Efficacy of peptide-based enamel coatings in the prevention of demineralization using fixed orthodontic brackets in a rat model.

INTRODUCTION: White spot lesions (WSLs) represent a prominent pathology encountered during orthodontic treatment, originating from enamel demineralization induced by the accumulation of bacterial biofilms. The previously developed bioinspired enamel coating form of self-assembling antimicrobial peptide D-GL13K exhibited antimicrobial activity and enhanced acid impermeability, offering a potential solution to prevent demineralization. The primary aim of this investigation is to assess the in vivo anti-demineralization properties and biocompatibility of the D-GL13K coating. METHODS: A rat model was developed to assess the antimicrobial enamel coating during fixed orthodontic treatment. The anti-demineralization efficacy attributed to the D-GL13K coating was evaluated by employing optical coherence tomography, Vickers microhardness testing, and scanning electron microscopy. The biocompatibility of the D-GL13K coating was investigated through histologic observations of vital organs and tissues using hematoxylin and eosin. RESULTS: The D-GL13K coating demonstrated significant anti-demineralization effects, evidenced by reduced demineralization depth analyzed through optical coherence tomography and enhanced Vickers hardness than in the noncoated control group, showcasing the coating's potential to protect teeth from WSLs. Scanning electron microscopy analysis further elucidated the diminished enamel damage observed in the group treated with D-GL13K. Importantly, histologic examination of vital organs and tissues using hematoxylin and eosin staining revealed no overt disparities between the D-GL13K coated group and the noncoated control group. CONCLUSIONS: The D-GL13K enamel coating demonstrated promising anti-demineralization and biocompatibility properties in a rat model, thereby suggesting its potential for averting WSLs after orthodontic interventions. Further research in human clinical settings is needed to evaluate the coating's long-term efficacy.

Robust paths to net greenhouse gas mitigation and negative emissions via advanced biofuels.

Biofuel and bioenergy systems are integral to most climate stabilization scenarios for displacement of transport sector fossil fuel use and for producing negative emissions via carbon capture and storage (CCS). However, the net greenhouse gas mitigation benefit of such pathways is controversial due to concerns around ecosystem carbon losses from land use change and foregone sequestration benefits from alternative land uses. Here, we couple bottom-up ecosystem simulation with models of cellulosic biofuel production and CCS in order to track ecosystem and supply chain carbon flows for current and future biofuel systems, with comparison to competing land-based biological mitigation schemes. Analyzing three contrasting US case study sites, we show that on land transitioning out of crops or pasture, switchgrass cultivation for cellulosic ethanol production has per-hectare mitigation potential comparable to reforestation and severalfold greater than grassland restoration. In contrast, harvesting and converting existing secondary forest at those sites incurs large initial carbon debt requiring long payback periods. We also highlight how plausible future improvements in energy crop yields and biorefining technology together with CCS would achieve mitigation potential 4 and 15 times greater than forest and grassland restoration, respectively. Finally, we show that recent estimates of induced land use change are small relative to the opportunities for improving system performance that we quantify here. While climate and other ecosystem service benefits cannot be taken for granted from cellulosic biofuel deployment, our scenarios illustrate how conventional and carbon-negative biofuel systems could make a near-term, robust, and distinctive contribution to the climate challenge.

Enzyme-Responsive Branched Glycopolymer-Based Nanoassembly for Co-Delivery of Paclitaxel and Akt Inhibitor toward Synergistic Therapy of Gastric Cancer.

Combined chemotherapy and targeted therapy holds immense potential in the management of advanced gastric cancer (GC). GC tissues exhibit an elevated expression level of protein kinase B (AKT), which contributes to disease progression and poor chemotherapeutic responsiveness. Inhibition of AKT expression through an AKT inhibitor, capivasertib (CAP), to enhance cytotoxicity of paclitaxel (PTX) toward GC cells is demonstrated in this study. A cathepsin B-responsive polymeric nanoparticle prodrug system is employed for co-delivery of PTX and CAP, resulting in a polymeric nano-drug BPGP@CAP. The release of PTX and CAP is triggered in an environment with overexpressed cathepsin B upon lysosomal uptake of BPGP@CAP. A synergistic therapeutic effect of PTX and CAP on killing GC cells is confirmed by in vitro and in vivo experiments. Mechanistic investigations suggested that CAP may inhibit AKT expression, leading to suppression of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway. Encouragingly, CAP can synergize with PTX to exert potent antitumor effects against GC after they are co-delivered via a polymeric drug delivery system, and this delivery system helped reduce their toxic side effects, which provides an effective therapeutic strategy for treating GC.

