Zhiyue Zhang

Spontaneous Protein Adsorption on Graphene Oxide Nanosheets Allowing Efficient Intracellular Vaccine Protein Delivery

Nanomaterials hold potential of altering the interaction between therapeutic molecules and target cells or tissues. High aspect ratio nanomaterials in particular have been reported to possess unprecedented properties and are intensively investigated for their interaction with biological systems. Graphene oxide (GOx) is a water-soluble graphene derivative that combines high aspect ratio dimension with functional groups that can be exploited for bioconjugation. Here, we demonstrate that GOx nanosheets can spontaneously adsorb proteins by a combination of interactions. This property is then explored for intracellular protein vaccine delivery, in view of the potential of GOx nanosheets to destabilize lipid membranes such as those of intracellular vesicles. Using a series of in vitro experiments, we show that GOx nanosheet adsorbed proteins are efficiently internalized by dendritic cells (DCs: the most potent class of antigen presenting cells of the immune system) and promote antigen cross-presentation to CD8 T cells. The latter is a hallmark in the induction of potent cellular antigen-specific immune responses against intracellular pathogens and cancer.

Poly(N-isopropylacrylamide) coated gold nanoparticles as colourimetric temperature and salt sensors

Thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) coated gold nanoparticles (AuNPs) are reported having residual citrate groups on the surface resulting from their synthesis via the Turkevich method. These PNIPAM coated AuNPs have dual stabilisation by the polymer chains and the charges, which we exploit for the development of temperature and salt sensors of which the sensing regime can be tuned by variation of salt concentration and temperature, respectively.

Hydrogen bonded multilayer films based on poly(2-oxazoline)s and tannic acid

In recent years, the layer-by-layer (LbL) assembly based on hydrogen bonding interactions is gaining popularity for the preparation of thin film coatings, especially for biomedical purposes, based on the use of neutral, non-toxic building blocks. The use of tannic acid (TA) as hydrogen bonding donor is especially interesting as it results in LbL films that are stable under physiological conditions. In this work, investigations on the LbL thin film preparation of TA with poly(2-oxazoline)s with varying hydrophilicity, namely poly(2-methyl-2-oxazoline) (PMeOx), poly(2-ethyl-2-oxazoline) (PEtOx) and poly(2-n-propyl-2-oxazoline) (PnPropOx), are reported. The LbL assembly process is investigated by quartz crystal microbalance and UV-vis spectroscopy revealing linear growth of the film thickness. Furthermore, isothermal titration calorimetry demonstrates the LbL assembly of TA, and PMeOx is found to be mostly enthalpy driven while the LbL assembly of TA with PEtOx and PnPropOx is mostly entropy driven. Finally, scanning electron microscopy and ellipsometry demonstrate the formation of smooth thin films for LbL assembly of TA with all three polymers. Such poly(2-oxazoline) coatings have high potential for use as anti-biofouling coatings.

Thermoresponsive polymer coated gold nanoparticles: from MADIX/RAFT copolymerization of N-vinylpyrrolidone and N-vinylcaprolactam to salt and temperature induced nanoparticle aggregation

In the present contribution, we report the MADIX/RAFT polymerization for the synthesis of thermoresponsive homo and statistical copolymers of N-vinylcaprolactam (NVCL) and N-vinylpyrrolidone (NVP). The conditions for the polymerization of NVP were optimized using an automated parallel synthesizer and these optimal conditions were applied for preparing copolymers with systematical variation in composition. The cloud point temperatures (TCPs) of aqueous solutions of PNVCL and P(NVCL-stat-NVP)’s (CP1–CP5) were found to be tuneable between 40 °C and >95 °C at 5 mg mL−1. Next, stable colloidal solutions of AuNPs coated with PNVCL and CP1–CP5 were obtained via an exchange reaction of pre-synthesized citrate stabilized AuNPs with PNVCL and CP1–CP5 by a direct ‘grafting to’ approach. The maximum absorbance wavelength (λmax) of the surface plasmon resonance (SPR) band and size of all the thermoresponsive polymer coated AuNPs were found to be almost unchanged up to 65 °C (above the TCP of PNVCL and CP5) in MilliQ water which is presumably due to electrostatic stabilization of the AuNPs by residual citrate groups on the surface. However, in 0.1 M NaCl aqueous solution the λmax of the thermoresponsive AuNPs were red shifted when heated up to 65 °C which is attributed to the screening of the citrate negative charges on the surface of AuNPs that suppress electrostatic stabilization enabling T-induced aggregation leading to a shift in the SPR band. These thermoresponsive AuNPs may find applications as colorimetric temperature and/or salt sensors.

