RESUMO
Aberrant splicing and protein interaction of Ras binding domain (RBD) are associated with melanoma drug resistance. Here, cobalt or nickel doped zinc oxide (ZnO) physiometacomposite (PMC) materials bind to RNA and peptide shown by Ninhydrin staining, UV-vis, Fourier transform infrared, and circular dichroism spectroscopy. PMCs deliver splice switching oligomer (SSO) into melanoma cells or 3-D tumor spheroids shown by flow cytometry, fluorescence microscopy, and bioluminescence. Stability in serum, liver, or tumor homogenate up to 48 h and B16F10 melanoma inhibition ≥98-99% is shown. These data suggest preclinical potential of PMC for delivery of SSO, RBD, or other nucleic acid therapeutic and anticancer peptides.
RESUMO
Physiological metals such as zinc, magnesium, and nickel facilitate nucleic acid and protein interactions and stability. In the nanoscale, the impact these have on nucleic acid structure-function is very poorly understood and was investigated here. Nanoparticles' (NP) RNA precipitation efficiency was in the order; NiOâ¯>â¯MgOâ¯>â¯ZnOâ¯>â¯CaOâ¯>â¯CaCO3>Cu. Gel mobility shift was observed for MgO and especially ZnO NP. Loss of staining intensity was shown for Cu suggesting this NP may denature RNA supported by the UV- and CD-spectroscopy patterns, change in area-under-the-curve (AUC) and abs260 nm measurements. Aptamer and triplex-forming oligomer (TFO) sequences were designed targeting RAS/Ras binding domain (RBD) and the impact of the NP on target interaction investigated. MgO NP promotes aptamer:RBD interaction and preserves triplex formation whereas NiO NP effects duplex migration and intensifies staining of the triplex suggesting a novel mechanism of interaction and conformation. These data strongly support the role of MgO, ZnO and NiO NP for nucleic acid nanobio interaction and suggest potential biomedical application for such novel interfaces.
Assuntos
Aptâmeros de Nucleotídeos/farmacologia , Nanopartículas Metálicas/química , Proteínas Proto-Oncogênicas c-raf/metabolismo , RNA/farmacologia , Aptâmeros de Nucleotídeos/química , Humanos , Óxido de Magnésio/química , Níquel/química , Conformação de Ácido Nucleico/efeitos dos fármacos , Óxidos/química , Domínios e Motivos de Interação entre Proteínas/efeitos dos fármacos , RNA/química , Óxido de Zinco/químicaRESUMO
The drug release profiles of doxorubicin-loaded electrospun fiber mats were investigated with regard to drug-polymer miscibility, fiber wettability and degradability. Doxorubicin in hydrophilic form (Dox-HCl) and hydrophobic free base form (Dox-base) was employed as model drugs, and an aliphatic polyester, poly(lactic acid) (PLA), was used as a drug-carrier matrix. When hydrophilic Dox-HCl was directly mixed with PLA solution, drug molecules formed large aggregates on the fiber surface or in the fiber core, due to poor drug-polymer compatibility. Drug aggregates on the fiber surface contributed to the rapid initial release. The hydrophobic form of Dox-base was dispersed better with PLA matrix compared to Dox-HCl. When dimethyl sulfoxide (DMSO) was used as the solvent for Dox-HCl, the miscibility of drug in the polymer matrix was significantly improved, forming a quasi-monolithic solution scheme. The drug release from this monolithic matrix was slowest, and this slow release led to a lower toxicity to hepatocellular carcinoma. When an enzyme was used to promote PLA degradation, the release rates were closely correlated with degradation rates, demonstrating degradation was the dominant release mechanism. The possible drug release mechanisms were speculated based on the release kinetics. The results suggest that manipulation of drug-polymer miscibility and polymer degradability can be an effective means of designing drug release profiles.
