Mixed-shell Polymeric Micelles Research Articles

One-pot synthesis of high-concentration mixed-shell polymeric micelles as nanochaperones for the renaturation of bulk proteins

A one-pot synthesis of high-concentration mixed-shell polymeric micelles and synthetic nanoparticles can be used to assist the refolding of bulk denatured proteins and stabilize native proteins for long-term storage.

Encapsulation of Photothermal Nanoparticles in Stealth and pH-Responsive Micelles for Eradication of Infectious Biofilms In Vitro and In Vivo.

Photothermal nanoparticles can be used for non-antibiotic-based eradication of infectious biofilms, but this may cause collateral damage to tissue surrounding an infection site. In order to prevent collateral tissue damage, we encapsulated photothermal polydopamine-nanoparticles (PDA-NPs) in mixed shell polymeric micelles, composed of stealth polyethylene glycol (PEG) and pH-sensitive poly(β-amino ester) (PAE). To achieve encapsulation, PDA-NPs were made hydrophobic by electrostatic binding of indocyanine green (ICG). Coupling of ICG enhanced the photothermal conversion efficacy of PDA-NPs from 33% to 47%. Photothermal conversion was not affected by micellar encapsulation. No cytotoxicity or hemolytic effects of PEG-PAE encapsulated PDA-ICG-NPs were observed. PEG-PAE encapsulated PDA-ICG-NPs showed good penetration and accumulation in a Staphylococcus aureus biofilm. Penetration and accumulation were absent when nanoparticles were encapsulated in PEG-micelles without a pH-responsive moiety. PDA-ICG-NPs encapsulated in PEG-PAE-micelles found their way through the blood circulation to a sub-cutaneous infection site after tail-vein injection in mice, yielding faster eradication of infections upon near-infrared (NIR) irradiation than could be achieved after encapsulation in PEG-micelles. Moreover, staphylococcal counts in surrounding tissue were reduced facilitating faster wound healing. Thus, the combined effect of targeting and localized NIR irradiation prevented collateral tissue damage while eradicating an infectious biofilm.

Self-Assembly Molecular Chaperone to Concurrently Inhibit the Production and Aggregation of Amyloid β Peptide Associated with Alzheimer's Disease.

Amyloid β peptide (Aβ) plays a crucial role in the pathogenesis of Alzheimer's disease (AD). Currently, decreasing Aβ production and preventing Aβ aggregation are thought to be important strategies in anti-AD therapy. However, inhibiting Aβ production or aggregation in isolation is not sufficient to reverse the neurodegenerative process of AD patients in clinical testing. Here, a self-assembly molecular chaperone (SAMC) consisting of γ-secretase inhibitor DAPT and mixed-shell polymeric micelles is devised, serving as a bifunctional suppressor of AD. This two-in-one combinational system can simultaneously inhibit Aβ production and aggregation, which would contribute to enhancing the therapeutic effect by decreasing Aβ levels. Decorating a neuron-specific RVG29 peptide onto the surface, the DAPT-incorporated SAMC can specifically target neuronal cells and, thus, will relieve the strong side effect of DAPT on normal cells. Therefore, this combination strategy holds great potential to open up an avenue for AD treatment.

Surface-Adaptive, Antimicrobially Loaded, Micellar Nanocarriers with Enhanced Penetration and Killing Efficiency in Staphylococcal Biofilms.

