Effect of oral administration of microcin Y on growth performance, intestinal barrier function and gut microbiota of chicks challenged with Salmonella Pullorum.

The lasso peptide microcin Y (MccY) effectively inhibits various serotypes of Salmonella in vitro, but the antibacterial effect against S. Pullorum in poultry is still unclear. This study was the first to evaluate the safety and anti-S. Pullorum infection of MccY in specific pathogen-free (SPF) chicks. The safety test showed that the body weight, IgA and IgM levels of serum, and cecal microbiota structure of 3 groups of chicks orally administrated with different doses of MccY (5 mg/kg, 10 mg/kg, 20 mg/kg) for 14 days were not significantly different from those of the control group. Then, the chicks were randomized into 3 groups for the experiment of anti-S. Pullorum infection: (I) negative control group (NC), (II) S. Pullorum-challenged group (SP, 5 × 108 CFU/bird), (III) MccY-treated group (MccY, 20 mg/kg). The results indicated that compared to the SP group, treatment of MccY increased body weight and average daily gain (P < 0.05), reduced S. Pullorum burden in feces, liver, and cecum (P < 0.05), enhanced the thymus, and decreased the spleen and liver index (P < 0.05). Additionally, MccY increased the jejunal villus height, lowered the jejunal and ileal crypt depth (P < 0.05), and upregulated the expression of IL-4, IL-10, ZO-1 in the jejunum and ileum, as well as CLDN-1 in the jejunum (P < 0.05) compared to the SP group. Furthermore, MccY increased probiotic flora (Barnesiella, etc.), while decreasing (P < 0.05) the relative abundance of pathogenic flora (Escherichia and Salmonella, etc.) compared to the SP group.

Protective effect of plantaricin bio-LP1 bacteriocin on multidrug-resistance Escherichia Coli infection by alleviate the inflammation and modulate of gut-microbiota in BALB/c mice model.

The rapid spread of multidrug-resistant pathogens with the low efficacy of common antibiotics for humans and animals in its clinical therapeutics are a global health concern. Therefore, there is a need to develop new treatment strategies to control them clinically. The study aimed to evaluate the effects of Plantaricin Bio-LP1 bacteriocin produced from Lactiplantibacillus plantarum NWAFU-BIO-BS29 to alleviate the inflammation caused by multidrug-resistance Escherichia Coli (MDR-E. coli) infection in BALB/c mice-model. The focus was given on aspects linked to the mechanism of the immune response. Results indicated that Bio-LP1 had highly promising effects on partially ameliorating MDR-E. coli infection by reducing the inflammatory response through inhibiting the overexpression of proinflammatory-cytokines such as secretion of tumor necrosis factor (TNF-α) and interleukin (IL-6 and IL-ß) and strongly regulated theTLR4 signaling-pathway. Additionally, avoided the villous destruct, colon length shortening, loss of intestinal barrier integrity, and increased disease activity index. Furthermore, significantly increased the relative abundance of beneficial-intestinal-bacteria including Ligilactobacillus, Enterorhabdus, Pervotellaceae, etc. Finally, improved the intestinal mucosal barrier to alleviate the pathological damages and promote the production of short-chain fatty acids (SCFAs) a source of energy for the proliferation. In conclusion, plantaricin Bio-LP1 bacteriocin can be considered a safe alternative to antibiotics against MDR-E. coli-induced intestinal inflammation.

A Strong Synergy Between the Thiopeptide Bacteriocin Micrococcin P1 and Rifampicin Against MRSA in a Murine Skin Infection Model.

