DNA Micelles Research Articles

Multimodal Shape Transformation of Dual-Responsive DNA Block Copolymers.

Herein, we report the self-assembly and multimodal shape transformation of dual-responsive DNA di- and triblock copolymers. Dual-responsive DNA diblock copolymer was synthesized by coupling a thermoresponsive polymer, poly(N-isopropylacrylamide (PNIPAM), and an oligonucleotide. DNA-b-PNIPAM possesses thermoresponsive properties of PNIPAM as well as molecular recognition properties of DNA. Thus, they undergo reversible temperature-triggered transition at lower critical solution temperature (LCST) between molecular DNA and polymer micelles with high density DNA corona. The hybridization of DNA-b-PNIPAM and DNA-modified nanoparticles generates functional nanoparticles showing unique temperature-dependent aggregation and disaggregation behaviors due to the dual-responsive nature of DNA-b-PNIPAM. DNA triblock copolymers of DNA-b-PNIPAM-b-PMA were synthesized by introducing a hydrophobic block, poly(methyl acrylate) (PMA), to DNA/PNIPAM block copolymers, which form spherical micelles at room temperature. They are capable of nanoscale shape transformation through the combination of thermal trigger and DNA binding. DNA-b-PNIPAM-b-PMA micelles undergo sphere-to-cylinder shape changes above LCST due to the conformational change of PNIPAM. The shape change is reversible, and fast cylinder-to-sphere transition occurs when the temperature is lowered below LCST. The low temperature spherical morphology can also be accessed while keeping the temperature above LCST by introducing complementary DNA strands with single stranded overhang regions. These results demonstrate the multidimensional shape changing capability of DNA-b-PNIPAM-b-PMA enabled by the dual-responsive property.

Precisely-controlled DNA-templated micelle as a multifunctional delivery platform for glioblastoma therapeutics

Event Abstract Back to Event Precisely-controlled DNA-templated micelle as a multifunctional delivery platform for glioblastoma therapeutics Yeh-Hsing Lao1, Smruthi Suryaprakash1, Kyle K. Phua2 and Kam W. Leong1 1 Columbia University, Department of Biomedical Engineering, United States 2 National University of Singapore, Department of Chemical & Biomolecular Engineering, Singapore Introduction: For cancer therapy, single drug treatment may not be sufficient to completely eliminate all the cancer cells due to their heterogeneity and insensitivity against the induced inhibitory signals. How to deliver multiple anti-cancer drugs in a controlled manner is a challenge in the field. Here we design a precisely-controlled DNA-templated micelle that is able to deliver various types of anti-cancer drugs, such as chemodrugs and nucleic acids. The DNA template comprises a DNA bridge with an amine-bearing PEG segment (Fig 1A). The DNA bridge can be loaded with chemodrugs and nucleic acid payloads through intercalation and Watson-Crick base paring, respectively. On the other hand, the PEG segment provides a functional group for ligand conjugation. Payloads and ligands are preloaded before DNA micelle formation; the loading efficiency and ligand density are therefore controlled in this system. DNA templates undergo a nanoprecipitation to form micelles, and the formed DNA micelles are narrowly dispersed, colloidally stable, and with low toxicity. We evaluated this micelle in an application as glioblastoma (GBM) therapeutics: micelle/cell cluster for tumor targeting. Mesenchymal stem cell (MSC) clusters were decorated with the micelles to co-deliver two types of anti-cancer drugs. The micelle decoration did not influence the homing ability of MSC toward GBM cells, and the clusters showed faster migration toward GBM cells compared with individual MSC. Materials and Methods: PEGylated DNA templates were purified by reversed-phased HPLC using an acetonitrile/PBS mixture as an eluent. The template concentration was quantitated by UV-VIS spectroscopy. The therapeutic payloads were loaded onto the DNA templates through a gradient annealing process with a cooling rate of 1oC/min. Payload-carrying DNA micelles were formed via nanoprecipitation. To decorate the MSC cluster for micelle/cell delivery, we slightly modified the protocol published previously[1]. Briefly, DNA-templated micelles were 1:1 mixed with an alginate-containing MSC suspension, and the clusters were subsequently formed via a microfluidic technology. Results and Discussion: In this study we optimized the micelle formulation for simultaneous drug and nucleic acid loading. The size of this DNA-templated micelle was tunable and ranged from 15 to 150 nm with a narrow polydispersity (PDI < 0.1). Fig 1B shows an example of the smallest micelle in the series. Furthermore, this micelle was colloidally stable in serum-containing environment (Fig 1C). The loading efficiency could reach nearly 100 % (Fig 1D), and the efficacy could be also enhanced by optimizing the ligand density (Fig 1E). To enhance the therapeutic efficacy on GBM treatment, we have developed a micelle/MSC hybrid cluster for codelivering multiple anti-cancer drugs. MSCs hold tumor homing property as they express tumor-associated cytokine receptors[2]. MSCs in the cluster form showed higher migration toward GBM cells. For decorating MSC clusters, we found that more than 50% of micelles were loaded on the ECM of the cluster and not internalized by the MSCs (Fig 1F), and the MSC migration was not affected by the decoration (Fig 1G). Conclusion: A DNA-templated delivery system is specifically designed for GBM therapy. These micelles are size-tunable, colloidally stable, and relatively monodispersed and non-toxic. It shows the flexibility and capability to integrate with other anti-cancer approaches, thereby making a contribution toward improving the efficacy on treating GBM. YHL and SS acknowledge the fellowship support from Taiwanese Ministry of Education and Schlumberger Foundation, respectively. This work is supported by the Department of Defenses (W81XWH-12-1-0261 to KWL) and the Grant-In-Aid of Research Program from Sigma Xi Research Society (G20131015280632 to YHL).

