ABSTRACT: PEGylated dendron-based copolymers (PDC) with different end-group functionalities (-NH(2), -COOH, and -Ac) were synthesized and self-assembled into dendron micelles to investigate the effect of terminal surface charges on size, morphology, and cellular interactions of the micelles. All of the dendron micelles exhibited similar sizes (20-60 nm) and spherical morphologies, as measured using dynamic light scattering and transmission electron microscopy, respectively. The cellular interactions of dendron micelles were evaluated using confocal microscopy and flow cytometry. Surprisingly, although amine-terminated dendrimers are known to strongly interact with cells non-specifically, all of the surface-modified dendron micelles exhibited charge-independent low-levels of cellular interaction. The unexpected results, particularly from the amine-terminated dendron micelles, could be attributed to: i) minimal end-group effects, as each PDC has an approximately 10-fold lower charge-number-to-molecular-weight ratio compared to the dendrimer; and ii) intra- and intermolecular hydrogen bonding between positively charged terminal groups with poly(ethylene glycol) (PEG) backbones, which leads to the sequestration of the charges, as demonstrated by atomistic molecular dynamics simulations. With the narrow size distribution, uniform morphologies, and low levels of non-specific cellular interactions, the dendron micelles offer a promising drug delivery platform.
Project description:To systematically investigate the relationship among surface charge, PEG chain length, and nano-bio interactions of dendron-based micelles (DMs), a series of PEGylated DMs with various end groups (-NH2, -Ac, and -COOH) and PEG chain lengths (600 and 2000 g/mol) are prepared and tested in vitro. The DMs with longer PEG chains (DM2K) do not interact with cells despite their positively charged surfaces. In sharp contrast, the DMs with shorter PEG chains (DM600) exhibit charge-dependent cellular interactions, as observed in both in vitro and molecular dynamics (MD) simulation results. Furthermore, all DMs with different charges display enhanced stability for hydrophobic dye encapsulation compared to conventional linear-block copolymer-based micelles, by allowing only a minimal leakage of the dye in vitro. Our results demonstrate the critical roles of the PEG chain length and polymeric architecture on the terminal charge effect and the stability of micelles, which provides an important design cue for polymeric micelles.
Project description:PEGylated dendron coils (PDCs) were investigated as a novel potential nanocarrier platform. PDCs self-assembled into micelles at lower CMCs than linear copolymer counterparts by 1-2 orders of magnitude, due to the unique architecture of dendrons. MD simulations also supported thermodynamically favourable self-assembly mediated by dendrons.
Project description:Engineering controllable cellular interactions into nanoscale drug delivery systems is key to enable their full potential. Here, using folic acid (FA) as a model targeting ligand and dendron micelles (DM) as a nanoparticle (NP) platform, we present a comprehensive experimental and modeling investigation of the structural properties of DMs that govern the formation of controllable, FA-mediated cellular interactions. Our experimental results demonstrate that a high level of control over the specific cell interactions of FA-targeted DMs can be achieved through modulation of the PEG corona length and the FA content. Using various molecular weight PEGs (0.6K, 1K, and 2K g/mol) and contents of dendron-FA conjugate incorporated into DMs (0, 5, 10, 25 wt %), the cell interactions of the targeted DMs could be controlled to exhibit minimal to >25-fold enhancement over nontargeted DMs. Molecular dynamics simulations indicated that structural characteristics, such as solvent accessible surface area of FA, local PEG density near FA, and FA mobility, account in part for the experimental differences in cellular interactions. The molecular structure that allows FA to depart from the surface of DMs to facilitate the initial cell surface binding was revealed to be the most important contributor for determining FA-mediated cellular interactions of DMs. The modular properties of DMs in controlling their specific cell interactions support the potential of DMs as a delivery platform and offer design cues for future development of targeted NPs.
