RESUMEN
Various diseases, including cancers and inflammatory diseases, are characterized by a disruption of redox homeostasis, suggesting the need for synergistic treatments involving co-delivery of gene therapies and free radical scavengers. In this report, polyethylenimine (PEI), nanoceria (NC), and DNA were complexed to form nanoparticles providing simultaneous delivery of a gene and an antioxidant. NC was coated in citric acid to provide stable, 4 nm particles that electrostatically bound PEI/DNA polyplexes. The resulting ternary particles transfected HeLa cells with similar efficiency to that of ternary polyplexes comprising 15 kDa poly-l-α-glutamic acid/PEI/DNA while providing smaller particle sizes by more than 100 nm. NC/PEI/DNA polyplexes exhibited enhanced radical-scavenging activity compared to free NC, and oxidative stress from the superoxide-generating agent, menadione, could be completely reversed by the delivery of NC/PEI/DNA polyplexes. Transfection by NC/PEI/DNA polyplexes was demonstrated to occur efficiently through caveolin-mediated endocytosis and macropinocytosis. Co-delivery of genes encoding reactive oxygen species-scavenging proteins, transcription factors, growth factors, tumor suppressors, or anti-inflammatory genes with NC, therefore, may be a promising strategy in synergistic therapeutics.
Asunto(s)
Antioxidantes , Polímeros , Humanos , Células HeLa , ADN/genética , ADN/metabolismoRESUMEN
Gene therapy aims to treat patients by altering or controlling gene expression. The field of gene therapy has had increasing success in recent years primarily using viral-based approaches; however, there is still significant interest toward the use of polymeric materials due to their potential as flexible, low-cost scaffolds for gene delivery that do not suffer the mutagenesis and immunogenicity concerns of viral vectors. To address the challenges of efficiency and biocompatibility, a series of zwitterion-like polyethylenimine derivatives (zPEIs) were produced via the succinylation of 2-11.5% of polyethylenimine (PEI) amines. With increasing modification, zPEI polyplexes exhibited decreased serum-protein aggregation and dissociated more easily in the presence of a competitor polyanion when compared to unmodified PEI. Surprisingly, the gene delivery mediated in the presence of serum showed that succinylation of as few as 2% of PEI amines resulted in transgene expression 260- to 480-fold higher than that of unmodified PEI and 50- to 65-fold higher than that of commercial PEI-PEG2k in HEK293 and HeLa cells, respectively. Remarkably, the same zPEIs also produced 16-fold greater efficiency of CRISPR/Cas9 gene knock-in compared to unmodified PEI in the presence of serum. In addition, we show that 2% succinylation does not significantly decrease polymer/DNA binding ability or serum protein interaction to a significant extent, yet this small modification is still sufficient to provide a remarkable increase in transgene expression and gene knock-in in the presence of serum.
Asunto(s)
Sistemas CRISPR-Cas/genética , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Polietileneimina/química , Técnicas de Sustitución del Gen , Células HEK293 , Células HeLa , Humanos , Polietileneimina/análogos & derivados , Reparación del ADN por RecombinaciónRESUMEN
Cyanuric chloride has been utilized in the development of new synthetic lipid compounds using two differing schemes. The resulting lipids, presented in this manuscript, were characterized and evaluated for their ability to form nanoparticles and subsequently tested for their utility in various biological applications, including gene delivery and immunization. Of the 12 lipids synthesized, 8 formed nanoparticles that remained stable, based on dynamic light scattering, for at least one month. The compounds were then assessed for their toxicity, and subsequently tested for their ability to encapsulate drugs, genes and peptides. While the compounds did not seem to encapsulate carboxyfluorescein, we demonstrate that these lipids are capable of plasmid delivery in vitro, and inducing antibody profiles similar to other hydrophobic anchors in liposomal peptide vaccines. This strategy for accessing diverse lipid compounds offers a way to easily optimize lipid-based therapeutics for research in an expedited manner.