DNA micelles as nanoreactors: efficient DNA functionalization with hydrophobic organic molecules.

We report a micelle-templated method to enhance the reactivity of DNA with highly hydrophobic molecules. Lipids, chromophores and polymers can be conjugated to DNA in high yield and under mild conditions. This method expands the range of DNA-templated reactions for DNA-encoded libraries, oligonucleotide and drug delivery, nanopore mimetics and DNA nanotechnology.

Investigation on the interaction of 1-amino-4-hydroxy-9,10-anthraquinone with calf thymus DNA and CTAB micelles

The interaction of 1-amino-4-hydroxy-9,10-anthraquinone (AHAQ), a simple analogue of anthracycline anticancer drugs with calf thymus DNA (ct-DNA) and cationic surfactant cetyltrimethylammonium bromide (CTAB) was investigated in aqueous solution at physiological pH (7.4) by cyclic voltammetry and UV–vis absorption spectroscopy. Both aspects of affinity of AHAQ molecule to DNA and cationic surfactant micelle, a model system for a biological membrane, are important in determining its biological action. By using different nonlinear fitting procedures, the intrinsic binding constant and the binding site size were evaluated for AHAQ–ct-DNA interaction and the results indicate a significant affinity of AHAQ towards ct-DNA. Binding constant, partition coefficient and the stoichiometry were determined for AHAQ–CTAB micelles interaction. The results showed that the hydrophobic interaction plays a major role in the binding of AHAQ to CTAB micelles. In addition, the hydrophobic interaction has an important role in the distribution of AHAQ between CTAB micelle — water phases. Gibbs free energy for the binding and distribution of AHAQ between the bulk aqueous medium and surfactant micelles were calculated.

Spectroscopic investigation of interactions of new potential anticancer drugs with DNA and non-ionic micelles.

