Tetrandrine for Targeting Therapy Resistance in Cancer.

During the last five decades, there has been tremendous development in our understanding of cancer biology and the development of new and novel therapeutics to target cancer. However, despite these advances, cancer remains the second leading cause of death across the globe. Most cancer deaths are attributed to the development of resistance to current therapies. There is an urgent and unmet need to address cancer therapy resistance. Tetrandrine, a bis-benzyl iso-quinoline, has shown a promising role as an anti-cancer agent. Recent work from our laboratory and others suggests that tetrandrine and its derivatives could be an excellent adjuvant to the current arsenal of anti-cancer drugs. Herein, we provide an overview of resistance mechanisms to current therapeutics and review the existing literature on the anti-cancer effects of tetrandrine and its potential use for overcoming therapy resistance in cancer.

An optimized polymeric delivery system for piggyBac transposition.

The piggyBac transposon/transposase system has been explored for long-term, stable gene expression to execute genomic integration of therapeutic genes, thus emerging as a strong alternative to viral transduction. Most studies with piggyBac transposition have employed physical methods for successful delivery of the necessary components of the piggyBac system into the cells. Very few studies have explored polymeric gene delivery systems. In this short communication, we report an effective delivery system based on low molecular polyethylenimine polymer with lipid substitution (PEI-L) capable of delivering three components, (i) a piggyBac transposon plasmid DNA carrying a gene encoding green fluorescence protein (PB-GFP), (ii) a piggyBac transposase plasmid DNA or mRNA, and (iii) a 2 kDa polyacrylic acid as additive for transfection enhancement, all in a single complex. We demonstrate an optimized formulation for stable GFP expression in two model cell lines, MDA-MB-231 and SUM149 recorded till day 108 (3.5 months) and day 43 (1.4 months), respectively, following a single treatment with very low cell number as starting material. Moreover, the stability of the transgene (GFP) expression mediated by piggyBac/PEI-L transposition was retained following three consecutive cryopreservation cycles. The success of this study highlights the feasibility and potential of employing a polymeric delivery system to obtain piggyBac-based stable expression of therapeutic genes.

Exploring the Potential of siRNA Delivery in Acute Myeloid Leukemia for Therapeutic Silencing.

We investigated the feasibility of using siRNA therapy for acute myeloid leukemia (AML) by developing macromolecular carriers that facilitated intracellular delivery of siRNA. The carriers were derived from low-molecular-weight (<2 kDa) polyethyleneimine (PEI) and modified with a range of aliphatic lipids. We identified linoleic acid and lauric acid-modified PEI as optimal carriers for siRNA delivery to AML cell lines KG1 and KG1a, as well as AML patient-derived mononuclear cells. As they have been proven to be potent targets in the treatment of AML, we examined the silencing of BCL2L12 and survivin and showed how it leads to the decrease in proliferation of KG1 and stem-cell-like KG1a cells. By optimizing the transfection schedule, we were able to enhance the effect of the siRNAs on proliferation over a period of 10 days. We additionally showed that with proper modifications of PEI, other genes, including MAP2K3, CDC20, and SOD-1, could be targeted to decrease the proliferation of AML cells. Our studies demonstrated the versatility of siRNA delivery with modified PEI to elicit an effect in leukemic cells that are difficult to transfect, offering an alternative to conventional drugs for more precise and targeted treatment options.

Safe and Effective Delivery of mRNA Using Modified PEI-Based Lipopolymers.

Chemically modified mRNA (modRNA) has proven to be a versatile tool for the treatment of various cancers and infectious diseases due to recent technological advancements. However, a safe and effective delivery system to overcome the complex extracellular and intracellular barriers is required in order to achieve higher therapeutic efficacy and broaden clinical applications. Here, we explored All-Fect and Leu-Fect C as novel transfection reagents derived from lipopolymers, which demonstrated excellent biocompatibility, efficient delivery capabilities, and a robust ability to escape the lysosomes. These properties directly increase mRNA stability by preventing mRNA degradation by nucleases and simultaneously promote efficient gene translation in vitro and in vivo. The modRNA delivered with lipopolymer vectors sustained effective transfection in mouse hearts following direct intramyocardial injection, as well as in major organs (liver and spleen) after systemic administration. No observable immune reactions or systemic toxicity were detected following the systemic administration of lipopolymer-mRNA complexes to additional solid organs. This study identified commercial reagents for the effective delivery of modRNA and may help facilitate the advancement of gene-based interventions involving the safe and effective delivery of nucleic acid drug substances.

