RESUMO
RNAi is a biological process that utilizes small interfering RNA (siRNA) to prevent the translation of mRNA to protein. This mechanism could be beneficial in preventing the overexpression of proteins in cancer. However, the cellular delivery of siRNA has proven to be challenging due to its inherent negative charge and relative instability. Here, we designed a multicomponent delivery system composed of a specifically designed peptide (linear or cyclic fatty acyl peptide conjugates and hybrid cyclic/linear peptides) and several lipids (DOTAP, DOPE, cholesterol, and phosphatidylcholine) to form a nanoparticle, which we have termed as peptide lipid-associated nucleic acids (PLANAs). Five formulations were prepared (a formulation with no peptide, which was named lipid-associated nucleic acid or LANA, and PLANA formulations A-D) using a mini extruder to form uniform nanoparticles around 100 nm in size with a slightly positive charge (less than +10 mv). Formulations were evaluated for peptide incorporation, siRNA encapsulation efficiency, release profile, toxicity, cellular uptake, and protein silencing. Our experiments showed effective encapsulation of siRNA (>95%), a controlled release profile, and negligible toxicity in formulations that did not contain a positively charged lipid. The results also revealed that PLANAs C and D exhibited optimum cellular uptake (with 80-90% siRNA-positive cells for most of the formulations). PLANA D formulation was selected to silence two model proteins (Src and RPS6KA5) in the triple-negative human breast cancer cell line MDA-MB-231, with promising silencing efficiency, which diminished the expression of RPS6KA5 and Src to approximately 29 and 38% compared to naïve cells, respectively. Many approaches have been investigated for safe and efficient delivery of nucleic acids in the last 20 years; however, many have failed due to the multifaceted challenges to overcome. Our results show a promising potential for a multicomponent design that incorporates different components for a variety of delivery tasks, which warrants further investigation of PLANAs in vivo.
Assuntos
Lipídeos/genética , Ácidos Nucleicos Peptídicos/genética , Peptídeos/genética , RNA Interferente Pequeno/genética , Linhagem Celular Tumoral , Inativação Gênica/fisiologia , Técnicas de Transferência de Genes , Humanos , Lipídeos/química , Nanopartículas/química , Ácidos Nucleicos Peptídicos/química , Peptídeos/química , Interferência de RNA/fisiologiaRESUMO
Cancer cells are known to be heterogeneous and plastic, which imparts innate and acquired abilities to resist molecular targeting by short interfering RNA (siRNA). Not all cancer cells in a population would show a similar responsiveness to targeting of genes critical for their survival and even the responders could quickly transform and switch to alternative mechanism(s) for their survival. This study was designed to look at this phenomenon by analyzing the effect of siRNA silencing of selected protein mRNAs involved in cell survival and proliferation on other protein mRNAs that could contribute to cell survival. We compared the gene expression profile of the initial population after siRNA silencing to the subpopulation that survived the siRNA silencing, to identify potential overexpressions that might explain the cell survival. Our studies show that silencing well-selected protein mRNAs simultaneously could offer advantages compared to individual siRNA silencing due to an additional impact on the expression level of certain protein mRNAs. We also demonstrate that overexpression of certain protein mRNAs could explain the innate unresponsiveness of a subpopulation of cells. These observations could be a stepping stone for further investigation of the possibility of significant synergistic effect for this combinational RNA interference strategy.
Assuntos
Biomarcadores Tumorais/antagonistas & inibidores , Neoplasias da Mama/tratamento farmacológico , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , RNA Interferente Pequeno/administração & dosagem , Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Terapia de Alvo Molecular , RNA Interferente Pequeno/genética , Transcriptoma , Células Tumorais CultivadasRESUMO
PURPOSE: An alternative cancer therapy based on RNA interference (RNAi) has shown considerable promise but the possibility of resistance development is not known. This study explored the possibility of therapeutic resistance against siRNA nanoparticles in human cancer cells. METHODS: Two approaches to siRNA treatment were undertaken using lipid-modified polyethylenimines, a single high concentration (shock) and repeated increasing concentrations (gradual). The targets were Mcl-1, RPS6KA5 and KSP in MDA-MB-435 cells. RESULTS: There was no evidence of resistance development in shock-treated cells, while the decrease in mRNA levels of targeted proteins was not as robust in naïve cells in gradual treatment. However, silencing efficiency was restored after a 7-day recovery period when expression of suppressed proteins returned to normal levels. Cellular uptake of siRNA was not affected by pre-treatments. Other mediators involved in cell survival and proliferation were altered in siRNA-treated cells, but only JUN silencing led to a heightened loss of viability. In vivo experiments demonstrated similar silencing efficiency at mRNA level after repeat doses. CONCLUSIONS: Human cancer cells responded to repeat siRNA nanoparticles in a similar fashion after a temporary initial alteration and little, if any, resistance was evident against repeated siRNA treatments.