Photophysical properties of some azinium iodides in aqueous solution of nanostructured systems as DNA and nonionic micelles were investigated using steady-state and ultrafast time-resolved spectroscopy. Spectrophotometric and fluorimetric titrations of the investigated compounds with salmon testes DNA supplied evidence of a good interaction between the salts and DNA with binding constants of 10(4)-10(6) M(-1), making them interesting for pharmaceutical applications. The interaction with DNA also changes the photobehavior of the compounds, increasing the radiative deactivation pathway to the detriment of internal conversion and slowing down the excited state dynamics. The interaction of the azinium salts with the nonionic surfactant Triton X-100 from premicellar to postmicellar concentration was studied by spectrophotometric and fluorimetric titrations evidencing the ability of the micelles to associate the studied salts in their hydrophobic portion and to release them in the presence of DNA, acting as promising drug carriers. Also transient absorption spectroscopy with femtosecond resolution demonstrated the insertion of the investigated compounds into micellar aggregates. Preliminary measurements by confocal fluorescence microscopy on MCF-7 cells in the presence of the studied azinium salts showed that they are able to cross the cellular membrane and that their cytotoxicity can be expressed through interaction with DNA (RNA). In fact, they showed a significant fluorescence signal in all cell compartments, particularly (for 2 and 3) into punctuate structures within the nuclei compatible with a localization into the nucleoli.

Water-soluble monodisperse core-shell nanorings: their tailorable preparation and interactions with oppositely charged spheres of a similar diameter.

Water-soluble monodisperse core-shell structured polymeric nanorings were robustly produced via precise self-assembly between a circular plasmid DNA (monodisperse) and monodisperse polymeric core-shell micelles; the structural parameters of the nanorings can be tailored by controlling the structural parameters of the DNA and the micelles. A study on the morphology-dependent properties of the obtained nanorings revealed that the nanorings exhibit a much higher binding affinity than their linear counterparts when interacting with oppositely charged spheres of a similar diameter. In addition, the formation of one-to-one nanoring/sphere complexes, in which the nanoring circles the equator of the sphere, was observed, which is manifested as a "host-guest" inclusion complex on the nanoscale.

Tuning DNA-amphiphile condensate architecture with strongly binding counterions.

Electrostatic self-assembly of colloidal and nanoparticles has attracted a lot of attention in recent years, since it offers the possibility of producing novel crystalline structures that have the potential to be used as advanced materials for photonic and other applications. The stoichiometry of these crystals is not constrained by charge neutrality of the two types of particles due to the presence of counterions, and hence a variety of three-dimensional structures have been observed depending on the relative sizes of the particles and their charge. Here we report structural polymorphism of two-dimensional crystals of oppositely charged linear macroions, namely DNA and self-assembled cylindrical micelles of cationic amphiphiles. Our system differs from those studied earlier in terms of the presence of a strongly binding counterion that competes with DNA to bind to the micelle. The presence of these counterions leads to novel structures of these crystals, such as a square lattice and a √3 x √3 superlattice of an underlying hexagonal lattice, determined from a detailed analysis of the small-angle diffraction data. These lower-dimensional equilibrium systems can play an important role in developing a deeper theoretical understanding of the stability of crystals of oppositely charged particles. Further, it should be possible to use the same design principles to fabricate structures on a longer length-scale by an appropriate choice of the two macroions.

DNA encapsulation via nanotemplates from cationic block copolymer micelles

We report on a method for the encapsulation of DNA into polymeric nanocapsules. The encapsulation procedure involves three steps: formation of polyplexes between DNA and cationic polymeric micelles; coating of polyplexes with a cross-linked shell; dissociation of polyplexes. In the first step copolymer micelles were obtained by self-assembly of an amphiphilic polystyrene-b-poly(quaternized 2-vinylpyridine) (PS-b-PQ2VP) block copolymer in aqueous solution. Nanosized polyplexes (Dh = 190 nm and ζ = −21 mV) were formed upon the addition of DNA to the micellar solution at a phosphate/amine group ratio of 3 : 1. In the second step the complexes were coated with a cross-linked shell formed by seeded radical polymerization of N-isopropylacrylamide which resulted in a slight shift of ζ potential of the particles to a less negative value. The dissociation of polyplexes and removal of PS-b-PQ2VP were achieved by addition of salt and solvent exchange. Following rehydration viadialysis against water, polymeric nanocapsules with entrapped DNA were obtained. The nanocapsules were visualized by transmission electron microscopy; they exhibited smaller dimensions compared to the initial polyplexes and ζ potential very close to that of the pristine DNA.