Antibiotic-resistant bacterial pathogens have become a serious threat worldwide. One of these pathogens is methicillin-resistant Staphylococcus aureus (MRSA), a major cause of skin and soft tissue infections. In this study we identified a strain of Staphylococcus equorum producing a substance with high antimicrobial activity against many Gram-positive bacteria, including MRSA. By mass spectrometry and whole genome sequencing the antimicrobial substance was identified as the thiopeptide bacteriocin micrococcin P1 (MP1). Based on its properties we developed a one-step purification protocol resulting in high yield (15 mg/L) and high purity (98%) of MP1. For shorter incubation times (5-7 h) MP1 was very potent against MRSA but the inhibitory effect was overshadowed by resistance development during longer incubation time (24h or more). To overcome this problem a synergy study was performed with a number of commercially available antibiotics. Among the antibiotics tested, the combination of MP1 and rifampicin gave the best synergistic effect, with MIC values 25 and 60 times lower than for the individual drugs, respectively. To assess the therapeutic potential of the MP1-rifampicin combination, we used a murine skin infection model based on the use of the multidrug-resistant luciferase-tagged MRSA strain Xen31. As expected, neither of the single antimicrobials (MP1 or rifampicin) could eradicate Xen31 from the wounds. By contrary, the MP1-rifampicin combination was efficient not only to eradicate but also to prevent the recurrence of Xen31 infection. Furthermore, compared to fucidin cream, which is commonly used in skin infection treatments, MP1-rifampicin combination was superior in terms of preventing resistance development. Our results show that combining MP1, and probably other thiopeptides, with antibiotics can be a promising strategy to treat SSTIs caused by MRSA and likely many other Gram-positive bacteria.

Pre-formulation and delivery strategies for the development of bacteriocins as next generation antibiotics.

Bacteriocins, a class of antimicrobial peptide produced by bacteria, may offer a potential alternative to traditional antibiotics, an important step towards mitigating the ever-increasing antimicrobial resistance crisis. They are active against a range of clinically relevant Gram-positive and Gram-negative bacteria. Bacteriocins have been discussed in the literature for over a century. Although they are used as preservatives in food, no medicine based on their antimicrobial activity exists on the market today. In order to formulate them into clinical antibiotics, pre-formulation studies on their biophysical and physicochemical properties that will influence their activity in vivo and their stability during manufacture must be elucidated. Thermal, pH and enzymatic stability of bacteriocins are commonly studied and regularly reported in the literature. Solubility, permeability and aggregation properties on the other hand are less frequently reported for many bacteriocins, which may contribute to their poor clinical progression. Promising cytotoxicity studies report that bacteriocins exhibit few cytotoxic effects on a variety of mammalian cell lines, at active concentrations. This review highlights the lack of quantitative data and in many cases even qualitative data, on bacteriocins' solubility, stability, aggregation, permeability and cytotoxicity. The formulation strategies that have been explored to date, proposed routes of administration, trends in in vitro/in vivo behaviour and efforts in clinical development are discussed. The future promise of bacteriocins as a new generation of antibiotics may require tailored local delivery strategies to fulfil their potential as a force to combat antimicrobial-resistant bacterial infections.

Biochemical, genetic and transcriptional characterization of multibacteriocin production by the anti-pneumococcal dairy strain Streptococcus infantarius LP90.

Streptococcus pneumoniae infections are one of the major causes of morbility and mortality worldwide. Although vaccination and antibiotherapy constitute fundamental and complementary strategies against pneumococcal infections, they present some limitations including the increase in non-vaccine serotypes and the emergence of multidrug-resistances, respectively. Ribosomally-synthesized antimicrobial peptides (i.e. bacteriocins) produced by Lactic Acid Bacteria (LAB) may represent an alternative or complementary strategy to antibiotics for the control of pneumococal infections. We tested the antimicrobial activity of 37 bacteriocinogenic LAB, isolated from food and other sources, against clinical S. pneumoniae strains. Streptococcus infantarius subsp. infantarius LP90, isolated from Venezuelan water-buffalo milk, was selected because of its broad and strong anti-pneumococcal spectrum. The in vitro safety assessment of S. infantarius LP90 revealed that it may be considered avirulent. The analysis of a 19,539-bp cluster showed the presence of 29 putative open reading frames (ORFs), including the genes encoding 8 new class II-bacteriocins, as well as the proteins involved in their secretion, immunity and regulation. Transcriptional analyses evidenced that the induction factor (IF) structural gene, the bacteriocin/IF transporter genes, the bacteriocin structural genes and most of the bacteriocin immunity genes were transcribed. MALDI-TOF analyses of peptides purified using different multichromatographic procedures revealed that the dairy strain S. infantarius LP90 produces at least 6 bacteriocins, including infantaricin A1, a novel anti-pneumococcal two-peptide bacteriocin.

