Minocycline and Silver Dual-Loaded Polyphosphoester-Based Nanoparticles for Treatment of Resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa has been detected in the lungs of ∼50% of patients with cystic fibrosis (CF), including 20% of adult CF patients. The majority of these adult patients harbor multi-drug resistant (MDR) strains, limiting the available treatment options. Silver has long been used as a broad-spectrum antimicrobial agent with a low incidence of resistance. Despite low toxicity, poor availability of silver cations mandates a high dosage to effectively eradicate infections. To address this shortcoming of silver, nanoparticles have been used as delivery devices to improve treatment outcomes. Furthermore, studies have demonstrated that synergistic combinations with careful dose calibrations and efficient delivery systems result in superior antimicrobial activity while avoiding potential side effects of both therapeutics. Here 4-epi-minocycline, a metabolite of minocycline, was identified as an active antimicrobial against P. aeruginosa using a high-throughput screen. The antimicrobial activities of 4-epi-minocycline, minocycline, and silver acetate against clinical isolates of P. aeruginosa obtained from CF patients were evaluated in vitro. Next, the synergistic activity of the silver/minocycline combination against P. aeruginosa isolates was investigated using checkerboard assays and identified with end-point colony forming unit determination assays. Finally, nanoparticles coloaded with minocycline and silver were evaluated in vitro for antimicrobial activity. The results demonstrated that both silver and minocycline are potent antimicrobials alone and that the combination allows a reduced dosage of both therapeutics to achieve the same antimicrobial effect. Furthermore, the proposed synergistic silver/minocycline combination can be coloaded into nanoparticles as a next-generation antibiotic to combat the threats presented by MDR pathogens.

Receptor-Mediated Attachment and Uptake of Hyaluronan Conjugates by Breast Cancer Cells.

Chemotherapy, a mainstay modality for cancer, is often hindered by systemic toxicity and side effects. With the emergence of nanomedicine, the development of drug therapy has shifted toward targeted therapy. Hyaluronan (HA) is an ideal molecule as a targeted delivery system because many carcinomas overexpress HA receptors. We have conjugated resveratrol, a natural polyphenol, and 3-(5-methoxy, 2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP), a chalcone, to HA with the goal of enhancing drug bioavailability and targeting triple negative breast cancers. We demonstrate the ability of HA conjugates to accumulate in the tumor interstitium within 6 h after tail vein injections. In vitro, these conjugates interact with their target receptors, which are overexpressed by triple negative breast cancer cells under static and physiological flow. These interactions result in enhanced uptake and efficacy of the therapeutic, as demonstrated by a reduced IC50 over that of nonconjugated drugs. Thus, HA offers a platform to solubilize, target, and enhance the efficacy of chemotherapeutics.

Antimicrobial activity of a natural compound and analogs against multi-drug-resistant Gram-positive pathogens.

The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) has sparked global concern due to the dwindling availability of effective antibiotics. To increase our treatment options, researchers have investigated naturally occurring antimicrobial compounds and have identified MC21-A (C58), which has potent antimicrobial activity against MRSA. Recently, we have devised total synthesis schemes for C58 and its chloro-analog, C59. Here, we report that both compounds eradicate 90% of the 39 MRSA isolates tested [MIC90 and minimum bactericidal concentration (MBC90)] at lower or comparable concentrations compared to several standard-of-care (SoC) antimicrobials including daptomycin, vancomycin, and linezolid. Furthermore, a stable, water-soluble sodium salt of C59, C59Na, demonstrates antimicrobial activity comparable to C59. C59, unlike vancomycin, kills stationary-phase MRSA in a dose-dependent manner and completely eradicates MRSA biofilms. In contrast to vancomycin, exposing MRSA to sub-MIC concentrations of C59 does not result in the emergence of spontaneous resistance. Similarly, in a multi-step study, C59 demonstrates a low propensity of resistance acquisition when compared to SoC antimicrobials, such as linezolid and clindamycin. Our findings suggest C58, C59, and C59Na are non-toxic to mammalian cells at concentrations that exert antimicrobial activity; the lethal dose at median cell viability (LD50) is at least fivefold higher than the MBC90 in the two mammalian cell lines tested. A morphological examination of the effects of C59 on a MRSA isolate suggests the inhibition of the cell division process as a mechanism of action. Our results demonstrate the potential of this naturally occurring compound and its analogs as non-toxic next-generation antimicrobials to combat MRSA infections. IMPORTANCE: The rapid emergence of methicillin-resistant Staphylococcus aureus (MRSA) isolates has precipitated a critical need for novel antibiotics. We have developed a one-pot synthesis method for naturally occurring compounds such as MC21-A (C58) and its chloro-analog, C59. Our findings demonstrate that these compounds kill MRSA isolates at lower or comparable concentrations to standard-of-care (SoC) antimicrobials. C59 eradicates MRSA cells in biofilms, which are notoriously difficult to treat with SoC antibiotics. Additionally, the lack of resistance development observed with C59 treatment is a significant advantage when compared to currently available antibiotics. Furthermore, these compounds are non-toxic to mammalian cell lines at effective concentrations. Our findings indicate the potential of these compounds to treat MRSA infections and underscore the importance of exploring natural products for novel antibiotics. Further investigation will be essential to fully realize the therapeutic potential of these next-generation antimicrobials to address the critical issue of antimicrobial resistance.