Comparison of Overall Immunity Levels among Workers at Grape Orchard, Rose Greenhouse, and Open-Field Onion Farm.

Background: Occupational hazards in crop farms vary diversely based on different field operations as soil management, harvesting processes, pesticide, or fertilizer application. We aimed at evaluating the immunological status of crop farmers, as limited systematic investigations on immune alteration involved with crop farming have been reported yet. Methods: Immunological parameters including plasma immunoglobulin level, major peripheral immune cells distribution, and level of cytokine production from activated T cell were conducted. Nineteen grape orchard, 48 onion open-field, and 21 rose greenhouse farmers were participated. Results: Significantly low proportion of natural killer (NK) cell, a core cell for innate immunity, was revealed in the grape farmers (19.8 ± 3.3%) in comparison to the onion farmers (26.4 ± 3.1%) and the rose farmers (26.9 ± 2.5%), whereas cytotoxic T lymphocyte proportion was lower in the grape and the onion farmers than the rose farmers. The proportion of NKT cell, an immune cell implicated with allergic response, was significantly higher in the grape (2.3 ± 0.3%) and the onion (1.6 ± 0.8%) farmers compared with the rose farmers (1.0 ± 0.4%). A significantly decreased interferon-gamma:interleukin-13 ratio was observed from ex vivo stimulated peripheral blood mononuclear cells of grape farmers compared with the other two groups. The grape farmers revealed the lowest levels of plasma IgG1 and IgG4, and their plasma IgE level was not significantly different from that of the onion or the rose farmers. Conclusion: Our finding suggests the high vulnerability of workplace-mediated allergic immunity in grape orchard farmers followed by open-field onion farmers and then the rose greenhouse farmers.

Comparison of Overall Immunity Levels among Workers at Grape Orchard, Rose Greenhouse, and Open-Field Onion Farm

Background@#Occupational hazards in crop farms vary diversely based on different field operations as soil management, harvesting processes, pesticide, or fertilizer application. We aimed at evaluating the immunological status of crop farmers, as limited systematic investigations on immune alteration involved with crop farming have been reported yet. @*Methods@#Immunological parameters including plasma immunoglobulin level, major peripheral immune cells distribution, and level of cytokine production from activated T cell were conducted. Nineteen grape orchard, 48 onion open-field, and 21 rose greenhouse farmers were participated. @*Results@#Significantly low proportion of natural killer (NK) cell, a core cell for innate immunity, was revealed in the grape farmers (19.8 ± 3.3%) in comparison to the onion farmers (26.4 ± 3.1%) and the rose farmers (26.9 ± 2.5%), whereas cytotoxic T lymphocyte proportion was lower in the grape and the onion farmers than the rose farmers. The proportion of NKT cell, an immune cell implicated with allergic response, was significantly higher in the grape (2.3 ± 0.3%) and the onion (1.6 ± 0.8%) farmers compared with the rose farmers (1.0 ± 0.4%). A significantly decreased interferon-gamma:interleukin-13 ratio was observed from ex vivo stimulated peripheral blood mononuclear cells of grape farmers compared with the other two groups. The grape farmers revealed the lowest levels of plasma IgG1 and IgG4, and their plasma IgE level was not significantly different from that of the onion or the rose farmers. @*Conclusion@#Our finding suggests the high vulnerability of workplace-mediated allergic immunity in grape orchard farmers followed by open-field onion farmers and then the rose greenhouse farmers.

A systematic comparison of lipopolymers for siRNA delivery to multiple breast cancer cell lines: In vitro studies.

