RESUMO
RNA therapeutics are of global interest because of their versatility in targeting a variety of intracellular and extracellular biomolecules. In that context, long double-stranded RNA (dsRNA) has been studied as an antitumor agent that activates the immune response. However, its performance is constrained by poor cancer selectivity and cell-penetration ability. Here, we designed and synthesized an oncolytic RNA hairpin pair (oHP) that was selectively cytotoxic toward cancer cells expressing abundant oncogenic microRNA-21 (miR-21). Although the structure of each hairpin RNA was thermodynamically metastable, catalytic miR-21 input triggered it to open to generate a long nicked dsRNA. We demonstrated that oHP functioned as a cytotoxic amplifier of information in the presence of miR-21 in various cancer cells and tumor-bearing mice. This work represents the first example of the use of short RNA molecules as build-up-type anticancer agents that are triggered by an oncogenic miRNA.
Assuntos
Antineoplásicos , MicroRNAs , Neoplasias , Animais , Camundongos , MicroRNAs/genética , RNA de Cadeia Dupla , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Antineoplásicos/química , Neoplasias/tratamento farmacológico , Neoplasias/genéticaRESUMO
Chemoresistance is a major cause of high mortality and poor survival in patients with ovarian cancer (OVCA). Understanding the mechanisms of chemoresistance is urgently required to develop effective therapeutic approaches to OVCA. Here, we show that expression of the long noncoding RNA, taurine upregulated gene 1 (TUG1), is markedly upregulated in samples from OVCA patients who developed resistance to primary platinum-based therapy. Depletion of TUG1 increased sensitivity to cisplatin in the OVCA cell lines, SKOV3 and KURAMOCHI. Combination therapy of cisplatin with antisense oligonucleotides targeting TUG1 coupled with a drug delivery system effectively relieved the tumor burden in xenograft mouse models. Mechanistically, TUG1 acts as a competing endogenous RNA by downregulating miR-4687-3p and miR-6088, both of which target DNA polymerase eta (POLH), an enzyme required for translesion DNA synthesis. Overexpression of POLH reversed the effect of TUG1 depletion on cisplatin-induced cytotoxicity. Our data suggest that TUG1 upregulation allows OVCA to tolerate DNA damage via upregulation of POLH; this provides a strong rationale for targeting TUG1 to overcome cisplatin resistance in OVCA.
Assuntos
Cisplatino , DNA Polimerase Dirigida por DNA , Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas , RNA Longo não Codificante , Animais , Feminino , Humanos , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Camundongos Nus , MicroRNAs/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , RNA Longo não Codificante/genética , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Currently, there is no effective treatment for glioblastoma multiforme (GBM), the most frequent and malignant type of brain tumor. The blood-brain (tumor) barrier (BB(T)B), which is composed of tightly connected endothelial cells and pericytes (with partial vasculature collapse), hampers nanomedicine accumulation in tumor tissues. We aimed to explore the effect of nanomedicine size on passive targeting of GBM. A series of size-tunable poly(ethylene glycol) (PEG)-grafted copolymers (gPEGs) were constructed with hydrodynamic diameters of 8-30 nm. Biodistribution studies using orthotopic brain tumor-bearing mice revealed that gPEG brain tumor accumulation was maximized at 10 nm with â¼14 doseâ¯%/g of tumor, which was 19 times higher than that in the normal brain region and 4.2 times higher than that of 30-nm gPEG. Notably, 10-nm gPEG exhibited substantially higher brain tumor accumulation than 11-nm linear PEG owing to the prolonged blood circulation property of gPEGs, which is derived from a densely PEG-packed structure. 