RESUMEN
Glioblastoma multiforme (GBM) is an aggressive tumor with no curative treatment. The tumor recurrence after resection often requires chemotherapy or radiation to delay the infiltration of tumor remnants. Intracerebral chemotherapies are preferentially being used to prevent tumor regrowth, but treatments remain unsuccessful because of the poor drug distribution in the brain. In this study, we investigated the therapeutic efficacy of cancer-targeting arginyl-glycyl-aspartic tripeptide (RGD) conjugated paclitaxel (PTX)-loaded nanoparticles (NPs) against GBM by nose-to-brain delivery. Our results demonstrated that RGD-modified PTX-loaded NPs showed cancer-specific delivery and enhanced anticancer effects in vivo. The intranasal (IN) inoculation of RGD-PTX-loaded NPs effectively controls the tumor burden (75 ± 12% reduction) by inducing apoptosis and/or inhibiting cancer cell proliferation without affecting the G0 stage of normal brain cells. Our data provide therapeutic evidence supporting the use of intranasally delivered cancer-targeted PTX-loaded NPs for GBM therapy.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Encéfalo/efectos de los fármacos , Glioblastoma/tratamiento farmacológico , Nanopartículas/química , Paclitaxel/farmacología , Péptidos/química , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Humanos , Masculino , Nariz , Paclitaxel/química , Ratas , Ratas Sprague-Dawley , Carga Tumoral/efectos de los fármacosRESUMEN
Effective delivery of DNA or siRNA into primary cells demands an efficient delivery system. However, the significant differences in physical and molecular characteristics of the two molecules generally necessitate distinct delivery systems or considerable differences in carrier formulation protocols for effective transfection. Arginine-grafted bioreducible poly (disulfide amine) (ABP) is a redox-sensitive, bioreducible, positively charged polymer which complexes with siRNA and DNA via charge interactions to form nanoplexes. ABP effectively mediates cytoplasmic delivery of both DNA and siRNA into multiple cell types, including primary cells like myoblast, human umbilical vein endothelial cells (HUVECs), and primary rat aorta vascular smooth muscle cells (SMCs) eliciting functional activity. In this chapter, we provide the detailed protocols for the synthesis of ABP as well as transfection of both siRNA and DNA using ABP.
Asunto(s)
Arginina/química , Polímeros/química , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , ADN/genética , Técnicas de Transferencia de Gen , Humanos , Peso Molecular , Resonancia Magnética Nuclear Biomolecular , Polímeros/toxicidad , ARN Interferente Pequeño/genética , Ratas , Transfección/métodosRESUMEN
RNAi-based gene therapy for cancer treatment has not shown significant clinical impact due to poor siRNA delivery to the target site. Here, we design a nonviral siRNA gene carrier using a combination of an arginine-grafted bioreducible polymer (ABP), microbubbles (MB), and ultrasound (US), for targeting vascular endothelial growth factor (VEGF) in a human ovarian cancer cell line. Newly designed MBs with a perfluorocrownether gas core show higher stability compared to controls. Further, MBs in combination with polyplexes show a significant higher loading capacity compared to naked siRNA. Lastly, only siRNA-ABP-MB (SAM) complexes in combination with US show significant VEGF knock down in A2780 human ovarian cancer cell line compared to naked siRNA when incubated for a short time after sonication treatment.
Asunto(s)
ARN Interferente Pequeño/administración & dosificación , Arginina/química , Línea Celular Tumoral , Portadores de Fármacos/química , Estabilidad de Medicamentos , Femenino , Técnicas de Silenciamiento del Gen , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Humanos , Microburbujas , Neoplasias Ováricas/genética , Neoplasias Ováricas/terapia , Polímeros/química , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Ultrasonido , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
The cardiomyocyte apoptosis plays a critical role in the development of myocardial injury after ischemia and reperfusion. Thus, alteration of the major apoptosis-regulatory factors during myocardial ischemia-reperfusion is expected to have favorable cardioprotective effects. Herein, we report ischemic-reperfused myocardial infarction (MI) repair with siRNA against Src homology region 2 domain-containing tyrosine phosphatase-1 (SHP-1), which is known as a key factor involved in regulating the progress of apoptosis in many cell types. A low molecular weight polyethyleneimine modified with deoxycholic acid (PEI1.8-DA)-based delivery strategy was suggested for the cardiac application of SHP-1 siRNA to overcome the poor gene delivery efficiency to myocardium due to the highly charged structures of the compact cardiac muscles. The PEI1.8-DA conjugates formed stable nanocomplexes with SHP-1 siRNA via electrostatic and hydrophobic interactions. The PEI1.8-DA/SHP-1 siRNA polyplexes effectively silenced SHP-1 gene expression in cardiomyocytes, leading to a significant inhibition of cardiomyocyte apoptosis under hypoxia. In comparison to conventional gene carriers, relatively large amounts of siRNA molecules remained after treatment with the PEI1.8-DA/SHP-1 siRNA polyplexes. Cardiac administration of the PEI1.8-DA/SHP-1 siRNA polyplexes resulted in substantial improvement in SHP-1 gene silencing, which can be explained by the enhancement of cardiac delivery efficiency of the PEI1.8-DA conjugates. In addition, in vivo treatment with the PEI1.8-DA/SHP-1 siRNA polyplexes induced a highly significant reduction in myocardial apoptosis and infarct size in rat MI models. These results demonstrate that the PEI1.8-DA/SHP-1 siRNA polyplex formulation is a useful system for efficient gene delivery into the compact myocardium that provides a fundamental advantage in treating ischemic-reperfused MI.
