Lipase and pH-responsive diblock copolymers featuring fluorocarbon and carboxyl betaine for methicillin-resistant staphylococcus aureus infections.

The emergence of multidrug-resistant bacteria along with their resilient biofilms necessitates the development of creative antimicrobial remedies. We designed versatile fluorinated polymer micelles with surface-charge-switchable properties, demonstrating enhanced efficacy against Methicillin-Resistant Staphylococcus Aureus (MRSA) in planktonic and biofilm states. Polymethacrylate diblock copolymers with pendant fluorocarbon chains and carboxyl betaine groups were prepared using reversible addition-fragmentation chain transfer polymerization. Amphiphilic fluorinated copolymers self-assembled into micelles, encapsulating ciprofloxacin in their cores (CIP@FCBMs) for antibacterial and antibiofilm applications. As a control, fluorine-free copolymer micelles loaded with ciprofloxacin (CIP@BCBMs) were prepared. Although both CIP@FCBMs and CIP@BCBMs exhibited pH-responsive surface charges and lipase-triggered drug release, CIP@FCBMs exhibited powerful antimicrobial and antibiofilm activities in vitro and in vivo, attributed to superior serum stability, higher drug loading, enhanced fluorination-facilitated cellular uptake, and lipase-triggered drug release. Collectively, reversing surface charge, on-demand antibiotic release, and fluorination-mediated nanoparticles hold promise for treating bacterial infections and biofilms.

Antibiotic-based small molecular micelles combined with photodynamic therapy for bacterial infections.

The appearance of multidrug-resistant bacteria and the formation of bacterial biofilms have necessitated the development of alternative antimicrobial therapeutics. Antibiotics conjugated with or embedded in nano-drug carriers show a great potential and advantage over free drugs, but the mass proportion of carriers generally exceeds 90% of the nano-drug, resulting in low drug loading and limited therapeutic output. Herein, we fabricated a nanocarrier using antibiotics as the building blocks, minimizing the use of carrier materials, significantly increasing the drug loading content and treatment effect. Firstly, we conjugated betaine carboxylate with ciprofloxacin (CIP) through an ester bond to form the amphiphilic conjugate (CIP-CB), which self-assembled into micelles (CIP-CBMs) in aqueous solutions, with a CIP loading content as high as 65.4% and pH-induced surface charge reversal properties. Secondly, a model photosensitizer (5, 10, 15, 20-tetraphenylporphyrin (TPP)) was encapsulated in CIP-CBMs, generating infection-targeted photodynamic/antibiotic combined nanomedicines (denoted as TPP@CIP-CBMs). Upon accumulation at infection sites or in deep bacterial biofilms, the ester bond between the betaine carboxylate and CIP is cleaved to release free TPP and CIP, leading to a synergetic antibacterial and antibiofilm activity in vitro and in vivo.

Counterion-induced antibiotic-based small-molecular micelles for methicillin-resistant Staphylococcus aureus infections.

A new counterion-induced small-molecule micelle (SM) with surface charge-switchable activities for methicillin-resistant Staphylococcus aureus (MRSA) infections is proposed. The amphiphilic molecule formed by zwitterionic compound and the antibiotic ciprofloxacin (CIP), via a "mild salifying reaction" of the amino and benzoic acid groups, can spontaneously assemble into counterion-induced SMs in water. Through vinyl groups designed on zwitterionic compound, the counterion-induced SMs could be readily cross-linked using mercapto-3, 6-dioxoheptane by click reaction, to create pH-sensitive cross-linked micelles (CSMs). Mercaptosuccinic acid was also decorated on the CSMs (DCSMs) by the same click reaction to afford charge-switchable activities, resulting in CSMs that were biocompatible with red blood cells and mammalian cells in normal tissues (pH 7.4), while having strong retention to negatively charged bacterial surfaces at infection sites, based on electrostatic interaction (pH 5.5). As a result, the DCSMs could penetrate deep into bacterial biofilms and then release drugs in response to the bacterial microenvironment, effectively killing the bacteria in the deeper biofilm. The new DCSMs have several advantages such as robust stability, a high drug loading content (∼ 30%), easy fabrication, and good structural control. Overall, the concept holds promise for the development of new products for clinical application. STATEMENT OF SIGNIFICANCE: We fabricated a new counterion-induced small-molecule micelle with surface charge-switchable activities (DCSMs) for methicillin-resistant Staphylococcus aureus (MRSA) infections. Compared with reported covalent systems, the DCSMs not only have improved stability, high drug loading content (∼ 30%), and good biosafety, but also have the environmental stimuli response, and antibacterial activity of the original drugs. As a result, the DCSMs exhibited enhanced antibacterial activities against MRSA both in vitro and in vivo. Overall, the concept holds promise for the development of new products for clinical application.