Assuntos
Neoplasias da Mama/terapia , Lipídeos/química , Nanopartículas/química , Polietilenoimina/química , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/uso terapêutico , Terapêutica com RNAi/métodos , Animais , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos Nus , Interferência de RNA , RNA Interferente Pequeno/genéticaRESUMO
With the first reports on the possibility of genome editing by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas)9 surfacing in 2005, the enthusiasm for protein silencing via nucleic acid delivery experienced a resurgence following a period of diminished enthusiasm due to challenges in delivering small interfering RNAs (siRNA), especially in vivo. However, delivering the components necessary for this approach into the nucleus is challenging, maybe even more than the cytoplasmic delivery of siRNA. We previously reported the birth of peptide/lipid-associated nucleic acids (PLANAs) for siRNA delivery. This project was designed to investigate the efficiency of these nanoparticles for in vitro delivery of CRISPR/Cas9 ribonucleoproteins. Our initial experiments indicated higher toxicity for PLANAs with the more efficient reverse transfection method. Therefore, polyethylene glycol (PEG) was added to the composition for PEGylation of the nanoparticles by partially replacing two of the lipid components with the PEG-conjugated counterparts. The results indicated a more significant reduction in the toxicity of the nanoparticle, less compromise in encapsulation efficiency and more PEGylation of the surface of the nanoparticles using DOPE-PEG2000 at 50â¯% replacement of the naïve lipid. The cell internalization and transfection efficiency showed a comparable efficiency for the PEGylated and non-PEGylated PLANAs and the commercially available Lipofectamine™ CRISPRMAX™. Next Generation Sequencing of the cloned cells showed a variety of indels in the transfected cell population. Overall, our results indicate the efficiency and safety of PEGylated PLANAs for in vitro transfection with CRISPR/Cas9 ribonucleoproteins. PEGylation has been studied extensively for in vivo delivery, and PEGylated PLANAs will be candidates for future in vivo studies.
Assuntos
Sistemas CRISPR-Cas , Ácidos Nucleicos Peptídicos , Ribonucleoproteínas/genética , RNA Interferente Pequeno , Polietilenoglicóis , Lipídeos , PeptídeosRESUMO
Combinatorial silencing of more than one protein via small interfering RNA (siRNA) is a new strategy that can enhance the effect of RNA interference on cell function. To explore this strategy, we selected JAK/STAT axis as a major signaling pathway that contributes to several mechanisms involved in cancer cell proliferation and survival. We focused on four proteins involved in this pathway to explore the possibility of identifying a combinatorial targeting strategy (as the proof of concept) with enhanced efficiency: gp 130 (a co-receptor for IL6 cytokines), JAK2, STAT3, and importin α3 (the nuclear transporter reportedly involved in translocation of activated STAT3 to nucleus). Selected proteins were targeted by siRNA in two selected Triple Negative Breast Cancer cell lines (MDA-MB-231 and MDA-MB-468). The effect of individual and selected combinations of siRNAs on selected downstream antiapoptotic proteins, pro-apoptosis proteins, and cell-cycle regulating proteins was explored. Combinatorial silencing of JAK2/gp 130 enhanced the effect of RNA interference on downstream proteins significantly, and demonstrated enhanced effect in reducing cell viability, cell migration, and the level of activation of STAT3. In conclusion, the promising results of simultaneous targeting of JAK2 and gp 130 might be an example for potential combinatorial silencing strategies in cancer treatment.