Asunto(s)
Terapia Genética , Infarto del Miocardio/terapia , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , ARN Interferente Pequeño/administración & dosificación , Daño por Reperfusión/terapia , Animales , Apoptosis , Ácido Desoxicólico/administración & dosificación , Ácido Desoxicólico/química , Silenciador del Gen , Masculino , Infarto del Miocardio/genética , Miocitos Cardíacos/metabolismo , Polietileneimina/administración & dosificación , Polietileneimina/química , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/genéticaRESUMEN
We developed a paclitaxel-conjugated polymeric micelle, ABP-PEG3.5k-Paclitaxel (APP) consisting of poly (ethylene glycol) (PEG) and arginine-grafted poly (cystaminebisacrylamide-diaminohexane) (ABP) for the co-delivery of gene and drug. The APP polymer self-assembled into cationic polymeric micelles with a critical micelle concentration (CMC) value of approximately 0.062 mg/mL, which was determined from measurements of the UV absorption of pyrene. The micelles have an average size of about 3 nm and a zeta potential of about +14 mV. Due to the positive surface charge, APP micelles formed polyplexes with plasmid DNA approximately 200 nm in diameter. The luciferase gene and mouse interleukin-12 (IL-12) gene was used to monitor gene delivery potency. APP polyplexes showed increased gene delivery efficiency and cellular uptake with higher anticancer potency than paclitaxel alone. These results demonstrate that an APP micelle-based delivery system is well suitable for the co-delivery of gene and drug.
Asunto(s)
Antineoplásicos Fitogénicos/administración & dosificación , Arginina/química , ADN/administración & dosificación , Micelas , Paclitaxel/administración & dosificación , Poliaminas/química , Polietilenglicoles/química , Animales , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , ADN/genética , Femenino , Glutatión/antagonistas & inhibidores , Glutatión/metabolismo , Humanos , Interleucina-12/genética , Ratones , Paclitaxel/química , Paclitaxel/farmacología , Tamaño de la Partícula , TransfecciónRESUMEN
Arginine-grafted bio-reducible poly(disulfide amine) (ABP) was incorporated into the poly(amido amine) (PAMAM) dendrimer, creating a high molecular weight bio-reducible polymer, PAM-ABP, to overcome the limitations of the low molecular weight ABP. The newly synthesized PAM-ABP was studied to determine its efficacy as a gene delivery carrier. The PAM-ABP demonstrated superior condensing ability for plasmid DNA through the formation of compact nanosized polyplexes. These compact polyplexes enhanced cellular uptake and were less susceptible to reducing agents, resulting in greater transfection efficiency compared to ABP alone. Based on these results, this newly developed PAM-ABP polyplex is a promising delivery system for clinical gene therapy.