Surface-Charge-Switchable and Size-Transformable Thermosensitive Nanocomposites for Chemo-Photothermal Eradication of Bacterial Biofilms in Vitro and in Vivo.

The appearance of multidrug-resistant bacteria and their biofilms presents a serious threat to modern medical systems. Herein, we fabricated a novel gold-nanorod-based chemo-photothermal-integrated antimicrobial platform with surface-charge-switchable and near-infrared (NIR)-induced size-transformable activities that show an enhanced killing efficiency against methicillin-resistant Staphylococcus aureus (MRSA) in both planktonic and biofilm phenotypes. The nanocomposites are prepared by in situ copolymerization using N-isopropyl acrylamide (NIPAM), acrylic acid (AA), and N-allylmethylamine (MAA) as monomers on the surfaces of gold nanorods (GNRs). Ciprofloxacin (CIP) is loaded onto polymer shells of nanocomposites with a loading content of 9.8%. The negatively charged nanocomposites switch to positive upon passive accumulation at the infectious sites, which promotes deep biofilm penetration and bacterial adhesion of the nanoparticles. Subsequently, NIR irradiation triggers the nanocomposites to rapidly shrink in volume, further increasing the depth of biofilm penetration. The NIR-triggered, ultrafast volume shrinkage causes an instant release of CIP on the bacterial surface, realizing the synergistic benefits of chemo-photothermal therapy. Both in vitro and in vivo evidence demonstrate that drug-loaded nanocomposites could eradicate clinical MRSA biofilms. Taken together, the multifunctional chemo-photothermal-integrated antimicrobial platform, as designed, is a promising antimicrobial agent against MRSA infections.

Ciprofloxacin conjugated gold nanorods with pH induced surface charge transformable activities to combat drug resistant bacteria and their biofilms.

The ever-growing threat of drug-resistant pathogens and their biofilms based persistent, chronic infections has created an urgent call for new strategies to deal with multidrug resistant bacteria (MDR). Near-infrared (NIR) laser-induced photothermal treatment (PTT) of gold nanorods (AuNRs) disinfects microbes by local heating with low possibility to develop resistant. However, PTT disinfection strategy of AuNRs alone shows less efficiency in killing multidrug resistant strains (i.e. Methicillin-resistant Staphylococcus aureus, MRSA) and their matured biofilms. Herein, a novel synergistic chemo-photothermal integrated antimicrobial platform (P(Cip-b-CB)-AuNRs) was fabricated which show enhanced killing efficiency against MRSA in both planktonic and biofilm phenotypes. Polymethacrylate copolymers with pendant ciprofloxacin (Cip) and the carboxyl betaine groups (P(Cip-b-CB)) were synthesized using reversible addition-fragmentation chain transfer (RAFT) polymerization. P(Cip-b-CB) was decorated onto AuNRs via gold-thiol bond which resulted in AuNRs with acidic-induced surface charge-switchable activities and lipase triggered Cip release properties (P(Cip-b-CB)-AuNRs). The lower pH value and overexpress of lipase are characteristics for microenvironment of microbial infections and their biofilms, which ensure the targeting on, penetration into and on-demand release of Cip from the nanocomposites in bacterial infection sites and their biofilms. The bacterial cell membrane was disrupt by photothermal therapy which could improve its permeability and sensitivity to antibiotics, meanwhile lipase-triggered release of Cip ensures a high concentration of antibiotics at the site of bacterial infection. Besides their NIR induced PTT disinfection activities, the increased local temperature generated by NIR light irradiation accelerated Cip release which further enhanced the antibacterial efficiency, leading to synergistic antibacterial activities of chemo-photothermal therapy. Taken together, the designed synergistic chemo-photothermal integrated antimicrobial platform is a promising antibacterial agent for fighting MDR bacterial infections and their biofilms.

Gold nanorods with surface charge-switchable activities for enhanced photothermal killing of bacteria and eradication of biofilm.