Assuntos
Fator de Transcrição STAT3 , Neoplasias de Mama Triplo Negativas , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Humanos , Janus Quinase 2/genética , Janus Quinase 2/metabolismo , Janus Quinase 2/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de SinaisRESUMO
Post-transcriptional silencing of antiapoptotic genes is a promising strategy for cancer therapy, but delivering short interfering RNA (siRNA) molecules against such targets is challenging due to inability of anionic siRNA to cross cellular membranes. Lipid substitution on small molecular weight, nontoxic polyethylenimine (PEI) has been investigated as a promising approach for effective siRNA delivery. In this study, we report on the ability of low molecular weight, lipid-substituted PEI to deliver siRNA against the antiapoptotic protein survivin. Toxicity of a library of lipid-substituted PEIs, as well as their siRNA delivery and survivin silencing efficiency, was evaluated in MDA-MB-231 human breast cancer cells. A significant increase in cellular delivery of siRNA was observed as a result of lipid substitution. Most significant downregulation of survivin was established by caprylic acid-substituted polymers, which resulted in significant levels of apoptosis induction and resultant loss of cell viability. Survivin downregulation prior to anticancer drug treatment decreased the IC(50) of several drugs by 50- to 120-fold. Our experiments indicated an effective downregulation of survivin, a cell protective protein upregulated in tumor cells, by delivering siRNA with hydrophobically modified PEI. This study introduces a promising delivery system for safe and effective siRNA delivery that will be suitable for further investigation in preclinical animal models.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Inativação Gênica , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/metabolismo , Antineoplásicos/agonistas , Antineoplásicos/farmacologia , Transporte Biológico , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caprilatos/química , Linhagem Celular Tumoral , Forma do Núcleo Celular , Sobrevivência Celular/efeitos dos fármacos , Estudos de Viabilidade , Feminino , Humanos , Interações Hidrofóbicas e Hidrofílicas , Iminas/efeitos adversos , Iminas/química , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Concentração Inibidora 50 , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Tamanho da Partícula , Polietilenos/efeitos adversos , Polietilenos/química , SurvivinaRESUMO
Doxorubicin (Dox) is used for breast cancer, leukemia, and lymphoma treatment as an effective chemotherapeutic agent. However, Dox use is restricted due to inherent and acquired resistance and an 8-fold increase in the risk of potentially fatal cardiotoxicity. Hybrid cyclic-linear peptide [R5K]W7A and linear peptide R5KW7A were conjugated with Dox through a glutarate linker to afford [R5K]W7A-Dox and R5KW7A-Dox conjugates to generate Dox derivatives. Alternatively, [R5K]W7C was conjugated with Dox via a disulfide linker to generate [R5K]W7C-S-S-Dox conjugate, where S-S is a disulfide bond. Comparative antiproliferative assays between conjugates [R5K]W7A-Dox, [R5K]W7C-S-S-Dox, linear R5KW7A-Dox, the corresponding physical mixtures of the peptides, and Dox were performed in normal and cancer cells. [R5K]W7A-Dox conjugate was 2-fold more efficient than R5KW7A-Dox, and [R5K]W7C-S-S-Dox conjugates in inhibiting the cell proliferation of human leukemia cells (CCRF-CEM). Therefore, hybrid cyclic-linear [R5K]W7A-Dox conjugate was selected for further studies and inhibited the cell viability of CCRF-CEM (84%), ovarian adenocarcinoma (SK-OV-3, 39%), and gastric carcinoma (AGS, 73%) at a concentration of 5 µM after 72 h of incubation, which was comparable to Dox (5 µM) efficacy (CCRF-CEM (85%), SK-OV-3 (33%), and AGS (87%)). While [R5K]W7A-Dox had a significant effect on the viability of cancer cells, it exhibited minimal cytotoxicity to normal kidney (LLC-PK1, 5-7%) and heart cells (H9C2, <9%) at concentrations of 5-10 µM (compared to free Dox at 5 µM that reduced the viability of kidney and heart cells by 85% and 44%, respectively). The fluorescence microscopy images were consistent with the cytotoxicity studies, indicating minimal uptake of the cyclic-linear [R5K]W7A-Dox (5 µM) in H9C2 cells. In comparison, Dox (5 µM) showed significant uptake, reduced cell viability, and changed the morphology of the cells after 24 h. [R5K]W7A-Dox showed 16-fold and 9.5-fold higher activity against Dox-resistant cells MDA231R and MES-SA/MX2 (lethal dose for 50% cell death or LC50 of 2.3 and 4.3 µM, respectively) compared to free Dox (LC50 of 36-41 µM, respectively). These data, along with the results obtained from the cell viability tests, indicate comparable efficiency of [R5K]W7A-Dox to free Dox in leukemia, ovarian, and gastric cancer cells, significantly reduced toxicity in normal kidney LLC-PK1 and heart H9C2 cells, and significantly higher efficiency in Dox-resistant cells. A number of endocytosis inhibitors did not affect the cellular uptake of [R5K]W7A-Dox.