Multifunctional nanocoating for enhanced titanium implant osseointegration.

Preventing bacterial infection and promoting osseointegration are essential for the long-term success of titanium (Ti) implants. In this study, we developed a multifunctional nanocoating on Ti mini-implants to simultaneously address these challenges. The nanocoating consists of self-assembled antimicrobial peptides GL13K and silver nanoparticles, referred to as Ag-GL. Our results showed that the Ag-GL coating did not alter the surface morphology of the mini-implants. Ag-GL coated mini-implants demonstrated a two orders of magnitude reduction in colony-forming unit (CFU) values compared to the noncoated eTi group, resulting in minimal inflammation and no apparent bone destruction in a bacterial infection in vivo model. When evaluating osseointegration properties, micro-CT analysis, histomorphometric analysis, and pull-out tests revealed that the Ag-GL coating significantly enhanced osseointegration and promoted new bone formation in vivo.

Ammonium sulfate gradient loading of brucine into liposomes: effect of phospholipid composition on entrapment efficiency and physicochemical properties in vitro.

BACKGROUND: Brucine, the major active alkaloid constituent extracted from traditional Chinese herbal medicine Nux vomica, had been found to possess remarkable antitumor, analgesic, and anti-inflammatory activities. In this study, we attempted to encapsulate brucine into liposomes to improve its therapeutic effects. The entrapment efficiency (EE) and the stability of liposomes are two key factors associated with the therapeutic effects of liposomal drugs. We developed a novel liposome-based brucine formulation that was composed of soybean phosphatidylcholine (SPC) and hydrogenated soybean phosphatidylcholine (HSPC). METHOD: The liposomes with different phospholipid composition were characterized for their EE, vesicle size, drug release profile, and leakage in vitro. RESULTS: The molar ratio of HSPC/SPC = 1:9 was determined as the optimum ratio. Compared with conventional liposomes composed of only SPC or HSPC, EE of the brucine-loaded novel liposomes was increased markedly, especially at high drug/lipid molar ratios. The results of drug release showed that the novel liposomes were more stable than the conventional SPC liposomes in the presence of fetal calf serum. In addition, the results of the leakage experiments revealed that the novel liposomes also had better stability in phosphate buffer solution (PBS) with respect to drug retention. Although the conventional HSPC liposomes is more stable than the novel liposomes, the novel liposomes composed of 10% HSPC and 90% SPC may still have promising application potential because HSPC is much more expensive than SPC. CONCLUSION: Taken together, efficient encapsulation of brucine into the novel liposomes, their improved stability, and the price of phospholipids indicate that the novel liposomes may act as promising carriers for active alkaloids such as brucine.

Synthesis of a novel polymer bile salts-(polyethylene glycol)2000-bile salts and its application to the liver-selective targeting of liposomal DDB.

PURPOSE: The objective of this study was to achieve a sustained and targeted delivery of liposome to the liver, by modifying the phospholipid [phosphatidylcholine (PC)/cholesterol (10 : 1) liposomes with a novel polymer bile salts-(polyethylene glycol)(2000)-bile salts (BP(2)B). METHODS: First, we generated a novel BP(2)B by N,N'-dicyclohexylcarbodiimide/4-dimethylaminopyridine esterification method and confirmed by Fourier transform infraredand (1) H-NMR spectra. Second, we prepared the BP(2)B-modified liposomes (BP(2)BL) that included BP(2)B, and the effect of the weight ratios of BP(2)B/PC on entrapment efficiency was investigated and BP(2)B/PC = 3% (w/w) was determined as the optimum ratio for the 4,4'-dimethoxy-5,6,5',6'-bi (methylenedioxy)-2,2'-bicarbomethoxybiphenyl liposomes. And then, the ability of the liver target of BP(2)BL was studied by calculating the targeted parameters. RESULTS AND DISCUSSION: All the results revealed that the introduction of polyoxyethylene chains could control interactions of bile salt moieties on liposome surfaces with the receptor compared with traditional liposomes (CL), marking BP(2)BL as a suitable carrier for hepatic parenchymal cell-specific and sustained targeting. It was suggested that liposomes containing such novel BP(2)B have great potential as drug delivery carriers for the liver-selective targeting that has targeted and sustained drug delivery.