The increasing growth and severity of bacterial biofilm infections and the appearance of multidrug-resistant bacteria pose alarming threats to public healthcare systems, mainly due to their formidable tolerance to conventional antibiotics. Different from the antibacterial mechanisms of antibiotics, gold nanorods (AuNRs) disinfect microbes by local heating induced by near-infrared (NIR) light irradiation; thus, they are potential disinfection agents. In an attempt to increase the biocompatibility and antibacterial activities of AuNRs against organisms in both planktonic and biofilm phenotypes, polymethacrylate with pendant carboxyl betaine groups was decorated on AuNRs (PCB-AuNRs) to afford AuNRs with pH-induced surface charge-transformable activities. The zwitterion-modified AuNRs demonstrated a pH-responsive transition from negative charge to positive charge; this confers the AuNRs with a change in functionality from biocompatible zwitterionic nanocomposites in healthy tissues (pH = ∼7.4) to enhanced antimicrobial cationic nanocomposites at acidic bacterial infection sites (pH = ∼5.5). AuNRs coated by polymethacrylate with pendant mPEG (PPEGMA-AuNRs) without surface charge transition activities were used for comparison. PCB-AuNRs presented better antimicrobial activity against Gram-negative bacteria (E. coli), Gram-positive bacteria (S. aureus) and their drug-resistant strains (MRSA and EBSL E. coli) than PPEGMA-AuNRs as a result of their pH-responsive surface charge transition activities. Moreover, PCB-AuNRs demonstrated deeper penetration into mature biofilms and better biofilm elimination activities than their non-surface charge-transformable counterparts. The results indicate that the designed zwitterion-coated AuNRs are a promising antibacterial agent for fighting bacterial infections.

Layer-by-Layer Assembled Multilayer Films with Multiple Antibacterial and pH-Induced Self-Cleaning Activities Based on Polyurethane Micelles.

Layer-by-layer assembled multilayer films with antifouling and pH induced self-cleaning activities were constructed by polyurethane micelles with dense PEG brush coronas (PEG-g-PUM) and polyethylenimine (bPEI). The dense PEG brush coronas and acidic induced surface charge transform activities of PEG-g-PUM rendered the multilayer films (PEG-g-PUM/bPEI)n with antifouling and self-cleaning activities, respectively. Multilayer films constructed from polyurethane micelles without surface charge switchable properties (PEG-c-PUM, PEG-b-PUM), showed negligible pH induced surface release of PU micelles. Furthermore, Triclosan encapsulation in the multilayer films ((PEG-g-PUM-TLS/bPEI)n) further improved the antibacterial performance, as a result of pH and lipase triggered release of payloads from the surfaces. The antifouling properties of PEG-g-PUM and the on-demand release of payloads and PU micelles, may account for their excellent multiple antibacterial performance and pH-induced self-cleaning activities.

Silver nanoparticles with pH induced surface charge switchable properties for antibacterial and antibiofilm applications.

Silver nanoparticles (AgNPs) are widely used as antibacterial agents because of their significant antimicrobial activities and little sign of antimicrobial resistance. However, the relatively high toxicity to healthy cells and low penetration efficiency into bacterial biofilms prevent their further use in biomedical applications. In order to decrease the cytotoxicity of the AgNPs to mammalian cells while increasing their antibacterial and antibiofilm efficiency, a novel nanocomposite composed of AgNPs decorated with carboxyl betaine groups (AgNPs-LA-OB) was prepared. The zwitterion modified AgNPs showed a pH responsive transition from a negative charge to a positive charge, which enabled the AgNPs to be compatible with mammalian cells and red blood cells (RBCs) in healthy tissues (pH ∼ 7.4), while strongly adhering quickly to negatively charged bacterial surfaces at infectious sites (pH ∼ 5.5) based on electrostatic attraction. The AgNPs penetrated deeply into bacterial biofilms and killed the bacteria living in an acidic environment. The results indicated that the designed zwitterion NPs for antibacterial applications and eradication of bacterial biofilms, which also had particles that did not harm the healthy cells showed promise for future use in humans. The satisfactory selectivity for bacteria compared to RBCs, together with their potent eradication of bacterial biofilms make AgNPs-LA-OB a promising antibacterial nanomedicine in biomedical fields.

Calpain inhibitor I attenuates atherosclerosis and inflammation in atherosclerotic rats through eNOS/NO/NF-κB pathway.

We previously reported that calpain, the Ca2+-sensitive cysteine protease, gets involved in atherogenesis. This study aimed to investigate the effects of calpain inhibitor I (CAI, 5 mg/kg per day) with or without NG-nitro-l-arginine-methyl ester (l-NAME) (100 mg/kg per day), the inhibitor of nitric oxide synthase (NOS), on atherosclerosis and inflammation in a rat model induced by high-cholesterol diet (HCD). The results demonstrated HCD increased protein expression of calpain-1 but not calpain-2 in aortic tissue. In addition, CAI reduced the thickness of atherosclerotic intima compared with HCD group, which was weakened by the l-NAME combination. CAI with or without l-NAME decreased the activity of calpain in the aorta. Also, CAI decreased the expressions of vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1), and monocyte chemoattractant protein-1 (MCP-1) in the aorta at the levels of both mRNA and protein. Furthermore, CAI increased the activity and the protein expression of endothelial NOS (eNOS) accompanied by increased content of NO and downregulated the protein expression of nuclear factor κB (NF-κB) of the nucleus in the aorta. However, the abovementioned effects were at least partly cancelled by l-NAME except for the protein expression of eNOS. The results suggested that CAI attenuated atherosclerosis and inflammation through eNOS/NO/NF-κB pathway.