In vivo gene delivery with L-tyrosine polyphosphate nanoparticles.

The concept of gene therapy is promising; however, the perceived risks and side effects associated with this technology have severely dampened the researchers' enthusiasm. Thus, the development of a nonviral gene vector without immunological effects and with high transfection efficiency is necessary. Currently, most nonviral vectors have failed to achieve the in vivo transfection efficiencies of viral vectors due to their toxicity, rapid clearance, and/or inappropriate release rates. Although our previous studies have successfully demonstrated the controlled-release of plasmid DNA (pDNA) polyplexes encapsulated into nanoparticles formulated with l-tyrosine polyphosphate (LTP-pDNA nanoparticles), the in vivo transfection capabilities and immunogenicity of this delivery system have yet to be examined. Thus, we evaluate LTP-pDNA nanoparticles in an in vivo setting via injection into rodent uterine tissue. Our results demonstrate through X-gal staining and immunohistochemistry of uterine tissue that transfection has successfully occurred after a nine-day incubation. In contrast, the results for the control nanoparticles show results similar to those of shams. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) from the injected tissues confirms the transfection in vivo. To examine the immunogenicity, the l-tyrosine polyphosphate (LTP) nanoparticles have been evaluated in a mouse model. No significant differences in the activation of the innate immune system are observed. These data provide the first report for the potential use of controlled-release nanoparticles formulated from an amino acid based polymer as an in vivo nonviral vector for gene therapy.

Synergistic Antimicrobial Effects of Ibuprofen Combined with Standard-of-Care Antibiotics against Cystic Fibrosis Pathogens.

Cystic fibrosis (CF) is a common life-shortening genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Lungs of CF patients are often colonized or infected with microorganisms requiring frequent courses of antibiotics. Antibiotic-resistant bacterial infections have been a growing concern in CF patients. Chronic bacterial infections and concomitant airway inflammation damage the lungs, ultimately leading to respiratory failure. Several clinical trials have demonstrated that high-dose ibuprofen reduces the rate of pulmonary function decline in CF patients. This beneficial effect has been attributed to the anti-inflammatory properties of ibuprofen. Previously, we have confirmed that high-dose ibuprofen demonstrates antimicrobial activity against P. aeruginosa both in vitro and in vivo. However, no study has examined the antimicrobial effect of combining ibuprofen with standard-of-care antimicrobials. Here, we evaluated the possible synergistic activity of combinations of common nonsteroidal anti-inflammatory drugs (NSAIDs), namely, ibuprofen, naproxen, and aspirin, with commonly used antibiotics for CF patients. The drug combinations were screened against different CF clinical isolates. Antibiotics that demonstrated increased efficacy in the presence of ibuprofen were further tested for potential synergistic effects between these NSAIDS and antimicrobials. Finally, a survival analysis of a P. aeruginosa murine infection model was used to demonstrate the efficacy of the most potent combination identified in in vitro screening. Our results suggest that combinations of ibuprofen with commonly used antibiotics demonstrate synergistic antimicrobial activity against drug-resistant, clinical bacterial strains in vitro. The efficacy of the combination of ceftazidime and ibuprofen against resistant P. aeruginosa was demonstrated in an in vivo pneumonia model.