Project description:Monitoring protein phosphorylation at the cellular level is important to understand the intracellular signaling. Among the phosphoproteomics methods, phosphokinase antibody arrays have emerged as preferred tools to measure well-characterized phosphorylation in the intracellular signaling. Here, we present a dendron-coated phosphokinase antibody array (DPA) in which the antibodies are immobilized on a dendron-coated glass slide. Self-assembly of conically shaped dendrons well-controlled in size and structure resulted in precisely controlled lateral spacing between the immobilized phosphosite-specific antibodies, leading to minimized steric hindrance and improved antigen-antibody binding kinetics. These features increased sensitivity, selectivity, and reproducibility in measured amounts of protein phosphorylation. To demonstrate the utility of the DPA, we generated the phosphorylation profiles of brain tissue samples obtained from Alzheimer's disease (AD) model mice. The analysis of the profiles revealed signaling pathways deregulated during the course of AD progression.
Project description:Poly(amidoamine) (PAMAM) dendrons were synthesized with c(RGDyK) peptide on the surface to create a scaffold for cellular targeting and multivalent binding. Binary dendron-RGD conjugates were synthesized with a single Alexa Fluor 488, biotin, methotrexate drug molecule, or additional functionalized dendron at the focal point. The targeted dendron platform was shown to specifically target ?v?3 integrin expressing human umbilical vein endothelial cells (HUVEC) and human glioblastoma cells (U87MG) in Vitro via flow cytometry. Specific targeting of the dendron-RGD platform was further confirmed by confocal microscopy. Biological activity of the targeted drug conjugate was confirmed via XTT assay. The orthogonal reaction chemistry used at the dendron focal point gives a precise 1:1 ratio of the attachment of multiple functionalities to a small-molecular-weight, chemically stable, high avidity molecule. These studies serve as a framework to selectively combine biologically relevant functions with enhanced specific binding activity to substitute for antibodies in many diagnostic and therapeutic applications.
Project description:The synthesis of a novel poly(propyleneimine) (PPI) dendron in gram scale as well as its use in the formation of a highly stable, dendronized gold nanoparticle (AuNP)-based drug delivery platform is described herein. The AuNP-based platform is composed of three complementary parts: (i) a 15 nm AuNP core, (ii) a heterofunctional thioctic acid-terminated tetraethylene glycol spacer, and (iii) a third-generation PPI dendron with a unique protonation profile and diverse end-group functionalization that allows for further derivatization. The prepared dendronized AuNPs are able to withstand several rounds of lyophilization cycles with no sign of aggregation, are stable in phosphate-buffered saline and Hanks' buffer as well as in serum, and are resistant to degradation by glutathione exchange reactions. This nanocarrier platform displays a dense coating, with >1400 dendrons/AuNPs, which will enable very high payload. Furthermore, while amine-terminated AuNPs expectedly showed cytotoxicity against the MCF-7 breast cancer cell line from a NP concentration of 1 nM, the mixed monolayer AuNPs (coated with 40/60 amine/carboxylate dendrons) interestingly did not exhibit any sign of toxicity at concentrations as high as 15 nM, similar to the carboxylate-terminated AuNPs. The described dendronized AuNPs address the current practical need for a stable NP-based drug delivery platform which is scalable and easily conjugable, has long-term stability in solution, and can be conveniently formulated as a powder and redispersed in desired buffer or serum.
Project description:A higher surface density of poly(ethylene glycol) (PEG) on polymeric micelles enhances their stability in serum, leading to improved plasma circulation. To obtain fundamental, mechanistic understanding of the PEG effect associated with polymeric architecture/configuration, we have synthesized PEGylated dendron-based copolymers (PDCs) and linear block copolymers (LBCs) with similar molecular weights. These copolymers formed dendron (hyperbranched) and linear micelles, respectively, which were compared in terms of their stabilities in serum, micelle-serum protein interactions, and in vivo biodistributions. Overall, the dendron micelles exhibited a better serum stability (longer half-life) and thus a slower release profile than the linear micelles. Fluorescence quenching assays and molecular dynamics (MD) simulations revealed that the high serum stability of the dendron micelles can be attributed to reduced micelle-serum protein interactions, owing to their dendritic, dense PEG outer shell. These results provide an important design cue for various polymeric micelles and nanoparticles.