Assuntos
Antibióticos Antineoplásicos/efeitos adversos , Doxorrubicina/efeitos adversos , Desenho de Fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia , Antibióticos Antineoplásicos/química , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Doxorrubicina/química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Estrutura Molecular , Peptídeos Cíclicos/síntese química , Peptídeos Cíclicos/química , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Portadores de Fármacos/química , Ácidos Graxos/química , Polietilenoimina/análogos & derivados , RNA Interferente Pequeno/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Clorpromazina/farmacologia , Portadores de Fármacos/metabolismo , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Ácidos Graxos/metabolismo , Inativação Gênica/efeitos dos fármacos , Genisteína/farmacologia , Humanos , Polietilenoimina/metabolismoRESUMO
Non-responsive subpopulation of tumor cells, and acquired resistance in initially responsive cells are major challenges for cancer therapy with molecularly-targeted drugs. While point mutations are considered the major contributing factor to acquired resistance, in this study we explored the role of heterogeneity and plasticity of selected human breast cancer cell lines (MDA-MB-231, MDA-MB-468, and AU565) in their initial and adjusted response, respectively, to ruxolitinib, everolimus, and erlotinib. After determination of lethal concentration for 50% cell death (LC50), cells were exposed to selected drugs using three different approaches: single exposure to 4 × LC50 and collection of surviving cells, multiple exposures to 1.5 × LC50 and monitoring the surviving population, and exposure to gradually increasing concentrations of selected drugs (range of concentrations equivalent to 10% of LC50 to 1.5 × LC50). Surviving cells were studied for adjustments in expression level of selected proteins using quantitative PCR and Western Blot. Our data indicated overexpression of a variety of proteins in resistant populations, which included cell membrane receptors EGFR and HER2, anti-apoptotic proteins Bcl-2 and BIRC8, and other proteins involved in cell signaling (e.g., Akt1, MAPK7, and RPS6KA5). Silencing the identified alternative proteins via siRNA resulted in significant drop in the LC50 of the selected molecularly-targeted drugs cells resistant to ruxolitinib (via targeting Akt), everolimus (via targeting EGFR, MAPK7, RPS6KA5, and HER2), and erlotinib (via silencing Bcl2 and BIRC8). Our data indicates that targeting well-selected alternative proteins could potentially sensitize the resistant cells to the effect of the molecularly-targeted treatment.
RESUMO
A number of amphiphilic cyclic peptides-[FR]4, [WR]5, and [WK]5-containing hydrophobic and positively-charged amino acids were synthesized by Fmoc/tBu solid-phase peptide methods and evaluated for their efficiency in intracellular delivery of siRNA to triple-negative breast cancer cell lines, MDA-MB-231 and MDA-MB-468, in the presence and absence of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Among the peptides, [WR]5, which contains alternate tryptophan (W) and arginine (R) residues, was found to be the most efficient in the delivery of siRNA by improving the delivery by more than 3-fold when compared to other synthesized cyclic peptides that were not efficient. The data also showed that co-formulation of [WR]5 with lipid DOPE significantly enhanced the efficiency of siRNA delivery by up to ~2-fold compared to peptide alone. Based on the data indicating the efficiency of [WR]5 in siRNA delivery, peptides containing arginine residues on the ring and tryptophan residues on the side chain, [R6K]W6 and [R5K]W5, were also evaluated, and demonstrated improved delivery of siRNA. The presence of DOPE again enhanced the siRNA delivery in most cases. [WR]5, [R5K]W5, and [R6K]W6 did not show any significant toxicity in MDA-MB-231, MDA-MB-468, and AU565 WT cells at N/P ratios of 20:1 or less, in the presence and absence of DOPE. Silencing of kinesin spindle protein (KSP) and Janus kinase 2 (JAK2) was evaluated in MDA-MB-231 cells in the presence of the peptides. The addition of DOPE significantly enhanced the silencing efficiency for all selected peptides. In conclusion, peptides containing tryptophan and arginine residues were found to enhance siRNA delivery and to generate silencing of targeted proteins in the presence of DOPE.
