Shell-crosslinked Nanoparticles Research Articles

Peptide-derivatized shell-cross-linked nanoparticles. 1. Synthesis and characterization.

The conjugation of the protein transduction domain (PTD) from the HIV-1 Tat protein to shell-cross-linked (SCK) nanoparticles is reported as a method to facilitate cell surface binding and transduction of SCK nanoparticles. Attaching increasing numbers of peptide sequences to SCK nanoparticles in a global solution-state functionalization strategy has been devised as a method for increasing the efficiency of the cell-penetrating process. The numbers of peptides per SCK were controlled through stoichiometric balance and measured experimentally by two independent methods, UV-visible spectroscopy and phenylglyoxal analysis. PTD was conjugated in (0.005, 0.01, and 0.02) molar ratios, relative to the acrylic acid residues in the shell, to the SCK nanoparticles resulting in SCK populations possessing nominally 52, 104, and 210 (41, 83, and 202 as measured by phenylglyoxal analysis) PTD peptides per particle, respectively. The methodologies for the block copolymer and nanoparticle syntheses, peptide derivatization, and characterization of peptide-functionalized SCK nanoparticles are reported and the feasibility and efficiency of intracellular internalization of the respective SCKs were quantified.

Peptide-derivatized shell-cross-linked nanoparticles. 2. Biocompatibility evaluation.

The conjugation of the protein transduction domain (PTD) from the HIV-1 Tat protein to shell cross-linked (SCK) nanoparticles is a method to facilitate cell surface binding and transduction. In the previous report, the preparation, derivatization, and characterization of peptide-functionalized SCK nanoparticles were reported in detail. Following assembly, the constructs were evaluated in vitro and in vivo to obtain a preliminary biocompatibility assessment. The effects of SCK exposure on cell viability were evaluated using a metabolic 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and a fluorescent apoptosis assay. Furthermore, stages of apoptosis were quantified by flow cytometry. Although higher levels of peptide functionalization resulted in decreased metabolic function as measured by MTT assay, significant apoptosis was not observed below 500 mg/L for all the samples. To evaluate the potential immunogenic response of the peptide-derivatized constructs, a real-time polymerase chain reaction (RT-PCR) system that allows for the in vitro analysis and quantification of the cellular inflammatory responses tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL1-beta) was utilized. The inflammatory response to the peptide-functionalized SCK nanoparticles as measured by RT-PCR show statistically significant increases in the levels of both TNF-alpha and IL1-beta relative to tissue culture polystyrene (TCPS). However, the measured cytokine levels did not preclude the further testing of SCKs in an in vivo mouse immunization protocol. In this limited assay, measured increases in immunoglobulin G (IgG) concentration in the sera were minimal with no specific interactions being isolated, and more importantly, none of the mice (>50) subjected to the three 100 microg immunization protocol have died. Additionally, no gross morphological changes were observed in postmortem organ histology examinations.

Well‐Defined Carbon Nanoparticles Prepared from Water‐Soluble Shell Cross‐linked Micelles that Contain Polyacrylonitrile Cores