DNA–Polymer Micelles as Nanoparticles with Recognition Ability

The Watson-Crick binding of DNA single strands is a powerful tool for the assembly of nanostructures. Our objective is to develop polymer nanoparticles equipped with DNA strands for surface-patterning applications, taking advantage of the DNA technology, in particular, recognition and reversibility. A hybrid DNA copolymer is synthesized through the conjugation of a ssDNA (22-mer) with a poly(ethylene oxide)-poly(caprolactone) diblock copolymer (PEO-b-PCl). It is shown that, in water, the PEO-b-PCl-ssDNA(22) polymer forms micelles with a PCl hydrophobic core and a hydrophilic corona made of PEO and DNA. The micelles are thoroughly characterized using electron microscopy (TEM and cryoTEM) and small-angle neutron scattering. The binding of these DNA micelles to a surface through DNA recognition is monitored using a quartz crystal microbalance and imaged by atomic force microscopy. The micelles can be released from the surface by a competitive displacement event.

Molecular aptamers for drug delivery

The active targeting of drugs in a cell-, tissue- or disease-specific manner represents a potentially powerful technology with widespread applications in medicine, including the treatment of cancers. Aptamers have properties such as high affinity and specificity for targets, easy chemical synthesis and modification, and rapid tissue penetration. They have become attractive molecules in diagnostics and therapeutics rivaling and, in some cases, surpassing other molecular probes, such as antibodies. In this review, we highlight the recent progress in aptamer-mediated delivery for therapeutics and disease-targeting based on aptamer integration with a variety of nanomaterials, such as gold nanorods, DNA micelles, DNA hydrogels and carbon nanotubes.

Pyrene-assisted efficient photolysis of disulfide bonds in DNA-based molecular engineering.

An efficient pyrene-assisted method has been developed for the photolysis of disulfide bonds, with 77% of disulfides cleaved after only 20 min of irradiation (0.3W) at 350 nm. By employing a DNA framework, it was possible to observe both a distance-dependent cleavage pathway and a radical-forming photoreaction mechanism. To demonstrate the biomedical applications of such pyrene disulfide molecular assemblies, a DNA micelle structure and DNAzyme analog were further studied. Rapid photodriven disassembly of DNA micelles was achieved, allowing the further design of controlled pharmaceutical release at the target region and at a specific time. The DNAzyme analog can carry out multiple turnover reactions that follow the Michaelis-Menten equation, with a kcat of 10.2 min(-1) and a KM of 46.3 μM (0.3W 350 nm light source), comparable to that of common DNAzymes, e.g., 8-17 DNAzyme.

Tunable Hydrophobicity in DNA Micelles: Design, Synthesis, and Characterization of a New Family of DNA Amphiphiles

This work describes the synthesis and characterization of a new family of DNA amphiphiles containing modified nucleobases. The hydrophobicity was imparted by the introduction of a dodec-1-yne chain at the 5-position of the uracil base, which allowed precise and simple tuning of the hydrophobic properties through solid-phase DNA synthesis. The micelles formed from these modified DNA sequences were characterized by atomic force microscopy, dynamic light scattering, and polyacrylamide gel electrophoresis. These experiments revealed the role of the quantity and location of the hydrophobic units in determining the morphology and stability of the micelles. The effects of hybridization on the physical characteristics of the DNA micelles were also studied; these results showed potential for the sequence-specific noncovalent functionalization of the self-assembled aggregates.

Virus-like Particles Templated by DNA Micelles: A General Method for Loading Virus Nanocarriers

DNA amphiphile particles template formation of virus capsids and enable their loading.

DNA-based micelles: synthesis, micellar properties and size-dependent cell permeability.

