RESUMEN
The piggyBac transposon/transposase system has been explored for long-term, stable gene expression to execute genomic integration of therapeutic genes, thus emerging as a strong alternative to viral transduction. Most studies with piggyBac transposition have employed physical methods for successful delivery of the necessary components of the piggyBac system into the cells. Very few studies have explored polymeric gene delivery systems. In this short communication, we report an effective delivery system based on low molecular polyethylenimine polymer with lipid substitution (PEI-L) capable of delivering three components, (i) a piggyBac transposon plasmid DNA carrying a gene encoding green fluorescence protein (PB-GFP), (ii) a piggyBac transposase plasmid DNA or mRNA, and (iii) a 2 kDa polyacrylic acid as additive for transfection enhancement, all in a single complex. We demonstrate an optimized formulation for stable GFP expression in two model cell lines, MDA-MB-231 and SUM149 recorded till day 108 (3.5 months) and day 43 (1.4 months), respectively, following a single treatment with very low cell number as starting material. Moreover, the stability of the transgene (GFP) expression mediated by piggyBac/PEI-L transposition was retained following three consecutive cryopreservation cycles. The success of this study highlights the feasibility and potential of employing a polymeric delivery system to obtain piggyBac-based stable expression of therapeutic genes.
Asunto(s)
ADN , Técnicas de Transferencia de Gen , Plásmidos , Línea Celular , Proteínas Fluorescentes Verdes/genética , Transposasas/genética , Transposasas/metabolismo , Elementos Transponibles de ADN/genética , Vectores GenéticosRESUMEN
The clinical development of tyrosine kinase inhibitors (TKI) has led to great strides in improving the survival of chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) patients. But even the new generation TKIs are rendered futile in the face of evolving landscape of acquired mutations leading to drug resistance, necessitating the pursuit of alternative therapeutic approaches. In contrast to exploiting proteins as targets like most conventional drugs and TKIs, RNA Interference (RNAi) exerts its therapeutic action towards disease-driving aberrant genes. To realize the potential of RNAi, the major challenge is to efficiently deliver the therapeutic mediator of RNAi, small interfering RNA (siRNA) molecules. In this study, we explored the feasibility of using aliphatic lipid (linoleic acid and lauric acid)-grafted polymers (lipopolymers) for the delivery of siRNAs against the FLT3 oncogene in AML and BCR-ABL oncogene in CML. The lipopolymer delivered siRNA potently suppressed the proliferation AML and CML cells via silencing of the targeted oncogenes. In both AML and CML subcutaneous xenografts generated in NCG mice, intravenously administered lipopolymer/siRNA complexes displayed significant inhibitory effect on tumor growth. Combining siFLT3 complexes with gilteritinib allowed for reduction of effective drug dosage, longer duration of remission, and enhanced survival after relapse, compared to gilteritinib monotherapy. Anti-leukemic activity of siBCR-ABL complexes was similar in wild-type and TKI-resistant cells, and therapeutic efficacy was confirmed in vivo through prolonged survival of the NCG hosts systemically implanted with TKI-resistant cells. These results demonstrate the preclinical efficacy of lipopolymer facilitated siRNA delivery, providing a novel therapeutic platform for myeloid leukemias.