RESUMEN
Polymeric materials provide particularly attractive scaffolds for the creation of supramolecular bioconjugates for the delivery of nucleic acids but typically lack the efficiency and biocompatibility to be clinically relevant. To address both issues, we produced zwitterion-like derivatives of polyethylenimine via succinylation of primary and secondary amines (zPEI). Polymers were generated with 9-55% of the amines modified (zPEI X, where X indicates the percentage of amines succinylated). Characterization of polymer/DNA interactions revealed that the presence of succinyl groups decreased the protonation constant of zPEI, resulting in both a decreased buffering capacity and polyplexes that dissociated in the presence of lower amounts of a competing counteranion compared to unmodified PEI. zPEI polyplexes also exhibited decreased aggregation in the presence of serum proteins. In the absence of serum, transfections with zPEI/DNA polyplexes exhibited similar or slightly improved transgene expression compared to unmodified PEI/DNA polyplexes. More importantly, zPEI 9-25 increased transgene expression up to 51-fold upon transfection in the presence of serum compared to PEI/DNA, while higher succinylation decreased gene delivery activity. Gene delivery mediated by zPEI 9/DNA polyplexes in the presence of serum was equal to or greater than unmodified PEI/DNA polyplexes in the absence of serum. The data suggest that succinylation increased gene transfection by decreasing polymer/DNA interaction strength, which may allow for more facile polyplex unpackaging, and/or increased stability of polyplex size and inhibition of aggregation in the presence of serum. However, it appears there exists a balance between the positive effects of succinylation and the need for sufficient polymer/DNA binding to condense and protect the cargo.
Asunto(s)
Neoplasias de la Mama/genética , ADN/administración & dosificación , Polietileneimina/química , Polímeros/química , Suero/química , Ácido Succínico/química , Transfección/métodos , Neoplasias de la Mama/patología , Supervivencia Celular , ADN/química , Femenino , Técnicas de Transferencia de Gen , Terapia Genética , Células HeLa , Humanos , Plásmidos/administración & dosificación , Plásmidos/química , Células Tumorales CultivadasRESUMEN
Basal-like breast cancer (BLBC) is a malignant carcinoma with aggressive motility and rapid growth. Accounting for 15% of breast cancers, BLBC often exhibits a poor prognosis and tends to metastasize to the brain and lungs. Because most BLBC display a triple-negative phenotype (ER-, PR-, and HER2-), conventional cytotoxic chemotherapy remains the only treatment option despite poor success and high rate of relapse. The overexpression of the forkhead-box transcription factor C1 (FOXC1) was recently identified as a biomarker of BLBC. Increased expression of FOXC1 was linked to excessive mobility and growth of BLBC cell lines, suggesting FOXC1 as a therapeutic target. In this study, siRNA-mediated knockdown of FOXC1 was confirmed to decrease the proliferation rate, migration, and invasion in a model BLBC-like cell line (4T1). 4T1 and 4T1-∆FOXC1 cells lacking FOXC1 expression (generated by CRISPR/Cas9) were used to evaluate the effects of FOXC1 expression in an orthotopic murine model of BLBC. No statistically significant difference in tumor volume was observed between 4T1 and 4T1-∆FOXC1 tumors. Furthermore, tumors metastasized to the liver and lungs to a similar degree regardless of FOXC1 expression. These data suggest that, despite positive results in vitro, FOXC1 may not be a promising therapeutic target for BLBC.
Asunto(s)
Factores de Transcripción Forkhead , Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Animales , Proteínas de Neoplasias , Animales , Línea Celular Tumoral , Femenino , Factores de Transcripción Forkhead/biosíntesis , Factores de Transcripción Forkhead/genética , Neoplasias Mamarias Animales/genética , Neoplasias Mamarias Animales/metabolismo , Neoplasias Mamarias Animales/patología , Neoplasias Mamarias Animales/terapia , Ratones , Metástasis de la Neoplasia , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genéticaRESUMEN
Small interfering RNA (siRNA) has established its reputation in the field of tissue engineering owing to its ability to silence the proteins that inhibit tissue regeneration. siRNA is capable of regulating cellular behavior during tissue regeneration processes. The concept of using siRNA technology in regenerative medicine derived from its ability to inhibit the expression of target genes involved in defective tissues and the possibility to induce the expression of tissue-inductive factors that improve the tissue regeneration process. To date, siRNA has been used as a suppressive biomolecule in different tissues, such as nervous tissue, bone, cartilage, heart, kidney, and liver. Moreover, various delivery systems have been applied in order to deliver siRNA to the target tissues. This review will provide an in-depth discussion on the development of siRNA and their delivery systems and mechanisms of action in different tissues.