Functional nanomaterials based on molecular self-assembly hold great promise for applications in biomedicine and biotechnology. However, their efficacy could be a problem and can be improved by precisely controlling the size, structure, and functions. This would require a molecular engineering design capable of producing monodispersed functional materials characterized by beneficial changes in size, shape, and chemical structure. To address this challenge, we have designed and constructed a series of amphiphilic oligonucleotide molecules. In aqueous solutions, the amphiphilic oligonucleotide molecules, consisting of a hydrophilic oligonucleotide covalently linked to hydrophobic diacyllipid tails, spontaneously self-assemble into monodispersed, three-dimensional micellar nanostructures with a lipid core and a DNA corona. These hierarchical architectures are results of intermolecular hydrophobic interactions. Experimental testing further showed that these types of micelles have excellent thermal stability and their size can be fine-tuned by changing the length of the DNA sequence. Moreover, in the micelle system, the molecular recognition properties of DNA are intact, thus, our DNA micelles can hybridize with complimentary sequences while retaining their structural integrity. Importantly, when interacting with cell membranes, the highly charged DNA micelles are able to disintegrate themselves and insert into the cell membrane, completing the process of internalization by endocytosis. Interestingly, the fluorescence was found accumulated in confined regions of cytosole. Finally, we show that the kinetics of this internalization process is size-dependent. Therefore, cell permeability, combined with small sizes and natural nontoxicity are all excellent features that make our DNA-micelles highly suitable for a variety of applications in nanobiotechnology, cell biology, and drug delivery systems.

Acid-transforming polypeptide micelles for targeted nonviral gene delivery

Efficient delivery of therapeutic genes requires overcoming key extracellular and intracellular barriers. These include stability during circulation, internalization by target cells, facilitated endosomal escape, and localization of genes in destined intracellular compartments (e.g., nucleus). Micelles that transform their structure in the mildly acidic endosome and release their cargo genes into the cytoplasm were synthesized by self-assembling DNA with PEG-conjugated poly(ketalized serine) [PEG-poly(kSer)]. It was confirmed that, upon acid-hydrolysis of ketal linkages, poly(kSer) converts to neutral and naturally occurring poly(serine), destabilizing PEG-poly(kSer)/DNA micelles. In vitro studies demonstrated that PEG-poly(kSer) micelles were able to transfect NIH 3T3 cells more efficiently than both PEG-poly(Lys)/DNA micelles and poly-L-lysine/DNA polyplexes through efficient DNA dissociation in the cytoplasm. In addition, the core of PEG-poly(kSer)/DNA micelles were cross-linked via acid-cleavable amine-bearing branches, and the resulting cross-linked PEG-poly(kSer)/DNA micelles showed improved transfection capability in the presence of serum. Conjugation of folic acids (FAs) at the PEG termini of the acid-transforming micelles resulted in selectively increased cellular internalization and transfection of FA receptor-expressing HeLa cells over NIH 3T3 cells, implicating the possibility of cancer-targeted gene delivery using FA-PEG-poly(kSer)/DNA micelles. This study demonstrates that the acid-transforming PEG-poly(kSer)/DNA micelles are promising nonviral vectors for stimuli-responsive, efficient, biocompatible, and targeted gene delivery.

Interactions of Nile Blue with Micelles, Reverse Micelles and a Genomic DNA

In this contribution we report studies on the nature of binding of a small ligand/drug Nile blue (NB) with sodium dodecyl sulfate (SDS) micelles, bis-(2-ethylehexyl) sulfosuccinate (AOT)/isooctane reverse micelles (RM) and a genomic DNA extracted from Salmon sperm. With detailed steady state and picosecond resolved optical spectroscopic techniques, we examined the fluorescence quenching of the ligand upon complexation with the SDS monomers and DNA. Polarization analyzed picosecond-resolved fluorescence measurements reveal geometrical restriction on the probe in SDS micelles, AOT-RM and DNA. Steady state and time resolved studies on the probe in nanocages of AOT RM with various degrees of hydration (w(0)) reveal the existence of NB as two distinct species namely, neutral and cationic. This study confirms that the emission of NB in aqueous micelles and DNA solution is due to the cationic form of the drug. Our experiments clearly identified non-specific electrostatic and intercalative modes of interaction of the probe with the DNA at lower and higher DNA concentrations respectively. The nature of binding of NB in presence of the DNA and SDS micelles reveals that the binding affinity of the probe is higher with the micelles than with the DNA. The complex rigidity of NB with DNA and its fluorescence quenching with DNA elucidate a strong recognition mechanism between NB and DNA.

