Monitoring the erosion of hydrolytically-degradable nanogels via multiangle light scattering coupled to asymmetrical flow field-flow fractionation.
ABSTRACT: We describe the synthesis and characterization of degradable nanogels that display bulk erosion under physiologic conditions (pH = 7.4, 37 degrees C). Erodible poly(N-isopropylmethacrylamide) nanogels were synthesized by copolymerization with N,O-(dimethacryloyl) hydroxylamine, a cross-linker previously used in the preparation of nontoxic and biodegradable bulk hydrogels. To monitor particle degradation, we employed multiangle light scattering and differential refractometry detection following asymmetrical flow field-flow fractionation. This approach allowed the detection of changes in nanogel molar mass and topology as a function of both temperature and pH. Particle erosion was evident from both an increase in nanogel swelling and a decrease in scattering intensity as a function of time. Following these analyses, the samples were recovered for subsequent characterization by direct particle tracking, which yields hydrodynamic size measurements and enables number density determination. Additionally, we confirmed the conservation of nanogel stimuli-responsivity through turbidity measurements. Thus, we have demonstrated the synthesis of degradable nanogels that erode under conditions and on time scales that are relevant for many drug delivery applications. The combined separation and light scattering detection method is demonstrated to be a versatile means to monitor erosion and should also find applicability in the characterization of other degradable particle constructs.
Project description:Stimuli-responsive nanogels are important drug and gene carriers that mediate the controlled release of therapeutic molecules. Herein, we report the synthesis of fully degradable disulfide cross-linked nanogel drug carriers formed by oxidative radical polymerization of 2,2'-(ethylenedioxy)diethanethiol (EDDET) as a monomer with different cross-linkers, including pentaerythritol tetramercaptoacetate (PETMA). Because the poly(EDDET) backbone repeat structure and cross-linking junctions are composed entirely of disulfide bonds, these nanogels specifically degrade to small molecule dithiols intracellularly in response to the reducing agent glutathione present inside of cells. Cross-linked nanogels were synthesized using controlled microfluidic mixing in the presence of a nonionic Pluronic surfactant PLU-127 to increase the nanogel stability. Adjusting the monomer to cross-linker ratio from 5?:?1 to 100?:?1 (mol/mol) tuned the cross-linking density, resulting in swelling ratios from 1.65 to >3. Increasing the amount of stabilizing Pluronic surfactant resulted in a decrease of nanogel diameter, as expected due to increased surface area of the resulting nanogels. The monomer to cross-linker ratio in the feed had no effect on the formed nanogel diameter, providing a way to control cross-linking density with constant nanogel size but tunable drug release kinetics. Nanogels exhibited an entrapment efficiency of up to 75% for loading of Rhodamine B dye. In vitro studies showed low cytotoxicity, quick uptake, and fast degradation kinetics. Due to the ease of synthesis, rapid gelation times, and tunable functionality, these non-toxic and fully degradable nanogels offer potential for use in a variety of drug delivery applications.
Project description:RAFT polymerization was utilized to prepare an amphiphilic block copolymer containing both hydrophilic and hydrophobic segments. The self-assembly behavior of the block copolymer into nano-scale particulate structures was studied in both water and polar organic solvents. Uniform micelle assemblies were stabilized by reaction within the hydrophobic core, which contained pendant azide groups, through copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry with a dialkyne crosslinker. The reaction preceded efficiently with negligible residual azide functionality and resulted in core-shell nanogel structures that were analyzed by a variety of techniques including light scattering, electron microscopy and the ability to take up hydrophobic molecules. Both thermo- and pH-responsive character of the nanogels and the linear polymers from which they were made were studied through cloud point testing at different pH levels. It was found that these nanogel dispersions in water exhibited the highest cloud point temperatures indicating a highly stable nanogel structure. The solvent-dispersed nanogels were used as building blocks to form extended polymer networks through the inter- as well as intra-particle reaction between hydroxyl groups within the hydrophilic domain of the nanogel shell by crosslinking with a diisocyanate. It was found that as little as 10 wt% nanogel dispersions in solvent reached the percolation threshold to yield highly porous macroscopic networks; while 50 wt% concentrations achieved densely packed and interdigitated nanogels to afford relatively homogeneous structures.