Small interfering RNA (siRNA) therapy is a promising approach for treatment of a wide range of cancers, including breast cancers that display variable phenotypic features. To explore the general utility of siRNA therapy to control aberrant expression of genes in breast cancer, we conducted a detailed analysis of siRNA delivery and silencing response in vitro in 6 separate breast cancer cell models (MDA-MB-231, MDA-MB-231-KRas-CRM, MCF-7, AU565, MDA-MB-435 and MDA-MB-468 cells). Using lipopolymers for siRNA complexation and delivery, we found a large variation in siRNA delivery efficiency depending on the specific lipopolymer used for siRNA complexation and delivery. Some lipopolymers were effective in all cell types used in this study, indicating the possibility of universal carriers for siRNA therapy. The delivery efficiency for effective lipopolymers was not correlated with dextran uptake in the cells tested, which indicated a receptor-mediated internalization for siRNA complexes with lipopolymers, unlike fluid-phase transfer associated with dextran uptake. Consistent with this, specific inhibitors involved in clathrin- and caveolin-mediated endocytosis significantly (>50%) reduced the internalization of siRNA complexes in all cell types. Using JAK2 and STAT3 silencing in MDA-MB-231 and MDA-MB-468 cells, a general correlation between the uptake and silencing efficiency at the mRNA level was evident, but it appeared that the choice of the target rather than the cell type was more critical for consistent silencing. We conclude that siRNA therapy with lipopolymers can be undertaken in multiple breast cancer cell phenotypes with similar efficiency, indicating the general applicability of non-viral RNAi in clinical management of molecularly heterogeneous breast cancers. STATEMENT OF SIGNIFICANCE: The manuscript investigated the efficacy of siRNA carriers across multiple breast cancer cell lines. The lipopolymeric carriers were capable of delivering effective dose of siRNA to a range of breast cancer cells. Despite some differences in uptake efficiency among cell types, the mechanism of delivery was similar, with CME and CvME significantly involved in the internalization of polyplexes, while fluid-phase endocytosis was not significant. Specific target silencing was correlated to delivery efficiency, but we did notice the presence of lipopolymers that achieved high silencing with minimal siRNA delivery. Silencing specific targets in different cell types were more uniformly achieved as compared to targeting different targets in the same cells. Our studies enhance the feasibility of delivering siRNA to different types of breast cancer cells.

Current attempts to implement siRNA-based RNAi in leukemia models.

Leukemias arise from genetic alterations in normal hematopoietic stem or progenitor cells, leading to abnormal blood population with transformed cells. With the advent of RNAi and its pharmacological mediator siRNA, it has become possible to downregulate specific drivers causing leukemias. In this review, we present unique aspects of RNAi-mediated therapy and delivery technologies. Recent updates on molecular targets and delivery systems are discussed emanating from in vitro cell models and preclinical animal models. We conclude with a view on the future of RNAi in leukemia therapy, emphasizing possible measures to achieve higher efficacy and improved safety.

Small hydrophobe substitution on polyethylenimine for plasmid DNA delivery: Optimal substitution is critical for effective delivery.