Asunto(s)
Arginina/administración & dosificación , Dendrímeros/administración & dosificación , Poliaminas/administración & dosificación , Transfección/métodos , Animales , Arginina/química , Benzoxazoles/administración & dosificación , Benzoxazoles/química , Butionina Sulfoximina , Línea Celular , Supervivencia Celular , ADN/administración & dosificación , ADN/química , Dendrímeros/química , Proteínas Fluorescentes Verdes/administración & dosificación , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Células HeLa , Humanos , Ratones , Células 3T3 NIH , Plásmidos , Poliaminas/química , Compuestos de Quinolinio/administración & dosificación , Compuestos de Quinolinio/químicaRESUMEN
Erythropoiesis-stimulating agents are widely used to treat anemia for chronic kidney disease (CKD) and cancer, however, several clinical limitations impede their effectiveness. Nonviral gene therapy systems are a novel solution to these problems as they provide stable and low immunogenic protein expression levels. Here, we show the application of an arginine-grafted bioreducible poly(disulfide amine) (ABP) polymer gene delivery system as a platform for in vivo transfer of human erythropoietin plasmid DNA (phEPO) to produce long-term, therapeutic erythropoiesis. A single systemic injection of phEPO/ABP polyplex led to higher hematocrit levels over a 60-day period accompanied with reticulocytosis and high hEPO protein expression. In addition, we found that the distinct temporal and spatial distribution of phEPO/ABP polyplexes contributed to increased erythropoietic effects compared to those of traditional EPO therapies. Overall, our study suggests that ABP polymer-based gene therapy provides a promising clinical strategy to reach effective therapeutic levels of hEPO gene.
Asunto(s)
Anemia/terapia , Arginina , Eritropoyesis , Eritropoyetina/genética , Terapia Genética , Poliaminas , Animales , ADN/genética , Eritrocitos/metabolismo , Eritropoyetina/biosíntesis , Técnicas de Transferencia de Gen , Vectores Genéticos , Hematócrito , Humanos , Riñón/metabolismo , Masculino , Nanopartículas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Distribución Aleatoria , Ratas , Reticulocitosis , TransfecciónRESUMEN
To deliver siRNA specifically to cardiomyocytes with a high transfection efficiency, primary cardiomyocyte-targeting (PCM) and/or cell-penetrating (Tat) peptides were incorporated into the siRNA. With the addition of plasmid DNA, these peptide-conjugated siRNAs were able to form compact and stable nanosized polyplex particles with bioreducible poly(CBA-DAH). The peptide-modified siRNA polyplexes enhanced the cellular uptake and the gene-silencing capacity of the siRNA in cardiomyocytes without significant immunogenicity or cytotoxicity. These findings demonstrate that the cell-targeting peptide and/or cell-penetrating peptide conjugation of siRNA may be a potentially important strategy for cell-specific gene therapy in gene-mediated disease states.
Asunto(s)
Péptidos de Penetración Celular/química , Silenciador del Gen , Miocitos Cardíacos/metabolismo , Polímeros/administración & dosificación , Polímeros/química , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/química , Transfección/métodos , Línea Celular , Terapia Genética , Humanos , Microscopía Electrónica de RastreoRESUMEN
OBJECTIVES: Paclitaxel coating of hemodialysis grafts is effective in suppressing neointimal hyperplasia in the graft and vascular anastomosis sites. However, paclitaxel can have unwanted effects on the surrounding tissues. To reduce such problems, we developed a method to coat the drug only on the luminal surface of the graft, with little loading on the outer surface. METHODS: A peristaltic pump and a double-solvent (water and acetone) system were used to achieve an inner coating of paclitaxel. At the ratio of 90% acetone, paclitaxel was homogeneously coated only on the luminal surface of the graft without changing the physical properties. To determine its effect, grafts were implanted between the common carotid artery and the external jugular vein in pigs using uncoated control grafts (n = 6) and low-dose (n = 6, 0.22 µg/mm(2)) and high-dose (n = 6, 0.69 µg/mm(2)) paclitaxel inner-coated grafts. Cross-sections of graft-venous anastomoses were analyzed histomorphometrically 6 weeks after placement to measure the patency rate, percentage of luminal stenosis, and neointimal area. RESULTS: No signs of infection or bacterial contamination were observed in the paclitaxel inner-coated groups. Only one of the six control grafts was patent, but all of the paclitaxel-coated grafts were patent, with little neointima. The mean ± standard error values of percentage luminal stenosis were 75.7% ± 12.7% (control), 17.5% ± 3.1% (low dose), and 19.7% ± 3.0% (high dose). The values for the neointimal area (in mm(2)) were 8.77 ± 1.66 (control), 3.53 ± 0.73 (lose dose), and 4.24 ± 0.99 (high dose). Compared with the control group, paclitaxel inner-coated vascular grafts significantly suppressed neointimal hyperplasia (low dose, P = .001; high dose, P = .002). Myofibroblast proliferation and migration into the graft interstices confirmed the firm attachment of the implanted graft to the surrounding tissue. CONCLUSIONS: Paclitaxel coating on the inner luminal surface of vascular grafts was effective in suppressing neointimal hyperplasia, with little inhibition of myofibroblast infiltration within the graft wall.