A case report on concurrent occurrence of systemic mastocytosis and myeloid sarcoma presenting with extensive skin involvements and the results of genetic study.

INTRODUCTION: Systemic mastocytosis is a rare disease due to mast cell accumulation in various extracutaneous sites. Systemic mastocytosis with an associated clonal hematologic non-MC lineage disease is the second most common subtype of systemic mastocytosis. The most common mutation associated with both systemic mastocytosis and myeloid sarcoma is mutation in Kit. Here, we identified the novel KIT D816V and ARID1A G1254S mutations co-occurring in systemic mastocytosis with myeloid sarcoma. PATIENT CONCERNS: A 33-year old male patient presented multiple skin lesions for 10 years. Symptoms accelerated in 2017 with decreased body weight. Physical examination revealed enlarged lymph nodes in his neck, axilla and inguinal region; conjunctival hemorrhage; gingival hyperplasia. Skin biopsy showed mast cell infiltration. Flow cytometry detected CD2, CD25 and CD117 positive cells in lymph nodes. Codon 816 KIT mutation D816V and codon 1245 ARID1A mutation G1254S were found in peripheral blood. MPO, CD117, CD68 positive cells in lymph nodes indicated co-existing myeloid sarcoma. DIAGNOSIS: Systemic mastocytosis with an associated clonal hematologic non-MC lineage disease of myeloid sarcoma INTERVENTIONS:: Cytarabine and daunorubicin for myeloid sarcoma and dasatinib for systemic mastocytosis were initiated. Anti-histamine and anti-leukotrienes therapy were used to prevent NSAIDs-induced shock. Platelets were infused to treat bone marrow suppression. OUTCOMES: Patient was discharged after recovered from bone marrow suppression. Dasatinib continued on outpatient. CONCLUSION: This is the first case of patient with systemic mastocytosis and myeloid sarcoma simultaneously presenting extensive skin involvements. Mutations of Kit and Arid1a emphasis the importance to notice possibility of various tumors occurring in patients with multiple mutations. In addition, cysteine-leukotrienes-receptor antagonists should always be used to prevent anaphylactic shock due to mast cell activation.

Stimuli-responsive polymer-doxorubicin conjugate: Antitumor mechanism and potential as nano-prodrug.

Polymer-drug conjugates has significantly improved the anti-tumor efficacy of chemotherapeutic drugs and alleviated their side effects. N-(1,3-dihydroxypropan-2-yl) methacrylamide (DHPMA) copolymer was synthesized via RAFT polymerization and polymer-doxorubicin (DOX) (diblock pDHPMA-DOX) were formed by conjugation, resulting in a self-aggregation-induced nanoprodrug with a favorable size of 21 nm and great stability. The nanoprodrug with a molecular weight (MW) of 95 kDa released drugs in response to tumor microenvironmental pH variations and they were enzymatically hydrolyzed into low MW segments (45 kDa). The nanoprodrug was transported through the endolysosomal pathway, released the drug into the cytoplasm and some was localized in the mitochondria, resulting in disruption of the cellular actin cytoskeleton. Cellular apoptosis was also associated with reduction in the mitochondrial potential caused by the nanoprodrug. Notably, the nanoprodrug had a significantly prolonged blood circulation time with an elimination half time of 9.8 h, displayed high accumulation within tumors, and improved the in vivo therapeutic efficacy against 4T1 xenograft tumors compared to free DOX. The tumor xenograft immunohistochemistry study clearly indicated tumor inhibition was through the inhibition of cell proliferation and antiangiogenic effects. Our studies demonstrated that the diblock pDHPMA-DOX nanoprodrug with a controlled molecular structure is promising to alleviate adverse effects of free DOX and have a great potential as an efficient anticancer agent. STATEMENT OF SIGNIFICANCE: In this work, we prepared a biodegradable diblock DHPMA polymer-doxorubicin conjugate via one-pot of RAFT polymerization and conjugate chemistry. The conjugate-based nanoprodrug was internalized by endocytosis to intracellularly release DOX and further induce disruption of mitochondrial functions, actin cytoskeleton alterations and cellular apoptosis. The nanoprodrug with a high molecular weight (MW) (95 kDa) showed a long blood circulation time and achieved high accumulation into tumors. The nanoprodrug was degraded into low MW (∼45 kDa) products below the renal threshold, which ensured its biosafety. Additionally, the multi-stimuli-responsive nanoprodrug demonstrated an enhanced antitumor efficacy against 4T1 breast tumors and alleviated side effects, showing a great potential as an efficient and safe anticancer agent.