RESUMO
Many bioactive molecules have intracellular targets, but have difficulty crossing the cell membrane to reach those targets. To address this difficulty, we fabricated arrays of nanoneedles to gently and simultaneously puncture 10(5) cells and thereby provide transient pathways for transport of molecules into the cells. The nanoneedles were microfabricated by etching silicon to create arrays of nanoneedles measuring 12 µm in height, tapering to a sharp tip less than 30 nm wide to facilitate puncture into cells and spaced 10 µm apart in order to have at least one nanoneedle puncture each cell in a confluent monolayer. These nanoneedles were used for intracellular delivery in two ways: puncture loading, in which nanoneedle arrays were pressed into cell monolayers, and centrifuge loading, in which cells in suspension were spun down onto nanoneedle arrays. The effects on intracellular uptake and cell viability were determined as a function of nanoneedle length and sharpness, puncture force and duration, and molecular weight of the molecule delivered. Under optimal conditions, intracellular uptake was seen in approximately 50 % of cells while maintaining high cell viability. Overall, this study provides a comparative analysis of intracellular delivery using nanoneedle arrays by two different loading methods over a range of operating parameters.
Assuntos
Sistemas de Liberação de Medicamentos/instrumentação , Sistemas de Liberação de Medicamentos/métodos , Nanoestruturas , Agulhas , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , MasculinoRESUMO
Effective public health responses to an influenza pandemic require an effective vaccine that can be manufactured and administered to large populations in the shortest possible time. In this study, we evaluated a method for vaccination against avian influenza virus that uses a DNA vaccine for rapid manufacturing and delivered by a microneedle skin patch for simplified administration and increased immunogenicity. We prepared patches containing 700-µm long microneedles coated with an avian H5 influenza hemagglutinin DNA vaccine from A/Viet Nam/1203/04 influenza virus. The coating DNA dose increased with DNA concentration in the coating solution and the number of dip-coating cycles. Coated DNA was released into the skin tissue by dissolution within minutes. Vaccination of mice using microneedles induced higher levels of antibody responses and hemagglutination inhibition titers, and improved protection against lethal infection with avian influenza as compared to conventional intramuscular delivery of the same dose of the DNA vaccine. Additional analysis showed that the microneedle coating solution containing carboxymethylcellulose and a surfactant may have negatively affected the immunogenicity of the DNA vaccine. Overall, this study shows that DNA vaccine delivery by microneedles can be a promising approach for improved vaccination to mitigate an influenza pandemic.
Assuntos
Vacinas contra Influenza/administração & dosagem , Vacinas contra Influenza/imunologia , Influenza Aviária/imunologia , Orthomyxoviridae/imunologia , Pele/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Animais , Formação de Anticorpos/imunologia , Aves , DNA Viral/administração & dosagem , DNA Viral/imunologia , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Imunoglobulina G/imunologia , Influenza Aviária/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Agulhas , Adesivo Transdérmico , Células Tumorais Cultivadas , Vacinação/métodosRESUMO
The impact of many biopharmaceuticals, including protein- and gene-based therapies, has been limited by the need for better methods of delivery into cells within tissues. Here, intracellular delivery of molecules and transfection with plasmid DNA by electroporation is presented using a novel microneedle electrode array designed for the targeted treatment of skin and other tissue surfaces. The microneedle array is molded out of polylactic acid. Electrodes and circuitry required for electroporation are applied to the microneedle array surface by a new metal-transfer micromolding method. The microneedle array maintains mechanical integrity after insertion into pig cadaver skin and is able to electroporate human prostate cancer cells in vitro. Quantitative measurements show that increasing electroporation pulse voltage increases uptake efficiency of calcein and bovine serum albumin, whereas increasing pulse length has lesser effects over the range studied. Uptake of molecules by up to 50% of cells and transfection of 12% of cells with a gene for green fluorescent protein is demonstrated at high cell viability. It is concluded that the microneedle electrode array is able to electroporate cells, resulting in intracellular uptake of molecules, and has potential applications to improve intracellular delivery of proteins, DNA, and other biopharmaceuticals.
