RESUMO
While limited drug loading continues to be problematic for chemotherapeutics formulated in nanoparticles, we found that we could take advantage of colloidal drug aggregation to achieve high loading when combined with polymeric excipients. We demonstrate this approach with two drugs, fulvestrant and pentyl-PABC doxazolidine (PPD; a prodrug of doxazolidine, which is a DNA cross-linking anthracycline), and two polymers, polysorbate 80 (UP80) and poly(d,l-lactide-co-2-methyl-2-carboxytrimethylene carbonate)-graft-poly(ethylene glycol) (PLAC-PEG; a custom-synthesized, self-assembling amphiphilic polymer). In both systems, drug-loaded nanoparticles had diameters < 200 nm and were stable for up to two days in buffered saline solution and for up to 24 h in serum-containing media at 37 °C. While colloidal drug aggregates alone are typically unstable in saline and serum-containing media, we attribute the colloid stability observed herein to the polymeric excipients and consequent decreased protein adsorption. We expect this strategy of polymer-stabilized colloidal drug aggregates to be broadly applicable in delivery formulations.
Assuntos
Coloides/química , Sistemas de Liberação de Medicamentos/métodos , Nanopartículas/química , Doxorrubicina/análogos & derivados , Doxorrubicina/química , Portadores de Fármacos/química , Micelas , Oxazóis/química , Polímeros/químicaRESUMO
While the formation of colloidal aggregates leads to artifacts in early drug discovery, their composition makes them attractive as nanoparticle formulations for targeted drug delivery as the entire nanoparticle is composed of drug. The typical transient stability of colloidal aggregates has inhibited exploiting this property. To overcome this limitation, we investigated a series of proteins to stabilize colloidal aggregates of the chemotherapeutic, fulvestrant, including the following: bovine serum albumin, a generic human immunoglobulin G, and trastuzumab, a therapeutic human epidermal growth factor receptor 2 antibody. Protein coronas reduced colloid size to <300 nm and improved their stability to over 48 h in both buffered saline and media containing serum protein. Unlike colloids stabilized with other proteins, trastuzumab-fulvestrant colloids were taken up by HER2 overexpressing cells and were cytotoxic. This new targeted formulation reimagines antibody-drug conjugates, delivering mM concentrations of drug to a cell.
Assuntos
Estradiol/química , Antineoplásicos , Neoplasias da Mama , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Estradiol/análogos & derivados , Fulvestranto , Humanos , Nanopartículas , Receptor ErbB-2 , TrastuzumabRESUMO
Colloidal aggregates of small molecules are the most common artifact in early drug discovery, sequestering and inhibiting target proteins without specificity. Understanding their structure and mechanism has been crucial to developing tools to control for, and occasionally even exploit, these particles. Unfortunately, their polydispersity and transient stability have prevented exploration of certain elementary properties, such as how they pack. Dye-stabilized colloidal aggregates exhibit enhanced homogeneity and stability when compared to conventional colloidal aggregates, enabling investigation of some of these properties. By small-angle X-ray scattering and multiangle light scattering, pair distance distribution functions suggest that the dye-stabilized colloids are filled, not hollow, spheres. Stability of the coformulated colloids enabled investigation of their preference for binding DNA, peptides, or folded proteins, and their ability to purify one from the other. The coformulated colloids showed little ability to bind DNA. Correspondingly, the colloids preferentially sequestered protein from even a 1600-fold excess of peptides that are themselves the result of a digest of the same protein. This may reflect the avidity advantage that a protein has in a surface-to-surface interaction with the colloids. For the first time, colloids could be shown to have preferences of up to 90-fold for particular proteins over others. Loaded onto the colloids, bound enzyme could be spun down, resuspended, and released back into buffer, regaining most of its activity. Implications of these observations for colloid mechanisms and utility will be considered.