Asunto(s)
Compuestos de Anilina , Leucemia Mielógena Crónica BCR-ABL Positiva , Leucemia Mieloide Aguda , Pirazinas , Humanos , Animales , Ratones , ARN Interferente Pequeño , Proteínas de Fusión bcr-abl/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Oncogenes , Modelos Animales , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/farmacología , Resistencia a AntineoplásicosRESUMEN
This study aimed at investigating the influence of commercial transfection reagents (Prime-Fect, Leu-Fect A, and Leu-Fect C) complexed with different siRNAs (CDC20, HSP90, Mcl-1 and Survivin) in MDA-MB-436 breast cancer cells and the impact of incorporating an anionic additive, Trans-Booster, into siRNA formulations for improving in vitro gene silencing and delivery efficiency. Gene silencing was quantitatively analyzed by real-time RT-PCR while cell proliferation and siRNA uptake were evaluated by the MTT assay and flow cytometry, respectively. Amongst the investigated siRNAs and transfection reagents, Mcl-1/Prime-Fect complexes showed the highest inhibition of cell viability and the most effective siRNA delivery. The effect of various formulations on transfection efficiency showed that the additive with 1:1 ratio with siRNA was optimal achieving the lowest cell viability compared to untreated cells and negative control siRNA treatment (p < 0.05). Furthermore, the combination of Mcl-1 and survivin siRNA suppressed the growth of MDA-MB-436 cells more effectively than treatment with the single siRNAs and resulted in cell viability as low as ~ 20% (vs. non-treated cells). This aligned well with the induction of apoptosis as analyzed by flow cytometry, which revealed higher apoptotic cells with the combination treatment group. We conclude that commercial transfection reagents formulated with Mcl-1/Survivin siRNA combination could serve as a potent anti-proliferation agent in the treatment of breast cancers.
Asunto(s)
Neoplasias de la Mama , Apoptosis , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Línea Celular Tumoral , Proliferación Celular , Femenino , Silenciador del Gen , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , ARN Interferente Pequeño/genética , Survivin/genética , Survivin/farmacología , TransfecciónRESUMEN
Overexpression and persistent activation of STAT5 play an important role in the development and progression of acute lymphoblastic leukemia (ALL), the most common pediatric cancer. Small interfering RNA (siRNA)-mediated downregulation of STAT5 represents a promising therapeutic approach for ALL to overcome the limitations of current treatment modalities such as high relapse rates and poor prognosis. However, to effectively transport siRNA molecules to target cells, development of potent carriers is of utmost importance to surpass hurdles of delivery. In this study, we investigated the use of lipopolymers as non-viral delivery systems derived from low molecular weight polyethylenimines (PEI) substituted with lauric acid (Lau), linoleic acid (LA) and stearic acid (StA) to deliver siRNA molecules to ALL cell lines and primary samples. Among the lipid-substituted polymers explored, Lau- and LA-substituted PEI displayed excellent siRNA delivery to SUP-B15 and RS4;11 cells. STAT5A gene expression was downregulated (36-92%) in SUP-B15 and (32%) in RS4;11 cells using the polymeric delivery systems, which consequently reduced cell growth and inhibited the formation of colonies in ALL cells. With regard to ALL primary cells, siRNA-mediated STAT5A gene silencing was observed in four of eight patient cells using our leading polymeric delivery system, 1.2PEI-Lau8, accompanied by the significant reduction in colony formation in three of eight patients. In both BCR-ABL positive and negative groups, three of five patients demonstrated marked cell growth inhibition in both MTT and trypan blue exclusion assays using 1.2PEI-Lau8/siRNA complexes in comparison with their control siRNA groups. Three patient samples did not show any positive results with our delivery systems. Differential therapeutic responses to siRNA therapy observed in different patients could result from variable genetic profiles and patient-to-patient variability in delivery. This study supports the potential of siRNA therapy and the designed lipopolymers as a delivery system in ALL therapy.