Cationic polymers have been turned into effective gene delivery agents by functionalizing with long-chain aliphatic lipids, but little information exists if small hydrophobic moieties can serve as effective substituents for this purpose. To explore this issue, we modified small molecular weight (1.2kDa) polyethylenimine (1.2PEI) by a small hydrophobe, propionic acid (PrA), through N-acylation and investigated the efficacy of resultant polymers to deliver plasmid DNA (pDNA) to breast cancer cells MDA-231 and MCF-7. A significant impact of PrA grafting was observed on physicochemical features of polymers and resultant pDNA complexes. pDNA binding capacity, as measured by BC50 (weight ratio for 50% binding), was decreased from 0.25 to 0.64 with PrA substitution. Hydrodynamic size of polymer/pDNA complexes was not altered, but the surface charge (ξ-potential) was increased with low PrA substitution and decreased at higher PrA substitutions. Similarly, in vitro pDNA transfection efficacy in MDA-231 and MCF-7 cells was significantly increased with PrA grafting and optimum efficacy was observed in polymers with modest substitution, 0.25-1.0 PrAs/PEI (mol/mol), but higher substitutions was detrimental to transfection. The transfection efficiency of PEI-PrAs was higher than aliphatic lipid (linoleic acid) substituted PEI and more stable than 25kDa branched PEI. However, unlike studies reported elsewhere, siRNA had no effect on transfection efficacy of pDNA/PEI-PrA complexes when used as an additive. We conclude that small hydrophobe substitution on low MW PEI converts it into effective pDNA delivery agent in breast cancer cells up to an optimal ratio, indicating that balancing hydrophobicity of polymer is critical for pDNA transfection. STATEMENT OF SIGNIFICANCE: This manuscript investigated the influence of small hydrophobe (propionic acid, PrA, 3 carbon) grafted onto small molecular weight polyethylenimine (1.2PEI) in pDNA delivery. We have explored this approach as an alternative of common strategies to graft long chain and/or bulky lipids [linoleic acid (18 carbon), cholesterol]. At optimal substitution, transfection efficiency of these polymers was significantly higher than long chain lipid substituted 1.2PEI, emphasizing a proper hydrophobic/hydrophilic balance for optimum gene delivery. The overall results establish the feasibility of using small hydrophobes to create functional carriers, as long as the polymers are engineered with optimal ratio of substituent. The reported studies should facilitate the efforts of biomaterials scientists and engineers to design new carriers for gene therapy.

In vitro selection of a peptide aptamer that changes fluorescence in response to verotoxin.

A peptide aptamer that changes fluorescence upon binding to verotoxin was selected in vitro using ribosome display with a tRNA carrying an environment-sensitive fluorescent probe. The aptamer specifically bound to verotoxin with a dissociation constant (K d) of 3.94 ± 1.6 µM, and the fluorescence decreased by 78% as the verotoxin concentration was increased. The selected peptide can be used for detection of verotoxin.

Redox potential ultrasensitive nanoparticle for the targeted delivery of camptothecin to HER2-positive cancer cells.

Ideal "smart" nanoparticles for drug delivery should enhance therapeutic efficacy without introducing side effects. To achieve that, we developed a drug delivery system (HCN) based on a polymer-drug conjugate of poly[2-(pyridin-2-yldisulfanyl)]-graft-poly(ethylene glycol) and camptothecin with an intracellularly cleavable linker and human epidermal growth factor receptor 2 (HER2) targeting ligands. An in vitro drug release study found that HCN was stable in the physiological environment and supersensitive to the stimulus of elevated intracellular redox potential, releasing all payloads in less than 30 min. Furthermore, confocal microscopy revealed that HCN could specifically enter HER2-positive cancer cells. As a consequence, HCN could effectively kill HER2-positive cancer cells while not affecting HER2-negative cells.

Multicompartment intracellular self-expanding nanogel for targeted delivery of drug cocktail.

A nano cocktail, NCPD, fabricated from a pH and redox dual responsive polymer shows a multicompartment structure. The NCPD nanogel is stable in physiological environments while intracellular spontaneous swelling and fast releasing its payload. NCPD displays much stronger synergism than its free drug counterpart, which suggests that NCPD could greatly attenuate the side effects of drug cocktails while boosting synergistic anticancer effects.

Design of serum compatible tetrary complexes for gene delivery.

A novel gene delivery system, called PoSC, consisting of PEI, PSP, and HA is described. In contrast to the DNA/PEI/HA ternary system whose transfection efficiency decreases significantly with increasing serum concentration, PoSC exhibits a high transfection efficiency of about 51 and 87% for NIH3T3 and HCT116 cells, respectively, at 50% serum concentration. Furthermore, PoSC shows no cytotoxic effect at its working concentration. The overall results suggest that HA adsorption on cationic complexes enhances the transfection efficiency, while PSP is essential for high transfection efficiency at higher serum concentration.

Lipid substitution on low molecular weight (0.6-2.0 kDa) polyethylenimine leads to a higher zeta potential of plasmid DNA and enhances transgene expression.