Salt-Driven Deposition of Thermoresponsive Polymer-Coated Metal Nanoparticles on Solid Substrates.

Here we report on a simple, generally applicable method for depositing metal nanoparticles on a wide variety of solid surfaces under all aqueous conditions. Noble-metal nanoparticles obtained by citrate reduction followed by coating with thermoresponsive polymers spontaneously form a monolayer-like structure on a wide variety of substrates in presence of sodium chloride whereas this phenomenon does not occur in salt-free medium. Interestingly, this phenomenon occurs below the cloud point temperature of the polymers and we hypothesize that salt ion-induced screening of electrostatic charges on the nanoparticle surface entropically favors hydrophobic association between the polymer-coated nanoparticles and a hydrophobic substrate.

Straightforward RAFT Procedure for the Synthesis of Heterotelechelic Poly(acrylamide)s

Heterotelechelic, hydrophilic polymers with a primary amine and thiol group at the α- and ω-chain end, respectively, are synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization in a straightforward and versatile way and subsequently used for the design of dual-responsive polymer/gold nanohybrids. Therefore, a phthalimido-containing chain transfer agent (CTA) is synthesized and used for the polymerization of the hydrophilic monomers N-isopropylacrylamide (NIPAM) and N,N-dimethylacrylamide (DMA). After polymerization, the trithiocarbonate functionality at the ω-chain end, originating from the CTA, is converted into a thiol upon aminolysis. In the next step, the phthalimido α-chain end is hydrolyzed into a primary amine, resulting in heterotelechelic, hydrophilic polymers. End-group conversions are monitored by (1)H NMR spectroscopy, MALDI-TOF MS analysis, and UV-Vis spectroscopy, confirming that quantitative modifications are obtained during each stage. The amino groups of these heterotelechelic polymer chains are modified with citraconic anhydride, after which the obtained polymers are grafted with the thiol group onto citrate-stabilized gold nanoparticles resulting in the creation of dual-temperature- and pH-responsive gold particles.

Squaric ester amides as hydrolysis-resistant functional groups for protein-conjugation of RAFT-derived polymers

We report on the synthesis of amine-reactive polymers, for the purpose of protein conjugation. Poly(N,N-dimethylacrylamide) (polyDMA) was prepared by RAFT polymerization using either a squaric ester amide functionalized chain transfer agent (CTA) or a phthalimide functionalized CTA which served as a precursor for post-polymerization introduction of a squaric ester amide moiety. Polymers were characterized for their end group fidelity by 1H-NMR and MALDI-TOF MS, which showed the use of a squaric ester amide functionalized CTA to be superior. The performance of these polymers for lysine-targeted protein modification was assessed using lysozyme as model protein and compared to N-hydroxysuccinimide (NHS)-functionalized polymers as the golden standard in bio-conjugation. We found that squaric ester amides were much less prone to hydrolysis, which yielded higher protein-conjugation efficiency in aqueous medium.

Linear polyethylenimine as (multi) functional initiator for organocatalytic L-lactide polymerization

The preparation of polyethylenimine (PEI)-polylactide (PLA) copolymer structures is promising as these materials may find use in gene and/or drug delivery applications. In the current work we have explored the utilization of linear polyethylenimine (L-PEI) as multifunctional initiator for the organocatalytic ring-opening polymerization of lactide. Evaluation of the effect of the amount of catalyst revealed that with high catalyst loadings mixtures of unmodified L-PEI and PEI-PLA were obtained while low catalyst loadings leads to efficient preparation of PEI-PLA graft copolymers. This difference is described to the enhanced polymerization time with lower catalyst loading enabling efficient initiation from up to every second ethylenimine unit. The resulting PEI-PLA were subsequently formulated into nanoparticles of ∼400 nm by nanoprecipitation, which could be efficiently labeled with rhodamine octadecylester as model hydrophobic drug. These nanoparticles were efficiently taken up by DC2.4 cells as demonstrated by flow cytometry and fluorescence microscopy demonstrating their potential for gene and/or drug delivery applications.