Asunto(s)
ARN Interferente Pequeño/farmacología , Medicina Regenerativa , Ingeniería de TejidosRESUMEN
Design of safe and effective synthetic nucleic acid delivery vectors such as polycation/DNA or polycation/siRNA complexes (polyplexes) will be facilitated by quantitative understanding of the mechanisms by which such materials escort cargo from the cell surface to the nucleus. In particular, the mechanisms of cellular internalization by various endocytosis pathways and subsequent endocytic vesicle trafficking have been shown to strongly affect nucleic acid delivery efficiency. Fluorescence microscopy and subcellular fractionation methods are commonly employed to follow intracellular trafficking of biomolecules and nanoparticulate delivery systems such as polyplexes. However, it is difficult to obtain quantitative data from microscopy and subcellular fractionation is experimentally difficult and low throughput. We have developed a method for quantifying the transport of polyplexes through important endocytic vesicles. The method is based on polymerization of 3,3'-diaminobenzidine by endocytosed horseradish peroxidase, causing an increase in the vesicle density, resistance to being solubilized by detergent and quenching of fluorophores within the vesicles, which makes them easy to separate and quantify. Using this method in HeLa cells, we have observed polyethylenimine/siRNA polyplexes initially appearing in early endosomes and rapidly moving to other compartments within 30min post-transfection. At the same time, we observed the kinetics of accumulation of the polyplexes in lysosomes at a similar rate. The results from the new method are consistent with similar measurements by confocal fluorescence microscopy and subcellular fractionation of endocytic vesicles on a Percoll gradient. The relative ease of this new method will aid investigation of gene delivery mechanisms by providing the means to rapidly quantify endocytic trafficking of polyplexes and other vectors.
Asunto(s)
Endocitosis , Poliaminas/metabolismo , Polietileneimina/metabolismo , ARN Interferente Pequeño/administración & dosificación , Transfección , Endosomas/metabolismo , Células HeLa , Humanos , Poliaminas/análisis , Polielectrolitos , Polietileneimina/análisis , Interferencia de ARN , ARN Interferente Pequeño/análisis , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacocinéticaRESUMEN
PURPOSE: siRNA may be delivered as electrostatic complexes with cationic lipids (lipoplexes) or polycations (polyplexes). The purpose of this project was to determine the effect of cellular internalization mechanism(s) on siRNA-mediated gene silencing efficiency. METHODS: Lipoplexes were formed comprising siRNA and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP), cholesterol and dioleoyl phosphatidylethanolamine (DOPE), and polyplexes comprised siRNA with polyethylenimine (PEI). During transfections, specific uptake mechanisms were inhibited by pharmacological agents and RNAi-mediated knockdown of proteins involved in various endocytosis pathways. Confocal fluorescence microscopy further elucidated the predominant endocytic pathways of siRNA delivery via colocalization of vectors with endocytic vesicle markers. RESULTS: Inhibition of macropinocytosis (MP), caveolin-mediated endocytosis (CvME), flotillin-mediated endocytosis (FME) and knockdown of ARF6 significantly decreased PEI/siRNA-mediated gene silencing. Inhibition of endocytosis pathways, however, had negligible effect on lipoplex uptake and gene silencing mediated by lipoplexes. Rather, internalization of lipoplexes and subsequent siRNA-mediated gene silencing occurred via an energy-independent process. CONCLUSIONS: MP, CvME and FME, but not the acidified clathrin-mediated pathway, lead to effective gene silencing by PEI/siRNA polyplexes. Lipoplexes, in contrast, deliver siRNA primarily by direct fusion of the liposomal and cellular membranes. These results provide a new understanding of the mechanisms of siRNA delivery materials in HeLa cells and may aid in design of more effective RNAi strategies.