Asunto(s)
Regulación hacia Abajo/efectos de los fármacos , Portadores de Fármacos/administración & dosificación , Polímeros/administración & dosificación , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , ARN Interferente Pequeño/administración & dosificación , Factor de Transcripción STAT5/genética , Proteínas Supresoras de Tumor/genética , Linfocitos B/efectos de los fármacos , Línea Celular Tumoral , Proteínas de Fusión bcr-abl/genética , Silenciador del Gen/efectos de los fármacos , Humanos , Ácido Linoleico/administración & dosificación , Polietileneimina/administración & dosificación , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Interferencia de ARN/efectos de los fármacos , ARN Bicatenario/genéticaRESUMEN
The discovery of RNA interference (RNAi) more than two decades ago opened avenues for avant-garde cancer treatments that possess the ability to evade issues hampering current chemotherapeutic strategies, owing to its specific gene sequence-driven mechanism of action. A potent short interfering RNA (siRNA) delivery vehicle designed to overcome physiological barriers is imperative for successful RNAi therapy. For this purpose, this study explored the characteristics and therapeutic efficacy of low-molecular weight (MW) polyethylenimine (PEI) with high cholesterol substitution, yielding water-insoluble polymers, in chronic myeloid leukemia (CML) K562 cells. A strong impact of cholesterol grafting on the physicochemical attributes of the resultant polymers and their corresponding complexes with siRNA was observed, with the siRNA binding capacity of polymers increasing and complex dissociation sensitivity decreasing with increase in cholesterol content of the polymers. The modified polymer complexes were significantly smaller in size and possessed higher cationic charge compared to the parent polymer. The interaction with anionic heparan sulfate preoteoglycans present on the cell surface was significant in cellular uptake of the complexes. The therapeutic efficacy of siRNA/polymer complexes was reflected in their ability to effectively silence the reporter green fluorescent protein gene and endogenous CML oncogene BCR-ABL as well as significantly inhibit colony formation by K562 cells post BCR-ABL silencing. The results of this study demonstrated beneficial effects of high levels of hydrophobic substitution on low MW PEI on their functional performance bestowing them the potential to be potent RNAi agents for CML therapy.
Asunto(s)
Leucemia Mielógena Crónica BCR-ABL Positiva , Polietileneimina , Interferencia de ARN , ARN Interferente Pequeño , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Polietileneimina/química , Polietileneimina/farmacología , ARN Interferente Pequeño/química , ARN Interferente Pequeño/farmacologíaRESUMEN
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.
Asunto(s)
Portadores de Fármacos/química , Ácidos Grasos/química , Polietileneimina/análogos & derivados , ARN Interferente Pequeño/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Clorpromazina/farmacología , Portadores de Fármacos/metabolismo , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Ácidos Grasos/metabolismo , Silenciador del Gen/efectos de los fármacos , Genisteína/farmacología , Humanos , Polietileneimina/metabolismoRESUMEN
Small interfering RNA (siRNA) delivered to silence overexpressed genes associated with malignancies is a promising targeted therapy to decrease the uncontrolled growth of malignant cells. To create potent delivery agents for siRNA, here we formulated additive polyplexes of siRNA using linoleic acid-substituted polyethylenimine and additive polymers (hyaluronic acid, poly(acrylic acid), dextran sulfate, and methyl cellulose) and characterized their physicochemical properties and effectiveness. Incorporating polyanionic polymer along with anionic siRNA in polyplexes was found to decrease the ζ-potential of polyplexes but enhance the cellular delivery of siRNA. The CDC20 and survivin siRNAs delivered by additive polyplexes showed promising efficacy in breast cancer MDA-MB-231, SUM149PT, MDA-MB-436, and MCF7 cells. However, the side effects of the siRNA delivery were observed in nonmalignant cells, and a careful formulation of siRNA/polymer polyplexes was needed to minimize side effects on normal cells. Because the efficacy of siRNA delivery by additive polyplexes was independent of breast cancer phenotypes used in this study, these polyplexes could be further developed to treat a wide range of breast cancers.