The Interactions between L-tyrosine based nanoparticles decorated with folic acid and cervical cancer cells under physiological flow.

Many anticancer drugs have been established clinically, but their efficacy can be compromised by nonspecific toxicity and an inability to reach the desired cancerous intracellular spaces. In order to address these issues, researchers have explored the use of folic acid as a targeted moiety to increase specificity of chemotherapeutic drugs. To expand upon such research, we have conjugated folic acid to functionalized poly(ethylene glycol) and subsequently decorated the surface of l-tyrosine polyphosphate (LTP) nanoparticles. These nanoparticles possess the appropriate size (100-500 nm) for internalization as shown by scanning electron microscopy and dynamic light scattering. Under simulated physiological flow, LTP nanoparticles decorated with folic acid (targeted nanoparticles) show a 10-fold greater attachment to HeLa, a cervical cancer cell line, compared to control nanoparticles and to human dermal fibroblasts. The attachment of these targeted nanoparticles progresses at a linear rate, and the strength of this nanoparticle attachment is shown to withstand shear stresses of 3.0 dyn/cm(2). These interactions of the targeted nanoparticles to HeLa are likely a result of a receptor-ligand binding, as a competition study with free folic acid inhibits the nanoparticle attachment. Finally, the targeted nanoparticles encapsulated with a silver based drug show increased efficacy in comparison to nondecorated (plain) nanoparticles and drug alone against HeLa cells. Thus, targeted nanoparticles are a promising delivery platform for developing anticancer therapies that overexpress the folate receptors (FRs).

Hetero-Multivalent Targeted Liposomal Drug Delivery to Treat Pseudomonas aeruginosa Infections.

Pseudomonas aeruginosa is the leading nosocomial and community-acquired pathogen causing a plethora of acute and chronic infections. The Centers for Disease Control and Prevention has designated multidrug-resistant isolates of P. aeruginosa as a serious threat. A novel delivery vehicle capable of specifically targeting  P. aeruginosa, and encapsulating antimicrobials, may address the challenges associated with these infections. We have developed hetero-multivalent targeted liposomes functionalized with host cell glycans to increase the delivery of antibiotics to the site of infection. Previously, we have demonstrated that compared with monovalent liposomes, these hetero-multivalent liposomes bind with higher affinity to P. aeruginosa. Here, compared with nontargeted liposomes, we have shown that greater numbers of targeted liposomes are found in the circulation, as well as at the site of P. aeruginosa (PAO1) infection in the thighs of CD-1 mice. No significant difference was found in the uptake of targeted, nontargeted, and PEGylated liposomes by J774.A1 macrophages. Ciprofloxacin-loaded liposomes were formulated and characterized for size, encapsulation, loading, and drug release. In vitro antimicrobial efficacy was assessed using CLSI broth microdilution assays and time-kill kinetics. Lastly, PAO1-inoculated mice treated with ciprofloxacin-loaded, hetero-multivalent targeted liposomes survived longer than mice treated with ciprofloxacin-loaded, monovalent targeted, or nontargeted liposomes and free ciprofloxacin. Thus, liposomes functionalized with host cell glycans target P. aeruginosa resulting in increased retention of the liposomes in the circulation, accumulation at the site of infection, and increased survival time in a mouse surgical site infection model. Consequently, this formulation strategy may improve outcomes in patients infected with P. aeruginosa.

Inequalities in Pediatric Fracture Care Timeline Based on Insurance Type.