Asunto(s)
Ácidos Grasos Monoinsaturados/química , Fosfatidiletanolaminas/química , Polietileneimina/química , Compuestos de Amonio Cuaternario/química , ARN Interferente Pequeño/metabolismo , Técnicas de Cultivo de Célula , Supervivencia Celular , Colesterol/química , Sistemas de Liberación de Medicamentos , Liberación de Fármacos , Endocitosis , Ácidos Grasos Monoinsaturados/metabolismo , Silenciador del Gen , Células HeLa , Humanos , Liposomas , Fosfatidiletanolaminas/metabolismo , Compuestos de Amonio Cuaternario/metabolismoRESUMEN
A mathematical reaction-diffusion model is defined to describe the gradual decomposition of polymer microspheres composed of poly(D,L-lactic-co-glycolic acid) (PLGA) that are used for pharmaceutical drug delivery over extended periods of time. The partial differential equation (PDE) model treats simultaneous first-order generation due to chemical reaction and diffusion of reaction products in spherical geometry to capture the microsphere-size-dependent effects of autocatalysis on PLGA erosion that occurs when the microspheres are exposed to aqueous media such as biological fluids. The model is solved analytically for the concentration of the autocatalytic carboxylic acid end groups of the polymer chains that comprise the microspheres as a function of radial position and time. The analytical solution for the reaction and transport of the autocatalytic chemical species is useful for predicting the conditions under which drug release from PLGA microspheres transitions from diffusion-controlled to erosion-controlled release, for understanding the dynamic coupling between the PLGA degradation and erosion mechanisms, and for designing drug release particles. The model is the first to provide an analytical prediction for the dynamics and spatial heterogeneities of PLGA degradation and erosion within a spherical particle. The analytical solution is applicable to other spherical systems with simultaneous diffusive transport and first-order generation by reaction.
Asunto(s)
Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos , Ácido Láctico/química , Microesferas , Modelos Teóricos , Ácido Poliglicólico/química , Catálisis , Copolímero de Ácido Poliláctico-Ácido PoliglicólicoRESUMEN
The design of biomaterials for increasingly complex tissue engineering applications often requires exogenous presentation of biomolecular signals. Integration of gene delivery vectors with a biomaterial scaffold offers the potential to bypass the use of expensive and relatively inefficient growth factor supplementation strategies to augment cell behavior. However, integration of cationic polymer based gene delivery vectors within three-dimensional biomaterials, particularly matrices which can carry significant surface charge, remains poorly explored. We examined the potential of polyethylenimine (PEI) as a gene delivery vector for three-dimensional collagen-glycosaminoglycan (CG) scaffolds under development for tendon repair. While acetylated versions of PEI have demonstrated improved transfection efficiency in 2D culture assays, we investigated translation of this effect to a 3D biomaterial that contains significant electrostatic charge. A reporter gene was used to examine the impact of polymer modification, polymer:DNA ratio, and the degree of sulfation of the biomaterial microenvironment on gene delivery in vitro. We observed highest transgene expression in acetylated and unmodified PEI at distinct polymer:DNA ratios; notably, the enhancement often seen in two-dimensional culture for acetylated PEI did not fully translate to three-dimensional scaffolds. We also found highly sulfated heparin-based CG scaffolds showed enhanced initial luciferase expression but not prolonged activity. While PEI constructs significantly reduced tenocyte metabolic health during the period of transfection, heparin-based CG scaffolds showed the greatest recovery in tenocyte metabolic health over the full 2 week culture. These results suggest that the electrostatic environment of three-dimensional biomaterials may be an important design criterion for cationic polymer-based gene delivery.