Assuntos
Peptídeos/química , Proteínas/química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Coloides , Corantes/química , Vermelho Congo/química , DNA/química , DNA/metabolismo , Difusão Dinâmica da Luz , Estradiol/análogos & derivados , Estradiol/química , Fluoresceínas/química , Fulvestranto , Niacinamida/análogos & derivados , Niacinamida/química , Peptídeos/metabolismo , Compostos de Fenilureia/química , Ligação Proteica , Dobramento de Proteína , Proteínas/metabolismo , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Espalhamento a Baixo Ângulo , Sorafenibe , Tripsina/química , Tripsina/metabolismo , Inibidores da Tripsina/química , Inibidores da Tripsina/metabolismo , Difração de Raios X , beta-Lactamases/química , beta-Lactamases/metabolismoRESUMO
Encapsulation of therapeutic molecules within polymer particles is a well-established method for achieving controlled release, yet challenges such as low loading, poor encapsulation efficiency, and loss of protein activity limit clinical translation. Despite this, the paradigm for the use of polymer particles in drug delivery has remained essentially unchanged for several decades. By taking advantage of the adsorption of protein therapeutics to poly(lactic-co-glycolic acid) (PLGA) nanoparticles, we demonstrate controlled release without encapsulation. In fact, we obtain identical, burst-free, extended-release profiles for three different protein therapeutics with and without encapsulation in PLGA nanoparticles embedded within a hydrogel. Using both positively and negatively charged proteins, we show that short-range electrostatic interactions between the proteins and the PLGA nanoparticles are the underlying mechanism for controlled release. Moreover, we demonstrate tunable release by modifying nanoparticle concentration, nanoparticle size, or environmental pH. These new insights obviate the need for encapsulation and offer promising, translatable strategies for a more effective delivery of therapeutic biomolecules.
Assuntos
Preparações de Ação Retardada , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Ácido Láctico , Nanopartículas , Ácido Poliglicólico , Adsorção , Fator Neurotrófico Derivado do Encéfalo/administração & dosagem , Fator Neurotrófico Derivado do Encéfalo/farmacocinética , Portadores de Fármacos/química , Composição de Medicamentos , Liberação Controlada de Fármacos , Concentração de Íons de Hidrogênio , Ácido Láctico/química , Método de Monte Carlo , Nanopartículas/química , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Proteínas/administração & dosagem , Proteínas/química , Proteínas/farmacocinética , Eletricidade EstáticaRESUMO
Small molecule aggregates are considered nuisance compounds in drug discovery, but their unusual properties as colloids could be exploited to form stable vehicles to preserve protein activity. We investigated the coaggregation of seven molecules chosen because they had been previously intensely studied as colloidal aggregators, coformulating them with bis-azo dyes. The coformulation reduced colloid sizes to <100 nm and improved uniformity of the particle size distribution. The new colloid formulations are more stable than previous aggregator particles. Specifically, coaggregation of Congo Red with sorafenib, tetraiodophenolphthalein (TIPT), or vemurafenib produced particles that are stable in solutions of high ionic strength and high protein concentrations. Like traditional, single compound colloidal aggregates, the stabilized colloids adsorbed and inhibited enzymes like ß-lactamase, malate dehydrogenase, and trypsin. Unlike traditional aggregates, the coformulated colloid-protein particles could be centrifuged and resuspended multiple times, and from resuspended particles, active trypsin could be released up to 72 h after adsorption. Unexpectedly, the stable colloidal formulations can sequester, stabilize, and isolate enzymes by spin-down, resuspension, and release.
Assuntos
Coloides/administração & dosagem , beta-Lactamases/metabolismo , Tamanho da PartículaRESUMO
Despite widespread clinical use, delivery of taxane chemotherapeutics remains a challenge due to poor solubility and lack of selectively. Polymeric nanomicelle strategies have been pursued to overcome these issues; however current formulations are often limited by low drug loading and poor serum stability. To achieve a drug delivery system that addresses these issues, poly(D,L-lactide-co-2-methyl-2-carboxytrimethylene carbonate)-g-poly(ethylene glycol) was covalently modified with the taxol binding peptide a peptide from the ß-tubulin-taxane binding site. This modification resulted in drug loadings five times higher than unmodified polymers, which is significantly higher than typical hydrophobic modifications, including with benzyl and docetaxel functionalization. Unlike many formulations with high drug loading, these nanomicelles were stable in serum for up to 24 h and maintained docetaxel cytotoxicity. By incorporating the taxane binding peptide into the polymer chemistry, a new twist was applied to an old problem, which is broadly applicable to other polymeric micelle systems and drug-peptide combinations in general.