10 nm gPEG exhibited deeper penetration into the brain tumor tissue than the larger gPEGs did (>10 nm). This study demonstrates, for the first time, the great potential of a nanomedicine downsizing strategy for passive GBM targeting.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Polietilenoglicóis , Glioblastoma/patologia , Glioblastoma/metabolismo , Animais , Polietilenoglicóis/química , Camundongos , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/metabolismo , Humanos , Barreira Hematoencefálica/metabolismo , Distribuição Tecidual , Linhagem Celular Tumoral , Tamanho da Partícula , Nanopartículas/química , Polímeros/químicaRESUMO
Various cationic polymers are used to deliver polyplex-mediated antisense oligonucleotides (ASOs). However, few studies have investigated the structural determinants of polyplex functionalities in polymers. This study focused on the polymer hydrophobicity. A series of amphiphilic polyaspartamide derivatives possessing various hydrophobic (R) moieties together with cationic diethylenetriamine (DET) moieties in the side chain (PAsp(DET/R)s) were synthesized to optimize the R moieties (or hydrophobicity) for locked nucleic acid (LNA) gapmer ASO delivery. The gene knockdown efficiencies of PAsp(DET/R) polyplexes were plotted against a hydrophobicity parameter, logD7.3, of PAsp(DET/R), revealing that the gene knockdown efficiency was substantially improved by PAsp(DET/R) with logD7.3 higher than -2.4. This was explained by the increased polyplex stability and improved cellular uptake of ASO payloads. After intratracheal administration, the polyplex samples with a higher logD7.3 than -2.4 induced a significantly higher gene knockdown in the lung tissue compared with counterparts with lower hydrophobicity and naked ASO. These results demonstrate that the hydrophobicity of PAsp(DET/R) is crucial for efficient ASO delivery in vitro and in vivo.
Assuntos
Oligonucleotídeos Antissenso , Polímeros , Polímeros/químicaRESUMO
Neointimal hyperplasia (NIH) after revascularization is a key unsolved clinical problem. Various studies have shown that attenuation of the acute inflammatory response on the vascular wall can prevent NIH. MicroRNA146a-5p (miR146a-5p) has been reported to show anti-inflammatory effects by inhibiting the NF-κB pathway, a well-known key player of inflammation of the vascular wall. Here, a nanomedicine, which can reach the vascular injury site, based on polymeric micelles was applied to deliver miR146a-5p in a rat carotid artery balloon injury model. In vitro studies using inflammation-induced vascular smooth muscle cell (VSMC) was performed. Results showed anti-inflammatory response as an inhibitor of the NF-κB pathway and VSMC migration, suppression of reactive oxygen species production, and proinflammatory cytokine gene expression in VSMCs. A single systemic administration of miR146a-5p attenuated NIH and vessel remodeling in a carotid artery balloon injury model in both male and female rats in vivo. MiR146a-5p reduced proinflammatory cytokine gene expression in injured arteries and monocyte/macrophage infiltration into the vascular wall. Therefore, miR146a-5p delivery to the injury site demonstrated therapeutic potential against NIH after revascularization.
