gene carrier, PEI-g-PEG-RGD. This targeted carrier was developed by the conjugation of the ανβ3/ανβ5 integrin-binding RGD peptide (ACDCRGDCFC) to the cationic polymer, branched polyethylenimine, with a hydrophilic polyethylene glycol (PEG) spacer. In this study, we used PEI-g-PEG-RGD to deliver siRNA against VEGFR1 into tumor site. The physicochemical properties of PEI-g-PEG-RGD/siRNA complexes was evaluated. Further, tumor growth profile was also investigated after systemic administration of PEI-g-PEG-RGD/siRNA complexes.     

Kinetic analysis of the role of zinc in the interaction of domain 5 of high-molecular weight kininogen (HK) with heparin

Previous investigations have shown that HK and its light chain bind heparin, preventing the enhancement of antithrombin inhibition of thrombin and potentiating the inhibition of plasma kallikrein by antithrombin. We found that both HK and HKa bound heparin, but HK exhibited a greater affinity. We therefore localized the binding sites for heparin on HK. HK domains 5 and 6 of the light chain as well as domain 3 from the heavy chain, expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli, were tested for binding to immobilized heparin by surface plasmon resonance using a BiaCore 2000 instrument. GST-D5, but not GST-D3, GST-D6, or GST, bound to heparin when the recombinant domains were present at a concentration of 70 nM. To localize more precisely the amino acid sequences on D5, both of the subdomains, histidine-glycine-rich GST-(K420-D474) and histidine-glycine-lysine-rich GST-(H475-S626), were expressed and tested for binding to immobilized heparin. The K(d) was much lower for GST-(K420-D474) than for GST-(H475-S626) in the presence or absence of Zn(2+). GST-(K420-D474) was effective in decreasing the rate of inactivation of thrombin by antithrombin in the presence of heparin and Zn(2+), while GST-(H475-S626) had no effect. We conclude that the binding of heparin to HK is a complex function of Zn(2+) interacting with histidines in the sequence K420-D474 to create high-affinity binding sites. HK has the potential to be an important modulator of heparin therapy.     

Simple modifications of branched PEI lead to highly efficient siRNA carriers with low toxicity

Polymer carriers like PEI which proved their efficiency in DNA delivery were found to be far less effective for the applications with siRNA. In the current study, we generated a number of nontoxic derivates of branched PEI through modification of amines by ethyl acrylate, acetylation of primary amines, or introduction of negatively charged propionic acid or succinic acid groups to the polymer structure. The resulting products showed high efficiency in siRNA-mediated knockdown of target gene. In particular, succinylation of branched PEI resulted in up to 10-fold lower polymer toxicity in comparison to unmodified PEI. Formulations of siRNA with succinylated PEI were able to induce remarkable knockdown (80% relative to untreated cells) of target luciferase gene at the lowest tested siRNA concentration of 50 nM in Neuro2ALuc cells. The polyplex stability assay revealed that the efficiency of formulations which are stable in physiological saline is independent of the affinity of siRNA to the polymer chain. The improved properties of modified PEI as siRNA carrier are largely a consequence of the lower polymer toxicity. In order to achieve significant knockdown of target gene, the PEI-based polymer has to be applied at higher concentrations, required most probably for sufficient accumulation and proton sponge effects in endosomes. Unmodified PEI is highly toxic at such polymer concentrations. In contrast, the far less toxic modified analogues can be applied in concentrations required for the knockdown of target genes without side effects.     

Effective delivery of siRNA to transgenic rice cells for enhanced transfection using PEI-based polyplexes

Various polymers were used as transfection factors for small interfering RNA (siRNA) to effectively suppress human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) gene in transgenic rice cells. Five kinds of polymers (PEI, PVA, PVP, and 8 and 20 kDa PEGs) were applied for delivery of siRNA with lipofectamine used as a control. In the cytotoxicity test, all polymers except 8 kDa PEG showed nontoxicity in relation to cell viability. For transfection efficiency, polyplexes composed of siRNA and PEG (20 kDa) did not significantly reduce production of intracellular hCTLA4Ig. On the other hand, siRNA + PEI polyplexes showed the most effective suppression efficiency with regards to production of intracellular hCTLA4Ig among all other polyplexes (PVA, PVP, and PEG (8 kDa)). Effects of molecular weight ratios of siRNA:PEI were investigated to obtain optimal transfection efficiency and avoid excessive damage to cells. PEI-based polyplexes with a 1:10 ratio of siRNA:PEI reduced production of intracellular hCTLA4Ig up to 70.6% without alteration of cell viability. These results demonstrate that PEI-based polyplexes are easy to prepare, inexpensive, non-toxic, and effective to deliver siRNA to transgenic plant cell cultures.     