Biofilms cause persistent bacterial infections and are extremely recalcitrant to antimicrobials, due in part to reduced penetration of antimicrobials into biofilms that allows bacteria residing in the depth of a biofilm to survive antimicrobial treatment. Here, we describe the preparation of surface-adaptive, Triclosan-loaded micellar nanocarriers showing (1) enhanced biofilm penetration and accumulation, (2) electrostatic targeting at acidic pH toward negatively charged bacterial cell surfaces in a biofilm, and (3) antimicrobial release due to degradation of the micelle core by bacterial lipases. First, it was established that mixed-shell-polymeric-micelles (MSPM) consisting of a hydrophilic poly(ethylene glycol) (PEG)-shell and pH-responsive poly(β-amino ester) become positively charged at pH 5.0, while being negatively charged at physiological pH. This is opposite to single-shell-polymeric-micelles (SSPM) possessing only a PEG-shell and remaining negatively charged at pH 5.0. The stealth properties of the PEG-shell combined with its surface-adaptive charge allow MSPMs to penetrate and accumulate in staphylococcal biofilms, as demonstrated for fluorescent Nile red loaded micelles using confocal-laser-scanning-microscopy. SSPMs, not adapting a positive charge at pH 5.0, could not be demonstrated to penetrate and accumulate in a biofilm. Once micellar nanocarriers are bound to a staphylococcal cell surface, bacterial enzymes degrade the MSPM core to release its antimicrobial content and kill bacteria over the depth of a biofilm. This constitutes a highly effective pathway to control blood-accessible staphylococcal biofilms using antimicrobials, bypassing biofilm recalcitrance to antimicrobial penetration.

Reversible Interactions of Proteins with Mixed Shell Polymeric Micelles: Tuning the Surface Hydrophobic/Hydrophilic Balance toward Efficient Artificial Chaperones.

Molecular chaperones can elegantly fine-tune its hydrophobic/hydrophilic balance to assist a broad spectrum of nascent polypeptide chains to fold properly. Such precious property is difficult to be achieved by chaperone mimicking materials due to limited control of their surface characteristics that dictate interactions with unfolded protein intermediates. Mixed shell polymeric micelles (MSPMs), which consist of two kinds of dissimilar polymeric chains in the micellar shell, offer a convenient way to fine-tune surface properties of polymeric nanoparticles. In the current work, we have fabricated ca. 30 kinds of MSPMs with finely tunable hydrophilic/hydrophobic surface properties. We investigated the respective roles of thermosensitive and hydrophilic polymeric chains in the thermodenaturation protection of proteins down to the molecular structure. Although the three kinds of thermosensitive polymers investigated herein can form collapsed hydrophobic domains on the micellar surface, we found distinct capability to capture and release unfolded protein intermediates, due to their respective affinity for proteins. Meanwhile, in terms of the hydrophilic polymeric chains in the micellar shell, poly(ethylene glycol) (PEG) excels in assisting unfolded protein intermediates to refold properly via interacting with the refolding intermediates, resulting in enhanced chaperone efficiency. However, another hydrophilic polymer-poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) severely deteriorates the chaperone efficiency of MSPMs, due to its protein-resistant properties. Judicious combination of thermosensitive and hydrophilic chains in the micellar shell lead to MSPM-based artificial chaperones with optimal efficacy.

Effect of the Surface Charge of Artificial Chaperones on the Refolding of Thermally Denatured Lysozymes.

Artificial chaperones are of great interest in fighting protein misfolding and aggregation for the protection of protein bioactivity. A comprehensive understanding of the interaction between artificial chaperones and proteins is critical for the effective utilization of these materials in biomedicine. In this work, we fabricated three kinds of artificial chaperones with different surface charges based on mixed-shell polymeric micelles (MSPMs), and investigated their protective effect for lysozymes under thermal stress. It was found that MSPMs with different surface charges showed distinct chaperone-like behavior, and the neutral MSPM with PEG shell and PMEO2MA hydrophobic domain at high temperature is superior to the negatively and positively charged one, because of the excessive electrostatic interactions between the protein and charged MSPMs. The results may benefit to optimize this kind of artificial chaperone with enhanced properties and expand their application in the future.

Thermo/pH Dual Responsive Mixed-Shell Polymeric Micelles Based on the Complementary Multiple Hydrogen Bonds for Drug Delivery.