INTRODUCTION: Socioeconomic and insurance status are often linked with limited access to health care. Despite several government-funded projects aimed at curtailing these barriers, pediatric orthopaedic patients continue to experience delays in receiving timely care for fracture treatments. This delay has been well-identified within the orthopaedic literature but, to our knowledge, has never been characterized based on timeline. Thus, the goal of this study is to evaluate the role of ethnicity, socioeconomic status, and insurance type on the timeline of pediatric patients to obtain orthopaedic care within our community. METHODS: Pediatric patients presenting to our clinic for the treatment of one of 21 most common fractures were included. Patient demographics and the timeline of patient care were collected by retrospective chart review. RESULTS: Government-funded insurance accounted for 60.6% of the 413 patients. These patients experienced significant (P < 0.001) delays in access to care when compared with commercial insurance patients; the time between injury and referral as well as the overall time from injury to orthopaedic evaluation was 2.8 and twofold greater at 4.4 days and 9.2 days, respectively. A strong correlation was established between income levels and insurance type. DISCUSSION: Pediatric patients with a lower socioeconomic status are more likely to rely on government-funded insurance and experience delays in fracture evaluation.

Antegrade Elastic Intramedullary Nailing Insertion Technique Results in Higher Incidence of Symptomatic Implants in Pediatric Ulnar Fractures.

Retrograde and antegrade nailing techniques are the two options available to a surgeon when using elastic stable intramedullary nailing; however, the literature comparing these two nailing techniques is scarce. Thus, we conducted a retrospective review of all pediatric and adolescent ulnar fractures treated with elastic stable intramedullary nailing at our facility. We hypothesize that the clinical outcomes (implant and wound complications) and the time between surgery and radiographic union will be similar for both techniques. Methods: A retrospective chart review of pediatric ulnar fracture patients treated at our facility was performed. Demographic and health information associated with the injury were collected, and the clinical outcomes of the two techniques were compared. Results: A total of 53 patients with 54 fractures were included in this study. Antegrade nail insertion was used to treat 59.2% fractures. Radiographic union was achieved in all patients. Nail insertion technique was not associated with postoperative wound complications, time to radiographic union or implant removal, or significant deficits in upper extremity rotation (P > 0.05). Antegrade nailing resulted in a symptomatic implantation 3.97 times more frequently than compared with retrograde nailing (P = 0.036). Discussion: Antegrade nailing demonstrates a similar healing profile but higher implant complications compared with the retrograde nailing technique in pediatric ulnar fractures.

N-Acetyl cysteine abrogates silver-induced reactive oxygen species in human cells without altering silver-based antimicrobial activity.

Silver-based antimicrobials are widely used topically to treat infections associated with multi-drug resistant (MDR) pathogens. Expanding this topical use to aerosols to treat lung infections requires understanding and preventing silver toxicity in the respiratory tract. A key mechanism resulting in silver-induced toxicity is the production of reactive oxygen species (ROS). In this study, we have verified ROS generation in silver-treated bronchial epithelial cells prompting evaluation of three antioxidants, N-acetyl cysteine (NAC), ascorbic acid, and melatonin, to identify potential prophylactic agents. Among them, NAC was the only candidate that abrogated the ROS generation in response to silver acetate exposure resulting in the rescue of these cells from silver-associated toxicity. Further, this protective effect directly translated to preservation of metabolic activity, as demonstrated by the normal levels of citric acid cycle metabolites in NAC-pretreated silver acetate-exposed cells. Because the citric acid cycle remained functional, silver-exposed cells pre-incubated with NAC demonstrated significantly higher levels of adenosine triphosphate levels compared with NAC-free controls. Moreover, we found that this prodigious capacity of NAC to rescue silver acetate-exposed cells was due not only to its antioxidant activity, but also to its ability to directly bind silver. Despite binding to silver, NAC did not alter the antimicrobial activity of silver acetate.

Pyoverdine-Dependent Virulence of Pseudomonas aeruginosa Isolates From Cystic Fibrosis Patients.

The development of therapies that modulate or prevent pathogen virulence may be a key strategy for circumventing antimicrobial resistance. Toward that end, we examined the production of pyoverdine, a key virulence determinant, in ∼70 Pseudomonas aeruginosa isolates from pediatric cystic fibrosis patients. Pyoverdine production was heterogeneous and showed a clear correlation with pathogenicity in Caenorhabditis elegans and an acute murine pneumonia model. Examination showed pyoverdine accumulation in host tissues, including extrapharyngeal tissues of C. elegans and lung tissues of mice, where accumulation correlated with host death. Many of the isolates tested were resistant to multiple antimicrobials, so we assayed the ability of pyoverdine inhibitors to mitigate virulence and rescue pyoverdine-mediated host pathology. Representatives from three different classes of pyoverdine inhibitors (gallium, fluoropyrimidines, and LK11) significantly improved survival. Our findings highlight the utility of targeting virulence factors in general, and pyoverdine in particular, as a promising method to control bacterial pathogenesis as the utility of antimicrobials continues to diminish.

