Nanochaperones Mediated Delivery of Insulin.

Insulin would undergo unfolding and fibrillation under stressed conditions, which may cause serious biotechnological and medical problems. Herein, by mimicking the structure and functions of natural chaperones HSP70s, self-assembled polymeric micelles are used as nanochaperones for the delivery of insulin. The confined hydrophobic domains on the surface of nanochaperones adsorb partially unfolded insulin, inhibiting the aggregation and fibrillation and enhancing the stability of insulin. The bioactivity of insulin is well-reserved after incubation with the nanochaperones at 37 °C for 7 d or heating at 70 °C for 1 h. The stealthy poly(ethylene glycol) chains around the confined domains protect the adsorbed insulin from enzymatic degradation and prolong the circulation time. More importantly, the excellent glucose sensitivity of the hydrophobic domains enables the nanochaperones to release and refold insulin in native form in response to hyperglycemia. This kind of nanochaperone may offer a hopeful strategy for the protection and delivery of insulin.

A Balance Between Capture and Release: How Nanochaperones Regulate Refolding of Thermally Denatured Proteins.

Nanochaperones have been designed and used for regulating the (re)folding of proteins, treating protein misfolding-related diseases, and, more recently, in drug delivery. Despite various successes, a complete understanding of the working mechanisms remains elusive, which represents a challenge for the realization of their full potential. Here, we thoroughly investigated the functioning of differently charged nanochaperones that regulate the refolding of thermally denatured lysozyme. We found that the balance between the capture and release of lysozyme clients that are controlled by nanochaperones plays a key role in regulating refolding. More importantly, the findings could be applied to other enzymes with various physicochemical properties. On the basis of these results, we could recover the activity of enzymes with high efficiency either after 20 days of storage at 40 °C or heating at high temperatures for 30-60 min. Our results provide important new design strategies for nanochaperone systems to improve their properties and expand their applications.

Coating of a Novel Antimicrobial Nanoparticle with a Macrophage Membrane for the Selective Entry into Infected Macrophages and Killing of Intracellular Staphylococci.

Internalization of Staphylococcus aureus by macrophages can inactivate bacterial killing mechanisms, allowing intracellular residence and dissemination of infection. Concurrently, these staphylococci can evade antibiotics that are frequently unable to pass mammalian cell membranes. A binary, amphiphilic conjugate composed of triclosan and ciprofloxacin is synthesized that self-assemble through micelle formation into antimicrobial nanoparticles (ANPs). These novel ANPs are stabilized through encapsulation in macrophage membranes, providing membrane-encapsulated, antimicrobial-conjugated NPs (Me-ANPs) with similar protein activity, Toll-like receptor expression and negative surface charge as their precursor murine macrophage/human monocyte cell lines. The combination of Toll-like receptors and negative surface charge allows uptake of Me-ANPs by infected macrophages/monocytes through positively charged, lysozyme-rich membrane scars created during staphylococcal engulfment. Me-ANPs are not engulfed by more negatively charged sterile cells possessing less lysozyme at their surface. The Me-ANPs kill staphylococci internalized in macrophages in vitro. Me-ANPs likewise kill staphylococci more effectively than ANPs without membrane-encapsulation or clinically used ciprofloxacin in a mouse peritoneal infection model. Similarly, organ infections in mice created by dissemination of infected macrophages through circulation in the blood are better eradicated by Me-ANPs than by ciprofloxacin. These unique antimicrobial properties of macrophage-monocyte Me-ANPs provide a promising direction for human clinical application to combat persistent infections.

NanoRNP Overcomes Tumor Heterogeneity in Cancer Treatment.

Tumor heterogeneity has been one of the most important factors leading to the failure of conventional cancer therapies due to the accumulation of genetically distinct tumor-cell subpopulations during the tumor development process. Due to the diversity of genetic mutations during tumor growth, combining the use of multiple drugs has only achieved limited success in combating heterogeneous tumors. Herein, we report a novel antitumor strategy that effectively addresses tumor heterogeneity by using a CRISPR/Cas9-based nanoRNP carrying a combination of sgRNAs. Such nanoRNP is synthesized from Cas9 ribonucleoprotein, any combinations of required sgRNAs, and a rationally designed responsive polymer that endows nanoRNP with high circulating stability, enhanced tumor accumulation, and the efficient gene editing in targeted tumor cells eventually. By carrying a combination of sgRNAs that targets STAT3 and RUNX1, the nanoRNP exhibited efficient gene expression disruptions on a heterogeneous tumor model with two subsets of cells whose proliferations were sensitive to the reduced expression of STAT3 and RUNX1, respectively, leading to the effective growth inhibition of the heterogeneous tumor. Considering the close relationship between tumor heterogeneity and cancer progression, resistance to therapy, and recurrences, nanoRNP provides a feasible strategy to overcome tumor heterogeneity in the development of more advanced cancer therapy against malignant tumors.