Thermo/pH dual responsive mixed-shell polymeric micelles based on multiple hydrogen bonding were prepared by self-assembly of diaminotriazine-terminated poly(ɛ-caprolactone) (DAT-PCL), uracil-terminated methoxy poly(ethylene glycol) (MPEG-U), and uracil-terminated poly(N-vinylcaprolactam) (PNVCL-U) at room temperature. PCL acted as the core and MPEG/PNVCL as the mixed shell. Increasing the temperature, PNVCL collapsed and enclosed the PCL core, while MPEG penetrated through the PNVCL shell, thereby leading to the formation of MPEG channels on the micelles surface. The low cytotoxicity of the mixed micelles was confirmed by an MTT assay against BGC-823 cells. Studies on the in vitro drug release showed that a much faster release rate was observed at pH 5.0 compared to physiological pH, owing to the dissociation of hydrogen bonds. Therefore, the mixed-shell polymeric micelles would be very promising candidates in drug delivery systems.

Artificial chaperones based on mixed shell polymeric micelles: insight into the mechanism of the interaction of the chaperone with substrate proteins using Förster resonance energy transfer.

Controlled and reversible interactions between polymeric nanoparticles and proteins have gained more and more attention with the hope to address many biological issues such as prevention of protein denaturation, interference of the fibrillation of disease relative proteins, removing of toxic biomolecules as well as targeting delivery of proteins, etc. In such cases, proper analytic techniques are needed to reveal the underlying mechanism of the particle-protein interactions. In the current work, Förster Resonance Energy Transfer (FRET) was used to investigate the interaction of our tailor designed artificial chaperone based on mixed shell polymeric micelles (MSPMs) with their substrate proteins. We designed a new kind of MSPMs with fluorescent acceptors precisely placed at the desired locations as well as hydrophobic domains which can adsorb unfolded proteins with a propensity to aggregate. Interactions of such model micelles with a donor-labeled protein-FITC-lysozyme, was monitored by FRET. The fabrication strategy of MSPMs makes it possible to control the accurate location of the acceptor, which is critical to reveal some unexpected insights of the micelle-protein interactions upon heating and cooling. Preadsorption of native proteins onto the hydrophobic domains of the MSPMs is a key step to prevent thermo-denaturation by diminishing interprotein aggregations. Reversible protein adsorption during heating and releasing during cooling have been confirmed. Conclusions from the FRET effect are in line with the measurement of residual enzymatic activity.

Thermosensitive mixed shell polymeric micelles decorated with gold nanoparticles at the outmost surface: tunable surface plasmon resonance and enhanced catalytic properties with excellent colloidal stability

Gold NPs are coupled to the outermost surface of mixed shell polymeric micelles with a PEG/PNIPAM shell, exhibit thermoresponsive surface plasmon resonance, enhanced catalytic properties and excellent colloidal stability.

Maintenance of Amyloid β Peptide Homeostasis by Artificial Chaperones Based on Mixed‐Shell Polymeric Micelles

AbstractThe disruption of Aβ homeostasis, which results in the accumulation of neurotoxic amyloids, is the fundamental cause of Alzheimer’s disease (AD). Molecular chaperones play a critical role in controlling undesired protein misfolding and maintaining intricate proteostasis in vivo. Inspired by a natural molecular chaperone, an artificial chaperone consisting of mixed‐shell polymeric micelles (MSPMs) has been devised with tunable surface properties, serving as a suppressor of AD. Taking advantage of biocompatibility, selectivity toward aberrant proteins, and long blood circulation, these MSPM‐based chaperones can maintain Aβ homeostasis by a combination of inhibiting Aβ fibrillation and facilitating Aβ aggregate clearance and simultaneously reducing Aβ‐mediated neurotoxicity. The balance of hydrophilic/hydrophobic moieties on the surface of MSPMs is important for their enhanced therapeutic effect.

Maintenance of amyloid β peptide homeostasis by artificial chaperones based on mixed-shell polymeric micelles.

