Enabling Combinatorial siRNA Delivery against Apoptosis-Related Proteins with Linoleic Acid and α-Linoleic Acid Substituted Low Molecular Weight Polyethylenimines.

PURPOSE: Short interfering RNA (siRNA) therapy promises a new era in treatment of breast cancers but effective delivery systems are needed for clinical use. Since silencing complementary targets may offer improved efficacy, this study was undertaken to identify non-viral carriers for combinatorial siRNA delivery for more effective therapy. METHODS: A library of lipid-substituted polymers from low molecular weight polyethyleneimine (PEI), linoleic acid (LA) and α-linoleic acid (αLA) with amide or thioester linkages was prepared and investigated for delivering Mcl-1, survivin and STAT5A siRNAs in breast cancer cells. RESULTS: The effective polymers formed 80-190 nm particles with similar zeta-potentials, but the serum stability was greater for complexes formed with amide-linked lipid conjugates. The LA and αLA substitutions, with the low molecular weight PEI (1.2 kDa and 2.0 kDa) were able to deliver siRNA effectively to cells and retarded the growth of breast cancer cells. The amide-linked lipid substituents showed higher cellular delivery of siRNA as compared to thioester linkages. Upon combinational delivery of siRNAs, growth of MCF-7 cells was inhibited to a greater extent with 2.0PEI-LA9 mediated delivery of Mcl-1 combined survivin siRNAs as compared to individual siRNAs. The qRT-PCR analysis confirmed the decrease in mRNA levels of target genes with specific siRNAs and 2.0PEI-LA9 was the most effective polymer for delivering siRNAs (either single or in combination). CONCLUSIONS: This study yielded effective siRNA carriers for combinational delivery of siRNAs. Careful choice of siRNA combinations will be critical since targeting individual genes might alter the expression of other critical mediators.

Novel targets for sensitizing breast cancer cells to TRAIL-induced apoptosis with siRNA delivery.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in variety of cancer cells without affecting most normal cells, which makes it a promising agent for cancer therapy. However, TRAIL therapy is clinically not effective due to resistance induction. To identify novel regulators of TRAIL that can aid in therapy, protein targets whose silencing sensitized breast cancer cells against TRAIL were screened with an siRNA library against 446 human apoptosis-related proteins in MDA-231 cells. Using a cationic lipopolymer (PEI-αLA) for delivery of library members, 16 siRNAs were identified that sensitized the TRAIL-induced death in MDA-231 cells. The siRNAs targeting BCL2L12 and SOD1 were further evaluated based on the novelty and their ability to sensitize TRAIL induced cell death. Silencing both targets sensitized TRAIL-mediated cell death in MDA-231 cells as well as TRAIL resistant breast cancer cells, MCF-7. Combination of TRAIL and siRNA silencing BCL2L12 had no effect in normal human umbilical vein cells and human bone marrow stromal cell. The silencing of BCL2L12 and SOD1 enhanced TRAIL-mediated apoptosis in MDA-231 cells via synergistically activating capsase-3 activity. Hence, here we report siRNAs targeting BCL2L12 and SOD1 as a novel regulator of TRAIL-induced cell death in breast cancer cells, providing a new approach for enhancing TRAIL therapy for breast cancer. The combination of siRNA targeting BCL2L12 and TRAIL can be a highly effective synergistic pair in breast cancer cells with minimal effect on the non-transformed cells.

Asialoglycoprotein Receptor-Mediated Gene Delivery to Hepatocytes Using Galactosylated Polymers.