Asunto(s)
Materiales Biocompatibles/química , Colágeno/genética , Glicosaminoglicanos/química , Polietileneimina/química , Tendones/citología , Colágeno/metabolismo , Técnicas de Transferencia de Gen , Luciferasas/genética , Luciferasas/metabolismo , Tendones/química , Ingeniería de Tejidos , Andamios del Tejido , TransfecciónRESUMEN
Viral and non-viral gene delivery vectors are in development for human gene therapy, but both exhibit disadvantages such as inadequate efficiency, lack of cell-specific targeting or safety concerns. We have recently reported the design of hybrid delivery vectors combining retrovirus-like particles with synthetic polymers or lipids that are efficient, provide sustained gene expression and are more stable compared to native retroviruses. To guide further development of this promising class of gene delivery vectors, we have investigated their mechanisms of intracellular trafficking. Moloney murine leukemia virus-like particles (M-VLPs) were complexed with chitosan (Chi) or liposomes (Lip) comprising DOTAP, DOPE and cholesterol to form the hybrid vectors (Chi/M-VLPs and Lip/M-VLPs, respectively). Transfection efficiency and cellular internalization of the vectors were quantified in the presence of a panel of inhibitors of various endocytic pathways. Intracellular transport and trafficking kinetics of the hybrid vectors were dependent on the synthetic component and used a combination of clathrin- and caveolar-dependent endocytosis and macropinocytosis. Chi/M-VLPs were slower to transfect compared to Lip/M-VLPs due to the delayed detachment of the synthetic component. The synthetic component of hybrid gene delivery vectors plays a significant role in their cellular interactions and processing and is a key parameter for the design of more efficient gene delivery vehicles.
Asunto(s)
Quitosano/metabolismo , Colesterol/metabolismo , Endocitosis , Ácidos Grasos Monoinsaturados/metabolismo , Vectores Genéticos , Virus de la Leucemia Murina de Moloney/metabolismo , Fosfatidiletanolaminas/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Transfección/métodos , Proteínas Sanguíneas/metabolismo , Caveolas/metabolismo , Quitosano/química , Colesterol/química , Vesículas Cubiertas por Clatrina/metabolismo , Endocitosis/efectos de los fármacos , Ácidos Grasos Monoinsaturados/química , Células HEK293 , Células HeLa , Humanos , Cinética , Liposomas , Microscopía Confocal , Virus de la Leucemia Murina de Moloney/genética , Fosfatidiletanolaminas/química , Pinocitosis , Compuestos de Amonio Cuaternario/química , Temperatura , Virión/genética , Virión/metabolismoRESUMEN
PURPOSE: Non-viral gene delivery vehicles such as polyethylenimine and polyamidoamine dendrimer effectively condense plasmid DNA, facilitate endocytosis, and deliver nucleic acid cargo to the nucleus in vitro. Better understanding of intracellular trafficking mechanisms involved in polymeric gene delivery is a prerequisite to clinical application. This study investigates the role of clathrin and caveolin endocytic pathways in cellular uptake and subsequent vector processing. METHODS: We formed 25-kD polyethylenimine (PEI) and generation 4 (G4) polyamidoamine (PAMAM) polyplexes at N/P 10 and evaluated internalization pathways and gene delivery in HeLa cells. Clathrin- and caveolin-dependent endocytosis inhibitors were used at varying concentrations to elucidate the roles of these important pathways. RESULTS: PEI and PAMAM polyplexes were internalized by both pathways. However, the amount of polyplex internalized poorly correlated with transgene expression. While the caveolin-dependent pathway generally led to effective gene delivery with both polymers, complete inhibition of the clathrin-dependent pathway was also deleterious to transfection with PEI polyplexes. Inhibition of one endocytic pathway may lead to an overall increase in uptake via unaffected pathways, suggesting the existence of compensatory endocytic mechanisms. CONCLUSIONS: The well-studied clathrin- and caveolin-dependent endocytosis pathways are not necessarily independent, and perturbing one mechanism of trafficking influences the larger trafficking network.