The disruption of Aβ homeostasis, which results in the accumulation of neurotoxic amyloids, is the fundamental cause of Alzheimer's disease (AD). Molecular chaperones play a critical role in controlling undesired protein misfolding and maintaining intricate proteostasis in vivo. Inspired by a natural molecular chaperone, an artificial chaperone consisting of mixed-shell polymeric micelles (MSPMs) has been devised with tunable surface properties, serving as a suppressor of AD. Taking advantage of biocompatibility, selectivity toward aberrant proteins, and long blood circulation, these MSPM-based chaperones can maintain Aβ homeostasis by a combination of inhibiting Aβ fibrillation and facilitating Aβ aggregate clearance and simultaneously reducing Aβ-mediated neurotoxicity. The balance of hydrophilic/hydrophobic moieties on the surface of MSPMs is important for their enhanced therapeutic effect.

Structure change of mixed shell polymeric micelles and its interaction with bio-targets as probed by the 1-anilino-8-naphthalene sulfonate (ANS) fluorescence

Multicompartment polymeric micelles (MPMs) have attracted broad interest, due to their intriguing advantages. Although a plethora of MPMs have been designed recently, characterization of the fine hierarchical compartmented structure and dynamic conformation change of MPMs are still challenging. In this contribution, we reported a strategy to detect thermo-induced structure rearrangement of one kind of MPMs--mixed shell polymeric micelles (MSPMs) and its interaction with bio-targets such as proteins by the means of the 1-anilino-8-naphthalene sulfonate (ANS) fluorescence. It is found that there exists a specific fluorescent emission phenomena characterized by a strong blue-shifted emission maximum and enhanced quantum yield when ANS interacts with MSPMs with a mixed shell consisting of homogeneously mixed poly(ethylene oxide) (PEG) and poly(N-isopropylacryamide) (PNIPAM). Such emission maximum was exploited to probe the structure evolution of MSPMs during the collapse of the thermo-sensitive PNIPAM component by heating. The variation of the emission behavior of ANS during heating is in line with the structure rearrangement of the MSPMs which critically depends on the nature of the micellar core. Binding of a model protein-carbonic anhydrase B (CAB) during heating induced denaturation to the hydrophobic PNIPAM domains of the MSPMs was also reflected by the change of the emission behavior of ANS.

Temperature‐Responsive Mixed‐Shell Polymeric Micelles for the Refolding of Thermally Denatured Proteins

We have fabricated a mixed-shell polymeric micelle (MSPM) that closely mimics the natural molecular chaperone GroEL-GroES complex in terms of structure and functionality. This MSPM, which possesses a shared PLA core and a homogeneously mixed PEG and PNIAPM shell, is constructed through the co-assembly of block copolymers poly(lactide-b-poly(ethylene oxide) (PLA-b-PEG) and poly(lactide)-b-poly(N-isopropylacryamide) (PLA-b-PNIPAM). Above the lower critical solution temperature (LCST) of PNIPAM, the MSPM evolves into a core-shell-corona micelle (CSCM), as a functional state with hydrophobic PNIPAM domains on its surface. Light scattering (LS), TEM, and fluorescence and circular dichroism (CD) spectroscopy were performed to investigate the working mechanism of the chaperone-like behavior of this system. Unfolded protein intermediates are captured by the hydrophobic PNIPAM domains of the CSCM, which prevent harmful protein aggregation. During cooling, PNIPAM reverts into its hydrophilic state, thereby inducing the release of the bound unfolded proteins. The refolding process of the released proteins is spontaneously accomplished by the presence of PEG in the mixed shell. Carbonic anhydrase B (CAB) was chosen as a model to investigate the refolding efficiency of the released proteins. In the presence of MSPM, almost 93 % CAB activity was recovered during cooling after complete denaturation at 70 °C. Further results reveal that this MSPM also works with a wide spectrum of proteins with more-complicated structures, including some multimeric proteins. Given the convenience and generality in preventing the thermal aggregation of proteins, this MSPM-based chaperone might be useful for preventing the toxic aggregation of misfolded proteins in some diseases.