Early prediction of tumor response after radiotherapy in combination with cetuximab in nasopharyngeal carcinoma using 99m Tc-duramycin imaging.

PURPOSE: 99mTc-duramycin imaging enables specific visualization of cell death qualitatively and quantitatively. This study aimed to investigate the potential of 99mTc-duramycin imaging in the early prediction of the curative effect of radiotherapy in combination with or without cetuximab in a nasopharyngeal carcinoma (NPC) model. METHODS: Male BALB/c mice bearing NPC xenografts were randomized into four groups (six mice each group). Group 1 received radiotherapy (RT, 15 Gy/mouse) in combination with cetuximab (CTX, 2 mg/mouse), group 2 received RT (15 Gy/mouse), group 3 was treated using CTX (2 mg/mouse), and group 4, the control group, was treated using a vehicle. 99mTc-duramycin imaging was performed before treatment and 24 h after treatment to evaluate tumor response. Tumor uptake of 99mTc-duramycin was validated ex vivo using γ-counting. Treatment response was further validated by cleaved caspase-3 (CC3) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL). Another four groups were treated parallelly under the same conditions to observe treatment response by tumor volume changes. RESULTS: After 24 h treatment, 99mTc-duramycin uptake in the NPC tumor models were significantly higher in group 1 than in group 2 (P < 0.05), group 3 (P < 0.05), or group 4 (P < 0.05); the uptake also increased notably in comparison with baseline values (P < 0.05). Compared with group 4, group 2 and group 3 both showed significant 99mTc-duramycin uptake in the tumors (P < 0.05). Although the 99mTc-duramycin uptake of group 2 was moderately higher than group 3, there were no significant differences between these two groups (P >0.05). There was a strong positive correlation between tumor 99mTc-duramycin uptake and CC3 (r = 0.893, p < 0.0001) and TUNEL (r = 0.918, P < 0.0001). Tumor volume decreased remarkably in the RT in combination with CTX group on day 5, in the RT alone group on day 7, and was inhibited on day 8 in the CTX alone group, whereas the tumors grew continuously in the control group. CONCLUSIONS: We demonstrated that RT in combination with CTX treatment significantly improved disease control in a NPC xenograft model compared with monotherapy with either. 99mTc-duramycin imaging might be able to reliably identify response to RT in combination with CTX as early as 24 h after therapy initiation in NPC xenograft models. This might help to isolate non-responding patients in a timely manner and avoid unnecessary side effects in the clinic in the future.

Lactobacillus plantarum USM8613 Aids in Wound Healing and Suppresses Staphylococcus aureus Infection at Wound Sites.

This study aimed to elucidate the targets and mechanisms of anti-staphylococcal effects from bioactive metabolites produced by lactic acid bacteria. We aimed to better understand the safety and efficacy of these bioactive metabolites in in vivo systems, typically at topical sites. The cell-free supernatant and protein-rich fraction from Lactobacillus plantarum USM8613 inhibited staphyloxanthin biosynthesis, reduced (p < 0.05) the cell number of Staphylococcus aureus by 106 CFU/mL and reduced biofilm thickness by 55% in S. aureus-infected porcine skins. Genome-wide analysis and gene expression analysis illustrated the production of several plantaricins, especially the plantaricins EF and JK that enhanced the anti-staphylococcal effects of L. plantarum USM8613. In vivo data using rats showed that the protein-rich fraction from L. plantarum USM8613 exerted wound healing properties via direct inhibition of S. aureus and promoted innate immunity, in which the expression of ß-defensin was significantly (p < 0.05) upregulated by 3.8-fold. The protein fraction from L. plantarum USM8613 also significantly enhanced (p < 0.05) the production of cytokines and chemokines through various stages of wound recovery. Using ∆atl S. aureus, the protein-rich fraction from L. plantarum USM8613 exerted inhibitory activity via targeting the atl gene in S. aureus. Taken altogether, our present study illustrates the potential of L. plantarum USM8613 in aiding wound healing, suppressing of S. aureus infection at wound sites and promoting host innate immunity.

Augmented therapeutic efficacy of 5-fluorouracil in conjunction with lantibiotic nisin against skin cancer.