Asunto(s)
Implantación de Prótesis Vascular/instrumentación , Prótesis Vascular , Fármacos Cardiovasculares/administración & dosificación , Materiales Biocompatibles Revestidos , Portadores de Fármacos , Oclusión de Injerto Vascular/prevención & control , Paclitaxel/administración & dosificación , Diálisis Renal , Túnica Íntima/efectos de los fármacos , Animales , Implantación de Prótesis Vascular/efectos adversos , Fármacos Cardiovasculares/química , Arteria Carótida Común/efectos de los fármacos , Arteria Carótida Común/patología , Arteria Carótida Común/cirugía , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Femenino , Oclusión de Injerto Vascular/etiología , Oclusión de Injerto Vascular/patología , Hiperplasia , Venas Yugulares/efectos de los fármacos , Venas Yugulares/patología , Venas Yugulares/cirugía , Miofibroblastos/efectos de los fármacos , Miofibroblastos/patología , Paclitaxel/química , Diseño de Prótesis , Solubilidad , Porcinos , Factores de Tiempo , Túnica Íntima/patología , Túnica Íntima/cirugía , Grado de Desobstrucción Vascular/efectos de los fármacosRESUMEN
Erythropoietin (EPO) plays a key regulatory role in the formation of new red blood cells (RBCs). Erythropoietin may also have a role as a therapeutic agent to counteract ischemic injury in neural, cardiac and endothelial cells. One of the limitations preventing the therapeutic application of EPO is its short half-life. The goal of this study was to develop a gene delivery system for the prolonged and controlled release of EPO. The arginine grafted bioreducible polymer (ABP) and its PEGylated version, ABP-PEG10, were utilized to study the expression efficiency and therapeutic effectiveness of this erythropoietin gene delivery system in vitro. Poly(ethylene glycol) (PEG) modification of the ABP was employed to inhibit the particle aggregation resulting from the interactions between cationic polyplexes and the negatively charged proteins typically present in serum. Both the ABP and the ABP-PEG10 carriers demonstrated efficient transfection and long-term production of EPO in a variety of cell types. The expressed EPO protein stimulated hematopoietic progenitor cells to form significant numbers of cell colonies in vitro. These data confirm that this EPO gene delivery system using a bioreducible polymeric carrier, either ABP or ABP-PEG 10, merits further testing as a potential therapeutic modality for a variety of clinically important disease states.
Asunto(s)
Arginina/administración & dosificación , ADN/administración & dosificación , Portadores de Fármacos/administración & dosificación , Eritropoyetina/genética , Nanopartículas/administración & dosificación , Polímeros/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Arginina/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , ADN/química , Portadores de Fármacos/química , Eritrocitos/efectos de los fármacos , Eritrocitos/patología , Hemólisis/efectos de los fármacos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Nanopartículas/química , Polímeros/química , Ratas , Ratas Sprague-Dawley , Transfección/métodosRESUMEN
As an effort to overcome limits of adenovirus (Ad) as a systemic delivery vector for cancer therapy, we developed a novel system using oncolytic Ad plasmid DNA with two bioreducible polymers: arginine-grafted bioreducible poly(disulfide amine)polymer (ABP) and PEG5k-conjugated ABP (ABP5k) in expectation of oncolytic effect caused by progeny viral production followed by replication. The linearized Ad DNAs for active viral replication polyplexed with each polymer were able to replicate only in human cancer cells and produce progeny viruses. The non-immunogenic polymers delivering the DNAs markedly elicited to evade the innate and adaptive immune response. The biodistribution ratio of the polyplexes administered systemically was approximately 99% decreased in liver when compared with naked Ad. Moreover, tumor-to-liver ratio of the Ad DNA delivered by ABP or ABP5k was significantly elevated at 229- or 419-fold greater than that of naked Ad, respectively. The ABP5k improved the chance of the DNA to localize within tumor versus liver with 1.8-fold increased ratio. In conclusion, the innovative and simple system for delivering oncolytic Ad plasmid DNA with the bioreducible polymers, skipping time-consuming steps such as generation and characterization of oncolytic Ad vectors, can be utilized as an alternative approach for cancer therapy.