Asunto(s)
Neoplasias de la Mama/terapia , Proteínas Cdc20/antagonistas & inhibidores , Ácido Hialurónico/química , Polietileneimina/química , Polímeros/química , ARN Interferente Pequeño/genética , Survivin/antagonistas & inhibidores , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Proteínas Cdc20/genética , Proliferación Celular , Femenino , Silenciador del Gen , Humanos , Polielectrolitos , ARN Interferente Pequeño/química , Survivin/genética , Células Tumorales CultivadasRESUMEN
Triple-negative breast cancer is an aggressive form of breast cancer with few therapeutic options if it recurs after adjuvant chemotherapy. RNA interference could be an alternative therapy for metastatic breast cancer, where small interfering RNA (siRNA) can silence the expression of aberrant genes critical for growth and migration of malignant cells. Here, we formulated a siRNA delivery system using lipid-substituted polyethylenimine (PEI) and hyaluronic acid (HA), and characterized the size, ζ-potential and cellular uptake of the nanoparticulate delivery system. Higher cellular uptake of siRNA by the tailored PEI/HA formulation suggested better interaction of complexes with breast cancer cells due to improved physicochemical characteristics of carrier and HA-binding CD44 receptors. The siRNAs against specific phosphatases that inhibited migration of MDA-MB-231 cells were then identified using library screen against 267 protein-tyrosine phosphatases, and siRNAs to inhibit cell migration were further validated. We then assessed the combinational delivery of a siRNA against CDC20 to decrease cell growth and a siRNA against several phosphatases shown to decrease migration of breast cancer cells. Combinational siRNA therapy against CDC20 and identified phosphatases PPP1R7, PTPN1, PTPN22, LHPP, PPP1R12A and DUPD1 successfully inhibited cell growth and migration, respectively, without interfering the functional effect of the co-delivered siRNA. The identified phosphatases could serve as potential targets to inhibit migration of highly aggressive metastatic breast cancer cells. Combinational siRNA delivery against cell cycle and phosphatases could be a promising strategy to inhibit both growth and migration of metastatic breast cancer cells, and potentially other types of metastatic cancer. STATEMENT OF SIGNIFICANCE: The manuscript investigated the efficacy of a tailored polymeric siRNA delivery system formulation as well as combinational siRNA therapy in metastatic breast cancer cells to inhibit malignant cell growth and migration. The siRNA delivery was undertaken by non-viral means with PEI/HA. We identified six phosphatases that could be critical targets to inhibit migration of highly aggressive metastatic breast cancer cells. We further report on specifically targeting cell cycle and phosphatase proteins to decrease both malignant cell growth and migration simultaneously. Clinical gene therapy against metastatic breast cancer with effective and safe delivery systems is urgently needed to realize the potential of molecular medicine in this deadly disease and our studies in this manuscript is intended to facilitate this endeavor.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Movimiento Celular , Técnicas Químicas Combinatorias , Ácido Hialurónico/química , Fosfoproteínas Fosfatasas/metabolismo , ARN Interferente Pequeño/administración & dosificación , Tensoactivos/química , Neoplasias de la Mama Triple Negativas/patología , Línea Celular Tumoral , Proliferación Celular , Silenciador del Gen , Humanos , Receptores de Hialuranos/metabolismo , Ácido Linoleico/química , Tamaño de la Partícula , Polietileneimina/química , Reproducibilidad de los Resultados , Electricidad Estática , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
The emerging molecular understanding of cancer cell behavior is leading to increasing possibilities to control unchecked cell growth and metastasis. On the other hand, development of multifunctional drug carriers at the 'nano'-scale is providing exciting new therapeutic strategies in clinical management of cancer beyond the conventional cytotoxic drugs. A new frontier in this regard is the combinational use of complementary agents based on nucleic acids to overcome the limitations of conventional therapy. The existence of tightly-integrated cross-talk through multiple signaling and effector pathways have been appreciated for some time, and the plasticity of such a network to overcome one-dimensional intervention is stimulating development of combinational therapy. The objective of this review is to underline the cutting edge technologies and opportunities employed in combination cancer therapy using nucleic acids therapeutics for successful clinical translation. Here, we provide a detailed analysis of the multifunctional carriers designed for different types of payloads, surveying the biomaterials used to construct the functional carriers. We then provide effective nucleic acid combinations employed to obtain more comprehensive outcomes, highlighting the critical factors involved in successful therapy. We conclude with an authors' perspective on the future of combinational therapy using nucleic acid therapeutics, articulating the main challenges to advance this promising approach to the clinical realm.