Chemotherapy, a gold standard for treating most of the cancers, involves drastic side-effects and multidrug resistance. An attractive alternative is development of combination therapy employing antimicrobial peptides with chemotherapeutic drugs. In vivo studies: Anti-cancer therapeutic efficacy of 5-fluororuacil (5-FU) in conjunction with nisin (50 mg/kg body weight) was evaluated against murine skin cancer, in terms of tumor biostatistics, histopathology, electron microscopy, infrared spectroscopy and transcriptional studies. In vitro studies: Dose and time dependent cytotoxicity of agents were assessed against A431 cell line using MTT assay, LDH assay and acridine orange/ethidium bromide dual staining. Significant percentage decrease(s) in mean tumor volume and tumor burden were observed in nisin+ 5-FU combination treated groups as compared to alone treated groups. Histoarchitecture of treated skins demonstrated restoration towards normal skin tissue (being highest in the combination group). Modulation of apoptotic, angiogenic and proliferative genes were observed in treated groups. IC50 of combination was found to be 2 µg/ml as compared to nisin alone (32µg/ml) and 5-FU alone (16µg/ml) with combination index of 0.188. Dual staining showed that rate of induction of apoptosis was higher in the combination group as compared to single agents. Nisin and 5-FU in combination were found to be synergistic both in vivo and in vitro.

Efficacy, toxicity study and antioxidant properties of plantaricin E and F recombinants against enteropathogenic Escherichia coli K1.1 (EPEC K1.1).

Enteropathogenic Escherichia coli (EPEC) is one of the resistance bacteria towards antibiotics and have been raising problem during treatments. Therefore, a new antibiotic candidate is required. Plantaricin E and F recombinant have been successfully produced by a GRAS host Lactococcus lactis. This study was aimed to evaluate the efficacy and toxicity of plantaricin E and F recombinant against EPEC K1.1 infection by in vivo assay. The production of plantaricin E and F recombinants from Lactococcus lactis was conducted and encapsulated. The in vivo study was carried out by inoculating the mice perorally with EPEC K1.1 for 7 days then treated with 100, 250, and 500 mg/kg body weight/day of recombinant plantaricin E and F for another 7 days. The toxicity assay were observed in ddY mice using various concentrations of treatment (50, 100, 1000, and 5000 mg/kg/body weight) doses perorally for 48 h. The result showed that the plantaricin E and F recombinant were successfully produced in Lactococcus lactis expression host with 3.7 kDa and 3.8 kDa in size. The efficacy study revealed the optimal doses of plantaricin E and F recombinant against EPEC K1.1 infection was 250 mg/kgBW for plantaricin E and 500 mg/kgBW for plantaricin F. The plantarisin E and F recombinant treatment showed improvement in leukocyte, hematocrit, and hemoglobin levels as well in decreasing malondialdehyde (MDA) level. Observation of the intestine histopathology showed small amounts of mononuclear inflammatory cell infiltration than the other groups of treatment. The acute toxicity assay showed that there was no mortality observed during the assay, even after 5000 mg/kg body weight of plantarisin E and F recombinant treatment (LD50 > 5000 mg/KgBW). The hematological and biochemical observations showed normal levels in leukocytes, erythrocytes, hematocrit, hemoglobin, platelets, urea, creatinine, and alanine transaminase aspartate transaminase (SGOT and SGPT) while histopathological observation shows a picture of normal liver and kidney cells. This study confirmed the application of bacteriocin for further academic and industrial purposes as a non-toxic substance for food preservative and antibiotic candidate.

Preclinical evaluation of the maximum tolerated dose and toxicokinetics of enteric-coated lantibiotic OG253 capsules.