Nanocomposites Inhibit the Formation, Mitigate the Neurotoxicity, and Facilitate the Removal of ß-Amyloid Aggregates in Alzheimer's Disease Mice.

Alzheimer's disease (AD) is a progressive and irreversible brain disorder. Recent studies revealed the pivotal role of ß-amyloid (Aß) in AD. However, there is no conclusive indication that the existing therapeutic strategies exerted any effect on the mitigation of Aß-induced neurotoxicity and the elimination of Aß aggregates simultaneously in vivo. Herein, we developed a novel nanocomposite that can eliminate toxic Aß aggregates and mitigate Aß-induced neurotoxicity in AD mice. This nanocomposite was designed to be a small-sized particle (14 ± 4 nm) with Aß-binding peptides (KLVFF) integrated on the surface. The nanocomposite was prepared by wrapping a protein molecule with a cross-linked KLVFF-containing polymer layer synthesized by in situ polymerization. The presence of the nanocomposite remarkably changed the morphology of Aß aggregates, which led to the formation of Aß/nanocomposite coassembled nanoclusters instead of Aß oligomers. With the reduction of the pathological Aß oligomers, the nanocomposites attenuated the Aß-induced neuron damages, regained endocranial microglia's capability to phagocytose Aß, and eventually protected hippocampal neurons against apoptosis. Thus, we anticipate that the small-sized nanocomposite will potentially offer a feasible strategy in the development of novel AD treatments.

Spatial Confined Synergistic Enzymes with Enhanced Uricolytic Performance and Reduced Toxicity for Effective Gout Treatment.

Confinement of urate oxidase with detoxifying enzymes into multienzyme architecture is an appealing approach for gout treatment due to its capability to decompose serum uric acid without generation of H2 O2 . However, most of these strategies involve chemical modifications to the enzymes and barely consider enhancing the stability of the multienzyme architectures particularly against proteolysis, which significantly dampened its catalytic activity and in vivo stability. Herein, a novel strategy to prepare multienzyme nanoclusters with highly uricolytic activity and enhanced stability is demonstrated. With the close proximation, catalase can effectively decompose the H2 O2 generated by uricase during uricolysis. Moreover, with a shell structure constructed with polyethylene glycol, the nanocluster achieves great performance in reducing the nonspecific serum protein adsorptions and proteases digestion, leading to an enhanced circulation time after the intravenous administration. Such complementary multienzyme nanoclusters realize the long-term therapeutic effect in the management of serum uric acid level, without any toxicity or undesired immune responses in vivo. This work mimics the synergistic effect of protein complex in nature and can be further developed to a general method for the construction of multienzyme nanoclusters, which provides new opportunities for utilizing therapeutic enzymes for the treatment of metabolic diseases.

Nitrilotriacetic Acid-Functionalized Glucose-Responsive Complex Micelles for the Efficient Encapsulation and Self-Regulated Release of Insulin.