Assuntos
Antineoplásicos/administração & dosagem , Portadores de Fármacos/metabolismo , Paclitaxel/metabolismo , Peptídeos/metabolismo , Taxoides/administração & dosagem , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Sítios de Ligação , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Linhagem Celular Tumoral , Docetaxel , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos , Humanos , Micelas , Dados de Sequência Molecular , Neoplasias/tratamento farmacológico , Peptídeos/química , Polietilenoglicóis/química , Polietilenoglicóis/metabolismo , Taxoides/metabolismo , Taxoides/farmacologiaRESUMO
Drug efficacy does not always increase sigmoidally with concentration, which has puzzled the community for decades. Unlike standard sigmoidal curves, bell-shaped concentration-response curves suggest more complex biological effects, such as multiple-binding sites or multiple targets. Here, we investigate a physical property-based mechanism for bell-shaped curves. Beginning with the observation that some drugs form colloidal aggregates at relevant concentrations, we determined concentration-response curves for three aggregating anticancer drugs, formulated both as colloids and as free monomer. Colloidal formulations exhibited bell-shaped curves, losing activity at higher concentrations, while monomeric formulations gave typical sigmoidal curves, sustaining a plateau of maximum activity. Inverting the question, we next asked if molecules with bell-shaped curves, reported in the literature, form colloidal aggregates at relevant concentrations. We selected 12 molecules reported to have bell-shaped concentration-response curves and found that five of these formed colloids. To understand the mechanism behind the loss of activity at concentrations where colloids are present, we investigated the diffusion of colloid-forming dye Evans blue into cells. We found that colloidal species are excluded from cells, which may explain the mechanism behind toxicological screens that use Evans blue, Trypan blue, and related dyes.
Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/química , Relação Dose-Resposta a Droga , Antineoplásicos/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Proliferação de Células/efeitos dos fármacos , Química Farmacêutica , Coloides , Azul Evans/administração & dosagem , Azul Evans/química , Humanos , Células MCF-7 , Estrutura MolecularRESUMO
RNA interference (RNAi) is a powerful therapeutic strategy that induces gene silencing by targeting disease-causing mRNA and can lead to their removal through degradation pathways. The potential of RNAi is especially relevant in cancer therapy, as it can be designed to regulate the expression of genes involved in all stages of tumor development (initiation, growth, and metastasis). We have generated gene silencing 3D DNA prisms that integrate antisense oligonucleotide therapeutics at 1, 2, 4, and 6 positions. Synthesis of these structures is readily achieved and leads to the assembly of highly monodisperse and well-characterized structures. We have shown that antisense strands scaffolded on DNA cages can readily induce gene silencing in mammalian cells and maintain gene knockdown levels more effectively than single and double stranded controls through increased stability of bound antisense units.
Assuntos
DNA/genética , Inativação Gênica/fisiologia , Células HeLa , Humanos , Luciferases de Vaga-Lume/genética , RNA Interferente Pequeno/genética , Análise de Sequência de DNARESUMO
Nature uses a combination of non-covalent interactions to create a hierarchy of complex systems from simple building blocks. One example is the selective association of the hydrophobic side chains that are a strong determinant of protein organization. Here, we report a parallel mode of assembly in DNA nanotechnology. Dendritic alkyl-DNA conjugates are hybridized to the edges of a DNA cube. When four amphiphiles are on one face, the hydrophobic residues of two neighbouring cubes engage in an intermolecular 'handshake', resulting in a dimer. When there are eight amphiphiles (four on the top and bottom cube faces, respectively), they engage in an intramolecular 'handshake' inside the cube. This forms the first example of a monodisperse micelle within a DNA nanostructure that encapsulates small molecules and releases them by DNA recognition. Creating a three-dimensional pattern of hydrophobic patches, like side chains in proteins, can result in specific, directed association of hydrophobic domains with orthogonal interactions to DNA base-pairing.
Assuntos
DNA/química , Dendrímeros/química , Eletroforese em Gel Bidimensional , Interações Hidrofóbicas e Hidrofílicas , Micelas , Modelos MolecularesRESUMO
Simple chemical modifications to oligonucleotide ends with hexaethylene glycol and hexanediol are shown to significantly increase nuclease resistance under serum conditions. The modified oligonucleotides were used to construct DNA prismatic cages in a single step and in quantitative yield. These cages further stabilize their strands towards nucleases, with lifetimes of 62 hours in serum. The cages contain a large number of single-stranded regions for functionalization, illustrating their versatility for biological applications.