Clostridium difficile associated disease (CDAD) is the leading infectious cause of antibiotic-associated diarrhea and colitis in the United States. Both the incidence and severity of CDAD have been increased over the past two decades. We evaluated the maximum tolerated dose (MTD) and toxicokinetics of OG253, a novel lantibiotic in development for the treatment of CDAD. OG253 was orally administered to Wistar Han rats as enteric-coated capsules in a one-day dose escalation study, followed by a seven-day repeated dose toxicokinetics study. All three doses of OG253 (6.75, 27 and 108 mg/day) were generally well-tolerated with no treatment-related clinical signs, alterations in body weight or food consumption in both one-day acute tolerability and seven-days repeated dose tolerability and toxicokinetics study. OG253 capsule administration neither significantly alter the weight of organs nor affect the hematology, coagulation, clinical biochemistry parameters and urine pH compared to placebo capsule administered rats. LC-MS/MS analysis did not detect OG253 in the plasma, indicating that OG253 is not absorbed into the blood from the rat gastrointestinal tract. Glandular atrophy of the rectal mucosa was noticed in two out of six rats administered with a high dose of OG253. Surprisingly, we found that OG253 treatment significantly lowered both serum cholesterol and triglyceride levels in both sexes of rats. Overall, there was a 29.8 and 61.38% decrease in the serum cholesterol and triglyceride levels, respectively as compared to placebo-treated rats. The well-tolerated high dose of OG253 (425.7 mg/kg/day) is recommended as the MTD for safety and efficacy studies. Further preclinical study is needed to evaluate the safety profile of OG253 under longer exposure.

Pharmacological, Toxicological, and Dose Range Assessment of OG716, a Novel Lantibiotic for the Treatment of Clostridium difficile-Associated Infection.

Lantibiotics present an attractive scaffold for the development of novel antibiotics. We report here a novel lantibiotic for the treatment of Clostridium difficile infection. The lead compounds were selected from a library of over 700 single- and multiple-substitution variants of the lantibiotic mutacin 1140 (MU1140). The best performers in vitro and in vivo were further used to challenge Golden Syrian hamsters orally in a Golden Syrian hamster model of Clostridium difficile-associated disease (CDAD) in a dose-response format, resulting in the selection of OG716 as the lead compound. This lantibiotic was characterized by a 50% effective dose of 23.85 mg/kg of body weight/day (10.97 µmol/kg/day) in this model. Upon oral administration of the maximum feasible dose (≥1,918 mg/kg/day), no observable toxicities or side effects were noted, and no effect on intestinal motility was observed. Compartmentalization to the gastrointestinal tract was confirmed. MU1140-derived variants offer a large pipeline for the development of novel antibiotics for the treatment of several indications and are particularly attractive considering their novel mechanism of action. Based on the currently available data, OG716 has an acceptable profile for further development for the treatment of CDAD.

Current developments in lantibiotic discovery for treating Clostridium difficile infection.

INTRODUCTION: Clostridium difficile is a major cause of healthcare-associated diarrhea linked to the misuse of antimicrobials and the corresponding deleterious impact they have on the protective microbiota of the gut. Resistance to agents used to treat C. difficile including metronizadole and vancomycin has been reported highlighting the need for novel agents. Lantibiotics represent a novel class of agents that many studies have highlighted as effective against C. difficile. Areas covered: In this review lantibiotics including nisin, actagardine, mersacidin, NAI-107 and MU-1140 that exhibit good activity against C.difficile, all of which are currently in the preclinical phase of investigation are discussed. The lantibiotic NVB302, which has completed phase I clinical trials for the treatment of C. difficile, is also described. Expert opinion: Lantibiotics represent promising candidates for the treatment of C. difficile infections due to their novel mode of action, which is thought to decrease the potential of resistance developing and the fact they often possess a less deleterious effect on the protective gut microbiota when compared to traditional agents. They are also extremely amenable to bioengineering approaches and the incorporation of synthetic biology to produce more potent variants.

Protecting bactofencin A to enable its antimicrobial activity using mesoporous matrices.