Insulin plays a significant role in diabetes treatment. Although a huge number of insulin-loaded, glucose-responsive nanocarriers have been developed in past decades, most of them showed a lower loading capacity and efficiency due to the weak interaction between insulin and nanocarriers. In this work, a novel insulin-encapsulated glucose-responsive polymeric complex micelle (CM) is devised, showing (i) enhanced insulin-loading efficiency owing to the zinc ions' chelation by nitrilotriacetic acid (NTA) groups of NTA-functioned glycopolymer and the histidine imidazole of insulin, (ii) the glucose-triggered pulse release of insulin, and (iii) long stability under physiological conditions. This CM was fabricated by the self-assembly of block copolymer PEG- b-P(Asp- co-AspPBA) and glycopolymer P(Asp- co-AspGA- co-AspNTA), resulting in complex micelles with a PEG shell and a cross-linked core composed of phenylboronic acid (PBA)/glucose complexations. Notably, the modified nitrilotriacetic acid (NTA) groups of CM could specifically bind insulin via chelated zinc ions, thus enhancing the loading efficacy of insulin compared to that of nonmodified CM. The dynamic PBA/glucose complexation core of CM dissociates under the trigger of high glucose concentration (>2 g/L) while being quite stable in low glucose concentrations (<2 g/L), as demonstrated by the pulse release of insulin in vitro. Finally, in a murine model of type 1 diabetes, NTA-modified complex micelles loading an insulin (NTA-CM-INS) group exhibited a long hypoglycemic effect which is superior to that of free insulin in the PBS (PBS-INS) group and insulin-loaded complex micelles without an NTA modification (CM-INS) group. This long-term effect benefited from Zn(II) chelation by NTA-modified complex micelles and could avoid hypoglycemia caused by the burst release of insulin. Taken together, this constitutes a highly effective way to encapsulate insulin and release insulin via an on-demand manner for blood glucose control in diabetes.

Photoswitchable Micelles for the Control of Singlet-Oxygen Generation in Photodynamic Therapies.

Inadvertent photosensitizer-activation and singlet-oxygen generation hampers clinical application of photodynamic therapies of superficial tumors or subcutaneous infections. Therefore, a reversible photoswitchable system was designed in micellar nanocarriers using ZnTPP as a photosensitizer and BDTE as a photoswitch. Singlet-oxygen generation upon irradiation didnot occur in closed-switch micelles with ZnTPP/BDTE feeding ratios >1:10. Deliberate switch closure/opening within 65-80 min was possible through thin layers of porcine tissue in vitro, increasing for thicker layers. Inadvertent opening of the switch by simulated daylight, took several tens of hours. Creating deliberate cell damage and prevention of inadvertent damage in vitro and in mice could be done at lower ZnTPP/BDTE feeding ratios (1:5) in open-switch micelles and at higher irradiation intensities than inferred from chemical clues to generate singlet-oxygen. The reduction of inadvertent photosensitizer activation in micellar nanocarriers, while maintaining the ability to kill tumor cells and infectious bacteria established here, brings photodynamic therapies closer to clinical application.

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.

Glucose-responsive polymer vesicles templated by α-CD/PEG inclusion complex.

Polymeric nanoparticles with glucose-responsiveness are of great interest in developing a self-regulated drug delivery system. In this work, glucose-responsive polymer vesicles were fabricated based on the complexation between a glucosamine (GA)-containing block copolymer PEG45-b-P(Asp-co-AspGA) and a phenylboronic acid (PBA)-containing block copolymer PEG114-b-P(Asp-co-AspPBA) with α-CD/PEG45 inclusion complex as the sacrificial template. The obtained polymer vesicles composed of cross-linked P(Asp-co-AspGA)/P(Asp-co-AspPBA) layer as wall and PEG chains as both inner and outer coronas. The vesicular morphology was observed by transmission electron microscopy (TEM), and the glucose-responsiveness was investigated by monitoring the variations of hydrodynamic diameter (Dh) and light scattering intensity (LSI) in the polymer vesicle solution with glucose using dynamic light scattering (DLS). Vancomycin as a model drug was encapsulated in the polymer vesicles and sugar-triggered drug release was carried out. This kind of polymer vesicle may be a promising candidate for glucose-responsive drug delivery.

Aggregation behavior of the template-removed 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin chiral array directed by poly(ethylene glycol)-block-poly(L-lysine).

Complexation between 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrin (TPPS) and poly(ethylene glycol)-block-poly(L-lysine) (PEG-b-PLL) was performed via electrostatic interaction. Two kinds of primary arrays of TPPS with different supramolecular chirality induced by PLL were obtained in the resultant complex by inverting the mixing procedure of the two components. These arrays could be displaced by poly(sodium-p-styrenesulfonate) (PSS) from the chiral PLL template through competitive electrostatic complexation, and then PSS formed a polyion complex micelle with PEG-b-PLL. The template-removed TPPS arrays preserved their induced chirality and served as primary subunits for the secondary aggregation of TPPS. The morphology of the secondary aggregates was strongly dependent upon the asymmetric primary supramolecular arrangement of TPPS. The rodlike nanostructure that was ∼200 nm in length was composed of the primary arrays that showed opposite exciton chirality between the J- and H-bands. In contrast, the micrometer-sized fibrils observed were composed of the arrays with the same exciton chirality at the J- and H-bands.