Asunto(s)
Neoplasias/terapia , Ácidos Nucleicos/administración & dosificación , Animales , Técnicas de Transferencia de Gen , Terapia Genética , HumanosRESUMEN
Bone healing after traumatic injuries or pathological diseases remains an important worldwide problem. In search of safer and more effective approaches to bone regeneration and repair, RNA-based therapeutic agents, specifically microRNAs (miRNAs) and short interfering RNA (siRNA), are beginning to be actively explored. In this review, we summarize current attempts to employ miRNAs and siRNAs in preclinical models of bone repair. We provide a summary of current limitations when attempting to utilize bioactive nucleic acids for therapeutic purposes and position the unique aspects of RNA reagents for clinical bone repair. Delivery strategies for RNA reagents are emphasized and nonviral carriers (biomaterial-based) employed to deliver such reagents are reviewed. Critical features of biomaterial carriers and various delivery technologies centered around nanoparticulate systems are highlighted. We conclude with the authors' perspectives on the future of the field, outlining main critical issues important to address as RNA reagents are explored for clinical applications.
RESUMEN
Conventional breast cancer therapies have significant limitations that warrant a search for alternative therapies. Short-interfering RNA (siRNA), delivered by polymeric biomaterials and capable of silencing specific genes critical for growth of cancer cells, holds great promise as an effective, and more specific therapy. Here, we employed amphiphilic polymers and silenced the expression of two cell cycle proteins, TTK and CDC20, and the anti-apoptosis protein survivin to determine the efficacy of polymer-mediated siRNA treatment in breast cancer cells as well as side effects in nonmalignant cells in vitro. We first identified effective siRNA carriers by screening a library of lipid-substituted polyethylenimines (PEI), and PEI substituted with linoleic acid (LA) emerged as the most effective carrier for selected siRNAs. Combinations of TTK/CDC20 and CDC20/Survivin siRNAs decreased the growth of MDA-MB-231 cells significantly, while only TTK/CDC20 combination inhibited MCF7 cell growth. The effects of combinational siRNA therapy was higher when complexes were formulated at lower siRNA:polymer ratio (1:2) compared to higher ratio (1:8) in nonmalignant cells. The lead polymer (1.2PEI-LA6) showed differential transfection efficiency based on the cell-type transfected. We conclude that the lipid-substituted polymers could serve as a viable platform for delivery of multiple siRNAs against critical targets in breast cancer therapy. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3031-3044, 2016.
Asunto(s)
Técnicas de Transferencia de Gen , Lípidos/química , Polietileneimina/química , ARN Interferente Pequeño/administración & dosificación , Tratamiento con ARN de Interferencia , Neoplasias de la Mama Triple Negativas/terapia , Proteínas Cdc20/genética , Proteínas de Ciclo Celular/genética , Línea Celular , Línea Celular Tumoral , Femenino , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/uso terapéutico , Survivin , Neoplasias de la Mama Triple Negativas/genéticaRESUMEN
A nano cocktail, NCPD, fabricated from a pH and redox dual responsive polymer shows a multicompartment structure. The NCPD nanogel is stable in physiological environments while intracellular spontaneous swelling and fast releasing its payload. NCPD displays much stronger synergism than its free drug counterpart, which suggests that NCPD could greatly attenuate the side effects of drug cocktails while boosting synergistic anticancer effects.