Effective Non-Viral Delivery of siRNA to Acute Myeloid Leukemia Cells with Lipid-Substituted Polyethylenimines

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.     

Development of a specific siRNA delivery system into HeLa cells using an IgG-binding fusion protein

Stable carriers are required for delivering siRNA to cells. The use of polyethyleneimine (PEI) as gene carrier has been researched extensively; however, it does not provide sufficient protection from RNase degradation and is not suitable for targeted siRNA delivery to specific cells. In this study, two repeats of Fc binding domain of protein G (C2) were used to introduce a specific antibody to PEI-based carrier of siRNA. In addition, we used the double-stranded RNA binding domain (DRBD) that can bind to siRNA. The complex, consisting of PEI, siRNA and constructed fusion protein, TrxC2DRBD including C2 and DRBD domains, could protect siRNA from RNase degradation. Furthermore, cell specific siRNA delivery into HeLa cells could be performed by the complex fusion with specific antibodies via C2 domain.     

Stability of [3H]ouabain binding to the (Na+ + K+)-ATPase solubilized with C12E8

The (Na+ + K+)-ATPase from dog kidney and partially purified membranes from HK dog erythrocytes were labeled with [3H]ouabain, solubilized with C12E8 and analyzed by HPLC through a TSK-GEL G3000SW column in the presence of C12E8, Mg2+, HPO4(2-) and glycerol at 20-23 degrees C. The peaks of [3H]ouabain bound to the enzyme from dog kidney and HK dog erythrocyte membranes corresponded to each other with apparent molecular weights of 470 000-490 000. In addition, these bindings of [3H]ouabain to the (Na+ + K+)-ATPase were observed to be stable at 20-23 degrees C for at least 18 h after the solubilization.     

Noncovalent interactions of poly(adenosine diphosphate ribose) with histones.

Covalent linkage of ADP-ribose polymers to proteins is generally considered essential for the posttranslational modification of protein function by poly(ADP-ribosyl)ation. Here we demonstrate an alternative way by which ADP-ribose polymers may modify protein function. Using a highly stringent binding assay in combination with DNA sequencing gels, we found that ADP-ribose polymers bind noncovalently to a specific group of chromatin proteins, i.e., histones H1, H2A, H2B, H3, and H4 and protamine. This binding resisted strong acids, chaotropes, detergents, and high salt concentrations but was readily reversible by DNA. When the interactions of variously sized linear and branched polymer molecules with individual histone species were tested, the hierarchies of binding were branched polymers greater than long, linear polymers greater than short, linear polymers and H1 greater than H2A greater than H2B = H3 greater than H4. For histone H1, the target of polymer binding was the carboxy-terminal domain, which is also the domain most effective in inducing higher order structure of chromatin. Thus, noncovalent interactions may be involved in the modification of histone functions in chromatin.     

Intracellular small interfering RNA delivery using genetically engineered double‐stranded RNA binding protein domain

Background

A variety of synthetic carriers, such as cationic polymers and lipids, have been used as nonviral carriers for small interfering RNA (siRNA) delivery. Although siRNA polyplexes and lipoplexes exhibited good gene silencing efficiencies, they often showed serious cytotoxicities, which are not useful for clinical applications. A double‐stranded RNA binding cellular protein with highly specific siRNA binding property and noncytotoxicity was used for siRNA delivery.

Methods

A double‐stranded RNA binding domain (dsRBD) of human double‐stranded RNA activated protein kinase R was genetically produced and utilized to complex siRNA for intracellular delivery. For characterization of the siRNA/dsRBD complexes, decomplexation assay and RNase protection assay were performed. Cytotoxicity and target gene inhibition ability were also examined using human carcinoma cell lines.

Results

The recombinantly produced polypeptide dsRBD exhibited its inherent binding activity for siRNA without sequence specificity, and the siRNA/dsRBD complexes protected siRNA from degradation by ribonucleases. Green fluorescent protein (GFP) siRNA/dsRBD complexes showed prominent down‐regulation of a target GFP gene, when an endosomal escape function was supplemented by addition of a fusogenic peptide, KALA, in the formulation.