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

The disruption of Aß 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 Aß homeostasis by a combination of inhibiting Aß fibrillation and facilitating Aß aggregate clearance and simultaneously reducing Aß-mediated neurotoxicity. The balance of hydrophilic/hydrophobic moieties on the surface of MSPMs is important for their enhanced therapeutic effect.

Cetyltrimethylammonium-chloride assisted in situ metabolic incorporation of nano-sized ROS-generating cascade-reaction containers in Gram-positive and Gram-negative peptidoglycan layers for the control of bacterially-induced sepsis.

Cascade-reaction containers generating reactive oxygen species (ROS) as an alternative for antibiotic-based strategies for bacterial infection control, require endogenous oxygen-sources and ROS-generation close to or preferably inside target bacteria. Here, this is achieved by cetyltrimethylammonium-chloride (CTAC) assisted in situ metabolic labeling and incorporation of mesoporous SiO2-nanoparticles, dual-loaded with glucose-oxidase and Fe3O4-nanoparticles as cascade-reaction containers, inside bacterial cell walls. First, azide-functionalized d-alanine (D-Ala-N3) was inserted in cell wall peptidoglycan layers of growing Gram-positive pathogens. In Gram-negatives, this could only be achieved after outer lipid-membrane permeabilization, using a low concentration of CTAC. Low concentrations of CTAC had no adverse effect on in vitro blood clotting or hemolysis nor on the health of mice when blood-injected. Next, dibenzocyclooctyne-polyethylene-glycol modified, SiO2-nanoparticles were in situ click-reacted with d-Ala-N3 in bacterial cell wall peptidoglycan layers. Herewith, a two-step cascade-reaction is facilitated inside bacteria, in which glucose-oxidase generates H2O2 at endogenously-available glucose concentrations, while subsequently Fe3O4-nanoparticles catalyze generation of •OH from the H2O2 generated. Generation of •OH inside bacterial cell walls by dual-loaded mesoporous SiO2-nanoparticles yielded more effective in vitro killing of both planktonic Gram-positive and Gram-negative bacteria suspended in 10 % plasma than SiO2-nanoparticles solely loaded with glucose-oxidase. Gram-positive or Gram-negative bacterially induced sepsis in mice could be effectively treated by in situ pre-treatment with tail-vein injected CTAC and d-Ala-N3, followed by injection of dual-loaded cascade-reaction containers without using antibiotics. This makes in situ metabolic incorporation of cascade-reaction containers as described attractive for further investigation with respect to the control of other types of infections comprising planktonic bacteria. STATEMENT OF SIGNIFICANCE: In situ metabolic-incorporation of cascade-reaction-containers loaded with glucose-oxidase and Fe3O4 nanoparticles into bacterial cell-wall peptidoglycan is described, yielding ROS-generation from endogenous glucose, non-antibiotically killing bacteria before ROS inactivates. Hitherto, only Gram-positives could be metabolically-labeled, because Gram-negatives possess two lipid-membranes. The outer membrane impedes direct access to the peptidoglycan. This problem was solved by outer-membrane permeabilization using a quaternary-ammonium compound. Several studies on metabolic-labeling perform crucial labeling steps during bacterial-culturing that in real-life should be part of a treatment. In situ metabolic-incorporation as described, can be applied in well-plates during in vitro experiments or in the body as during in vivo animal experiments. Surprisingly, metabolic-incorporation proceeded unhampered in blood and a murine, bacterially-induced sepsis could be well treated.

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.

pH/sugar dual responsive core-cross-linked PIC micelles for enhanced intracellular protein delivery.