Assuntos
Eletroporação/métodos , Proteínas/administração & dosagem , Transfecção/métodos , Animais , Bovinos , Linhagem Celular Tumoral , Sobrevivência Celular , Eletrodos , Fluoresceínas/administração & dosagem , Fluoresceínas/metabolismo , Humanos , Masculino , Proteínas/genética , Soroalbumina Bovina/administração & dosagem , Soroalbumina Bovina/genéticaRESUMO
Peptides and polypeptides have important pharmacological properties but only a limited number have been exploited as therapeutics because of problems related to their delivery. Most of these drugs require a parenteral delivery system which introduces the problems of pain, possible infection, and expertise required to carry out an injection. The aim of this study was to develop a transdermal patch containing microneedles (MNs) coated with a peptide drug, salmon calcitonin (sCT), as an alternative to traditional subcutaneous and nasal delivery routes. Quantitative analysis of sCT after coating and drying onto microneedles was performed with a validated HPLC method. In vivo studies were carried out on hairless rats and serum levels of sCT were determined by ELISA. The AUC value of MNs coated with a trehalose-containing formulation (250 ± 83 ng/mL min) was not significantly different as compared to subcutaneous injections (403 ± 253 ng/mL min), but approximately 13 times higher than nasal administration (18.4 ± 14.5 ng/mL min). T(max) (7.5 ± 5 min) values for MN mediated administration were 50% shorter than subcutaneous injections (15 min), possibly due to rapid sCT dissolution and absorption by dermal capillaries. These results suggest that with further optimization of coating formulations, microneedles may enable administration of sCT and other peptides without the need for hypodermic injections.
Assuntos
Calcitonina/administração & dosagem , Materiais Revestidos Biocompatíveis , Portadores de Fármacos , Administração Intranasal , Animais , Disponibilidade Biológica , Calcitonina/sangue , Calcitonina/química , Calcitonina/farmacocinética , Química Farmacêutica , Cromatografia Líquida de Alta Pressão , Ensaio de Imunoadsorção Enzimática , Desenho de Equipamento , Injeções Intradérmicas , Injeções Intravenosas , Injeções Subcutâneas , Masculino , Microinjeções , Miniaturização , Agulhas , Ratos , Ratos Pelados , Ratos Sprague-Dawley , Solubilidade , Tecnologia Farmacêutica/métodos , Adesivo TransdérmicoRESUMO
Tuberculosis (TB) caused by Mycobacterium tuberculosis continues to be a leading cause of mortality among bacterial diseases, and the bacillus Calmette-Guérin (BCG) is the only licensed vaccine for human use against this disease. TB prevention and control would benefit from an improved method of BCG vaccination that simplifies logistics and eliminates dangers posed by hypodermic needles without compromising immunogenicity. Here, we report the design and engineering of a BCG-coated microneedle vaccine patch for a simple and improved intradermal delivery of the vaccine. The microneedle vaccine patch induced a robust cell-mediated immune response in both the lungs and the spleen of guinea pigs. The response was comparable to the traditional hypodermic needle based intradermal BCG vaccination and was characterized by a strong antigen specific lymphocyte proliferation and IFN-γ levels with high frequencies of CD4(+)IFN-γ(+), CD4(+)TNF-α(+) and CD4(+)IFN-γ(+)TNF-α(+) T cells. The BCG-coated microneedle vaccine patch was highly immunogenic in guinea pigs and supports further exploration of this new technology as a simpler, safer, and compliant vaccination that could facilitate increased coverage, especially in developing countries that lack adequate healthcare infrastructure.
Assuntos
Vacina BCG/administração & dosagem , Vacina BCG/imunologia , Vacinação/instrumentação , Vacinação/métodos , Administração Cutânea , Animais , Linfócitos T CD4-Positivos/imunologia , Proliferação de Células , Feminino , Cobaias , Interferon gama/biossíntese , Pulmão/imunologia , Baço/imunologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
We have designed and fabricated a microneedle array with electrical functionality with the final goal of electroporating skin's epidermal cells to increase their transfection by DNA vaccines. The microneedle array was made of polymethylmethacrylate (PMMA) by micromolding technology from a polydimethylsiloxane (PDMS) mold, followed by metal deposition, patterning using laser ablation, and electrodeposition. This microneedle array possessed sufficient mechanical strength to penetrate human skin in vivo and was also able to electroporate both red blood cells and human prostate cancer cells as an in vitro model to demonstrate cell membrane permeabilization. A computational model to predict the effective volume for electroporation with respect to applied voltages was constructed from finite element simulation. This study demonstrates the mechanical and electrical functionalities of the first MEMS-fabricated microneedle array for electroporation, designed for DNA vaccine delivery.