Carbon-based materials have traditionally played an important role in modern technologies, ranging from the manufacture of everyday-use products such as automobile tires (carbon fillers), through reinforcements in composites (carbon fibers), electrode materials for a variety of processes, to activated carbons widely used in separation techniques, to name just a few. In addition to those commodity materials, in recent decades there has been a dynamic growth in the area of advanced engineering carbon materials. Seminal events in this area include the development of synthetic diamond and highly oriented pyrolytic graphite in 1960. 2] In the last two decades, this field has been particularly reenergized by the discovery of fullerenes in 1985 and carbon nanotubes in 1991. In addition to generating tremendous fundamental interest, these nanostructured forms of carbon have found, or are expected to find, numerous applications such as advanced fillers, materials for energy and gas storage, templates, nanoprobes and sensors, and elements for molecular electronics devices. 11] Two main groups of strategies for the preparation of engineering carbon materials include: 1) pyrolysis of organic precursors (mostly polymeric) under inert atmosphere to yield large-scale engineering carbon materials and 2) physical/chemical vapor deposition techniques to produce well-defined nanostructured forms of carbon. Whereas techniques from the first group are applicable to large-scale production, they offer very limited control of the carbon (nano)structure. Techniques from the second group, while allowing for atomic-scale precision in nanostructure control, are relatively expensive, have limited yield, and require complex equipment. Recently, we developed a novel, low-cost route to welldefined nanostructured carbon materials based on the pyrolysis of block copolymer precursors that contain polyacrylonitrile (PAN) and a sacrificial block (e.g., poly(n-butyl acrylate). By this method, the carbon nanostructure is templated after the nanostructure of the PAN domains, which are formed through self-assembly and driven by phase separation between PAN and the sacrificial block. Upon pyrolysis, PAN domains are converted into increasingly graphitic carbon, whereas the sacrificial phase is volatilized. The prerequisite for this approach is the ability of the PAN domains to retain their nanostructure upon thermal treatment. The necessary stabilization is achieved through thermal treatment (heating in the presence of air to 230 8C), a process well-known in the field of carbon fibers, which causes the PAN precursor to form cyclic, ladder, and eventually crosslinked species. Nanostructured carbon materials derived through this novel route hold considerable promise in many areas such as: field emitters, supercapacitors, and photovoltaic cells. One appealing advantage of this approach is the possibility of combining it with currently used devicefabrication techniques that rely on thin-film processing and lithography. One of the potential issues here, however, is the processability of the precursors: PAN and its block copolymers are soluble in a narrow range of solvents such as: N,Ndimethylformamide (DMF), ethylene carbonate (EC), dimethylsulfoxide and N-methylpyrrolidone (NMP). As one of the possible ways of addressing this challenge, herein we present an alternative approach, which now relies on the use of covalently stabilized micellar precursors that are soluble in aqueous systems. These precursors belong to a class of shell cross-linked nanoparticles (shell cross-linked knedels, SCKs), formed by self-assembly and covalent stabilization of amphiphilic block copolymers. PAN block constitutes the core of the SCKs and the sacrificial block (e.g., poly(acrylic acid), PAA) forms a water-soluble shell, and some of the carboxylic acid groups from the PAA block within the shell layer are covalently cross-linked following the micellization process, which occurs in a mixture of DMF and water. The use of nanostructured amphiphilic block copolymers as a template to form inorganic nanostructures has numerous precedents. The use of SCKs rather than simple micelles in this study is motivated by their colloidal stability and their ability to retain their nanostructure upon processing into thin and ultrathin films, and, as demonstrated below, upon subsequent thermal treatment. Processability into thin films is of particular importance when nanostructures are to be patterned or organized into ordered arrays. [*] C. Tang, Prof. K. Matyjaszewski, Prof. T. Kowalewski Department of Chemistry Carnegie Mellon University 4400 Fifth Avenue, Pittsburgh, PA 15213 (USA) Fax: (+1)412-268-6897 E-mail: tomek@andrew.cmu.edu

Determination of the bioavailability of biotin conjugated onto shell cross-linked (SCK) nanoparticles.

Shell cross-linked nanoparticles (SCKs) presenting surface- and bioavailable biotin functional groups were synthesized via a mixed micelle methodology, whereby co-micellization of chain terminal biotinylated poly(acrylic acid)-b-poly(methyl acrylate) (PAA-b-PMA) and nonbiotinylated PAA-b-PMA were cross-linked in an intramicellar fashion within the shell layer of the mixed micelles, between the carboxylic acid groups of PAA and the amine functionalities of 2,2'-(ethylenedioxy)diethylamine. The hydrodynamic diameters (D(h)) of the micelles and the SCKs with different biotinylated block copolymer contents were determined by dynamic light scattering (DLS), and the dimensions of the SCKs were characterized with tapping-mode atomic force microscopy (AFM) and transmission electron microscopy (TEM). The amount of surface-available biotin was tuned by varying the stoichiometric ratio of the biotinylated PAA-b-PMA versus the nonbiotinylated PAA-b-PMA, as demonstrated with solution-state, binding interaction analyses, an avidin/HABA (avidin/4'-hydroxyazobenzene-2-carboxylic acid) competitive binding assay, and fluorescence correlation spectroscopy (FCS). The avidin/HABA assay found the amount of available biotin at the surface of the biotinylated SCK nanoparticles to increase with increasing biotin-terminated block copolymer incorporation, but to be less than 25% of the theoretical value. FCS measurements showed the same trend.