Project description:We have developed a novel and simple synthesis route to create nanosized (?5nm) silver nanoparticles (Ag NPs) embedded in a biocompatible nanogel (NG) comprising degradable, natural polymers, namely dextran and lysozyme. In this study, we prepared hybrid nanogels with varying lysozyme content, evaluated their potential to reduce Ag NPs in situ (using ultraviolet-visible spectroscopy, cryo-transmission electronic microscopy, thermogravimetric analysis and Fourier transform infrared spectroscopy) and determined their antibacterial properties against Escherichia coli and Staphylococcus aureus. Lysozyme was found to enhance nucleation and stabilization of Ag NPs while limiting their growth. As lysozyme concentration increased, larger nanogels with greater loading of smaller Ag NPs were obtained. The antibacterial properties of hybrid NGs were found to depend upon nanogel type and bacterial conditions. Hybrid nanogels with the largest Ag NPs showed the lowest minimum inhibition concentration. However, the greatest bacterial killing efficiency (up to 100%) occurred within 1h if the bacteria were exposed to hybrid nanogels with smaller Ag NPs while agitating the medium. These results suggest that nanogel properties as well as antibacterial activity can be tuned by varying the lysozyme content. By targeting drug delivery (e.g. ligand grafted surface), these nanogels can be used to prevent biofilm formation and control infection without the complications (i.e. overexposure) associated with classical antibiotic delivery platforms.
Project description:The covalent conjugation of bovine serum albumin (BSA) to disulfide cross-linked polymeric nanogels is reported. Polymeric nanogel precursors were synthesized via a reversible addition-fragmentation chain transfer (RAFT) random copolymerization of poly(ethylene glycol) methyl ether methacrylate (PEGMA) and pyridyl disulfide methacrylate (PDSMA). Reaction of the p(PEGMA-co-PDSMA) with dithiothreitol resulted in the formation of nanogels. PDSMA serves as both a crosslinking agent and a reactive handle for the surface modification of the nanogels. Lipophilic dye, DiI, was sequestered within the nanogels by performing the crosslinking reaction in the presence of the hydrophobic molecule. Thiol-enriched BSA was conjugated to nanogels loaded with DiI via a disulfide reaction between the BSA and the surface exposed nanogel pyridyl disulfides. Conjugation was confirmed by fast protein liquid chromatography, dynamic light scattering, and agarose and polyacrylamide gel electrophoresis. We expect that this methodology is generally applicable to the preparation of nanogel-protein therapeutics.
Project description:PURPOSE:The aim of the study is to synthesize and characterize nanogel carriers composed of amphiphilic polymers and cationic polyethylenimine for encapsulation and delivery of cytotoxic nucleoside analogs 5'-triphosphates (NTPs) into cancer cells. METHODS:Nanogels were synthesized by a novel micellar approach and compared with carriers prepared by the emulsification/evaporation method. Complexes of nanogels with NTP were prepared; particle size and in vitro drug release were characterized. Resistance of the nanogel-encapsulated NTP to enzymatic hydrolysis was analyzed by ion-pair high-performance liquid chromatography. Binding to isolated cellular membranes, cellular accumulation and cytotoxicity were compared using breast carcinoma cell lines CL-66, MCF-7, and MDA-MB-231. In vivo biodistribution of the 3H-labeled NTP encapsulated in different types of nanogels was evaluated in comparison to the injected NTP alone. RESULTS:Nanogels with a particle size of 100-300 nm in the unloaded form and less than 140 nm in the NTP-loaded form were prepared. An in vitro release of NTP was >50% during the first 24 h. Nanogel formulations ensured increased NTP drug stability against enzymatic hydrolysis as compared to the drug alone. Pluronic-based nanogels NG(F68), NG(F127), NG(P85), and NGM(P123) demonstrated 2-2.5 times enhanced interaction with cellular membranes and association with various cancer cells compared to NG(PEG). Among them, NG(F68) and NG(F127) exhibited the lowest cytotoxicity. Injection of nanogel-formulated NTP significantly modulated the drug accumulation in different mouse organs. CONCLUSIONS:Nanogels composed of Pluronic F68 and P123 were shown to display certain advanced properties compared to NG(PEG) as a drug delivery system for NTP analogs. Formulations of nucleoside analogs in active NTP form with these nanogels will improve the delivery of these cytotoxic drugs to cancer cells and the therapeutic potential of this anticancer chemotherapy.