Assuntos
DNA/metabolismo , Nanoestruturas/química , Animais , Bovinos , DNA/sangue , DNA/química , Desoxirribonucleases/metabolismo , Etilenoglicóis/química , Desnaturação de Ácido Nucleico , Oligonucleotídeos/químicaRESUMO
The C-linked phenolic adduct, C8-(2â³-hydroxyphenyl)-2'-deoxyguanosine (o-PhOHdG), has been employed to study the impact of N7-metalation of 2'-deoxyguanosine (dG) within duplex DNA. The phenolic group of o-PhOHdG assists selective metal ion coordination by the N7-site of the attached dG moiety, which is the most important metal binding site in duplex DNA. The biaryl nucleobase probe o-PhOHdG is highly fluorescent in water (Φ(fl) = 0.44), and changes in its absorption and emission were used to determine apparent association constants (K(a)) for binding to Cu(II), Ni(II), and Zn(II). The nucleoside was found to bind Cu(II) (log K(a) = 4.59) and Ni(II) (log K(a) = 3.65) effectively, but it showed relatively poor affinity for Zn(II) (log K(a) = 2.55). The fluorescent nucleobase o-PhOHdG was incorporated into a pyrimidine-rich oligonucleotide substrate (ODN1) and a purine-rich (ODN2) substrate to monitor selective binding of Cu(II) through fluorescence quenching of the enol emission of o-PhOHdG within the DNA substrates. The pyrimidine-rich substrate ODN1 was found to possess greater affinity for Cu(II) than the free nucleobase, while the purine-rich substrate ODN2 exhibited diminished Cu(II) binding affinity. The impact of Cu(II) on duplex stability and structure was determined using UV melting temperature analysis and circular dichroism (CD) measurements. These studies highlight the syn preference for Cu(II)-bound o-PhOHdG within ODN1 duplexes and demonstrate competitive Cu(II) binding by other natural dG nucleobases within ODN2. The metal binding properties of o-PhOHdG are compared to the structurally similar 2-(2'-hydroxyphenyl)benzoxazole (HBO) derivatives and the nucleoside C8-(2-pyridyl)-dG (2PydG) that has also been used to control N7-metal coordination in DNA. Our results show certain advantages to the use of o-PhOHdG that stem from its highly fluorescent nature in aqueous media and provide additional tools for studying the effects of N7-metalation on the structure and stability of duplex DNA.
Assuntos
Carbono/química , Adutos de DNA/química , Desoxiguanosina/química , Corantes Fluorescentes/química , Metais Pesados/química , Nitrogênio/química , Fenóis/química , Sequência de Bases , Adutos de DNA/genética , Adutos de DNA/metabolismo , Desoxiguanosina/metabolismo , Metais Pesados/metabolismo , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Especificidade por SubstratoRESUMO
Here, we introduce a 3D-DNA construction method that assembles a minimum number of DNA strands in quantitative yield, to give a scaffold with a large number of single-stranded arms. This DNA frame is used as a core structure to organize other functional materials in 3D as the shell. We use the ring-opening metathesis polymerization (ROMP) to generate block copolymers that are covalently attached to DNA strands. Site-specific hybridization of these DNA-polymer chains on the single-stranded arms of the 3D-DNA scaffold gives efficient access to DNA-block copolymer cages. These biohybrid cages possess polymer chains that are programmably positioned in three dimensions on a DNA core and display increased nuclease resistance as compared to unfunctionalized DNA cages.
Assuntos
DNA/química , Polímeros/química , Modelos Moleculares , Estrutura Molecular , Tamanho da Partícula , Propriedades de SuperfícieRESUMO
The powerful self-assembly features of DNA make it a unique template to finely organize and control matter on the nanometre scale. While DNA alone offers a high degree of fidelity in its self-assembly, a new area of research termed 'supramolecular DNA assembly' has recently emerged. This field combines DNA building blocks with synthetic organic, inorganic and polymeric structures. It thus brings together the toolbox of supramolecular chemistry with the predictable and programmable nature of DNA. The result of this molecular partnership is a variety of hybrid architectures, that expand DNA assembly beyond the boundaries of Watson-Crick base pairing into new structural and functional properties. In this tutorial review we outline this emerging field of study, and describe recent research aiming to synergistically combine the properties inherent to DNA with those of a number of supramolecular scaffolds. This ultimately creates structures with numerous potential applications in materials science, catalysis and medicine.