Nanoscale Cage-like Structures Derived from Polyisoprene-Containing Shell Cross-linked Nanoparticle Templates

Hollowed materials of nanoscopic dimensions offer exciting possibilities in a number of fields such as therapeutics and imaging. The production of nanoscopic cage-like materials is described, based upon the excavation of shell cross-linked nanoparticle (SCK) templates. This report focuses upon extensive characterization of the ozonolysis reaction conditions used to degrade the core material, which demonstrates that thirty minutes of ozone treatment is optimal. The resulting nanocage is also shown to undergo pH tunable hydrodynamic diameter variations and to demonstrate a loss of hydrophobic guest interactions, relative to the SCK precursors.

Synthesis, characterization, and bioavailability of mannosylated shell cross-linked nanoparticles.

Saccharide-functionalized shell cross-linked (SCK) polymer micelles designed as polyvalent nanoscaffolds for selective interactions with receptors on Gram negative bacteria were constructed from mixed micelles composed of poly(acrylic acid-b-methyl acrylate) and mannosylated poly(acrylic acid-b-methyl acrylate). The mannose unit was conjugated to the hydrophilic chain terminus of the amphiphilic diblock copolymer precursor, from which the SCK nanoparticles were derived, by the growth of the diblock copolymer from a mannoside functionalized atom transfer radical polymerization (ATRP) initiator. Mixed micelle formation between the amphiphilic diblock copolymer and mannosylated amphiphilic diblock copolymer was followed by condensation-based cross-linking between the acrylic acid residues present in the periphery of the polymer micelles to afford SCK nanoparticles. SCKs presenting variable numbers of mannose functionalities were prepared from mixed micelles of controlled stoichiometric ratios of mannosylated and nonmannosylated diblock copolymers. The polymer micelles and SCKs were characterized by dynamic light scattering (DLS), electrophoretic light scattering, atomic force microscopy (AFM), transmission electron microscopy (TEM), and analytical ultracentrifugation (AU). Surface availability and bioactivity of the mannose units were evaluated by interactions of the nanostructures with the model lectin Concanavalin A via DLS studies, with red blood cells (rabbit) via agglutination inhibition assays and with bacterial cells (E. coli) via TEM imaging.

Thermal shaping of shell-crosslinked (SCK) nanoparticles, facilitated by nanoconfinement of fluid-like cores