RESUMO
This study was designed to develop an optimized co-solvent evaporation procedure for the efficient encapsulation of hydrophobic drugs in polymeric micelles of methoxy poly(ethylene oxide)-block-poly(epsilon-caprolactone) (MePEO-b-PCL). MePEO-b-PCL block copolymers having varied MePEO and PCL molecular weights were synthesized, assembled to polymeric micelles, and used for the encapsulation of cyclosporine A (CyA) by a co-solvent evaporation method. The co-solvent composition was varied by changing the type of organic co-solvent (using acetone, acetonitrile and tetrahydrofuran), the ratio of organic to aqueous phase, and their order of addition. Carrier size, morphology and encapsulated CyA levels were defined by dynamic light scattering (DLS), transmission electron microscopy (TEM) and HPLC, respectively, and the effect of co-solvent composition on micellar properties and loaded CyA levels was evaluated. Application of acetone and acetonitrile as the selective co-solvent for the core-forming block led to a decrease in the average diameter of self-assembled structures. When acetone was added to water, a decrease in the ratio of organic to aqueous phase led to an increase in the loading efficiency of CyA in MePEO-b-PCL micelles. A similar trend in CyA loading was observed for MePEO-b-PCL micelles of varied MePEO and PCL block lengths. The ratio of organic to aqueous phase did not affect CyA loading when water was added to acetone. Irrespective of the order of addition, the decrease in the organic to aqueous phase ratio caused a reduction in the average diameter of the empty and CyA loaded micelles. We conclude that the co-solvent evaporation method may be optimized to improve the efficiency of drug encapsulation in polymeric micelles. For CyA encapsulation in MePEO-b-PCL micelles, addition of acetone to water at lower organic to aqueous phase ratio is shown to be the optimum procedure leading to higher drug encapsulation and smaller average diameter for the self-assembled structures.
Assuntos
Ciclosporina/administração & dosagem , Sistemas de Liberação de Medicamentos , Lactonas , Micelas , Polietilenoglicóis , Solventes , Interações Hidrofóbicas e Hidrofílicas , SolubilidadeRESUMO
A number of amphiphilic difatty acyl linear and cyclic R5K2 peptide conjugates were synthesized by solid-phase peptide methods to enhance the interaction with the hydrophobic cellular phospholipid bilayer and to improve siRNA delivery and silencing. Binding to siRNA molecules was significantly less for the cyclic peptide conjugates. A gradual decrease was observed in the particle size of the complexes with increasing peptide/siRNA ratio for most of the synthesized peptides, suggesting the complex formation. Most of the complexes showed a particle size of less than 200 nm, which is considered an appropriate size for in vitro siRNA delivery. A number of fatty acyl-conjugated peptides, such as LP-C16 and LP-C18, displayed near complete protection against serum degradation. Flow cytometry studies demonstrated significantly higher internalization of fluorescence-labeled siRNA (FAM-siRNA) in the presence of LP-C16, LP-C18, and CP-C16 with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) addition. Confocal microscopy confirmed the cellular internalization of fluorescence-labeled siRNA in the presence of LP-C16 and LP-C18 with DOPE when compared with cells exposed to DOPE/FAM-siRNA. While C16- and C18-conjugated peptides (especially linear peptides) showed silencing against kinesin spindle protein (KSP) and janus kinase 2 (JAK2) proteins, the addition of DOPE enhanced the silencing efficiency significantly for all selected peptides, except for CP-C16. In conclusion, C16 and C18 difatty acyl peptide conjugates were found to enhance siRNA delivery and generate silencing of targeted proteins in the presence of DOPE. This study provides insights for the design and potential application of optimized difatty acyl peptide/lipid nanoparticles for effective siRNA delivery.