Assuntos
Lesões das Artérias Carótidas , MicroRNAs , Animais , Anti-Inflamatórios/metabolismo , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Artérias , Lesões das Artérias Carótidas/metabolismo , Proliferação de Células , Citocinas/metabolismo , Feminino , Hiperplasia/metabolismo , Inflamação/metabolismo , Masculino , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , NF-kappa B/metabolismo , Nanomedicina , Neointima/tratamento farmacológico , Neointima/metabolismo , Neointima/prevenção & controle , RatosRESUMO
To stabilize small interfering RNA (siRNA) in the bloodstream for systemic RNAi therapeutics, we previously fabricated ultrasmall siRNA nanocarriers that were sub-20 nm in hydrodynamic diameter, named as unit polyion complexes (uPICs), using two-branched poly(ethylene glycol)-b-poly(l-lysine) (bPEG-PLys). The blood retention time of uPICs is dramatically increased in the presence of free bPEG-PLys, suggesting dynamic stabilization of uPICs by free bPEG-PLys based on their equilibrium. Herein, we examined how the degree of polymerization of PLys (DPPLys) affected the dynamic stability of uPICs in the bloodstream during prolonged circulation. We prepared a series of bPEG-PLys with DPPLys values of 10, 13, 20, 40, and 80 for the uPIC formation and siRNA with 40 negative charges. These bPEG-PLys were then evaluated in physicochemical characterization and pharmacokinetic analyses. Structural analyses revealed that the uPIC size and association numbers were mainly determined by the molecular weights of PEG and DPPLys, respectively. Under bPEG-PLys-rich conditions, the hydrodynamic diameters of uPICs were 15-20 nm, which were comparable to that of the bPEG block (i.e., â¼18 nm). Importantly, DPPLys significantly affected the association constant of bPEG-PLys to siRNA (Ka) and blood retention of free bPEG-PLys. A smaller DPPLys resulted in a lower Ka and a longer blood retention time of free bPEG-PLys. Thus, DPPLys can control the dynamic stability of uPICs, i.e., the balance between Ka and blood concentration of free bPEG-PLys. Ultimately, the bPEG-PLys with DPPLys values of 14 and 19 prolonged the blood circulation of siRNA-loaded uPICs with relatively small amounts of free bPEG-PLys. This study revealed that the uPIC formation between siRNA and bPEG-PLys can be controlled by their charges, which may be helpful for designing PIC-based delivery systems.
Assuntos
Lisina , Polietilenoglicóis , Cátions , Lisina/análogos & derivados , Polietilenoglicóis/química , RNA Interferente Pequeno/químicaRESUMO
RNA interference (RNAi) by small interfering RNAs (siRNAs) is a promising therapeutic approach. Because siRNA has limited intracellular access and is rapidly cleared in vivo, the success of RNAi depends on efficient delivery technologies. Particularly, polyion complexation between block catiomers and siRNA is a versatile approach for constructing effective carriers, such as unit polyion complexes (uPIC), core-shell polyion complex (PIC) micelles and vesicular siRNAsomes, by engineering the structure of block catiomers. In this regard, the flexibility of block catiomers could be an important parameter in the formation of PIC nanostructures with siRNA, though its effect remains unknown. Here, we studied the influence of block catiomer flexibility on the assembly of PIC structures with siRNA using a complementary polymeric system, i.e. poly(ethylene glycol)-poly(L-lysine) (PEG-PLL) and PEG-poly(glycidylbutylamine) (PEG-PGBA), which has a relatively more flexible polycation segment than PEG-PLL. Mixing PEG-PGBA with siRNA at molar ratios of primary amines in polymer to phosphates in the siRNA (N/P ratios) higher than 1.5 promoted the multimolecular association of uPICs, whereas PEG-PLL formed uPIC at all N/P ratios higher than 1. Moreover, uPICs from PEG-PGBA were more stable against counter polyanion exchange than uPICs from PEG-PLL, probably due to a favorable complexation process, as suggested by computational studies of siRNA/block catiomer binding. In in vitro experiments, PEG-PGBA uPICs promoted effective intracellular delivery of siRNA and efficient gene knockdown. Our results indicate the significance of polycation flexibility on assembling PIC structures with siRNA, and its potential for developing innovative delivery systems.
RESUMO
Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer compared with luminal or epidermal growth factor receptor 2 subtypes, thus effective therapeutic options for TNBC are yet to be developed. Nowadays, oncogenic long noncoding RNAs (lncRNAs) are applied to cancer management as a new class of therapeutic targets. We previously showed that thymopoietin antisense transcript 1 (TMPO-AS1) is a proliferation-associated lncRNA that contributes to hormone-dependent breast cancer progression by stabilizing estrogen receptor-α mRNA. We here showed that TMPO-AS1 is abundantly expressed in basal-like breast cancer subtype based on the transcriptomic data in The Cancer Genome Atlas as well as in TNBC cell lines and patient-derived cells. Small interfering RNA-based loss-of-function analyses showed that TMPO-AS1 knockdown substantially represses the proliferation and migration of TNBC cells. Expression microarray analysis showed that TMPO-AS1 alters gene signatures related to transforming growth factor-ß signaling in addition to proliferative E2F signaling pathways. TMPO-AS1-targeted siRNA treatment through engineered drug delivery systems using cancer-targeted polyion complex micelle or nanoball technology significantly impaired the in vivo growth of primary and metastatic TNBC xenograft tumors. Our findings suggest that TMPO-AS1 plays a key role in TNBC pathophysiology and could be a potential therapeutic target for TNBC.