Project description:Temperature-responsive nanocarrier systems using external stimuli are one of the most widely investigated stimuli-responsive strategies because heat is easy and safe to use for hyperthermia and controlled drug delivery. Polyamidoamine dendron lipids (PAMAM-DLs) composed of PAMAM dendron as head group and two alkyl chains can exhibit temperature-responsive morphological change through the attachment of suitable moieties to terminal of PAMAM dendron. In this study, oligo(ethylene glycol)s including ethoxy- or methoxy-diethylene glycols were attached to the terminals of PAMAM-DL, and temperature-responsive properties of their self-assemblies were evaluated by calorimetric and turbidity measurements. In the evaluation of temperature-responsive properties, ethoxy diethylene glycol (EDEG)-attached PAMAM-DL composed of two saturated alkyl chains and PAMAM dendron with 1st generation had lipid bilayer structure and suitable cloud point for the application as drug carrier. In vitro performances of the assemblies combining EDEG-attached PAMAM-DLs with cholesteryl-oxy-poly(ethylene glycol) (PEG-Chol) was evaluated using doxorubicin (DOX) as an anticancer drug. Cellular uptake of DOX-loaded EDEG-attached PAMAM-DL/PEG-Chol assemblies was promoted at 42 °C rather than 37 °C, resulting in an effective decrease in cell viability.
Project description:Luminescent colloidal nanocrystals (NCs) are emerging as a new tool in neuroscience field, representing superior optical probes for cellular imaging and medical diagnosis of neurological disorders with respect to organic fluorophores. However, only a limited number of studies have, so far, explored NC applications in primary neurons, glia and related cells. Indeed astrocytes, as resident cells in the central nervous system (CNS), play an important pathogenic role in several neurodegenerative and neuroinflammatory diseases, therefore enhanced imaging tools for their thorough investigation are strongly amenable. Here, a comprehensive and systematic study on the in vitro toxicological effect of core-shell type luminescent CdSe@ZnS NCs incorporated in polyethylene glycol (PEG) terminated phospholipid micelles on primary cultures of rat astrocytes was carried out. Cytotoxicity response of empty micelles based on PEG modified phospholipids was compared to that of their NC containing counterpart, in order to investigate the effect on cell viability of both inorganic NCs and micelles protecting NC surface. Furthermore, since the surface charge and chemistry influence cell interaction and toxicity, effect of two different functional groups terminating PEG-modified phospholipid micelles, namely amine and carboxyl group, respectively, was evaluated against bare micelles, showing that carboxyl group was less toxic. The ability of PEG-lipid micelles to be internalized into the cells was qualitatively and quantitatively assessed by fluorescence microscopy and photoluminescence (PL) assay. The results of the experiments clearly demonstrate that, once incorporated into the micelles, a low, not toxic, concentration of NCs is sufficient to be distinctly detected within cells. The overall study provides essential indications to define the optimal experimental conditions to effectively and profitably use the proposed luminescent colloidal NCs as optical probe for future in vivo experiments.
Project description:The rational design of materials with tailored properties is of paramount importance for a wide variety of biological, medical, electronic and optical applications. Here we report molecular level control over the spatial distribution of functional groups on surfaces utilizing self-assembled monolayers (SAMs) of pH-switchable surface-appended pseudorotaxanes. The supramolecular systems were constructed from a poly(aryl ether) dendron-containing a dibenzocrown-8 (DB24C8) macrocycle and a thiol ligand-containing a dibenzylammonium recognition site and a fluorine end group. The dendron establishes the space (dendritic effect) that each pseudorotaxane occupies on the SAM. Following SAM formation, the dendron is released from the surface by switching off the noncovalent interactions upon pH stimulation, generating surface materials with tailored physical and chemical properties.