Enzyme-Responsive Copolymer as a Theranostic Prodrug for Tumor In Vivo Imaging and Efficient Chemotherapy.

With the advance in nanomedicine, diagnostic and therapeutic nanoscale prodrugs have been rapidly developed in the field of cancer treatment. In this study, we constructed an enzyme-responsive polymer-paclitaxel (PTX) prodrug with a biocompatible saccharide-containing polymer backbone through the reversible addition-fragmentation chain transfer (RAFT) polymerization. A near-infrared fluorescent molecule (pheophorbide a) and magnetic resonance imaging (MRI) contrast agent (gadolinium-tetraazacyclododecanetetraacetic acid) were further conjugated onto the copolymer backbone to impart the ability of multimode imaging and tracing, forming the final diagnosis and treatment polymeric prodrug. This prodrug was amphiphilic and was able to self-assemble into uniform-size nanoparticles (80.1 nm). With the specific catalysis of enzymes, the anti-cancer drug, PTX, in the nanoparticles could be effectively released to kill cancer cells. The results of near-infrared fluorescence imaging and MRI showed that the diagnostic prodrug was preferentially concentrated at the tumor site compared with the free imaging reagents, suggesting improved and durable tumor imaging effects, which are beneficial for precise cancer diagnosis. The tumor growth in the mice could be effectively retarded after the administration of the prodrug. The tumor almost completely disappeared till the final treatment, and the tumor inhibition rate was as high as 96.4%. Immunohistochemical analysis indicated that the high anti-tumor effects might be attributed to the result that the prodrug not only induced the apoptosis of tumor cells, but also inhibited the formation of new blood vessels in the tumor environment. Therefore, this theranostic prodrug, which is based on the saccharide-containing polymer, holds potency for the development of a robust nanoscale platform for the diagnosis and treatment of cancer.

Tunable Hydrophile-Lipophile Balance for Manipulating Structural Stability and Tumor Retention of Amphiphilic Nanoparticles.

Hydrophile-lipophile balance (HLB) has a great influence on the self-assembly and physicochemical properties of amphiphiles, thus affecting their biological effects. It is shown that amphiphilic nanoparticles (NPs) with a moderate HLB value display enhanced stability and highly efficient tumor retention. 2,2-Bis(hydroxymethyl)propionic acid hyperbranched poly(ethylene glycol) (PEG)-pyropheophorbide-a (Ppa) amphiphiles (G320P, G310P, G220P, and G210P) are synthesized with a tunable HLB value from 6.1 to 9.9 by manipulating the number of generation of dendrons (G2 or G3) and the molecular weight of PEG chains (10 or 20 kDa). Molecular dynamics simulations reveal that G320P and G210P with a moderate HLB value (8.0 and 7.8) self-assemble into very stable NPs with a small solvent accessible surface area and high nonbonding interactions. G320P with a moderate HLB value (8.0) and a long PEG chain excels against other NPs in prolonging the blood circulation time of Ppa (up to 13-fold), penetrating deeply into multicellular tumor spheroids and accumulating in tumors, and enhancing the PDT efficacy with a tumor growth inhibition of 96.0%. Rational design of NPs with a moderate HLB value may be implemented in these NP-derived nanomedicines to achieve high levels of retention in tumors.

Hydrogen bond guidance and aromatic stacking drive liquid-liquid phase separation of intrinsically disordered histidine-rich peptides.