Assuntos
DNA/química , Nanotecnologia , Elementos de Transição/químicaRESUMO
We describe a rapid and quantitative method to generate DNA cages of deliberately designed geometry from readily available starting strands. Balancing the incorporation of sequence uniqueness and symmetry in a face-centered approach to 3D construction can result in triangular (TP), rectangular (RP), and pentagonal prisms (PP) without compromising the potential for nanostructure addressability.
Assuntos
DNA/química , Nanoestruturas/química , Conformação de Ácido Nucleico , TemperaturaRESUMO
The site-specific insertion of organic and inorganic molecules into DNA nanostructures can provide unique structural and functional capabilities. We have demonstrated the inclusion of two types of molecules. The first is a diphenylphenanthroline (dpp, 1) molecule that is site specifically inserted into DNA strands and which can be used as a template to create metal-coordinating pockets. These building blocks can then be used to assemble metal-DNA 2D and 3D structures, including metal-DNA triangles, described here. The second insertion is a triaryl molecule that provides geometric control in the preparation of 2D single-stranded DNA templates. These can be designed to further assemble into geometrically well-defined nanotubes. Here, we detail the steps involved in the construction of metal-DNA triangles and DNA nanotubes using these methods.
Assuntos
DNA/química , Metais/química , Nanotecnologia/métodos , Nanotubos/química , Microscopia de Força Atômica , Conformação de Ácido Nucleico , Fenantrolinas/químicaRESUMO
Nanotubes hold promise for a number of biological and materials applications because of their high aspect ratio and encapsulation potential. A particularly attractive goal is to access nanotubes that exert well-defined control over their cargo, such as selective encapsulation, precise positioning of the guests along the nanotube length and triggered release of this cargo in response to specific external stimuli. Here, we report the construction of DNA nanotubes with longitudinal variation and alternating larger and smaller capsules along the tube length. Size-selective encapsulation of gold nanoparticles into the large capsules of these tubes leads to 'nanopeapod' particle lines with positioning of the particles 65 nm apart. These nanotubes can then be opened when specific DNA strands are added to release their particle cargo spontaneously. This approach could lead to new applications of self-assembled nanotubes, such as in the precise organization of one-dimensional nanomaterials, gene-triggered selective delivery of drugs and biological sensing.
Assuntos
DNA/química , Nanotubos/química , Cápsulas , DNA/metabolismo , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Ouro/química , Cinética , Nanopartículas Metálicas/química , Modelos Moleculares , Conformação de Ácido Nucleico , Tamanho da Partícula , Análise Espectral , Especificidade por SubstratoRESUMO
A straightforward strategy to stably anchor one or more gold nanoparticles (AuNPs) at both the internal and terminal positions of single-stranded DNA is presented. Discrete DNA-AuNP conjugates are isolated using a noncovalent extension strand strategy, that helps to resolve prepared species by agarose gel electrophoresis (AGE). These are used to assemble well-defined AuNP squares and rectangles. Two complementary bis-AuNP-labeled DNA conjugates are then prepared. One of these places two smaller (5 nm) AuNPs at defined internal positions within a DNA strand, and the other places two larger (13 nm) AuNPs at each of its terminal positions. We show the self-assembly of these bis-AuNP conjugates into a tapered tetrameric gold nanoparticle "antenna" structures of direct relevance to engineered "hot spots" and surface enhanced Raman scattering (SERS) substrates.
Assuntos
DNA/química , Ouro/química , Nanopartículas Metálicas/química , Sítios de Ligação , Tamanho da Partícula , Análise Espectral Raman , Compostos de Sulfidrila/química , Propriedades de SuperfícieRESUMO
DNA nanotubes can template the growth of nanowires, orient transmembrane proteins for nuclear magnetic resonance determination, and can potentially act as stiff interconnects, tracks for molecular motors and nanoscale drug carriers. Current methods for the construction of DNA nanotubes result in symmetrical and cylindrical assemblies that are entirely double-stranded. Here, we report a modular approach to DNA nanotube synthesis that provides access to geometrically well-defined triangular and square-shaped DNA nanotubes. We also construct the first nanotube assemblies that can exist in double- and single-stranded forms with significantly different stiffness. This approach allows for parameters such as geometry, stiffness, and single- or double-stranded character to be fine-tuned, and could enable the creation of designer nanotubes for a range of applications, including the growth of nanowires of controlled shape, the loading and release of cargo, and the real-time modulation of stiffness and persistence length within DNA interconnects.