Asunto(s)
Adenoviridae/genética , Arginina/administración & dosificación , ADN Viral/administración & dosificación , Virus Oncolíticos/genética , Poliaminas/administración & dosificación , Polietilenglicoles/administración & dosificación , Animales , Arginina/farmacocinética , Línea Celular Tumoral , ADN Viral/farmacocinética , Femenino , Vectores Genéticos/administración & dosificación , Células HEK293 , Humanos , Interleucina-6/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Viroterapia Oncolítica , Plásmidos , Poliaminas/farmacocinética , Polietilenglicoles/farmacocinética , Distribución TisularRESUMEN
Small interfering RNA (siRNA) represent an interesting class of developmental nucleic acid-based therapeutics. Cationic carriers for deoxyribonucleic acids (DNA) are potential vehicles for siRNA delivery. However, in contrast to supercoiled plasmid DNA, the physical properties of siRNA molecules induces the formation of larger, loosely-packed complexes with most polycationic carriers, and consequently, poor target silencing. Here, we investigate a gene delivery agent, arginine-grafted bioreducible poly (disulfide amine) polymer (ABP) for siRNA delivery as it contains arginine residues with siRNA binding properties. ABP combines the attributes of polycations and poly disulfide-amines namely- excellent cell-penetrability and rapid release after disulphide bond reduction in the intracellular compartment. ABP bound siRNA, assembled into stable 150 nm sized nanoparticles and efficiently released complexed siRNA upon cellular entry. We investigated the utility of ABP in a combinatorial RNAi strategy for solid cancer therapy. Systemic administration of ABP-siRNA resulted in a preferential and enhanced accumulation of carrier-siRNA complexes in the tumor tissue. Two administrations of the formulation with a siRNA cocktail targeting Bcl-2, VEGF and Myc at 0.3 mg total siRNA/kg body weight could effectively regress advanced stage tumors. Our results establish the promise of ABP as a common systemic delivery platform for both siRNA and DNA therapeutics.
Asunto(s)
Arginina/química , Materiales Biocompatibles/química , Técnicas de Transferencia de Gen , Polímeros/química , ARN Interferente Pequeño/administración & dosificación , Animales , Línea Celular Tumoral , Proliferación Celular , Fenómenos Químicos , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Melanoma/patología , Melanoma/terapia , Ratones , ARN Interferente Pequeño/metabolismoRESUMEN
Systemic administration of adenovirus (Ad) vectors is complicated by host immune responses and viral accumulation in the liver, resulting in a short circulatory virus half-life, low efficacy, and host side effects. Ad surface modification is thus required to enhance safety and therapeutic efficacy. An arginine-grafted bioreducible polymer (ABP) was chemically conjugated to the Ad surface, generating Ad-ΔE1/GFP-ABP. A hepatocellular carcinoma [HCC]-selective oncolytic Ad complex, YKL-1001-ABP, was also generated. Transduction efficiency of Ad-ΔE1/GFP-ABP was enhanced compared to naked Ad-ΔE1/GFP. YKL-1001-ABP elicited an enhanced and specific killing effect in liver cancer cells (Huh7 and HepG2) expressing α-fetoprotein (AFP). Compared with naked Ad, systemic administration of ABP-conjugated Ad resulted in reduced liver toxicity and interleukin (IL)-6 production in vitro and in vivo. Ad-ΔE1/GFP-ABP was more resistant to the neutralizing effects of human serum compared to naked Ad-ΔE1/GFP. ABP conjugation extended blood circulation time 45-fold and reduced anti-Ad Ab neutralization. Moreover, systemic administration of YKL-1001-ABP markedly suppressed growth of Huh7 hepatocellular carcinoma. These results demonstrate that chemical conjugation of ABP to the Ad surface improves safety and efficacy, indicating that ABP-conjugated Ad is a potentially useful cancer therapeutic agent to target cancer via systemic administration.