Cationic polymers are desirable gene carriers because of their better safety profiles than viral delivery systems. Low molecular weight (MW) polymers are particularly attractive, since they display little cytotoxicity, but they are also ineffective for gene delivery. To create effective carriers from low MW polymers palmitic acid (PA) was substituted on 0.6-2.0 kDa polyethylenimines (PEIs) and their efficiency for plasmid DNA (pDNA) delivery was evaluated. The extent of lipid substitution was dependent on the lipid/PEI feed ratio and the polymer MW. While the hydrodynamic size of the polymer/pDNA complexes (polyplexes) increased or decreased depending on the extent of lipid substitution, the ζ potential of the assembled complexes was consistently higher as a result of lipid substitution. Lipid substitution generally increased the in vitro toxicity of the PEIs, but it was significantly lower than that of the 25 kDa branched PEI. The in vitro transfection efficiency of the lipid-substituted polymers was higher than that of native PEIs, which were not at all effective. The delivery efficiency was proportional to the extent of lipid substitution as well as the polymer MW. This correlated with the increased uptake of lipid-substituted polyplexes, based on confocal microscopic investigations with FITC-labeled pDNA. The addition of chloroquine further increased the transfection efficiency of lipid-substituted PEIs, indicating that endosomal release was a limiting factor affecting the efficiency of these carriers. This study indicates that lipid substitution on low MW PEIs makes their assembly more effective, resulting in better delivery of pDNA into mammalian cells.

A comparative evaluation of disulfide-linked and hydrophobically-modified PEI for plasmid delivery.

Non-viral gene therapy has become an important approach for treatment of hereditary and acquired diseases as a result of better understanding of molecular mechanisms involved in disease development. To design more effective gene carriers, plasmid DNA (pDNA) delivery to 293T cells was investigated by using two types of polymeric carriers; polymer constructed with disulfide (-S-S-) linkages and polymers modified with hydrophobic moieties. The base polymer used for this study was 2-kDa poly(ethylene imine) (PEI2), a relatively cell-compatible but ineffective gene carrier. The -S-S- linking was achieved via Michael addition reaction using cystamine bisacrylamide (CBA), whereas hydrophobic modification by N-acylation of PEI2 amines with palmitoyl chloride (PA). The cytotoxicity of the polymers was found to be lower than that of the 25-kDa branched PEI, but both types of modifications increased the toxicity of PEI2 to some extent. The polymers were able to form polyplexes with pDNA with variable hydrodynamic sizes (130-600 nm) and ζ-potential (3.6-20.9 mV). Based on the expression of the reporter gene Enhanced Green Fluorescent Protein (EGFP), disulfide linking significantly increased the efficiency of native PEI2, which was not effective on its own. The PA-modified PEI2 was also effective for gene delivery, but disulfide linkage of this polymer did not increase its efficiency any further. Our results showed that hydrophobic modification of 2-kDa PEI significantly improved its transfection efficiency but improvements in transfection efficiency as a result of disulfide linking was dependent on the nature of the polymeric building blocks.

Improving position-specific predictions of protein functional sites using phylogenetic motifs.

MOTIVATION: Accurate computational prediction of protein functional sites is critical to maximizing the utility of recent high-throughput sequencing efforts. Among the available approaches, position-specific conservation scores remain among the most popular due to their accuracy and ease of computation. Unfortunately, high false positive rates remain a limiting factor. Using phylogenetic motifs (PMs), we have developed two combined (conservation + PMs) prediction schemes that significantly improve prediction accuracy. RESULTS: Our first approach, called position-specific MINER (psMINER), rank orders alignment columns by conservation. Subsequently, positions that are also not identified as PMs are excluded from the prediction set. This approach improves prediction accuracy, in a statistically significant way, compared to the underlying conservation scores. Increased accuracy is a general result, meaning improvement is observed over several different conservation scores that span a continuum of complexity. In addition, a hybrid MINER (hMINER) that quantitatively considers both scoring regimes provides further improvement. More importantly, it provides critical insight into the relative importance of phylogeny versus alignment conservation. Both methods outperform other common prediction algorithms that also utilize phylogenetic concepts. Finally, we demonstrate that the presented results are critically sensitive to functional site definition, thus highlighting the need for more complete benchmarks within the prediction community.