Conclusions

The results suggest that dsRBD‐based protein carriers could be successfully applied for a wide range of therapeutic siRNAs for intracellular gene inhibition without showing any cytotoxicity. Copyright © 2009 John Wiley & Sons, Ltd.     

Activated Hepatic Stellate Cells Are Dependent on Self-collagen,Cleaved by Membrane Type 1 Matrix Metalloproteinase for Their Growth

Stellate cells are distributed throughout organs, where, upon chronic damage, they become activated and proliferate to secrete collagen, which results in organ fibrosis. An intriguing property of hepatic stellate cells (HSCs) is that they undergo apoptosis when collagen is resolved by stopping tissue damage or by treatment, even though the mechanisms are unknown. Here we disclose the fact that HSCs, normal diploid cells, acquired dependence on collagen for their growth during the transition from quiescent to active states. The intramolecular RGD motifs of collagen were exposed by cleavage with their own membrane type 1 matrix metalloproteinase (MT1-MMP). The following evidence supports this conclusion. When rat activated HSCs (aHSCs) were transduced with siRNA against the collagen-specific chaperone gp46 to inhibit collagen secretion, the cells underwent autophagy followed by apoptosis. Concomitantly, the growth of aHSCs was suppressed, whereas that of quiescent HSCs was not. These in vitro results are compatible with the in vivo observation that apoptosis of aHSCs was induced in cirrhotic livers of rats treated with siRNAgp46. siRNA against MT1-MMP and addition of tissue inhibitor of metalloproteinase 2 (TIMP-2), which mainly inhibits MT1-MMP, also significantly suppressed the growth of aHSCs in vitro. The RGD inhibitors echistatin and GRGDS peptide and siRNA against the RGD receptor αVβ1 resulted in the inhibition of aHSCs growth. Transduction of siRNAs against gp46, αVβ1, and MT1-MMP to aHSCs inhibited the survival signal of PI3K/AKT/IκB. These results could provide novel antifibrosis strategies.     

Inhibition of vitronectin-mediated haptotaxis and haptoinvasion of MG-63 cells by domain 5 (D5(H)) of human high-molecular-weight kininogen and identification of a minimal amino acid sequence.

We found that human kinin-free high-molecular-weight kininogen (kf-HK) significantly inhibited vitronectin-mediated migration (haptotaxis) and invasive potentiation (haptoinvasion) of osteosarcoma (MG-63) cells but that HK, LK, the common heavy chain of HK and LK, and the light chain (D6(H)) of HK had no inhibitory effect. Recombinant GST-D5(H) (histidine-rich region of HK) obtained from Escherichia coli. (BL21) also inhibited both haptotaxis and haptoinvasion to about 30% of the control level in a dose-dependent manner. These findings suggest that a specific region of D5(H) is responsible for the inhibition of cell haptotaxis and haptoinvasion. Among the seven synthetic peptides covering D5(H), peptide H(479)KHGHGHGKHKNKGK(493) (P-5) inhibited both haptotaxis and haptoinvasion in a dose-dependent manner, suggesting that P-5 could possibly be utilized to prevent primary and secondary metastases of tumor cells.     

Lid2 is required for coordinating H3K4 and H3K9 methylation of heterochromatin and euchromatin

In most eukaryotes, histone methylation patterns regulate chromatin architecture and function: methylation of histone H3 lysine-9 (H3K9) demarcates heterochromatin, whereas H3K4 methylation demarcates euchromatin. We show here that the S. pombe JmjC-domain protein Lid2 is a trimethyl H3K4 demethylase responsible for H3K4 hypomethylation in heterochromatin. Lid2 interacts with the histone lysine-9 methyltransferase, Clr4, through the Dos1/Clr8-Rik1 complex, which also functions in the RNA interference pathway. Disruption of the JmjC domain alone results in severe heterochromatin defects and depletion of siRNA, whereas overexpressing Lid2 enhances heterochromatin silencing. The physical and functional link between H3K4 demethylation and H3K9 methylation suggests that the two reactions act in a coordinated manner. Surprisingly, crossregulation of H3K4 and H3K9 methylation in euchromatin also requires Lid2. We suggest that Lid2 enzymatic activity in euchromatin is regulated through a dynamic interplay with other histone-modification enzymes. Our findings provide mechanistic insight into the coordination of H3K4 and H3K9 methylation.