Asunto(s)
Adenoviridae/fisiología , Materiales Biocompatibles/farmacología , Carcinoma Hepatocelular/terapia , Neoplasias Hepáticas/terapia , Virus Oncolíticos/efectos de los fármacos , Polímeros/farmacología , Inmunidad Adaptativa/efectos de los fármacos , Adenoviridae/efectos de los fármacos , Animales , Arginina/administración & dosificación , Arginina/farmacocinética , Arginina/farmacología , Materiales Biocompatibles/administración & dosificación , Materiales Biocompatibles/farmacocinética , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Ditiotreitol/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Evasión Inmune/efectos de los fármacos , Inmunidad Innata/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/patología , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos BALB C , Tamaño de la Partícula , Polímeros/administración & dosificación , Polímeros/farmacocinética , Electricidad Estática , Transducción Genética , Resultado del TratamientoRESUMEN
Even though oncolytic adenovirus (Ad) has been highlighted in the field of cancer gene therapy, transductional targeting and immune privilege still remain difficult challenges. The recent reports have noted the increasing tendency of adenoviral surface shielding with polymer to overcome the limits of its practical application. We previously reported the potential of the biodegradable polymer, poly(CBA-DAH) (CD) as a promising candidate for efficient gene delivery. To endow the selective-targeting moiety of tumor vasculature to CD, cRGDfC well-known as a ligand for cell-surface integrins on tumor endothelium was conjugated to CD using hetero-bifunctional cross-linker SM (PEG)(n). The cytopathic effects of oncolytic Ad coated with the polymers were much more enhanced dose-dependently when compared with that of naked Ad in cancer cells selectively. Above all, the most potent oncolytic effect was assessed with the treatment of Ad/CD-PEG(500)-RGD in all cancer cells. The enhanced cytopathic effect of Ad/RGD-conjugated polymer was specifically inhibited by blocking antibodies to integrins, but not by blocking antibody to CAR. HT1080 cells treated with Ad/CD-PEG(500)-RGD showed strong induction of apoptosis and suppression of IL-8 and VEGF expression as well. These results suggest that RGD-conjugated bioreducible polymer might be used to deliver oncolytic Ad safely and efficiently for tumor therapy.
Asunto(s)
Adenoviridae/metabolismo , Interleucina-8/genética , Oligopéptidos/metabolismo , Virus Oncolíticos/metabolismo , Polímeros/metabolismo , ARN Interferente Pequeño/metabolismo , Adenoviridae/genética , Apoptosis , Línea Celular Tumoral , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Integrinas/genética , Integrinas/metabolismo , Interleucina-8/metabolismo , Ensayo de Materiales , Estructura Molecular , Neoplasias/genética , Neoplasias/terapia , Oligopéptidos/genética , Virus Oncolíticos/genética , Tamaño de la Partícula , Polímeros/química , ARN Interferente Pequeño/genética , Receptores Virales/genética , Receptores Virales/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
The primary cardiomyocyte-specific peptide (PCM) and the cell-penetrating peptide (CPP), HIV-Tat (49-57), were incorporated into the polymer, cystamine bisacrylamide-diaminohexane (CBA-DAH), to increase the delivery of RNAi to target cells, specifically cardiomyocytes. Interestingly, the impact of PCM and Tat conjugation on cellular uptake and transfection efficiency was greater in H9C2 rat cardiomyocytes than in NIH 3T3 cells. We examined the potential for siRNA targeting SHP-1 or Fas to inhibit the apoptosis of cardiomyocytes under hypoxic conditions using PCM and Tat-modified poly(CBA-DAH), (PCM-CD-Tat). To evaluate for efficacy in inhibiting apoptosis, either Fas siRNA/polymer or SHP-1 siRNA/polymer were transfected into cardiomyocytes treated under hypoxic and serum-deprived conditions. After incubation under hypoxic conditions, treatment with either the SHP-1 siRNA complex or the Fas siRNA complex resulted in an increase in cell viability and a reduction in LDH-cytotoxicity. The cells transfected with either of the siRNA polyplexes had a lower incidence of apoptosis as demonstrated by Annexin V-FITC/PI staining. Both the SHP-1 siRNA/PCM-CD-Tat complex and the Fas siRNA/PCM-CD-Tat complex warrant further investigation as therapeutic agents to inhibit the apoptosis of cardiomyocytes.
Asunto(s)
Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Polímeros/química , Polímeros/metabolismo , ARN Interferente Pequeño/metabolismo , Transfección/métodos , Acrilamidas/química , Animales , Apoptosis , Cistamina/química , Silenciador del Gen , Hexanos/química , Ensayo de Materiales , Ratones , Estructura Molecular , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Células 3T3 NIH , Polímeros/síntesis química , Proteína Tirosina Fosfatasa no Receptora Tipo 6/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 6/inmunología , Interferencia de ARN , ARN Interferente Pequeño/genética , RatasRESUMEN
Implantation of skeletal myoblasts to the heart has been investigated as a means to regenerate and protect the myocardium from damage after myocardial infarction. While several animal studies utilizing skeletal myoblasts have reported positive findings, results from clinical studies have been mixed. In this study we utilize a newly developed bioreducible polymer system to transfect skeletal myoblasts with a plasmid encoding vascular endothelial growth factor (VEGF) prior to implantation into acutely ischemic myocardium. VEGF has been demonstrated to promote revascularization of the myocardium following myocardial infarction. We report that implanting VEGF expressing skeletal myoblasts into acutely ischemic myocardium produces superior results compared to implantation of untransfected skeletal myoblasts. Skeletal myoblasts expressing secreted VEGF were able to restore cardiac function to non-diseased levels as measured by ejection fraction, to limit remodeling of the heart chamber as measured by end systolic and diastolic volumes, and to prevent myocardial wall thinning. Additionally, arteriole and capillary formation, retention of viable cardiomyocytes, and prevention of apoptosis was significantly improved by VEGF expressing skeletal myoblasts compared to untransfected myoblasts. This work demonstrates the feasibility of using bioreducible cationic polymers to create engineered skeletal myoblasts to treat acutely ischemic myocardium.