Assuntos
Biomarcadores Tumorais , RNA Longo não Codificante/genética , Neoplasias de Mama Triplo Negativas/genética , Animais , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Biologia Computacional/métodos , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Terapia de Alvo Molecular , Interferência de RNA , RNA Interferente Pequeno/genética , Neoplasias de Mama Triplo Negativas/tratamento farmacológicoRESUMO
Whereas small siRNA nanocarriers with a size of 10-20 nm exert high tissue-permeability, they encounter the challenge of inefficient adsorption on the cell surface, resulting in poor cellular uptake of siRNA. To solve this dilemma, this study aims to control the hydrophobicity of a small siRNA nanocarrier, unimer polyion complex (uPIC), with a size of â¼10 nm. The uPICs are fabricated to consist of a single pair between siRNA and a smart triblock copolymer comprising hydrophilic poly(2-ethyl-2-oxazoline) (PEtOx), thermoswitchable poly(2-n-propyl-2-oxazoline) (PnPrOx), and cationic poly(l-lysine) (PLL). The PnPrOx segment is dehydrated at 37 °C (>lower critical solution temperature) to enhance the hydrophobicity of uPICs. The uPICs with a hydrophobic domain facilitates cellular uptake of the siRNA payload through stronger binding to the cell surface, compared with control uPICs without a PnPrOx segment, leading to a significantly enhanced gene silencing effect in cultured cancer cells.
Assuntos
Portadores de Fármacos/química , Interações Hidrofóbicas e Hidrofílicas , Nanoestruturas/química , Polímeros/química , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Temperatura , Transporte Biológico , Inativação Gênica , Células HeLa , Humanos , RNA Interferente Pequeno/genéticaRESUMO
For the simultaneous delivery of antisense oligonucleotides and their effector enzymes into cells, nanosized vesicular polyion complexes (PICs) were fabricated from oppositely charged polyion pairs of oligonucleotides and poly(ethylene glycol) (PEG)-b-polypeptides. First, the polyion component structures were carefully designed to facilitate a multimolecular (or secondary) association of unit PICs for noncovalent (or chemical cross-linking-free) stabilization of vesicular PICs. Chemically modified, single-stranded oligonucleotides (SSOs) dramatically stabilized the multimolecular associates under physiological conditions, compared to control SSOs without chemical modifications and duplex oligonucleotides. In addition, a high degree of guanidino groups in the polypeptide segment was also crucial for the high stability of multimolecular associates. Dynamic light scattering and transmission electron microscopy revealed the stabilized multimolecular associates to have a 100 nm sized vesicular architecture with a narrow size distribution. The loading number of SSOs per nanovesicle was determined to be â¼2500 using fluorescence correlation spectroscopic analyses with fluorescently labeled SSOs. Furthermore, the nanovesicle stably encapsulated ribonuclease H (RNase H) as an effector enzyme at â¼10 per nanovesicle through simple vortex-mixing with preformed nanovesicles. Ultimately, the RNase H-encapsulated nanovesicle efficiently delivered SSOs with RNase H into cultured cancer cells, thereby eliciting the significantly higher gene knockdown compared with empty nanovesicles (without RNase H) or a mixture of nanovesicles with RNase H without encapsulation. These results demonstrate the great potential of noncovalently stabilized nanovesicles for the codelivery of two varying bio-macromolecule payloads for ensuring their cooperative biological activity.