RESUMO
The cell cycle proteins are key regulators of cell cycle progression whose deregulation is one of the causes of breast cancer. RNA interference (RNAi) is an endogenous mechanism to regulate gene expression and it could serve as the basis of regulating aberrant proteins including cell cycle proteins. Since the delivery of small interfering RNA (siRNA) is a main barrier for implementation of RNAi therapy, we explored the potential of a non-viral delivery system, 2.0 kDa polyethylenimines substituted with linoleic acid and caprylic acid, for this purpose. Using a library of siRNAs against cell cycle proteins, we identified cell division cycle protein 20 (CDC20), a recombinase RAD51, and serine-threonine protein kinase CHEK1 as effective targets for breast cancer therapy, and demonstrated their therapeutic potential in breast cancer MDA-MB-435, MDA-MB-231, and MCF7 cells with respect to another well-studied cell cycle protein, kinesin spindle protein. We also explored the efficacy of dicer-substrate siRNA (DsiRNA) against CDC20, RAD51, and CHEK1, where a particular DsiRNA against CDC20 showed an exceptionally high inhibition of cell growth in vitro. There was no apparent effect of silencing selected cell cycle proteins on the potency of the chemotherapy drug doxorubicin. The efficacy of DsiRNA against CDC20 was subsequently assessed in a xenograft model, which indicated a reduced tumor growth as a result of CDC20 DsiRNA therapy. The presented study highlighted specific cell cycle protein targets critical for breast cancer therapy, and provided a polymeric delivery system for their effective down-regulation.
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Locally delivered plasmid DNA (pDNA) is currently pursued as gene-based therapy for regenerative medicine, but important information on in situ pDNA pharmacokinetics and transgene expression is lacking in animal models. To investigate pDNA pharmacokinetics in implants, low molecular weight (2 kDa) polyethylenimine (PEI) and linoleic acid substituted 2 kDa PEI (PEI-LA) were used for pDNA delivery in gelatin sponges. An efficient pDNA extraction method combined with quantitative PCR (qPCR) was found to give equivalent quantitation of naked and polymer-bound pDNA, making it suitable to assess pDNA polyplexes in implants. Naked pDNA implanted in a rat subcutaneous model was >98% lost after 24 hours whereas PEI and PEI-LA delivered pDNA remained intact in implants for 2 and 4 weeks, respectively. Using a plasmid expressing DsRed as a reporter gene, mRNA and protein expression was observed only for PEI-LA despite the extended retention and cellular uptake of PEI complexes. The in vivo data were in agreement with in vitro results showing that only PEI-LA was an effective transfection agent even though both PEI and PEI-LA complexes were internalized by the cells. Dose dependence was observed for mRNA expression, with a 20 µg dose giving faster onset and higher expression levels compared to a 5 µg pDNA dose. The mRNA expression after PEI-LA mediated delivery was sustained for at least 4 weeks and a significant correlation between pDNA retention in sponges and mRNA expression was observed. In addition to establishing a promising gene carrier for gene delivery, these studies provided important information about the retention and transgene expression by implanted non-viral carriers.
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Chemotherapy is an effective approach to curb uncontrolled proliferation of malignant cells. However, most drugs rapidly lose their efficacy as a result of resistance development. We explored the potential of combinational siRNA silencing to prevent growth of drug-resistant breast cancer cells independent of chemotherapy. Resistance was induced in two breast cancer lines by chronic exposure to doxorubicin. Microarray analysis of apoptosis-related proteins showed Bcl2, survivin, NF B, and Mcl1 to be prominently up-regulated in drug-resistant cells. Human siRNA libraries against apoptosis-related proteins and kinases were screened using lipid-substituted polymers as non-viral carrier, and siRNAs were selected to diminish cell growth without affecting growth of skin fibroblasts. Surprisingly, the selected siRNAs led to similar responses in wild-type and drug-resistant cells, despite their phenotypic differences. Promising kinase siRNAs were co-delivered with anti-apoptotic Mcl-1 siRNA and Ribosomal Protein S6 Kinase (RPS6KA5) was found the most promising candidate for simultaneous silencing with Mcl-1. In both MDA435 wild type (WT) and MDA435 resistant (R) xenografts in nude mice, double silencing of Mcl-1/RPS6KA5 also led to improved inhibition of tumor growth in the absence of chemotherapy. We conclude that combinational silencing of well-selected targets could be a feasible therapeutic strategy in the absence of drug therapy and could provide a new avenue for therapy of drug-resistant breast cancers.
Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Doxorrubicina/uso terapêutico , RNA Interferente Pequeno/uso terapêutico , Animais , Antibióticos Antineoplásicos/farmacologia , Apoptose , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Terapia Combinada , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , RNA Interferente Pequeno/genética , Proteínas Quinases S6 Ribossômicas/genéticaRESUMO
RNAi represents a new alternative for treatment of chronic myeloid leukemia (CML) to overcome the difficulties of current drug treatments such as the acquired resistance. However, potent carriers that can overcome delivery barriers to RNAi agents and have therapeutic efficacy especially in difficult-to-transfect CML cells are needed. Here, we explored the use of lipid-modified polyethylenimines (PEI) of low molecular weights (0.6, 1.2 and 2.0kDa) in K562 cells and showed that the delivery efficiency was dependent on the type of lipid used for polymer modification, degree of lipid substitution and polymer molecular weight. Among the lipid-substituted polymers investigated, palmitic acid (PA)-substituted 1.2kDa PEI (~2 lipids/PEI) has proven to be highly efficient in delivering siRNA and silencing of the reporter gene green fluorescent protein (GFP). The silencing efficacy achieved with this polymer was found to be higher than the 25kDa PEI and is similar to commercial reagent Lipofectamine™ 2000. Moreover, when BCR-ABL protein was targeted in K562 cells, a reduction in the corresponding mRNA levels was observed, as well as an induction of early and late stage apoptosis. The results of this study demonstrated that PA-substitutions on low MW polymers could be useful for siRNA delivery in CML cells for therapeutic purposes.
Assuntos
Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Polietilenoimina/química , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , Transfecção/métodos , Regulação para Baixo , Humanos , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêuticoRESUMO
Breast Cancer Resistance Protein (BCRP, ABCG2) is an efflux protein whose aberrant activity has been linked to multidrug resistance in cancer. Although siRNA delivery to down-regulate BCRP expression is promising to sensitize tumor cells against drugs, therapeutic use of siRNA requires effective carriers that can deliver siRNA intracellularly with minimal toxicity on target cells. This study explored the feasibility of special class of cationic polymers, namely lipid-substituted low molecular weight (2kDa) polyethyleneimine (PEI), as a carrier for siRNA-mediated BCRP down-regulation. Structure-function studies methodically evaluated the effect of a range of lipophilic substitutions for siRNA delivery and BCRP down-regulation. Our results showed a significant increase in siRNA delivery as a function of lipid substitution for a range of lipids ranging from C8 to C18. The BCRP silencing was correlated to siRNA delivery efficiency of the polymers, and effectively lasted for â¼5days after a single treatment of siRNA. BCRP down-regulation sensitized the drug-resistant cells to cytotoxic effect of mitoxantrone by a â¼14-fold decrease in the IC(50) value, whose effect was evident even after 14days. This study demonstrated the possibility of functional siRNA delivery by lipid-modified low molecular weight PEI and highlighted the importance of the extent and nature of lipid substitution in effective siRNA delivery.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Regulação para Baixo , Proteínas de Neoplasias/genética , Polímeros/química , RNA Interferente Pequeno/administração & dosagem , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Linhagem Celular , Cães , Resistencia a Medicamentos Antineoplásicos , Estudos de Viabilidade , Inativação Gênica , Concentração Inibidora 50 , Lipídeos/química , Mitoxantrona/administração & dosagem , Mitoxantrona/farmacologia , Peso Molecular , Fatores de TempoRESUMO
PURPOSE. Updated information on the stability of compounded thioguanine oral suspensions prepared with currently available ingredients, as well as results of testing to determine if the addition of an antioxidant could extend shelf life by inhibiting formation of guanine, are presented. METHODS. Using triturated thioguanine tablets, three compounded suspensions were prepared: (1) a reference formulation containing methylcellulose and simple syrup, (2) an equivalent formulation using Ora-Plus and Ora-Sweet, and (3) an antioxidant-containing formulation prepared by adding ascorbic acid to the equivalent formulation. The compounded batches were stored at room temperature (19-23 °C). The chemical stability of the suspensions was evaluated immediately after compounding and at weekly intervals by a validated liquid chromatography-mass spectrometry (LCMS) assay method; physical stability was evaluated by regular visual checks and weekly pH testing. RESULTS. As demonstrated by serial LCMS testing, mean thioguanine levels in sampled batches of all three suspensions remained above accepted standards and mean guanine formation remained within acceptable limits for up to 63 days. The addition of ascorbic acid appeared to slow guanine formation but did not significantly extend the shelf life of the suspension. CONCLUSION. Compounded oral suspensions of thioguanine 20 mg/mL exhibited acceptable chemical and physical stability for up to nine weeks at 19-23 °C. The addition of ascorbic acid at a concentration of 0.1% to the suspension was not effective in consistently increasing the shelf life of the thioguanine suspensions.