Highly efficient, specific, and nontoxic gene delivery vector is required for gene therapy to the liver. Hepatocytes exclusively express asialoglycoprotein receptor (ASGPR), which can recognize and bind to galactose or N-acetylgalactosamine. Galactosylated polymers are therefore explored for targeted gene delivery to the liver. A library of safe and stable galactose-based glycopolymers that can specifically deliver genes to hepatocytes were synthesized having different architectures, compositions, and molecular weights via the reversible addition-fragmentation chain transfer process. The physical and chemical properties of these polymers have a great impact on gene delivery efficacy into hepatocytes, as such block copolymers are found to form more stable complexes with plasmid and have high gene delivery efficiency into ASGPR expressing hepatocytes. Transfection efficiency and uptake of polyplexes with these polymers decreased significantly by preincubation of hepatocytes with free asialofetuin or by adding free asialofetuin together with polyplexes into hepatocytes. The results confirmed that polyplexes with these polymers were taken up specifically by hepatocytes via ASGPR-mediated endocytosis. The results from transfection efficiency and uptake of these polymers in cells without ASGPR, such as SK Hep1 and HeLa cells, further support this mechanism. Since in vitro cytotoxicity assays prove these glycopolymers to be nontoxic, they may be useful for delivery of clinically important genes specifically to the liver.

pH and redox dual responsive nanoparticle for nuclear targeted drug delivery.

To mimic the clinic dosing pattern, initially administering high loading dose and then low maintenance dose, we designed a novel poly(2-(pyridin-2-yldisulfanyl)ethyl acrylate) (PDS) based nanoparticle delivery system. Side chain functional PDS was synthesized by free radical polymerization. Polyethylene glycol and cyclo(Arg-Gly-Asp-d-Phe-Cys) (cRGD) peptide was conjugated to PDS through thiol-disulfide exchange reaction to achieve RPDSG polymer. RPDSG/DOX, RPDSG nanoparticle loaded with doxorubicin, was fabricated by cosolvent dialysis method. The size of the nanoparticles was 50.13 ± 0.5 nm in PBS. The RPDSG/DOX nanoparticle is stable in physiological condition while quickly releasing doxorubicin with the trigger of acidic pH and redox potential. Furthermore, it shows a two-phase release kinetics, providing both loading dose and maintenance dose for cancer therapy. The conjugation of RGD peptide enhanced the cellular uptake and nuclear localization of the RPDSG/DOX nanoparticles. RPDSG/DOX exhibits IC(50) close to that of free doxorubicin for HCT-116 colon cancer cells. Due to the synergetic effect of RGD targeting effect and its two-phase release kinetics, RPDSG/DOX nanoparticles display significantly higher anticancer efficacy than that of free DOX at concentrations higher than 5 µM. These results suggest that RPDSG/DOX could be a promising nanotherapeutic for tumor-targeted chemotherapy.

TRAIL therapy and prospective developments for cancer treatment.

Tumor Necrosis Factor (TNF) Related Apoptosis-Inducing Ligand (TRAIL), an immune cytokine of TNF-family, has received much attention in late 1990s as a potential cancer therapeutics due to its selective ability to induce apoptosis in cancer cells. TRAIL binds to cell surface death receptors, TRAIL-R1 (DR4) and TRAIL-R2 (DR5) and facilitates formation of death-inducing signaling complex (DISC), eventually activating the p53-independent apoptotic cascade. This unique mechanism makes the TRAIL a potential anticancer therapeutic especially for p53-mutated tumors. However, recombinant human TRAIL protein (rhTRAIL) and TRAIL-R agonist monoclonal antibodies (mAb) failed to exert robust anticancer activities due to inherent and/or acquired resistance, poor pharmacokinetics and weak potencies for apoptosis induction. To get TRAIL back on track as a cancer therapeutic, multiple strategies including protein modification, combinatorial approach and TRAIL gene therapy are being extensively explored. These strategies aim to enhance the half-life and bioavailability of TRAIL and synergize with TRAIL action ultimately sensitizing the resistant and non-responsive cells. We summarize emerging strategies for enhanced TRAIL therapy in this review and cover a wide range of recent technologies that will provide impetus to rejuvenate the TRAIL therapeutics in the clinical realm.

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.

Cholesterol grafted cationic lipopolymers: Potential siRNA carriers for selective chronic myeloid leukemia therapy.