The controlled manipulation of the shape of shell-crosslinked (SCK) nanoparticles is described as a novel methodology for the construction of complex nanostructured materials. The presence of a hydrophobic, fluid-like (low Tg) core domain in the SCK allows for substrate-induced shaping of the nanoparticle. The SCK nanoparticles are prepared by the self-assembly of poly(acrylic acid)-block-polyisoprene (PAA-b-PI) diblock copolymers into polymer micelles, followed by crosslinking of the hydrophilic shell layer via amidation reactions between the acrylic acid functionalities and 2,2′-(ethylenedioxy)bis(ethylamine). The synthesis of two amphiphilic diblock copolymers, one consisting of an unmodified PI block (PAA-b-PI, PI having 86% 4,1-microstructure, Tg(PI) = −75 °C) and the other composed of a hydrochlorinated PI block (PAA-b-PI(HCl), Tg(PI(HCl)) = 27 °C), the assembly of the copolymers into polymer micelles and the subsequent shell-crosslinking of the micelles into SCKs is described. The synthesis of a poly(tert-butyl acrylate)-block-polyisoprene (PtBA-b-PI) precursor via nitroxide-mediated radical polymerization (NMRP) and the subsequent methanesulfonic acid catalyzed cleavage of the precursor's tert-butyl ester groups is discussed. Treatment of poly(tert-butyl acrylate)-block-polyisoprene (PAA-b-PI) with HCl(g) is also described as a method for hydrolysis of the tert-butyl esters and for hydrochlorination of the unsaturated groups of PI to yield PAA-b-PI(HCl) in one step. The results of the analyses of solution properties are presented and these findings, in conjunction with transmission electron microscopy (TEM) and atomic force microscopy (AFM) size measurements, indicate spherical shapes for the SCKs in aqueous solution. Using a combination of TEM and AFM dimension determinations, an SCK containing a PI core (predominantly cis-4,1-repeat units) of Tg = −63 °C is shown to deform, at room temperature, from the solution-state spherical shape upon deposition onto a mica substrate. By contrast, deposition of an SCK containing a PI(HCl) core (Tg = 33 °C) shows little deformation at room temperature from spherical shape as determined by its particle aspect ratio of 1.4 (sphere = 1.0). Thermally-driven particle shape manipulation, behavior analogous to the processing of nanoscopic thermoplastic materials, is demonstrated with the hydrochlorinated SCK (PAA-b-PI(HCl)). A large decrease in the average AFM particle height of this SCK is found upon heating above the Tg of the hydrochlorinated PI core (33 °C). This finding demonstrates the major role played by the core (glassy vs. fluid-like) in defining the equilibrium shape of the SCK. Crosslinking within the core domain (in addition to the initial shell crosslinking) is also shown to dramatically increase the rigidity of the nanoparticles and prevent nanoparticle shape deformation when in contact with substrates.

Folic acid-conjugated nanostructured materials designed for cancer cell targeting.

Shell cross-linked nanoparticles (SCKs) constitute a unique class of materials with amphiphilic core-shell morphology; SCKs are characterised by their structural integrity and available functionality to attach receptor-recognising or receptor-specific ligands on the shell surface and, therefore, hold great potential in drug delivery applications; in an attempt to develop novel, cancer cell specific delivery vehicles, folate receptor targeted SCKs have been prepared.

Environmentally-Responsive, Entirely Hydrophilic, Shell Cross-linked (SCK) Nanoparticles

Precise tuning of the chemical composition of shell cross-linked (SCK) nanoparticles provides systematic control over their environmental interactions, as is demonstrated by regioselective modification of the internal and external chemistries. Hydrolysis of the poly(methyl acrylate) core domain of SCKs composed of poly(acrylic acid-co-acrylamide) shell layers produced entirely hydrophilic, pH responsive nanocage-like structures presenting poly(acrylic acid) chains from the inner cage surface. Two-dimensional, colloidally crystallized nanoarrays of the SCKs and nanocages when sorbed at solid substrate surfaces in the presence of water were substrate dependent and pH tunable.

Nanostructured materials designed for cell binding and transduction.