Downregulations of CD36 and Calpain-1, Inflammation, and Atherosclerosis by Simvastatin in Apolipoprotein E Knockout Mice.

BACKGROUND: In the previous in vitro study, we found that simvastatin decreased the protein expression of CD36, the scavenger receptor, and calpain-1, the Ca2+-sensitive cysteine protease, in oxidized low-density lipoprotein (oxLDL)-treated macrophages. In this in vivo study, we investigated whether simvastatin downregulates the expression of CD36 and calpain-1 and inhibits the inflammation and atherosclerosis in apolipoprotein E knockout (ApoE KO) mice. METHODS: Twenty male 6-week-old ApoE KO mice were divided into 2 groups: the ApoE KO group and the ApoE KO + simvastatin (ApoE KO + Sim) group. Atherosclerotic lesions were evaluated and the expressions of CD68, CD36, and calpain-1 in aorta were examined. RESULTS: Simvastatin inhibited the atherosclerotic lesion in ApoE KO mice. In addition, simvastatin reduced the contents of oxLDL, thiobarbituric acid reactive substances, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in serum, decreased the mRNA and protein expressions of CD36 and reduced the mRNA expression of TNF-α and IL-6 in the aortas. Furthermore, simvastatin reduced the calpain activity and the protein expression of calpain-1 in the aorta. CONCLUSION: The results suggested that the attenuation of atherosclerotic lesions in ApoE KO mice by simvastatin might be associated with the downregulations of CD36 and calpain-1 and with inflammation.

Simvastatin inhibited oxLDL-induced proatherogenic effects through calpain-1-PPARγ-CD36 pathway.

We previously reported that simvastatin, an inhibitor of HMG-CoA reductase, inhibits atherosclerosis in rats. The present study was designed to investigate the effect of simvastatin on mouse peritoneal macrophage foam cell formation, the early feature of atherosclerosis, and explore its mechanisms. The results showed that simvastatin decreased cholesterol content and DiI-oxLDL (1,1'-didodecyl 3,3,3',3'-indocarbocyanine perchlorate - oxidized low-density lipoprotein) uptake, reduced the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the medium, down-regulated the mRNA and protein expression of CD36 (a fatty acid receptor), and reduced the mRNA expressions of peroxisome proliferator-activated receptor gamma (PPARγ), TNF-α, and IL-6 in macrophages treated with oxLDL. However, PPARγ agonist troglitazone partly abolished the effects of simvastatin on foam cells. In addition, simvastatin reduced the protein expression of calpain-1, a Ca2+-sensitive cysteine protease, in oxLDL-treated macrophages. Furthermore, PD150606, a specific calpain inhibitor, reduced mRNA expressions of PPARγ and CD36 in macrophages treated with oxLDL. Combination of simvastatin and PD150606 had no further effect on mRNA expression of PPARγ and CD36 compared with either alone. However, over-expression of calpain-1 in macrophages partly reversed the simvastatin effects, including cell cholesterol content, mRNA expressions of PPARγ, and CD36. The results suggested that simvastatin inhibits foam cell formation of oxLDL-treated macrophages through a calpain-1-PPARγ-CD36 pathway.

Application of micro-solid-phase extraction for the determination of persistent organic pollutants in tissue samples.

A novel, simple and efficient method for determining persistent organic pollutants (POPs) in tissue samples has been developed. This technique involves the use of simultaneous microwave-assisted digestion (MAD) and micro-solid-phase extraction (micro-SPE), in which the sorbent is held within a propylene membrane envelope, with gas chromatographic-mass spectrometric (GC-MS) analysis. The POPs studied included eleven organochlorine pesticides and five polychlorinated biphenyl congeners. Optimization of the MAD-micro-SPE parameters was performed. The relative standard deviations (RSDs) of the method ranged from 0.14 to 12.7%. Correlation coefficients up to 0.9999 were obtained across a concentration range of 1.25-50 ng g(-1). The method detection limits for POPs ranged from 0.002 to 0.009 ng g(-1). A preliminary study applying the MAD-micro-SPE procedure to human ovarian cancer tissue showed that it was capable of detecting the presence of a wide spectrum of different POPs in benign and malignant tumors.