Asunto(s)
Antineoplásicos/farmacología , Doxorrubicina/farmacología , Sistemas de Liberación de Medicamentos , Paclitaxel/farmacología , Polietilenglicoles/farmacología , Polietileneimina/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Doxorrubicina/síntesis química , Doxorrubicina/química , Ensayos de Selección de Medicamentos Antitumorales , Células HCT116 , Humanos , Estructura Molecular , Nanogeles , Paclitaxel/síntesis química , Paclitaxel/química , Tamaño de la Partícula , Polietilenglicoles/síntesis química , Polietilenglicoles/química , Polietileneimina/síntesis química , Polietileneimina/química , Relación Estructura-Actividad , Propiedades de SuperficieRESUMEN
To mimic the clinic dosing pattern, initially administering high loading dose and then low maintenance dose, we designed a novel poly(2-(pyridin-2-yldisulfanyl)ethyl acrylate) (PDS) based nanoparticle delivery system. Side chain functional PDS was synthesized by free radical polymerization. Polyethylene glycol and cyclo(Arg-Gly-Asp-d-Phe-Cys) (cRGD) peptide was conjugated to PDS through thiol-disulfide exchange reaction to achieve RPDSG polymer. RPDSG/DOX, RPDSG nanoparticle loaded with doxorubicin, was fabricated by cosolvent dialysis method. The size of the nanoparticles was 50.13 ± 0.5 nm in PBS. The RPDSG/DOX nanoparticle is stable in physiological condition while quickly releasing doxorubicin with the trigger of acidic pH and redox potential. Furthermore, it shows a two-phase release kinetics, providing both loading dose and maintenance dose for cancer therapy. The conjugation of RGD peptide enhanced the cellular uptake and nuclear localization of the RPDSG/DOX nanoparticles. RPDSG/DOX exhibits IC(50) close to that of free doxorubicin for HCT-116 colon cancer cells. Due to the synergetic effect of RGD targeting effect and its two-phase release kinetics, RPDSG/DOX nanoparticles display significantly higher anticancer efficacy than that of free DOX at concentrations higher than 5 µM. These results suggest that RPDSG/DOX could be a promising nanotherapeutic for tumor-targeted chemotherapy.
Asunto(s)
Doxorrubicina/química , Nanopartículas/administración & dosificación , Nanopartículas/química , Antineoplásicos/administración & dosificación , Antineoplásicos/química , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Sistemas de Liberación de Medicamentos/métodos , Células HCT116 , Humanos , Concentración de Iones de Hidrógeno , Cinética , Oligopéptidos/administración & dosificación , Oligopéptidos/química , Oxidación-Reducción , Tamaño de la Partícula , Polietilenglicoles/administración & dosificación , Polietilenglicoles/química , Polímeros/administración & dosificación , Polímeros/químicaRESUMEN
A novel gene delivery system, called PoSC, consisting of PEI, PSP, and HA is described. In contrast to the DNA/PEI/HA ternary system whose transfection efficiency decreases significantly with increasing serum concentration, PoSC exhibits a high transfection efficiency of about 51 and 87% for NIH3T3 and HCT116 cells, respectively, at 50% serum concentration. Furthermore, PoSC shows no cytotoxic effect at its working concentration. The overall results suggest that HA adsorption on cationic complexes enhances the transfection efficiency, while PSP is essential for high transfection efficiency at higher serum concentration.
Asunto(s)
ADN/química , Técnicas de Transferencia de Gen , Vectores Genéticos/química , Ácido Hialurónico/química , Polietileneimina/química , Espermina/análogos & derivados , Animales , Línea Celular , Supervivencia Celular/fisiología , ADN/genética , Endocitosis/fisiología , Expresión Génica , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Humanos , Espectroscopía de Resonancia Magnética , Ratones , Microscopía Fluorescente , Plásmidos/química , Plásmidos/genética , TransfecciónRESUMEN
Cationic polymers are desirable gene carriers because of their better safety profiles than viral delivery systems. Low molecular weight (MW) polymers are particularly attractive, since they display little cytotoxicity, but they are also ineffective for gene delivery. To create effective carriers from low MW polymers palmitic acid (PA) was substituted on 0.6-2.0 kDa polyethylenimines (PEIs) and their efficiency for plasmid DNA (pDNA) delivery was evaluated. The extent of lipid substitution was dependent on the lipid/PEI feed ratio and the polymer MW. While the hydrodynamic size of the polymer/pDNA complexes (polyplexes) increased or decreased depending on the extent of lipid substitution, the ζ potential of the assembled complexes was consistently higher as a result of lipid substitution. Lipid substitution generally increased the in vitro toxicity of the PEIs, but it was significantly lower than that of the 25 kDa branched PEI. The in vitro transfection efficiency of the lipid-substituted polymers was higher than that of native PEIs, which were not at all effective. The delivery efficiency was proportional to the extent of lipid substitution as well as the polymer MW. This correlated with the increased uptake of lipid-substituted polyplexes, based on confocal microscopic investigations with FITC-labeled pDNA. The addition of chloroquine further increased the transfection efficiency of lipid-substituted PEIs, indicating that endosomal release was a limiting factor affecting the efficiency of these carriers. This study indicates that lipid substitution on low MW PEIs makes their assembly more effective, resulting in better delivery of pDNA into mammalian cells.