Liquid-liquid phase separation (LLPS) of intrinsically disordered proteins (IDPs) is involved in both intracellular membraneless organelles and extracellular tissues. Despite growing understanding of LLPS, molecular-level mechanisms behind this process are still not fully established. Here, we use histidine-rich squid beak proteins (HBPs) as model IDPs to shed light on molecular interactions governing LLPS. We show that LLPS of HBPs is mediated though specific modular repeats. The morphology of separated phases (liquid-like versus hydrogels) correlates with the repeats' hydrophobicity. Solution-state NMR indicates that LLPS is a multistep process initiated by deprotonation of histidine residues, followed by transient hydrogen bonding with tyrosine, and eventually by hydrophobic interactions. The microdroplets are stabilized by aromatic clustering of tyrosine residues exhibiting restricted molecular mobility in the nano-to-microsecond timescale according to solid-state NMR experiments. Our findings provide guidelines to rationally design pH-responsive peptides with LLPS ability for various applications, including bioinspired protocells and smart drug-delivery systems.

Enzyme/pH-sensitive polyHPMA-DOX conjugate as a biocompatible and efficient anticancer agent.

In this study, to enhance the therapeutic function and reduce the side-effects of doxorubicin (DOX), a biodegradable N-(2-hydroxypropyl) methacrylamide (HPMA) polymer-DOX conjugate has been prepared through reversible addition fragmentation chain transfer (RAFT) polymerization and conjugation chemistry, and the anticancer agent DOX was covalently linked to the polymeric vehicle through a pH-responsive hydrazone bond. The cellular mechanisms of the conjugate were explored, and the therapeutic indexes were studied as well. The high molecular weight (MW) polymeric conjugate (94 kDa) was degraded into products with low MW (45 kDa) in the presence of lysosomal cathepsin B and also showed pH-responsive drug release behavior. In vitro cellular mechanism studies revealed that the polymeric conjugate was uptaken by the 4T1 cells, leading to cell apoptosis and cytotoxicity to cancer cells, while the polymeric conjugate demonstrated excellent in vivo biosafety even at a high dose. Compared to free DOX, the conjugate has a much longer half-life in pharmacokinetics and accumulates in tumors with a much higher amount. The conjugate therefore has a much greater in vivo anticancer efficacy against 4T1 xenograft tumors and shows subtle side-effects, which were confirmed via tumor size and weight, immunohistochemistry and histological studies. Overall, this polymeric conjugate may be used as an enzyme/pH-sensitive anticancer agent.

PEGylated Multistimuli-Responsive Dendritic Prodrug-Based Nanoscale System for Enhanced Anticancer Activity.

A PEGylated multistimuli-responsive dendritic copolymer-doxorubicin (DOX) prodrug-based nanoscale system was developed as a delivery model for hydrophobic drugs. In this system, PEGylation did not only prolong circulation of the nanoscale system in the body (average half-life of 14.6 h, four times longer than that of the free drug), but also allowed the system to aggregate into nanoparticles (NPs) because of interactions between hydrophilic (polyethylene glycol) and hydrophobic (dendritic prodrug) moieties for better uptake through endocytosis (around 150 nm of particle size with a neutrally charged surface for the PEGylated dendritic prodrug with 12.1 wt % of DOX). The dendritic structure was built by bridging poly[ N-(2-hydroxypropyl)methacrylamide] segments with enzyme-responsive GFLG (Gly-Phe-Leu-Gly tetrapeptide) linkers. DOX was released by hydrolyzing the hydrazone bond between DOX and the copolymer framework in the acidic endosomes/lysosomes. In vitro studies on DOX released from the NPs induced mitochondrial dysfunction during apoptosis. By imaging the main organs and tumor tissues from mice treated with the NPs, boosted accumulation of this nanoscale medicine was found in tumor tissues, leading to a decrease in toxicity and side effects to normal tissues and enhancement in drug tolerance. In the 4T1 breast cancer model, these NPs exhibited a superior antitumor efficacy confirmed by inhibiting angiogenesis, proliferation of tumor tissues, and inducing procedural apoptosis of tumor cells. The highest tumor growth inhibition value mediated by the NPs was up to 86.5%. Therefore, this PEGylated multistimuli-responsive dendritic copolymer-DOX prodrug-based nanoscale system may be further explored as an alternative to traditional chemotherapy for breast cancer treatment.