Assuntos
Oligonucleotídeos , Peptídeos , Técnicas de Silenciamento de Genes , Micelas , Oligonucleotídeos/genética , PolietilenoglicóisRESUMO
Polymeric micelles are demonstrating high potential as nanomedicines capable of controlling the distribution and function of loaded bioactive agents in the body, effectively overcoming biological barriers, and various formulations are engaged in intensive preclinical and clinical testing. This Review focuses on polymeric micelles assembled through multimolecular interactions between block copolymers and the loaded drugs, proteins, or nucleic acids as translationable nanomedicines. The aspects involved in the design of successful micellar carriers are described in detail on the basis of the type of polymer/payload interaction, as well as the interplay of micelles with the biological interface, emphasizing on the chemistry and engineering of the block copolymers. By shaping these features, polymeric micelles have been propitious for delivering a wide range of therapeutics through effective sensing of targets in the body and adjustment of their properties in response to particular stimuli, modulating the activity of the loaded drugs at the targeted sites, even at the subcellular level. Finally, the future perspectives and imminent challenges for polymeric micelles as nanomedicines are discussed, anticipating to spur further innovations.
Assuntos
Micelas , Nanomedicina , Portadores de Fármacos/química , Composição de Medicamentos , Humanos , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Preparações Farmacêuticas/química , Preparações Farmacêuticas/metabolismo , Polímeros/químicaRESUMO
To understand the role of RAS-signaling networks in the pathogenesis of renal cell carcisnoma, we clarified the relationship between miR-143 and RAS. The expression of miR-143 was extremely downregulated in tumor tissues from renal cell carcinoma patients compared with that in the adjacent normal tissues and Caki-1 cells. We developed a synthetic miR-143#12, and we found that the ectopic expression of it inhibited cell growth with autophagy in Caki-1 cells. Also, the expression level of c-Myc was markedly decreased, resulting in the perturbation of cancer-specific energy metabolism by negatively modulating the expression of GLUT1 and the PTBP1/PKMs axis. A partial metabolic shift from glycolysis to oxidative phosphorylation induced autophagy through increasing the intracellular level of reactive oxygen species (ROS). In an in vivo study, the potent anti-tumor activity of polyion complex (PIC)-loaded miR-143#12 (miR-143#12/PIC) was shown by systemic administration of it to Caki-1 cell-xenografted mice. Higher levels of miR-143 were found in both blood and tumor tissues after the systemic administration with miR-143#12/PIC compared to those with lipoplexes in the xenografted mice. These findings indicated that this synthetic miR-143#12 induced a marked growth inhibition by impairing K-RAS-signaling networks in vitro and in vivo.
Assuntos
Carcinoma de Células Renais/genética , Carcinoma de Células Renais/terapia , MicroRNAs/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Autofagia/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Transportador de Glucose Tipo 1/genética , Glicólise/genética , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Camundongos , MicroRNAs/farmacologia , Fosforilação Oxidativa/efeitos dos fármacos , Proteína de Ligação a Regiões Ricas em Polipirimidinas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Current antisense oligonucleotide (ASO) therapies for the treatment of central nervous system (CNS) disorders are performed through invasive administration, thereby placing a major burden on patients. To alleviate this burden, we herein report systemic ASO delivery to the brain by crossing the blood-brain barrier using glycemic control as an external trigger. Glucose-coated polymeric nanocarriers, which can be bound by glucose transporter-1 expressed on the brain capillary endothelial cells, are designed for stable encapsulation of ASOs, with a particle size of about 45â nm and an adequate glucose-ligand density. The optimized nanocarrier efficiently accumulates in the brain tissue 1â h after intravenous administration and exhibits significant knockdown of a target long non-coding RNA in various brain regions, including the cerebral cortex and hippocampus. These results demonstrate that the glucose-modified polymeric nanocarriers enable noninvasive ASO administration to the brain for the treatment of CNS disorders.