The surface-functionalization of shell cross-linked (SCK) nanoparticles with the oligomeric peptide sequence YGRKKRRQRRR, the protein transduction domain (PTD) from the human immunodeficiency virus TAT protein, is described, and the cell binding interactions these nanobioconjugates exhibit are demonstrated. A convergent synthetic strategy was employed, whereby the SCK nanoparticles and the PTD were prepared independently and then coupled together during immobilization of the PTD component on a solid support. The SCK nanoparticles were prepared by the micellization of amphiphilic block copolymers of poly(epsilon-caprolactone-b-acrylic acid), followed by amidation-based cross-linking of the acrylic acid residues located within the micellar corona. The PTD sequence was constructed upon a solid support, from C-terminus to N-terminus, followed by extension with four glycine residues, leaving the amino chain end for subsequent coupling with remaining acrylic acid functionalities present on the surface of the SCK. Finally, cleavage from the solid support was performed, which also facilitated deprotection of the peptide side chain functionalities as well as hydrolysis of the poly(epsilon-caprolactone) segments composing the SCK core domain, to yield PTD-derivatized nanocage structures (PTD-nanocage). Covalent labeling of the SCK precursor with fluorescein-5-thiosemicarbazide provided fluorescently tagged PTD-nanocage nanobioconjugates to allow for their detection by fluorescence microscopy. The fluorescent PTD-nanocage bioconjugates were found to interact with CHO cells and HeLa cells, whereas the analogous structure lacking the PTD component did not. CHO cells bound with fluorescent PTD-nanocage bioconjugates were analyzed using flow cytometry and fluorescence activated cell sorting (FACS). Fluorescence confocal microscopy of isolated bioconjugate-bound CHO cells indicated that the bioconjugated nanoparticles were primarily located near the cell periphery; however, transduction of the nanoparticle into the cells also occurred.

Fundamental design aspects of amphiphilic shell-crosslinked nanoparticles for controlled release applications

A unique method is developed for the controlled release of the hydrophobic polymer chains from the core of the shell crosslinked nanoparticles (SCKs) by selective cleavage of labile C–ON bonds present at the core–shell interface; this represents a methodology to probe the permeability of nanoscopic membranes and a means for applications in the controlled release of macromolecular species.

Shell Cross-Linked Nanoparticles Containing Hydrolytically Degradable, Crystalline Core Domains

Shell cross-linked knedel-like nanoparticles (SCKs) possessing an amphiphilic core−shell morphology consisting of a cross-linked shell and a hydrolytically degradable, crystalline core domain were synthesized from poly(e-caprolactone)-b-poly(acrylic acid) (PCL-b-PAA) amphiphilic diblock copolymers via a two-step process: self-assembly of PCL-b-PAA into polymer micelles followed by cross-linking of the hydrophilic shell layer via condensation reactions between the carboxylic acid functionalities of PAA and the amine groups of 2,2‘-(ethylenedioxy)bis(ethylamine). PCL-b-PAA was prepared from the selective hydrolysis of a poly(e-caprolactone)-b-poly(tert-butyl acrylate) (PCL-b-PtBA) precursor, which was synthesized by ring opening polymerization (ROP) of e-caprolactone (e-CL) followed by atom transfer radical polymerization (ATRP) of tert-butyl acrylate (tBA). Selective hydrolysis of the tert-butyl ester groups of the PtBA block by reaction with trimethylsilyl iodide (TMSI), followed by reaction with aqueous...

Solution and Surface Charge Properties of Shell Cross-Linked Knedel Nanoparticles

Solution-state characterizations of poly(4-vinylpyridine):polystyrene (PVP:PS) shell cross-linked knedel nanoparticles (SCK's) are described. A combination of techniques, including dynamic light scattering, electrophoretic light scattering, and analytical ultracentrifugation, are used to determine the hydrodynamic diameter, surface charge characteristics, molecular weight, and aggregation number of the SCK's and their micelle precursors. The use of analytical ultracentrifugation is highlighted because of the wealth of information it provides in characterizing SCK solution behavior. The analysis of SCK's indicates that particle diameter, molecular weight, and aggregation increase with increasing PS chain length of the PVP:PS block copolymer used to prepare the SCK. Measurements of electrophoretic mobility and zeta potential via electrophoretic light scattering point to partial burial of charge below the particle surface of quaternized PVP units comprising the shell of the SCK. By contrast, the characterization of corresponding solution properties for the non-cross-linked micelle precursors is problematic due to significant particle heterogeneity and nonideal solution behavior. The comparison of solution data for SCK's and micelle precursors clearly indicates stabilization of SCK particles is achieved through shell cross-linking.