Enzyme-Sensitive and Amphiphilic PEGylated Dendrimer-Paclitaxel Prodrug-Based Nanoparticles for Enhanced Stability and Anticancer Efficacy.

In this study, we prepared a smart polymeric vehicle for the hydrophobic drug paclitaxel (PTX) that allowed a maximum steady-state circulation and a fast intracellular release in tumors. PTX was linked to the Janus PEGylated (PEG = poly(ethylene glycol)) peptide dendrimer via an enzyme-sensitive linker glycylphenylalanylleucylglycine tetrapeptide by efficient click reaction, resulting in Janus dendritic prodrug with 20.9% PTX content. The prodrug self-assembled into nanoscale particles with appropriate nanosizes, compact morphology, and negative surface charge. In addition to high stability during circulation, as demonstrated by protein adsorption assays and drug release studies in the cancer's intracellular environment, the nanoparticles were able to quickly release the drug intact in its original molecular structure, as verified via high-performance liquid chromatography and mass spectrometry analyses. Compared to free PTX, the enzyme-responsive feature of nanoparticles promoted higher cytotoxicity against 4T1 cancer cells and much lower cytotoxicity against normal cells. The nanoparticles accumulated in the tumor and were retained for an extended period of time, as confirmed by fluorescence imaging. Therefore, these nanoparticles exhibited significantly enhanced antitumor efficiency in the 4T1 breast cancer model as indicated by the observed inhibition of angiogenesis and proliferation as well as induction of apoptosis. Moreover, the nanoparticles reduced the occurrence of side effects, particularly dose-limited toxicities, as monitored by body weight and hematological features. Hence, our Janus PEGylated dendrimer-PTX prodrug-based nanoparticles may potentially serve as nanoscale vehicles for breast cancer therapy.

Stimuli-Sensitive Biodegradable and Amphiphilic Block Copolymer-Gemcitabine Conjugates Self-Assemble into a Nanoscale Vehicle for Cancer Therapy.

The availability and the stability of current anticancer agents, particularly water-insoluble drugs, are still far from satisfactory. A widely used anticancer drug, gemcitabine (GEM), is so poorly stable in circulation that some polymeric drug-delivery systems have been under development for some time to improve its therapeutic index. Herein, we designed, prepared, and characterized a biodegradable amphiphilic block N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer-GEM conjugate-based nanoscale and stimuli-sensitive drug-delivery vehicle. An enzyme-sensitive oligopeptide sequence glycylphenylalanylleucylglycine (GFLG) was introduced to the main chain with hydrophilic and hydrophobic blocks via the reversible addition-fragmentation chain transfer (RAFT) polymerization. Likewise, GEM was conjugated to the copolymer via the enzyme-sensitive peptide GFLG, producing a high molecular weight (MW) product (90 kDa) that can be degraded into smaller MW segments (<50 kDa), and ensuring potential rapid site-specific release and stability in vivo. The amphiphilic copolymer-GEM conjugate can self-assemble into compact nanoparticles. NIR fluorescent images demonstrated that the conjugate-based nanoparticles could accumulate and be retained within tumors, resulting in significant increased antitumor efficacy compared to free GEM. The conjugate was not toxic to organs of the mice as measured by body weight reductions and histological analysis. In summary, this biodegradable amphiphilic block HPMA copolymer-gemcitabine conjugate has the potential to be a stimuli-sensitive and nanoscale drug-delivery vehicle.

Cellulosic ethanol: status and innovation.

Although the purchase price of cellulosic feedstocks is competitive with petroleum on an energy basis, the cost of lignocellulose conversion to ethanol using today's technology is high. Cost reductions can be pursued via either in-paradigm or new-paradigm innovation. As an example of new-paradigm innovation, consolidated bioprocessing using thermophilic bacteria combined with milling during fermentation (cotreatment) is analyzed. Acknowledging the nascent state of this approach, our analysis indicates potential for radically improved cost competitiveness and feasibility at smaller scale compared to current technology, arising from (a) R&D-driven advances (consolidated bioprocessing with cotreatment in lieu of thermochemical pretreatment and added fungal cellulase), and (b) configurational changes (fuel pellet coproduction instead of electricity, gas boiler(s) in lieu of a solid fuel boiler).