Assuntos
Antimetabólitos Antineoplásicos/análise , Tioguanina/análise , Antioxidantes/farmacologia , Cromatografia Líquida , Composição de Medicamentos/métodos , Estabilidade de Medicamentos , Armazenamento de Medicamentos/métodos , Guanina/antagonistas & inibidores , Humanos , Suspensões/análiseRESUMO
The purpose of this study was to design an in vitro experiment that can assess the stability of polymeric micellar formulations of hydrophobic drugs such as cyclosporine A (CyA) in blood, and predict the in vivo performance of the examined delivery system. Poly(ethylene oxide)-block-poly(epsilon-caprolactone) (PEO-b-PCL) copolymers were assembled to polymeric nano-containers for the physical encapsulation of CyA by a co-solvent evaporation method using different loading conditions. CyA-loaded micelles were prepared and compared to commercially available intravenous formulation of CyA (Sandimmune) for in vitro release, protein binding, and pharmacokinetic parameters in Sprague-Dawley rats. The unbound fraction (fu) of CyA was determined using an erythrocyte vs. plasma and buffer partitioning technique. Different polymeric micellar formulations of CyA did not show any significant difference in CyA release when dialyzed against bovine serum albumin. The fu experiments, however, revealed a significant decrease in the fu of the loaded drug with an increase in the drug/polymer loading ratio, while the fu of all micellar formulations were significantly lower than Sandimmune. The pharmacokinetic study showed that fu of CyA in each formulation correlated with its in vivo performance determined by pharmacokinetic parameters: the lower fu of the formulation, translated to a higher area under the concentration versus time curve (AUC), and a lower clearance (CL) and volume of distribution (Vd). In conclusion, determination of the unbound fraction of encapsulated drug can be used to predict the in vivo stability of polymeric micellar nano-containers. PEO-b-PCL micelles containing higher CyA-loaded levels are shown to be more stable changing the pharmacokinetics of the encapsulated CyA to a higher extent.
Assuntos
Ciclosporina/química , Ciclosporina/farmacocinética , Lactonas/química , Lactonas/farmacocinética , Nanopartículas/química , Polietilenoglicóis/química , Polietilenoglicóis/farmacocinética , Animais , Ciclosporina/administração & dosagem , Ciclosporina/sangue , Estabilidade de Medicamentos , Eritrócitos/química , Eritrócitos/metabolismo , Lactonas/administração & dosagem , Micelas , Nanopartículas/administração & dosagem , Polietilenoglicóis/administração & dosagem , Ligação Proteica , Distribuição Aleatória , Ratos , Ratos Sprague-DawleyRESUMO
The purpose of this study was to assess the effect of hyperlipoproteinemia on the biodistribution of cyclosporine A (CyA), an extensively lipoprotein bound immunosuppressant, in a rat model and to determine the potential toxicological significance of this effect. Normolipidemic and hyperlipoproteinemic rats were given a single 5 mg/kg dose of CyA as intravenous bolus and at selected times postdose, tissues, blood, and plasma were harvested and assayed for CyA content. Hyperlipoproteinemia was induced by intraperitoneal injection of 1 g/kg poloxamer 407. Compared with normolipidemic animals, hyperlipoproteinemic rats had higher plasma, blood, kidney, and liver CyA concentrations. In contrast, in heart and spleen the concentrations were decreased in hyperlipoproteinemia. The nephrotoxic effect of CyA was also evaluated in normolipidemic and hyperlipoproteinemic rats after 7 days of dosing with 20 mg/kg/day. In both groups of animals, repeated doses of CyA were associated with equivalent decreases in creatinine and urea clearances compared with matching control and predose baseline measures. The concentrations of drug in kidney were equivalent at the conclusion of the study. However, despite these similarities there was microscopic evidence of more severe changes in the kidneys in the hyperlipoproteinemic rats, which also experienced a significant decrease in body weight compared with the normolipedemic animals. In conclusion, the distribution of CyA to kidneys was enhanced in poloxamer 407-induced hyperlipoproteinemic rats after single doses, and with repeated doses there was an apparent greater adverse effect on these animals compared with normolipidemic animals.