Asunto(s)
ADN/genética , Expresión Génica , Lípidos/química , Plásmidos/genética , Polietileneimina/química , Electricidad Estática , Transgenes/genética , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cloroquina/farmacología , Expresión Génica/efectos de los fármacos , Humanos , Hidrodinámica , Microscopía Confocal , Peso Molecular , Pirenos/química , Solubilidad/efectos de los fármacos , TransfecciónRESUMEN
In this study, we synthesized empty core-shell structured nanocapsules of Pluronic F127 and chitosan and characterized the thermal responsiveness of the nanocapsules in size and wall-permeability. Moreover, we determined the feasibility of using the nanocapsules to encapsulate small molecules for temperature-controlled release and intracellular delivery. The nanocapsules are â¼37 nm at 37 °C and expand to â¼240 nm when cooled to 4 °C in aqueous solutions, exhibiting >200 times change in volume. Moreover, the permeability of the nanocapsule wall is high at 4 °C (when the nanocapsules are swollen), allowing free diffusion of small molecules (ethidium bromide, MW = 394.3 Da) across the wall, while at 37 °C (when the nanocapsules are swollen), the wall-permeability is so low that the small molecules can be effectively withheld in the nanocapsule for hours. As a result of their thermal responsiveness in size and wall-permeability, the nanocapsules are capable of encapsulating the small molecules for temperature-controlled release and intracellular delivery into the cytosol of both cancerous (MCF-7) and noncancerous (C3H10T1/2) mammalian cells. The cancerous cells were found to take up the nanocapsules much faster than the noncancerous cells during 45 min incubation at 37 °C. Moreover, toxicity of the nanocapsules as a delivery vehicle was found to be negligible. The Pluronic F127-chitosan nanocapsules should be very useful for encapsulating small therapeutic agents to treat diseases particularly when it is combined with cryotherapy where the process of cooling and heating between 37 °C and hypothermic temperatures is naturally done.
Asunto(s)
Quitosano/química , Etidio/metabolismo , Espacio Intracelular/metabolismo , Nanocápsulas/química , Poloxámero/química , Poloxámero/síntesis química , Temperatura , Animales , Materiales Biocompatibles/síntesis química , Materiales Biocompatibles/química , Materiales Biocompatibles/metabolismo , Materiales Biocompatibles/toxicidad , Transporte Biológico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Preparaciones de Acción Retardada , Fluoresceína-5-Isotiocianato/química , Humanos , Ratones , Nanocápsulas/toxicidad , Poloxámero/metabolismo , Poloxámero/toxicidad , Propiedades de SuperficieRESUMEN
This study deals with the synthesis of cysteine capped gold nanoparticles with an average size of 12 nm by borohydride reduction and spectroscopic identification of SAu interaction. We have studied the interaction of thiol with gold nanoparticles in aqueous medium by employing UV-vis, Raman, NMR, and FT-IR spectroscopy. The shifting of gold plasmon resonance in the UV-vis spectra shows the stabilization of gold nanoparticles by cysteine. The disappearance of S-H stretching in both the IR and Raman spectra and the shifting of the NMR signals of the protons in close proximity to the metal center supported the existence of the S-Au interaction in cysteine capped gold nanoparticles. The TEM images shows cysteine capped gold nanoparticles as distinct and spherical entities as compared to free colloidal gold nanoparticles.