Asunto(s)
Materiales Biocompatibles/química , Mioblastos Esqueléticos/metabolismo , Isquemia Miocárdica/terapia , Polímeros/química , Transfección/métodos , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Células Cultivadas , Terapia Genética/métodos , Inmunohistoquímica , Imagen por Resonancia Magnética , Masculino , Ratas , Ratas Sprague-Dawley , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
Although RNA interference (RNAi)-mediated gene silencing provides a powerful strategy for modulating specific gene functions, difficulties associated with siRNA delivery have impeded the development of efficient therapeutic applications. In particular, the efficacy of siRNA delivery into neurons has been limited by extremely low transfection efficiencies. e-PAM-R is a biodegradable arginine ester of PAMAM dendrimer, which is readily degradable under physiological conditions (pH 7.4, 37 degrees C). In the present study, we investigated the efficiency of siRNA delivery by e-PAM-R in primary cortical cultures and in rat brain. e-PAM-R/siRNA complexes showed high transfection efficiencies and low cytotoxicities in primary cortical cultures. Localization of fluorescence-tagged siRNA revealed that siRNA was delivered not only into the nucleus and cytoplasm, but also along the processes of the neuron. e-PAM-R/siRNA complex-mediated target gene reduction was observed in over 40% of cells and it was persistent for over 48 h. The potential use of e-PAM-R was demonstrated by gene knockdown after transfecting High mobility group box-1 (HMGB1, a novel cytokine-like molecule) siRNA into H(2)O(2)- or NMDA-treated primary cortical cultures. In these cells, HMGB1 siRNA delivery successfully reduced both basal and H(2)O(2)- or NMDA-induced HMGB1 levels, and as a result of that, neuronal cell death was significantly suppressed in both cases. Furthermore, we showed that e-PAM-R successfully delivered HMGB1 siRNA into the rat brain, wherein HMGB1 expression was depleted in over 40% of neurons and astrocytes of the normal brain. Moreover, e-PAM-R-mediated HMGB1 siRNA delivery notably reduced infarct volume in the postischemic rat brain, which is generated by occluding the middle cerebral artery for 60 min. These results indicate that e-PAM-R, a novel biodegradable nonviral gene carrier, offers an efficient means of transfecting siRNA into primary neuronal cells and in the brain and of performing siRNA-mediated gene knockdown.
Asunto(s)
Materiales Biocompatibles/química , Isquemia Encefálica/terapia , Corteza Cerebral/efectos de los fármacos , Dendrímeros/química , Portadores de Fármacos/química , Proteína HMGB1/genética , Fármacos Neuroprotectores/administración & dosificación , ARN Interferente Pequeño/administración & dosificación , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/patología , Infarto Encefálico/genética , Infarto Encefálico/patología , Infarto Encefálico/terapia , Isquemia Encefálica/genética , Isquemia Encefálica/patología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Células Cultivadas , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Electroforesis en Gel de Poliacrilamida , Silenciador del Gen/efectos de los fármacos , Immunoblotting , Inmunohistoquímica , Masculino , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Nylons/química , Poliésteres , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
OBJECTIVE: To evaluate the efficacy of the curcumin-coating stent (CCS) on the inhibition of restenosis in a rabbit iliac artery stent model. RESULTS: Curcumin, pigment naturally acquired from the rhizome of the plant curcuma longa, is known to have antiproliferative, antimigratory, and anti-inflammatory effects. However, it is still unclear that curcumin can inhibit neointimal proliferation of the injured vessel. METHODS: Dose-dependent inhibition of cell growth was observed over a dose range from 10 nM to 10 microM. CCS was prepared by a dip-coating method (high-dose: HD, low-dose: LD). The release profile of the HD CCS showed that drug release persisted until day 21. Scanning electron microscopy of the CCS showed an intact surface of the stent even after expansion. To test the efficacy of CCS in vivo, LD CCS, HD CCS, and bare metal stents (BMS) were implanted in random order in one iliac artery (N = 30 arteries) of male New Zealand White rabbits (N = 15). RESULTS: After 28 days, the LD and HD CCS groups had a 43% and 55% reduction in the neointimal area, compared with the BMS group (BMS 3.3 +/- 1.0 mm(2), LD 1.9 +/- 0.8 mm(2), and HD 0.9 +/- 0.5 mm(2), P < 0.05). There appeared to be no cytotoxicity related to curcumin at the indicated doses. CONCLUSIONS: Curcumin, a natural compound in the human diet, seems to be a safe and effective candidate drug for use in a drug-eluting stent for the prevention of stent restenosis following angioplasty.