Assuntos
Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Glucose/química , Nanoestruturas/química , Oligonucleotídeos Antissenso/química , Polímeros/química , Animais , Linhagem Celular Tumoral , Portadores de Fármacos/química , Corantes Fluorescentes/química , Humanos , Camundongos , Oligonucleotídeos Antissenso/metabolismo , Tamanho da Partícula , RNA Longo não Codificante/antagonistas & inibidores , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismoRESUMO
Vesicular polyion complexes (PICs) were fabricated through self-assembly of rigid cylindrical molecules, small interfering RNAs (siRNAs), with flexible block catiomers of poly(ethylene glycol) (2 kDa) and cationic polyaspartamide derivative (70 units) bearing a 5-aminopentyl side chain. 100 nm-sized siRNA-assembled vesicular PICs, termed siRNAsomes, were fabricated in specific mixing ranges between siRNA and block catiomer. The siRNAsome membrane was revealed to consist of PIC units fulfilling a simple molar ratio (1:2 or 2:3) of block catiomer and siRNA. These ratios correspond to the minimal integer molar ratio to maximally compensate the charge imbalance of PIC, because the numbers of charges per block catiomer and siRNA are +70 and -40, respectively. Accordingly, the ζ-potentials of siRNAsomes prepared at 1:2 and 2:3 were negative and positive, respectively. Cross-section transmission electron microscopic observation clarified that the membrane thicknesses of 1:2 and 2:3 siRNAsomes were 11.0 and 17.2 nm, respectively. Considering that a calculated long-axial length of siRNA is 5.9 nm, these thickness values correspond to the membrane models of two (11.8 nm) and three (17.7 nm) tandemly aligned siRNAs associating with one and two block catiomers, respectively. For biological application, siRNAsomes were stabilized through membrane-cross-linking with glutaraldehyde. The positively charged and cross-linked siRNAsome facilitated siRNA internalization into cultured cancer cells, eliciting significant gene silencing with negligible cytotoxicity. The siRNAsome stably encapsulated dextran as a model cargo macromolecule in the cavity by simple vortex mixing. Confocal laser scanning microscopic observation displayed that both of the payloads were internalized together into cultured cells. These results demonstrate the potential of siRNAsomes as a versatile platform for codelivery of siRNA with other cargo macromolecules.
Assuntos
Polietilenoglicóis/química , Interferência de RNA , RNA Interferente Pequeno/química , Linhagem Celular Tumoral , Inativação Gênica , Humanos , Íons/síntese química , Íons/química , Substâncias Macromoleculares/química , Estrutura Molecular , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
Degradability of polycations under physiological conditions is an attractive feature for their use in biomedical applications, such as the delivery of nucleic acids. This study aims to design polycations with tunable nonenzymatic degradability. A series of cationic N-substituted polyaspartamides were prepared to possess primary amine via various lengths of alkyl spacers in side chains. The degradation rate of each polyaspartamide derivative was determined by size exclusion chromatography under different pH conditions. The N-substituted polyaspartamide containing a 2-aminoethyl moiety in the side chain (PAsp(AE)) showed considerable degradability under physiological conditions (pH 7.4, 37 °C). In contrast, the N-substituted polyaspartamides bearing a longer alkyl spacer in the side chain, i.e. the 3-aminopropyl (PAsp(AP)) and 4-aminobutyl moieties (PAsp(AB)), more strongly suppressed degradation. Further, a positive correlation was observed between the degradation rate of N-substituted polyaspartamides and a deprotonation degree of primary amines in their side chains. Therefore, we conclude that the deprotonated primary amine in the side chain of N-substituted polyaspartamides can induce the degradation of the main chain through the activation of amide nitrogen in the side chain. When N-substituted polyaspartamides were utilized as a messenger RNA (mRNA) delivery vehicle via formation of polyion complexes (PICs), degradable PAsp(AE) elicited significantly higher mRNA expression efficiency in cultured cells compared to PAsp(AP) and PAsp(AB). The higher efficiency of PAsp(AE) might be due to the facilitated destabilization of PICs within the cells, directed toward mRNA release. Additionally, degradation of PAsp(AE) considerably reduced its cytotoxicity. Thus, our study highlights a useful design of well-defined cationic poly(amino acid)s with tunable nonenzymatic degradability.