Herein, a series of biocompatible, robust, pH/sugar-sensitive, core-cross-linked, polyion complex (PIC) micelles based on phenylboronic acid-catechol interaction were developed for protein intracellular delivery. The rationally designed poly(ethylene glycol)-b-poly(glutamic acid-co-glutamicamidophenylboronic acid) (PEG-b-P(Glu-co-GluPBA)) and poly(ethylene glycol)-b-poly(l-lysine-co-ε-3,4-dihydroxyphenylcarboxyl-L-lysine) (PEG-b-P(Lys-co-LysCA)) copolymers were successfully synthesized and self-assembled under neutral aqueous condition to form uniform micelles. These micelles possessed a distinct core-cross-linked core-shell structure comprised of the PEG outer shell and the PGlu/PLys polyion complex core bearing boronate ester cross-linking bonds. The cross-linked micelles displayed superior physiological stabilities compared with their non-cross-linked counterparts while swelling and disassembling in the presence of excess fructose or at endosomal pH. Notably, either negatively or positively charged proteins can be encapsulated into the micelles efficiently under mild conditions. The in vitro release studies showed that the release of protein cargoes under physiological conditions was minimized, while a burst release occurred in response to excess fructose or endosomal pH. The cytotoxicity of micelles was determined by cck-8 assay in HepG2 cells. The cytochrome C loaded micelles could efficiently delivery proteins into HepG2 cells and exhibited enhanced apoptosis ability. Hence, this type of core-cross-linked PIC micelles has opened a new avenue to intracellular protein delivery.

Holdase/Foldase Mimetic Nanochaperone Improves Antibody-Based Cancer Immunotherapy.

Despite unprecedented successes of antibody-based cancer immunotherapy, the serious side effects and rapid clearance following systemic administration remain big challenges to realize its full potential. At the same time, combination immunotherapy using multiple antibodies has shown particularly promising in cancer treatment. It is noticed that the working mechanisms of natural holdase and foldase chaperone are desirable to overcome the limitations of therapeutic antibodies. Holdase chaperone stabilizes unfolded client and prevents it from activation and degradation, while foldase chaperone assists unfolded client to its native state to function. Here a holdase/foldase mimetic nanochaperone (H/F-nChap) to co-delivery two types of monoclonal antibodies (mAbs), αCD16 and αPDL1, and resiquimod (R848) is developed, which significantly improves cancer immunotherapy. The H/F-nChap presents holdase activity in blood and normal tissues that hides and protects mAbs from unnecessary targeted activation and degradation, thereby prolonging blood circulation and reducing immunotoxicity in vivo. Furthermore, H/F-nChap switches to foldase activity in the tumor microenvironment that exposes mAbs and releases R848 to enhance the engagement between NK cells and tumor cells and promote immune activation, respectively. The H/F-nChap represents a strategy for safe and spatiotemporal delivery of multiple mAbs, providing a promising platform for improved cancer immunotherapy.

Investigation of the interaction between the functionalized mesoporous silica nanocarriers and bovine serum albumin via multi-spectroscopy.

It is well known that the physicochemical properties of nanocarriers, which are closely related to the surface modification of nanoparticles, have crucial impacts on their biological effects. Herein, the interaction between functionalized degradable dendritic mesoporous silica nanoparticles (DDMSNs) and bovine serum albumin (BSA) was investigated for probing into the nanocarriers' potential toxicity using multi-spectroscopy such as ultraviolet/visible (UV/Vis), synchronous fluorescence, Raman and circular dichroism (CD) spectroscopy. BSA, owing to its structural homology and high sequence similarity with HSA, was employed as the model protein to study the interactions with DDMSNs, amino-modified DDMSNs (DDMSNs-NH2) and hyaluronic acid (HA) coated nanoparticles (DDMSNs-NH2-HA). It was found that the static quenching behavior of DDMSNs-NH2-HA to BSA was accompanied by an endothermic and hydrophobic force-driven thermodynamic process, which was confirmed by fluorescence quenching spectroscopic studies and thermodynamic analysis. Furthermore, the conformational variations of BSA upon interaction with nanocarriers were observed by combination of UV/Vis, synchronous fluorescence, Raman and CD spectroscopy. The microstructure of amino residues in BSA changed due to the existence of nanoparticles, for example, the amino residues and hydrophobic groups exposed to microenvironment and the alpha helix (α-helix) content of BSA decreased. Specially, through thermodynamic analysis, the diverse binding modes and driving forces between nanoparticles and BSA were discovered because of different surface modifications on DDMSNs, DDMSNs-NH2 and DDMSNs-NH2-HA. We believe that this work can promote the interpretation of mutual impact between nanoparticles and biomolecules, which will be in favor of predicting the biological toxicity of nano-DDS and engineering functionalized nanocarriers.