Project description:We report the design, synthesis and efficacy of a new class of gel-like nano-carrier, or 'nanogel', prepared via templated electrostatic assembly of anionic hyaluronic acid (HA) polysaccharides with the cationic peptide amphiphile poly-L-lysine (PLL). Small molecules and proteins present during nanogel assembly become directly encapsulated within the carrier and are precisely released by tuning the nanogel HA:PLL ratio to control particle swelling. Remarkably, nanogels exhibit versatile and complimentary mechanisms of cargo delivery depending on the biologic context. For example, in mammalian cells, nanogels are rapidly internalized and escape the endosome to both deliver membrane-impermeable protein cargo into the cytoplasm and improve chemotherapeutic potency in drug resistant cancer cells. In bacteria, nanogels permeabilize microbial membranes to sensitize bacterial pathogens to the action of a loaded antibiotic. Thus, peptide nanogels represent a versatile, readily scalable and bio-responsive carrier capable of augmenting and enhancing the utility of a broad range of biomolecular cargoes.
Project description:P?AE polymers have emerged as highly promising candidates for biomedical and drug delivery applications owing to their tunable, degradable and pH sensitive properties. These polymeric systems can serve as prodrug carriers for the delivery of bioactive compounds which suffer from poor aqueous solubility, low bioavailability and are biologically unstable, such as the antioxidant, quercetin. Using acrylate functionalized quercetin, it is possible to incorporate the polyphenol into the backbone of the polymer matrix, permitting slow release of the intact molecule which is perfectly timed with the polymer degradation. While formulating these quercetin conjugated P?AE matrix into nanocarriers would allow for multiple delivery routes (oral, intravenous, inhalation etc.), well known oil-water nano-emulsion formulation methods are not amenable to the crosslinked hydrolytically sensitive nanoparticle/nanogel. In this work, a single-phase reaction-precipitation method was developed to formulate quercetin conjugated P?AE nanogels (QNG) via reaction of acrylated quercetin (4-5 acrylate groups) with a secondary diamine under dilute conditions using acetonitrile as the reaction medium, resulting in a self-stabilized suspension. The proposed approach permits the post synthesis modification of the spherical nanogels with a PEGylated coating, enhancing their aqueous stability and stealth characteristics. Nanogel size was controlled by varying feed reactant concentrations, achieving drug loadings of 25-38wt%. Uniform release of quercetin over 45-48h was observed upon P?AE ester hydrolysis under physiological conditions with its retained antioxidant activity over the extended times.Here we present the first demonstration of using poly(beta amino ester) chemistry to form nanogels composed of a bioactive polyphenol for the control of cellular oxidative stress. Previous nanogel and nanoparticle approaches, which use a water phase, are not readily amenable to PBAE chemistry due to their hydrolytic sensitivity. Here we demonstrate a simple approach to control particle size, modify surface chemistry and achieve highly regulated controlled release of active antioxidants, which can protect cells against external oxidative stress signals. This work has importance in the area of controlling material biocompatibility through augmenting the antioxidant status of cells.