Asunto(s)
Caveolinas/metabolismo , Clatrina/metabolismo , Dendrímeros/química , Endocitosis , Plásmidos/metabolismo , Polietileneimina/química , Transfección/métodos , Relación Dosis-Respuesta a Droga , Endocitosis/efectos de los fármacos , Regulación de la Expresión Génica , Genes Reporteros , Células HeLa , Humanos , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/genética , Plásmidos/químicaRESUMEN
Recombinant retroviruses provide highly efficient gene delivery and the potential for sustained gene expression, but suffer from significant disadvantages including low titer, expensive production, poor stability and limited flexibility for modification of tropism. In contrast, polymer-based vectors are more robust and allow cell- and tissue-specific deliveries via conjugation of ligands, but are comparatively inefficient. The design of hybrid gene delivery agents comprising both virally derived and synthetic materials (nanobiovectors) represents a promising approach to development of safe and efficient gene therapy vectors. Non-infectious murine leukemia virus-like particles (M-VLPs) were electrostatically complexed with chitosan (χ) to replace the function of the viral envelope protein. At optimal fabrication conditions and compositions, ranging from 6 to 9µg chitosan/10(9) M-VLPs at 10×10(9)M-VLPs/ml to 40µg chitosan/10(9) M-VLPs at 2.5×10(9)M-VLPs/ml, χ/M-VLPs were ~300-350nm in diameter and exhibited efficient transfection similar to amphotropic MLV vectors. In addition, these nanobiovectors were non-cytotoxic and provided sustained transgene expression for at least three weeks in vitro. This combination of biocompatible synthetic agents with inactive viral particles to form a highly efficient hybrid vector is a significant extension in the development of novel gene delivery platforms.
Asunto(s)
Quitosano/química , Vectores Genéticos/química , Virus de la Leucemia Murina/química , Transfección/métodos , Virión/química , Vectores Genéticos/genética , Células HEK293 , Humanos , Virus de la Leucemia Murina/genética , Virión/genética , Virión/ultraestructuraRESUMEN
PURPOSE: Pulsatile delivery of proteins, in which release occurs over a short time after a period of little or no release, is desirable for many applications. This paper investigates the effect of biodegradable polymer shell thickness on pulsatile protein release from biodegradable polymer microcapsules. METHODS: Using precision particle fabrication (PPF) technology, monodisperse microcapsules were fabricated encapsulating bovine serum albumin (BSA) in a liquid core surrounded by a drug-free poly(lactide-co-glycolide) (PLG) shell of uniform, controlled thickness from 14 to 19 µm. RESULTS: When using high molecular weight PLG (Mw 88 kDa), microparticles exhibited the desired core-shell structure with high BSA loading and encapsulation efficiency (55-65%). These particles exhibited very slow release of BSA for several weeks followed by rapid release of 80-90% of the encapsulated BSA within 7 days. Importantly, with increasing shell thickness the starting time of the pulsatile release could be controlled from 25 to 35 days. CONCLUSIONS: Biodegradable polymer microcapsules with precisely controlled shell thickness provide pulsatile release with enhanced control of release profiles.
Asunto(s)
Cápsulas/química , Poliglactina 910/química , Proteínas/química , Plásticos Biodegradables/química , Composición de Medicamentos/métodos , Peso Molecular , Tamaño de la Partícula , Polímeros/química , Albúmina Sérica Bovina/químicaRESUMEN
In-stent restenosis occurs in coronary arteries after implantation of drug-eluting stents with non-uniform restenosis thickness distribution in the artery cross section. Knowledge of the spatio-temporal drug uptake in the arterial wall is useful for investigating restenosis growth but may often be very expensive/difficult to acquire experimentally. In this study, local delivery of a hydrophobic drug from a drug-eluting stent implanted in a coronary artery is mathematically modelled to investigate the drug release and spatio-temporal drug distribution in the arterial wall. The model integrates drug diffusion in the coating and drug diffusion with reversible binding in the arterial wall. The model is solved by the finite volume method for both high and low drug loadings relative to its solubility in the stent coating with varied isotropic-anisotropic vascular drug diffusivities. Drug release profiles in the coating are observed to depend not only on the coating drug diffusivity but also on the properties of the surrounding arterial wall. Time dependencies of the spatially averaged free- and bound-drug levels in the arterial wall on the coating and vascular drug diffusivities are discussed. Anisotropic vascular drug diffusivities result in slightly different average drug levels in the arterial wall but with very different spatial distributions. Higher circumferential vascular diffusivity results in more uniform drug loading in the upper layers and is potentially beneficial in reducing in-stent restenosis. An analytical expression is derived which can be used to determine regions in the arterial with higher free-drug concentration than bound-drug concentration.