There is huge global concern surrounding the emergence of antimicrobial resistant bacteria and this is resulting in an inability to treat infectious diseases. This is due to a lack of new antimicrobials coming to the market and irresponsible use of traditional antibiotics. Bactofencin A, a novel antimicrobial peptide which shows potential as an antibiotic, is susceptible to enzyme degradation. To improve its solution stability and inherent activity, bactofencin A was loaded onto a traditional silica mesoporous matrix, SBA-15, and a periodic mesoporous organosilane, MSE. The loading of bactofencin A was considerably higher onto SBA-15 than MSE due to the hydrophilic nature of SBA-15. While there was no detectable peptide released from SBA-15 into phosphate buffered saline and only 20% of the peptide loaded onto MSE was released, the loaded matrices showed enhanced activity compared to the free peptide during in vitro antimicrobial assays. In addition, the mesoporous matrices were found to protect bactofencin A against enzymatic degradation where results showed that the SBA-15 and MSE with loaded bactofencin A exposed to trypsin inhibited the growth of S. aureus while a large decrease in activity was observed for free bactofencin upon exposure to trypsin. Thus, the activity and stability of bactofencin A can be enhanced using mesoporous matrices and these matrices may enable its potential development as a novel antibiotic. This work also shows that in silico studies looking at surface functional group and size complementarity between the peptide and the protective matrix could enable the systemic selection of a mesoporous matrix for individual bacteriocins with potential antimicrobial therapeutic properties.

Risks Related to High-Dosage Recombinant Antimicrobial Peptide Microcin J25 in Mice Model: Intestinal Microbiota, Intestinal Barrier Function, and Immune Regulation.

Antimicrobial peptide (AMP) can be a promising alternative in various domains. However, further risk information is required. In this study, mice were orally administrated different dosages of recombinant AMP microcin J25 (4.55, 9.1, and 18.2 mg/kg; MccJ25) for 1 week, and the toxicity risk impacts were examined. We evidenced that middle-dosage administration mice had a lower inflammation, better body weight, and ameliorated mucosal morphology, accompanied by reduced intestinal permeability and tighter intestinal barrier. Fecal microbiota composition analysis in middle- or low-dosage mice revealed the Bifidobacterium count was increased and the coliform bacteria count was decreased, and increased in short-chain fatty acid levels. Unexpectedly, there was a risk that high-dosage mice increased intestinal permeability and imbalance of intestinal bacteria. Taken together, these data indicated a safe threshold for usage of MccJ25 in clinical practice. Such studies can effectively enhance the safety of various aspects such as food preservative and drug.

Campylobacter control measures in indoor broiler chicken: critical re-assessment of cost-utility and putative barriers to implementation.

As campylobacteriosis is one of the most important foodborne infections, a European Union (EU)-27 level cost-effectiveness model has been developed on the socio-economic costs and benefits of applying certain control measures for the reduction of Campylobacter in broiler meat. This is expected to be a gold standard for food safety policymakers in the EU; hence, the validity of its modelling assumptions is essential. The authors of the present paper conducted an independent review of model input parameters on health and economic burden and found that the model most probably overestimated the burden of human campylobacteriosis. A discounted, quality-adjusted life year (QALY)-based European estimate has been developed for human campylobacteriosis and resulted in 15.23 QALY loss per 1000 human gastroenteritis cases. Country-specific cost of illness estimates have been developed for various countries in the EU-27. Based on these model adaptations, a selected Campylobacter control strategy was re-assessed and its high cost-effectiveness was confirmed at the EU level, and also in all but three Member States. Bacteriocin treatment or vaccination of the animals, two alternative control measures were also re-evaluated, and these strategies seemed to be far less cost-effective than the investigated strategy. Putative barriers to the rapid implementation of the investigated Campylobacter control strategy are discussed, and potential solutions are proposed. Further research is required on stakeholder perspectives pertaining to the realistic barriers and implementation opportunities.

OG716: Designing a fit-for-purpose lantibiotic for the treatment of Clostridium difficile infections.

Lantibiotics continue to offer an untapped pipeline for the development of novel antibiotics. We report here the discovery of a novel lantibiotic for the treatment of C. difficile infection (CDI). The leads were selected from a library of over 300 multiple substitution variants of the lantibiotic Mutacin 1140 (MU1140). Top performers were selected based on testing for superior potency, solubility, manufacturability, and physicochemical and/or metabolic stability in biologically-relevant systems. The best performers in vitro were further evaluated orally in the Golden Syrian hamster model of CDAD. In vivo testing ultimately identified OG716 as the lead compound, which conferred 100% survival and no relapse at 3 weeks post infection. MU1140-derived variants are particularly attractive for further clinical development considering their novel mechanism of action.

SPECT/CT imaging of chemotherapy-induced tumor apoptosis using 99mTc-labeled dendrimer-entrapped gold nanoparticles.