Synthetic siRNA technology has emerged as a promising approach for molecular therapy of cancer but, despite its potential for post-transcriptional gene silencing, there is an urgent need to develop efficient delivery systems particularly for difficult-to-transfect, anchorage-independent cells. In this study, we designed highly hydrophobic cationic lipopolymers by grafting cholesterol (Chol) onto low-molecular weight (0.6, 1.2, and 2.0 kDa) polyethylenimines (PEIs) to enable specific siRNA therapy to chronic myeloid leukemia (CML) cells. The siRNA binding by PEI-Chol led to nano-sized (100-200 nm diameter) polyplexes with enhanced ζ-potential (+20 to +35 mV) and ability to protect the loaded siRNA completely in fresh serum. The siRNA delivery to CML (K562) cells was proportional to degree of substitution and, unexpectedly, inversely proportional to molecular size of the polymeric backbone. Chol grafting with as little as ~1.0 Chol/PEI on 0.6 and 1.2 kDa PEIs enabled silencing of the reporter Green Fluorescent Protein gene as well as the endogenous BCR-Abl oncogene in K562 cells. The PEI-Chol mediated delivery of siRNAs specific for BCR-Abl and KSP genes significantly arrested the growth the cells which was significantly reflected in colony formation potency of K562 cells. BCR-Able siRNA mediated therapeutic efficacy was also observed in significantly increased caspase activity and apoptosis of K562 cells. Thus, Chol-grafted low-molecular weight PEIs appear to be unique siRNA carriers to realize the molecular therapy in CML cells.

siRNA Library Screening to Identify Complementary Therapeutic Pairs in Triple-Negative Breast Cancer Cells.

The existence of tightly integrated cross talk through multiple signaling and effector pathways has been appreciated in malignant cells. The realization of the plasticity of such networks is stimulating the development of combinational therapy to overcome the limitations of one-dimensional therapies. Synergistic pairs of siRNAs or siRNA and drug combinations are the new frontiers in identifying effective therapeutic combinations. To elucidate effective combinations, we developed a versatile protocol to screen siRNA libraries in triple-negative breast cancer cell models. This protocol outlines the steps to identify synergistic combinations of siRNA-siRNA or siRNA-drug combinations using siRNA libraries via a robotic screen. By focusing on smaller functional siRNA libraries, we present methodologies to identify synergistic siRNA pairings against cancerous cell growth and molecular targets to augment the activity of pro-apoptotic TRAIL protein. Here, we summarize the critical steps to undertake such combinational target identification, emphasizing critical factors that affect the outcome of the screens. Our experience suggests that siRNA library screening is an efficient protocol to identify complementary therapeutic pairs of new or already-existing drugs. This protocol is simple, robust and can be completed within a 1-week working period.

BCR-Abl Silencing by siRNA: A Potent Approach to Sensitize Chronic Myeloid Leukemia Cells to Tyrosine Kinase Inhibitor Therapy.

Nonviral gene therapy with specific short interfering RNAs (siRNAs) against BCR-Abl can be an alternative and/or supportive therapy of chronic myeloid leukemia (CML) with tyrosine kinase inhibitors (TKIs), given the often observed resistance to TKIs in clinical setting. In this study, we explored the feasibility of BCR-Abl siRNA therapy in CML K562 cells in vitro by employing a cationic polymer derived from cholesterol (Chol) grafted low-molecular weight polyethyleneimine (PEI). The first generation TKI imatinib upregulated the expression of BCR-Abl in K562 cells as expected. Delivery of BCR-Abl siRNA in both drug-sensitive and drug-resistant K562 cells significantly downregulated the mRNA levels in both cell types. Similarly, the BCR-Abl siRNA treatment arrested the growth of both drug-sensitive and drug-resistant K562 cells with no obvious differences despite a large difference in drug responsiveness. The BCR-Abl gene silencing in combination with TKI treatments exhibited significant synergism in drug-resistant K562 cells in generating substantial antileukemic activity, where the TKIs on their own were not effective. The effect of BCR-Abl siRNA and TKIs on non-CML cells (Jurkat and primary fibroblast) was negligible, indicating the specificity of the proposed therapy. This strategy can significantly overcome TKI resistance in CML cells, suggesting a feasible and effective treatment model for CML patients suffering from clinical resistances.