RESUMO
Small interfering RNA (siRNA) drugs have been considered to treat various diseases in major organs. However, siRNA drugs developed for cancer therapy are hindered from proceeding to the clinic. To date, various delivery formulations have been developed from cationic lipids, polymers, and/or inorganic nanoparticles for systemic siRNA delivery to solid tumors. Most of these delivery vehicles do not generate small particle sizes and pharmacokinetics required for accumulation in target cancer cells compared with clinically tested anticancer drug-loaded polymeric micelles. This review describes the significance of small, long-circulating vehicles for efficient delivery of siRNA to cancer tissues via the enhanced permeability and retention (EPR) effect. We summarize recent biological evidence that supports the size effect of delivery vehicles in tumor microenvironments and introduce promising strategies for the construction of small vehicles with sizes of 10-50 nm. We then discuss the feasibility of these delivery vehicles with respect to translation to clinical trials.
Assuntos
Técnicas de Transferência de Genes , Neoplasias/terapia , Terapêutica com RNAi/métodos , Animais , Marcação de Genes/métodos , Humanos , Nanoconjugados/químicaRESUMO
Antibody fragment (Fab')-installed polyion complex (PIC) micelles were constructed to improve targetability of small interfering RNA (siRNA) delivery to pancreatic cancer cells. To this end, we synthesized a block copolymer of azide-functionalized poly(ethylene glycol) and poly(l-lysine) and prepared PIC micelles with siRNA. Then, a dibenzylcyclooctyne (DBCO)-modified antihuman tissue factor (TF) Fab' was conjugated to azido groups on the micellar surface. A fluorescence correlation spectroscopic analysis revealed that 1, 2, or 3 molecule(s) of Fab'(s) were installed onto one micellar nanoparticle according to the feeding ratio of Fab' (or DBCO) to micelle (or azide). The resulting micelles exhibited â¼40 nm in hydrodynamic diameter, similar to that of the parent micelles before Fab' conjugation. Flow cytometric analysis showed that three molecules of Fab'-installed PIC micelles (3(Fab')-micelles) had the highest binding affinity to cultured pancreatic cancer BxPC3 cells, which are known to overexpress TF on their surface. The 3(Fab')-micelles also exhibited the most efficient gene silencing activity against polo-like kinase 1 mRNA in the cultured cancer cells. Furthermore, the 3(Fab')-micelles exhibited high penetrability and the highest cellular internalization amounts in BxPC3 spheroids compared with one or two molecule(s) of Fab'-installed PIC micelles. These results demonstrate the potential of anti-TF Fab'-installed PIC micelles for active targeting of stroma-rich pancreatic tumors.