Asunto(s)
Angioplastia de Balón/instrumentación , Arteriopatías Oclusivas/prevención & control , Fármacos Cardiovasculares/administración & dosificación , Materiales Biocompatibles Revestidos , Curcumina/administración & dosificación , Stents Liberadores de Fármacos/efectos adversos , Hipercolesterolemia/complicaciones , Arteria Ilíaca/efectos de los fármacos , Angioplastia de Balón/efectos adversos , Animales , Arteriopatías Oclusivas/etiología , Arteriopatías Oclusivas/patología , Becaplermina , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Constricción Patológica , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Arteria Ilíaca/patología , Masculino , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Miocitos del Músculo Liso/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Diseño de Prótesis , Proteínas Proto-Oncogénicas c-sis , Conejos , Ratas , Ratas Sprague-Dawley , Propiedades de Superficie , Factores de TiempoRESUMEN
We developed a polyethylene glycol (PEG)-based biodegradable hydrogel through disulfide crosslinking of polyethylene oxide sulfide (PEOS). The crosslinking rate was highly dependent on temperature, and incubation at about 40-50 degrees C was required for efficient crosslinking. The crosslinked PEOS hydrogel showed glutathione-dependent dissolution and corresponding controlled release of a model drug-fluorescein isothiocyanate (FITC)-labeled dextran-because the disulfide bond, the main linker, is selectively degraded in response to the high concentration of glutathione. The temperature-sensitive crosslinking and the hydrogel formation have the potential for use as an injectable biogel precursor, which was confirmed by in situ gel formation in mice.
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Dextranos/administración & dosificación , Fluoresceína-5-Isotiocianato/análogos & derivados , Polietilenglicoles/química , Sulfuros/química , Animales , Reactivos de Enlaces Cruzados/química , Preparaciones de Acción Retardada , Portadores de Fármacos/química , Fluoresceína-5-Isotiocianato/administración & dosificación , Glutatión/metabolismo , Hidrogeles , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos C57BL , TemperaturaRESUMEN
We synthesized biodegradable polycationic PAMAM (polyamidoamine) esters (e-PAM-R, e-PAM-K) that contain arginines or lysines at the peripheral ends of PAMAM-OH dendrimer through ester bond linkages. The PAMAM esters were readily degradable under physiological conditions (pH 7.4, 37 degrees C), with more than 50% of the grafted amino acids hydrolyzed within 5h. However, polyplexes were very stable and were hardly degraded in the endosomal pH range. Moreover, these amino-acid-modified polymers showed excellent buffering capacities between pH 5.1 and 7.4, facilitating endosomal escape of polyplexes. While the lysine-grafted PAMAM ester did not display significant improvement in transfection efficiency, the arginine-conjugated PAMAM ester-mediated transfection of a luciferase gene showed better transfection efficiency than the branched 25 kDa PEI (polyethylenimine) and PAM-R (peptide bond), and lower cytotoxicity, especially with primary cells such as HUVECs (human umbilical vein endothelial cells) and SMCs (primary rat aorta vascular smooth muscle cells). Furthermore, after DNA release, free e-PAM-R degraded completely into nontoxic PAMAM-OH and arginines by hydrolysis, which resulted in lower cytotoxicity in contrast to the poorly degradable arginine-modified PAMAM with amide bonds. These findings demonstrated that the arginine-grafted biodegradable PAMAM dendrimer, e-PAM-R, is a potential candidate as a safe and efficient gene delivery carrier for gene therapy.