Controlled release of ionic drugs from complex micelles with charged channels.

Oral administration of ionic drugs generally encounters with significant fluctuation in plasma concentration due to the large variation of pH value in the gastrointestinal tract and the pH-dependent solubility of ionic drugs. Polymeric complex micelles with charged channels on the surface provided us with an effective way to reduce the difference in the drug release rate upon change in pH value. The complex micelles were prepared by self-assembly of PCL-b-PAsp and PCL-b-PNIPAM in water at room temperature with PCL as the core and PAsp/PNIPAM as the mixed shell. With an increase in temperature, PNIPAM collapsed and enclosed the PCL core, while PAsp penetrated through the PNIPAM shell, leading to the formation of negatively charged PAsp channels on the micelle surface. Release behavior of ionic drugs from the complex micelles was remarkably different from that of usual core-shell micelles where diffusion and solubility of drugs played a key role. Specifically, it was mainly dependent on the conformation of the PAsp chains and the electrostatic interaction between PAsp and drugs, which could partially counteract the influence of pH-dependent diffusion and solubility of drugs. As a result, the variation of drug release rate with pH value was suppressed, which was favorable for acquiring relatively steady plasma drug concentration.

Phenylboronic acid-based complex micelles with enhanced glucose-responsiveness at physiological pH by complexation with glycopolymer.

Polymeric nanoparticles with glucose-responsiveness under physiological conditions are of great interests in developing drug delivery system for the treatment of diabetes. Herein, glucose-responsive complex micelles were prepared by self-assembly of a phenylboronic acid-contained block copolymer PEG-b-P(AA-co-APBA) and a glycopolymer P(AA-co-AGA) based on the covalent complexation between phenylboronic acid and glycosyl. The formation of the complex micelles with a P(AA-co-APBA)/P(AA-co-AGA) core and a PEG shell was confirmed by HNMR analysis. The glucose-responsiveness of the complex micelles was investigated by monitoring the light scattering intensity and the fluorescence (ARS) of the micelle solutions. The complex micelles displayed an enhanced glucose-responsiveness compared to the simple PEG-b-P(AA-co-APBA) micelles and the sensitivity of the complex micelles to glucose increased with the decrease of the amount of P(AA-co-AGA) in the compositions. The cytotoxicity of the polymers and the complex micelles was also evaluated by MTT assay. This kind of complex micelles may be an excellent candidate for insulin delivery and may find application in the treatment of diabetes.

A Guanosine-Quadruplex Hydrogel as Cascade Reaction Container Consuming Endogenous Glucose for Infected Wound Treatment-A Study in Diabetic Mice.

Diabetic foot ulcers infected with antibiotic-resistant bacteria form a severe complication of diabetes. Antimicrobial-loaded hydrogels are used as a dressing for infected wounds, but the ongoing rise in the number of antimicrobial-resistant infections necessitates new, nonantibiotic based designs. Here, a guanosine-quadruplex (G4 )-hydrogel composed of guanosine, 2-formylphenylboronic acid, and putrescine is designed and used as a cascade-reaction container. The G4 -hydrogel is loaded with glucose-oxidase and hemin. The first cascade-reaction, initiated by glucose-oxidase, transforms glucose and O2  into gluconic acid and H2 O2 . In vitro, this reaction is most influential on killing Staphylococcus aureus or Pseudomonas aeruginosa in suspension, but showed limited killing of bacteria in biofilm-modes of growth. The second cascade-reaction, however, transforming H2 O2  into reactive-oxygen-species (ROS), also enhances killing of biofilm bacteria due to hemin penetration into biofilms and interaction with eDNA G-quadruplexes in the biofilm matrix. Therewith, the second cascade-reaction generates ROS close to the target bacteria, facilitating killing despite the short life-time of ROS. Healing of infected wounds in diabetic mice proceeds faster upon coverage by these G4 -hydrogels than by clinically common ciprofloxacin irrigation. Moreover, local glucose concentrations around infected wounds decrease. Concluding, a G4 -hydrogel loaded with glucose-oxidase and hemin is a good candidate for infected wound dressings, particularly in diabetic patients.