Asunto(s)
Vasos Coronarios/metabolismo , Stents Liberadores de Fármacos , Modelos Biológicos , Anisotropía , Difusión , FarmacocinéticaRESUMEN
Polymeric composite microspheres consisting of a poly(D,L-lactic-co-glycolic acid) (PLGA) core surrounded by a poly(D,L-lactic acid) (PDLLA) shell layer were successfully fabricated by coaxial electrohydrodynamic atomization (CEHDA) process. Process conditions, including nozzle voltage and polymer solution flow rates, as well as solution parameters, such as polymer concentrations, were investigated to ensure the formation of composite microspheres with a doxorubicin-loaded PLGA core surrounded by a relatively drug-free PDLLA shell layer. Various microsphere formulations were fabricated and characterized in terms of their drug distribution, encapsulation efficiency and in vitro release. Numerical simulation of CEHDA process was performed based on a computational fluid dynamics (CFD) model in Fluent by employing the process conditions and fluid properties used in the experiments. The simulation results were compared with the experimental work to illustrate the capability of the CFD model to predict the production of consistent compound droplets, and hence, the expected core-shell structured microspheres.
RESUMEN
Biodegradable polymer microparticles are promising delivery depots for protein therapeutics due to their relatively simple fabrication and facile administration. Double-wall microspheres (DWMS) comprising a core and shell made of two distinct polymers may provide enhanced control of the drug release profiles. Using precision particle fabrication (PPF) technology, monodisperse DWMS were fabricated with model protein bovine serum albumin (BSA)-loaded poly(lactide-co-glycolide) (PLG) core and drug-free poly(d,l-lactic acid) (PDLL) shell of uniform thickness. Monolithic single-wall microspheres were also fabricated to mimic the BSA-loaded PLG core. Using ethyl acetate and dichloromethane as shell- and core-phase solvents, respectively, BSA was encapsulated selectively in the core region within DWMS with higher loading and encapsulation efficiency compared to using dichloromethane as core and shell solvents. BSA in vitro release rates were retarded by the presence of the drug-free PDLL shell. Moreover, increasing PDLL shell thickness resulted in decreasing BSA release rate. With a 14-µm thick PDLL shell, an extended period of constant-rate release was achieved.
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Materiales Biocompatibles/química , Preparaciones de Acción Retardada/química , Ácido Láctico/química , Ácido Poliglicólico/química , Albúmina Sérica Bovina/administración & dosificación , Animales , Bovinos , Microesferas , Tamaño de la Partícula , Poliésteres , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Polímeros/química , PorosidadRESUMEN
The therapeutic efficiency of combined chemotherapy and gene therapy on human hepatocellular carcinoma HepG2 cells was investigated using double-walled microspheres that consisted of a poly(D,L-lactic-co-glycolic acid) (PLGA) core surrounded by a poly(L-lactic acid) (PLLA) shell layer and fabricated via the precision particle fabrication (PPF) technique. Here, double-walled microspheres were used to deliver doxorubicin (Dox) and/or chitosan-DNA nanoparticles containing the gene encoding the p53 tumor suppressor protein (chi-p53), loaded in the core and shell phases, respectively. Preliminary studies on chi-DNA nanoparticles were performed to optimize gene transfer to HepG2 cells. The transfection efficiency of chi-DNA nanoparticles was optimal at an N/P ratio of 7. In comparison to the 25-kDa branched polyethylenimine (PEI), chitosan showed no inherent toxicity towards the cells. Next, the therapeutic efficiencies of Dox and/or chi-p53 in microsphere formulations were compared to free drug(s) and evaluated in terms of growth inhibition, and cellular expression of tumor suppressor p53 and apoptotic caspase 3 proteins. Overall, the combined Dox and chi-p53 treatment exhibited enhanced cytotoxicity as compared to either Dox or chi-p53 treatments alone. Moreover, the antiproliferative effect was more substantial when cells were treated with microspheres than those treated with free drugs. High p53 expression was maintained during a five-day period, and was largely due to the controlled and sustained release of the microspheres. Moreover, increased activation of caspase 3 was observed, and was likely to have been facilitated by high levels of p53 expression. Overall, double-walled microspheres present a promising dual anticancer delivery system for combined chemotherapy and gene therapy.