A novel in vitro metric predicts in vivo efficacy of inhaled silver-based antimicrobials in a murine Pseudomonas aeruginosa pneumonia model.

To address the escalating problem of antimicrobial resistance and the dwindling antimicrobial pipeline, we have developed a library of novel aerosolizable silver-based antimicrobials, particularly for the treatment of pulmonary infections. To rapidly screen this library and identify promising candidates, we have devised a novel in vitro metric, named the "drug efficacy metric" (DEM), which integrates both the antibacterial activity and the on-target, host cell cytotoxicity. DEMs calculated using an on-target human bronchial epithelial cell-line correlates well (R2 > 0.99) with in vivo efficacy, as measured by median survival hours in a Pseudomonas aeruginosa pneumonia mouse model following aerosolized antimicrobial treatment. In contrast, DEMs derived using off-target primary human dermal fibroblasts correlate poorly (R2 = 0.0595), which confirms our hypothesis. SCC1 and SCC22 have been identified as promising drug candidates through these studies, and SCC22 demonstrates a dose-dependent survival advantage compared to sham treatment. Finally, silver-bearing biodegradable nanoparticles were predicted to exhibit excellent in vivo efficacy based on its in vitro DEM value, which was confirmed in our mouse pneumonia model. Thus, the DEM successfully predicted the efficacy of various silver-based antimicrobials, and may serve as an excellent tool for the rapid screening of potential antimicrobial candidates without the need for extensive animal experimentation.

Phytochemicals potently inhibit migration of metastatic breast cancer cells.

Cell migration is a major process that drives metastatic progression of cancers, the major cause of cancer death. Existing chemotherapeutic drugs have limited efficacy to prevent and/or treat metastasis, emphasizing the need for new treatments. We focus on triple negative breast cancer (TNBC), the subtype of breast cancer with worst prognosis and no standard chemotherapy protocols. Here we demonstrate that a group of natural compounds, known as phytochemicals, effectively block migration of metastatic TNBC cells. Using a novel cell micropatterning technology, we generate consistent migration niches in standard 96-well plates where each well contains a cell-excluded gap within a uniform monolayer of cells. Over time, cells migrate into and occupy the gap. Treating TNBC cells with non-toxic concentrations of phytochemicals significantly blocks motility of cells. Using a molecular analysis approach, we show that anti-migratory property of phytochemicals is partly due to their inhibitory effects on phosphorylation of ERK1/2. This study provides a framework for future studies to understand molecular targets of phytochemicals and evaluate their effectiveness in inhibiting metastasis in animal models of cancer.

Microstereolithography and characterization of poly(propylene fumarate)-based drug-loaded microneedle arrays.

Drug-loaded microneedle arrays for transdermal delivery of a chemotherapeutic drug were fabricated using multi-material microstereolithography (µSL). These arrays consisted of twenty-five poly(propylene fumarate) (PPF) microneedles, which were precisely orientated on the same polymeric substrate. To control the viscosity and improve the mechanical properties of the PPF, diethyl fumarate (DEF) was mixed with the polymer. Dacarbazine, which is widely used for skin cancer, was uniformly blended into the PPF/DEF solution prior to crosslinking. Each microneedle has a cylindrical base with a height of 700 µm and a conical tip with a height of 300 µm. Compression test results and characterization of the elastic moduli of the PPF/DEF (50:50) and PPF/drug mixtures indicated that the failure force was much larger than the theoretical skin insertion force. The release kinetics showed that dacarbazine can be released at a controlled rate for five weeks. The results demonstrated that the PPF-based drug-loaded microneedles are a potential method to treat skin carcinomas. In addition, µSL is an attractive manufacturing technique for biomedical applications, especially for micron-scale manufacturing.