Project description:Characterization of responsive hydrogels and their interaction with other molecules have significantly expanded our understanding of the functional materials. We here report on the response of poly(N-isopropylacrylamide-co-acrylic acid) (pNIPAm-co-AAc) nanogels to the addition of the poly(allylamine hydrochloride) (PAH) in aqueous dispersions. We find that the hydrodynamic radius and stability of nanogels are dependent on the PAH/nanogel stoichiometry. If the nanogel solution is titrated with very small aliquots of PAH, the nanogels decrease in radius until the equivalence point, followed by aggregation at suprastoichiometric PAH additions. Conversely, when titrated with large aliquots, the nanogel charge switches rapidly from anionic to cationic, and no aggregation is observed. This behavior correlates well with electrophoretic mobility measurements, which shows the nanogel charge transitioning from negative to positive upon PAH addition. The volume phase transition temperature (VPTT) of the nanogels is also measured to discover the effect of polyelectrolyte complexation on the deswelling thermodynamics. These data show that charge neutralization upon PAH addition decreases the VPTT of the nanogel at pH 6.5. However, if an excess amount of PAH is added to the nanogel solution, the VPTT shifts back to higher temperatures due to the formation of a net positive charge in the nanogel network.
Project description:Detailed characterization of hydrogel particle erosion revealed critical physicochemical differences between spheres, where network decomposition was informative of network structure. Real-time, in situ monitoring of the triggered erosion of colloidal hydrogels (microgels) was performed via multiangle light scattering. The solution-average molar mass and root-mean-square radii of eroding particles were measured as a function of time for microgels prepared from N-isopropylacrylamide (NIPAm) or N-isopropylmethacrylamide (NIPMAm), copolymerized with a chemically labile cross-linker (1,2-dihydroxylethylene)bisacrylamide (DHEA). Precipitation polymerization was employed to yield particles of comparable dimensions but with distinct topological features. Heterogeneous cross-linker incorporation resulted in a heterogeneous network structure for pNIPAm microgels. During the erosion reaction, mass loss proceeded from the exterior toward the interior of the polymer. In contrast, pNIPMAm microgels had a more homogeneous network structure, which resulted in a more uniform mass loss throughout the particle during erosion. Although both particle types degraded into low molar mass products, pNIPAm microgels were incapable of complete dissolution due to the presence of nondegradable cross-links arising from chain transfer and branching during particle synthesis. The observations described herein provide insight into key design parameters associated with the synthesis of degradable hydrogel particles, which may be of use in various biotechnological applications.
Project description:<h4>Background</h4>Emulsions stabilized by colloidal particles are known as Pickering emulsions. To date, soft microgel particles as well as inorganic and organic particles have been utilized as Pickering emulsifiers. Although cyclodextrin (CD) works as an attractive emulsion stabilizer through the formation of a CD-oil complex at the oil-water interface, a high concentration of CD is normally required. Our research focuses on an effective Pickering emulsifier based on a soft colloidal CD polymer (CD nanogel) with a unique surface-active property.<h4>Results</h4>CD nanogels were prepared by crosslinking heptakis(2,6-di-O-methyl)-?-cyclodextrin with phenyl diisocyanate and subsequent immersion of the resulting polymer in water. A dynamic light scattering study shows that primary CD nanogels with 30-50 nm diameter assemble into larger CD nanogels with 120 nm diameter by an increase in the concentration of CD nanogel from 0.01 to 0.1 wt %. The CD nanogel has a surface-active property at the air-water interface, which reduces the surface tension of water. The CD nanogel works as an effective Pickering emulsion stabilizer even at a low concentration (0.1 wt %), forming stable oil-in-water emulsions through interfacial adsorption by the CD nanogels.<h4>Conclusion</h4>Soft CD nanogel particles adsorb at the oil-water interface with an effective coverage by forming a strong interconnected network and form a stable Pickering emulsion. The adsorption property of CD nanogels on the droplet surface has great potential to become new microcapsule building blocks with porous surfaces. These microcapsules may act as stimuli-responsive nanocarriers and nanocontainers.