Asunto(s)
Carcinoma Hepatocelular/tratamiento farmacológico , Quitosano/química , Doxorrubicina/uso terapéutico , Terapia Genética , Neoplasias Hepáticas/tratamiento farmacológico , Microesferas , Proteína p53 Supresora de Tumor/genética , Carcinoma Hepatocelular/patología , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Terapia Combinada , ADN/metabolismo , Doxorrubicina/farmacología , Técnica del Anticuerpo Fluorescente , Células Hep G2 , Humanos , Neoplasias Hepáticas/patología , Luciferasas/metabolismo , Nanopartículas/química , Proteína p53 Supresora de Tumor/uso terapéuticoRESUMEN
The drug release and degradation behavior of two double-walled microsphere formulations consisting of a doxorubicin-loaded poly(d,l-lactic-co-glycolic acid) (PLGA) core (â¼46 kDa) surrounded by a poly(d,l-lactic acid) (PDLLA) shell layer (â¼55 and 116 kDa) were examined. It was postulated that different molecular weights of the shell layer could modulate the erosion of the outer coating and limit the occurrence of water penetration into the inner drug-loaded core on various time scales, and therefore control the drug release from the microspheres. For both microsphere formulations, the drug release profiles were observed to be similar. The degradation of the microspheres was monitored for a period of about nine weeks and analyzed using scanning electron microscopy, laser scanning confocal microscopy, and gel permeation chromatography. Interestingly, both microsphere formulations exhibited occurrence of bulk erosion of PDLLA on a similar time scale despite different PDLLA molecular weights forming the shell layer. The shell layer of the double-walled microspheres served as an effective diffusion barrier during the initial lag phase period and controlled the release rate of the hydrophilic drug independent of the molecular weight of the shell layer.
Asunto(s)
Doxorrubicina/farmacología , Ácido Láctico/química , Microesferas , Ácido Poliglicólico/química , Composición de Medicamentos , Fluorescencia , Microscopía Electrónica de Rastreo , Peso Molecular , Imagen Óptica , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Porosidad , Reología/efectos de los fármacos , Factores de TiempoRESUMEN
Biodegradable polymer double-wall microspheres (DWMS) are promising vehicles for macromolecular therapeutics such as proteins and peptides. Using precision particle fabrication (PPF) technology, uniform DWMS with outer diameter approximately 55 µm were fabricated comprising poly(lactide-co-glycolide) cores encapsulating bovine serum albumin (BSA) and approximately 10 µm thick, drug-free, poly(lactic acid) (PLA) shells of varying PLA molecular weight. Also, monolithic single-wall microspheres (SWMS) were fabricated to mimic the BSA-loaded core. The use of relatively fast-extracting ethyl acetate and slowly extracting dichloromethane as shell- and core-phase solvents, respectively, was found to produce DWMS with well-defined core-shell structure, high BSA encapsulation efficiency, and the desired localization of protein in the particle core. Initial protein distribution, particle erosion, and in vitro protein release from DWMS and SWMS were examined. The presence of a BSA-free shell in DWMS decreased the protein release rate and extended the duration of release from approximately 50 days to 70-80 days, demonstrating the capacity of such DWMS to provide enhanced control of protein delivery rates.