Breathing New Life into TRAIL for Breast Cancer Therapy: Co-Delivery of pTRAIL and Complementary siRNAs Using Lipopolymers.

Preclinical studies showed that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) therapy is safe and effective to combat cancers, but clinical outcomes have been less than optimal due to short half-life of TRAIL protein, insufficient induction of apoptosis, and TRAIL resistance displayed in many tumors. In this study, we explored co-delivery of a TRAIL expressing plasmid (pTRAIL) and complementary small interfering RNAs (siRNAs) (silencing Bcl2-like 12 [BCL2L12] and superoxide dismutase 1 [SOD1]) to improve the response of breast cancer cells against TRAIL therapy. It is desirable to co-deliver the pDNA along with siRNA using a single delivery agent, but this is challenging given different structures of long/flexible pDNA and short/rigid siRNA. Toward this goal, we identified an aliphatic lipid-grafted low-molecular weight polyethylenimine (PEI) that accommodated both pDNA and siRNA in a single complex. The co-delivery of pTRAIL with BCL2L12- or SOD1-specific siRNAs resulted more significant cell death in different breast cancer cells compared with separate delivery without affecting nonmalignant cells viability. Ternary complexes of lipopolymer with pTRAIL and BCL2L12 siRNA significantly retarded the growth of breast cancer xenografts in mice. The enhanced anticancer activity was attributed to increased in situ secretion of TRAIL and sensitization of breast cancer cells against TRAIL by the co-delivered siRNAs. The lipid-grafted PEIs capable of co-delivering multiple types of nucleic acids can serve as powerful carriers for more effective complementary therapeutics. Graphical Abstract [Figure: see text].

Nucleic acid combinations: A new frontier for cancer treatment.

The emerging molecular understanding of cancer cell behavior is leading to increasing possibilities to control unchecked cell growth and metastasis. On the other hand, development of multifunctional drug carriers at the 'nano'-scale is providing exciting new therapeutic strategies in clinical management of cancer beyond the conventional cytotoxic drugs. A new frontier in this regard is the combinational use of complementary agents based on nucleic acids to overcome the limitations of conventional therapy. The existence of tightly-integrated cross-talk through multiple signaling and effector pathways have been appreciated for some time, and the plasticity of such a network to overcome one-dimensional intervention is stimulating development of combinational therapy. The objective of this review is to underline the cutting edge technologies and opportunities employed in combination cancer therapy using nucleic acids therapeutics for successful clinical translation. Here, we provide a detailed analysis of the multifunctional carriers designed for different types of payloads, surveying the biomaterials used to construct the functional carriers. We then provide effective nucleic acid combinations employed to obtain more comprehensive outcomes, highlighting the critical factors involved in successful therapy. We conclude with an authors' perspective on the future of combinational therapy using nucleic acid therapeutics, articulating the main challenges to advance this promising approach to the clinical realm.

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.

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.

High-performance liquid chromatography analysis of capsaicin content in 16 Capsicum fruits from Nepal.

Capsicum fruit, a popular spice as chili pepper, is an important ingredient of the formulations used in traditional medicines. Moreover, Capsicum fruit is listed as an official drug in several pharmacopoeias. Capsaicin, the most abundant component in Capsicum fruit, exhibits its therapeutic and adverse effects in a dose-dependent manner. Therefore, the known capsaicin content is the prerequisite for optimizing any formulation based on Capsicum fruit as a crude drug. We studied 16 samples of Capsicum fruits grown at different altitudes in Nepal and determined their capsaicin content by high-performance liquid chromatography. The capsaicin content was found to range from 2.19 to 19.73 mg/g of dry weight of Capsicum fruits. Capsaicin content in pericarp was found to be higher than in seeds. No correlation was found between the shape or size of the fruits and its capsaicin content. Our findings indicate that many of the formulations prepared from Capsicum fruit, even as described in pharmacopoeias, may vary in their strength, therapeutic activity, and possible side effects if the capsaicin content in Capsicum fruit is not standardized.