Non-invasive imaging of apoptosis in tumors induced by chemotherapy is of great value in the evaluation of therapeutic efficiency. In this study, we report the synthesis, characterization, and utilization of radionuclide technetium-99m (99mTc)-labeled dendrimer-entrapped gold nanoparticles (Au DENPs) for targeted SPECT/CT imaging of chemotherapy-induced tumor apoptosis. Generation five poly(amidoamine) (PAMAM) dendrimers (G5.NH2) were sequentially conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), polyethylene glycol (PEG) modified duramycin, PEG monomethyl ether, and fluorescein isothiocyanate (FI) to form the multifunctional dendrimers, which were then utilized as templates to entrap gold nanoparticles. Followed by acetylation of the remaining dendrimer surface amines and radiolabeling of 99mTc, the SPECT/CT dual mode nanoprobe of tumor apoptosis was constructed. The developed multifunctional Au DENPs before and after 99mTc radiolabeling were well characterized. The results demonstrate that the multifunctional Au DENPs display favorable colloidal stability under different conditions, own good cytocompatibility in the given concentration range, and can be effectively labeled by 99mTc with high radiochemical stability. Furthermore, the multifunctional nanoprobe enables the targeted SPECT/CT imaging of apoptotic cancer cells in vitro and tumor apoptosis after doxorubicin (DOX) treatment in the established subcutaneous tumor model in vivo. The designed duramycin-functionalized Au DENPs might have the potential to be employed as a nanoplatform for the detection of apoptosis and early tumor response to chemotherapy.

Molecular Imaging of Apoptosis in Ischemia Reperfusion Injury With Radiolabeled Duramycin Targeting Phosphatidylethanolamine: Effective Target Uptake and Reduced Nontarget Organ Radiation Burden.

OBJECTIVES: The purpose of this study was to evaluate the feasibility of imaging apoptosis in experimental ischemia-reperfusion model by technetium-99m (99mTc)-labeled Duramycin, and compare it to an established tracer, 99mTc-labeled Annexin-V, which has a relative disadvantage of high radiation burden to nontarget organs. BACKGROUND: During apoptosis, the cell membrane phospholipids-phosphatidylserine (PS) and phosphatidylethanolamine (PE) are exposed and can be targeted by Annexin-V and Duramycin, respectively, for in vivo imaging. Identification of a reversible cell death process should permit therapeutic intervention to help reduce myocyte loss and left ventricle dysfunction. METHODS: In a 40-min left coronary artery ischemia-reperfusion model in 17 rabbits, 7 mCi of 99mTc-labeled Duramycin (n = 10), 99mTc-linear Duramycin (a negative tracer control; n = 3), or 99mTc-Annexin-V (a positive tracer-control; n = 4) were intravenously administered 30 min after reperfusion. Of the 10 Duramycin group animals, 4 animals were treated with an antiapoptotic agent, minocycline at the time of reperfusion. In vivo and ex vivo micro-single-photon emission computed tomography (µSPECT) and micro-computed tomography (µCT) imaging was performed 3 h after reperfusion, followed by quantitative assessment of tracer uptake and pathological characterization. Fluorescent Duramycin and Annexin-V were injected in 4 rats to visualize colocalization in infarct areas in a 40-min left coronary artery occlusion and 30-min reperfusion model. RESULTS: Intense uptake of Duramycin and Annexin-V was observed in the apical (infarcted) areas. The percent injected dose per gram uptake of Duramycin in apical region (0.751 ± 0.262%) was significantly higher than remote area in same animals (0.045 ± 0.029%; p < 0.01). Duramycin uptake was insignificantly lower than Annexin-V uptake (1.23 ± 0.304%; p > 0.01) but demonstrated substantially lower radiation burden to kidneys (0.358 ± 0.210% vs. 1.58 ± 0.316%, respectively; p < 0.001). Fluorescence studies with Duramycin and Annexin V showed colocalization in infarct areas. Minocycline treatment substantially resolved Duramycin uptake (0.354% ± 0.0624%; p < 0.01). CONCLUSIONS: Duramycin is similarly effective in imaging apoptotic cell death as Annexin-V with lower nontarget organ radiation. Clinical feasibility of apoptosis imaging with a PE-seeking tracer should be tested.