Line optical tweezers instrument for measuring nanoscale interactions and kinetics

We describe an optical tweezers instrument for measuring short-ranged colloidal interactions, based on a combination of a continuous wave line optical tweezers, high speed video microscopy, and laser illumination. Our implementation can measure the separation of two nearly contacting microspheres to better than 4nm at rates in excess of 10kHz. A simple image analysis algorithm allows us to sensibly remove effects from diffraction blurring and microsphere image overlap for separations ranging from contact to at least 100nm. The result is a versatile instrument for measuring steric, chemical and single-molecular interactions and dynamics, with a force resolution significantly better than achievable with current atomic force microscopy. We demonstrate the effectiveness of the instrument with measurements of the pair interactions and dynamics of microspheres in the presence of transient molecular bridges of DNA or surfactant micelles.

Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer.

Gene therapy is based on the vectorization of genes to target cells and their subsequent expression. Cationic amphiphile-mediated delivery of plasmid DNA is the nonviral gene transfer method most often used. We examined the supramolecular structure of lipopolyamine/plasmid DNA complexes under various condensing conditions. Plasmid DNA complexation with lipopolyamine micelles whose mean diameter was 5 nm revealed three domains, depending on the lipopolyamine/plasmid DNA ratio. These domains respectively corresponded to negatively, neutrally, and positively charged complexes. Transmission electron microscopy and x-ray scattering experiments on complexes originating from these three domains showed that although their morphology depends on the lipopolyamine/plasmid DNA ratio, their particle structure consists of ordered domains characterized by even spacing of 80 A, irrespective of the lipid/DNA ratio. The most active lipopolyamine/DNA complexes for gene transfer were positively charged. They were characterized by fully condensed DNA inside spherical particles (diameter: 50 nm) sandwiched between lipid bilayers. These results show that supercoiled plasmid DNA is able to transform lipopolyamine micelles into a supramolecular organization characterized by ordered lamellar domains.

Dielectric Relaxation of Biological Water

Dielectric relaxation and NMR spectrum of water in biological systems such as proteins, DNA, and reverse micelles can often be described by two widely different time constants, one of which is in the picosecond while the other is in the nanosecond regime. Although it is widely believed that the bimodal relaxation arises from water at the hydration shell, a quantitative understanding of this important phenomenon is lacking. In this article we present a theory of dielectric relaxation of biological water. The time dependent relaxation of biological water is described in terms of a dynamic equilibrium between the free and bound water molecules. It is assumed that only the free water molecules undergo orientational motion; the bound water contribution enters only through the rotation of the biomolecule, which is also considered. The dielectric relaxation is then determined by the equilibrium constant between the two species and the rate of conversion from bound to free state and vice versa. However, the dielectric relaxation in such complex biomolecular systems depends on several parameters such as the rotational time constant of the protein molecule, the dimension of the hydration shell, the strength of the hydrogen bond, the static dielectric constant of the water bound to the biomolecule, etc. The present theory includes all these aspects in a consistent way. The results are shown to be in very good agreement with all the known results. The present study can be helpful in understanding the solvation of biomolecules such as proteins.

Form dichroism and the study of molecular shape

Form dichroism is the absorptive analogue of form birefringence and can be observed in anisotropic media which have complex permittivity. Two theoretical treatments of form dichroism are presented: first, for a uniaxial crystal using a simple polariton formalism, secondly for a solution of partially oriented ellipsoidal molecules by using an earlier flow birefringence model. The two descriptions can be made equivalent by introducing relative permittivities, which takes into account the medium surrounding the molecules in the crystal, and by defining the crystal anisotropy so that it describes the shape of the material distribution. The results show that form dichroism provides a method for determining the shape of macromolecules that are built up of isotropic or isotropically oriented chromophoric units. Compact DNA and detergent micelles are discussed as examples.