Assuntos
Anticorpos Antineoplásicos , Proteínas de Ciclo Celular/antagonistas & inibidores , Sistemas de Liberação de Medicamentos , Inativação Gênica , Fragmentos Fab das Imunoglobulinas , Micelas , Neoplasias Pancreáticas/tratamento farmacológico , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/antagonistas & inibidores , RNA Interferente Pequeno , Tromboplastina/antagonistas & inibidores , Anticorpos Antineoplásicos/química , Anticorpos Antineoplásicos/farmacologia , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/farmacologia , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Polilisina/química , Polilisina/farmacologia , Proteínas Serina-Treonina Quinases/biossíntese , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Tromboplastina/metabolismo , Quinase 1 Polo-LikeRESUMO
Pancreatic cancer is one of the most lethal types of cancer, with aggressive properties characterized by metastasis, recurrence and drug resistance. Cancer stem cells are considered to be responsible for these properties. PRDM14, a transcriptional regulator that maintains pluripotency in embryonic stem cells, is overexpressed in some cancers. Here, we assessed PRDM14 expression and the effects of PRDM14 knockdown on cancer stem-like phenotypes in pancreatic cancer. We observed that PRDM14 protein was overexpressed in pancreatic cancer tissues compared with normal pancreatic tissues. Using lentiviral shRNA-transduced pancreatic cancer cells, we found that PRDM14 knockdown decreased sphere formation, number of side population and cell surface marker-positive cells and subcutaneous xenograft tumors and liver metastasis in mice. This was accompanied by upregulation of some microRNAs (miRNAs), including miR-125a-3p. miR-125a-3p, a tumor suppressor that is down-regulated in pancreatic cancer, has been suggested to regulate the expression of the Src-family kinase, Fyn. In PRDM14-knockdown cells, Fyn was expressed at lower levels and downstream proteins were less activated. These changes were considered to cause suppression of the above cancer phenotypes. In addition, we used small interfering RNA (siRNA)-based therapy targeting PRDM14 in a mouse model of liver metastasis induced using MIA-PaCa2 cells, and this treatment significantly decreased metastasis and in vitro migration. Taken together, these results suggest that targeting the overexpression of PRDM14 suppresses cancer stem-like phenotypes, including liver metastasis, via miRNA regulation and siRNA-based therapy targeting it shows promise as a treatment for patients with pancreatic cancer.
Assuntos
Neoplasias Hepáticas/prevenção & controle , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/prevenção & controle , Fatores de Transcrição/antagonistas & inibidores , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/prevenção & controle , Proteínas de Ligação a DNA , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs , Recidiva Local de Neoplasia/genética , Estadiamento de Neoplasias , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , RNA Interferente Pequeno , Proteínas de Ligação a RNA , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Achieving precise control over the morphology and function of polymeric nanostructures during self-assembly remains a challenge in materials as well as biomedical science, especially when independent control over particle properties is desired. Herein, we report on nanostructures derived from amphiphilic block copolypept(o)ides by secondary-structure-directed self-assembly, presenting a strategy to adjust core polarity and function separately from particle preparation in a bioreversible manner. The peptide-inherent process of secondary-structure formation allows for the synthesis of spherical and worm-like core-cross-linked architectures from the same block copolymer, introducing a simple yet powerful approach to versatile peptide-based core-shell nanostructures.
RESUMO
Polyion complexes (PICs) of mRNA with synthetic polyamines are receiving increasing attention as mRNA delivery vehicles, and the search for polyamine structure maximizing the translational efficiency of complexed mRNA becomes a critical research topic. Herein, we discovered that fine-tuning of the protonation status of synthetic polyamines can regulate mRNA translation through the preservative binding of eukaryotic initiation factor 4E to m(7)GpppN (cap structure) on the 5' end of mRNA. A series of polyamines with varied numbers of aminoethylene repeats in their side chains were prepared by an aminolysis reaction of poly(ß-benzyl-l-aspartate) and paired with mRNA to form PICs. PICs formed from polyamines with higher numbers of aminoethylene repeats preserved the original translational efficiency to naked mRNA, whereas the efficiency significantly dropped by decreasing the number of aminoethylene repeats in the polyamines. Immunoprecipitation assays using anti-eIF4E antibodies revealed that the binding affinity of eIF4E to the cap structure of mRNA in the PIC was sensitive to the number of charged aminoethylene repeats in the polyamine side chain and was strongly correlated with their translational efficiency. These results indicate that the fine-tuning of the polyamine structure plays a critical role in maximizing the translational efficiency of mRNA in the PICs having potential utility as mRNA delivery vehicles.