Stabilization of gold nanoparticles by hydrophobically-modified polycations.

Surface-modified gold nanoparticles have pronounced benefits in the biomedical field due to their significant interaction with delivery materials. In the present study we used hydrophobically-modified polycations (i.e., N-acylated chitosan) to stabilize gold nanoparticles. Aliphatic hydrophobic groups, having carbon chains of different lengths, were first grafted onto the backbone of chitosan by N-acylation with fatty-acid chlorides in order to increase its hydrophobicity. Gold nanoparticles stabilized with native chitosan and N-acylated chitosan were prepared by the graft-onto approach. Chemical modification and its quantification were studied by Fourier-transform infrared (FT-IR) spectroscopy. Further, the stabilized gold nanoparticles were characterized by different physico-chemical techniques such as UV-Vis, FT-IR, TEM, TGA and DLS. Spectral studies of gold nanoparticles show the backbone and the side chain functional groups of chitosan were not cleaved during the conjugation process. TEM observations revealed that the modified chitosan gold nanoparticles were well dispersed and spherical in shape with average size around 10-12 nm in triply-distilled water at pH 7.4, whereas the native chitosan gold nanoparticles appeared as clusters with 9.9 nm as average diameter and were dispersed only in dilute HCl. The size of modified chitosan gold nanoparticles varied depending on the length of grafting molecules.

Protein threading with profiles and distance constraints using clique based algorithms.

With the advent of experimental technologies like chemical cross-linking, it has become possible to obtain distances between specific residues of a newly sequenced protein. These types of experiments usually are less time consuming than X-ray crystallography or NMR. Consequently, it is highly desired to develop a method that incorporates this distance information to improve the performance of protein threading methods. However, protein threading with profiles in which constraints on distances between residues are given is known to be NP-hard. By using the notion of a maximum edge-weight clique finding algorithm, we introduce a more efficient method called FTHREAD for profile threading with distance constraints that is 18 times faster than its predecessor CLIQUETHREAD. Moreover, we also present a novel practical algorithm NTHREAD for profile threading with Non-strict constraints. The overall performance of FTHREAD on a data set shows that although our algorithm uses a simple threading function, our algorithm performs equally well as some of the existing methods. Particularly, when there are some unsatisfied constraints, NTHREAD (Non-strict constraints threading algorithm) performs better than threading with FTHREAD (Strict constraints threading algorithm). We have also analyzed the effects of using a number of distance constraints. This algorithm helps the enhancement of alignment quality between the query sequence and template structure, once the corresponding template structure is determined for the target sequence.

Study of electrolyte induced aggregation of gold nanoparticles capped by amino acids.

This work reports the electrolyte induced aggregation of gold nanoparticles directly conjugated to amino acid by chemical reduction in aqueous solution. The study was focused on three different classes of amino acids depending on the nature of alpha substituent, viz. l-cysteine, l-leucine, and l-asparagine. The band broadening and the red shift of surface plasmon band with increase in flocculation parameter showed the aggregation of gold nanoparticles with increase in electrolyte concentration and decrease in pH as monitored by UV-visible spectrophotometer. The (1)H NMR spectrum demonstrates that the sulfide bond of cysteine and alpha amino group of leucine and asparagine interact with nanoparticles surface. Furthermore, transmission electron microscope (TEM) and thermogravimetric analysis (TGA) were performed to characterize and to support the fate of stabilization of the gold nanoparticles by amino acid.

Multiple methods for protein side chain packing using maximum weight cliques.

In this paper, we present several methods for computing a solution to the protein side chain packing problem, with all methods having a common solution approach of breaking the polymer into subpolymers and using maximum edge weight cliques to prune the search space for the optimal side chain packing. We characterize the graph sizes generated for each method and compare their prediction accuracies. These methods are demonstrated for computing proteins up to approximately 8000 residues. In addition, we update a result published previously.