Enhancing 2-Pyrone Synthase Efficiency by High-Throughput Mass-Spectrometric Quantification and In†Vitro/In†Vivo Catalytic Performance Correlation.

Engineering efficient biocatalysts is essential for metabolic engineering to produce valuable bioproducts from renewable resources. However, due to the complexity of cellular metabolic networks, it is challenging to translate success in vitro into high performance in cells. To meet such a challenge, an accurate and efficient quantification method is necessary to screen a large set of mutants from complex cell culture and a careful correlation between the catalysis parameters in vitro and performance in cells is required. In this study, we employed a mass-spectrometry based high-throughput quantitative method to screen new mutants of 2-pyrone synthase (2PS) for triacetic acid lactone (TAL) biosynthesis through directed evolution in E. coli. From the process, we discovered two mutants with the highest improvement (46 fold) in titer and the fastest kcat (44 fold) over the wild type 2PS, respectively, among those reported in the literature. A careful examination of the correlation between intracellular substrate concentration, Michaelis-Menten parameters and TAL titer for these two mutants reveals that a fast reaction rate under limiting intracellular substrate concentrations is important for in-cell biocatalysis. Such properties can be tuned by protein engineering and synthetic biology to adopt these engineered proteins for the maximum activities in different intracellular environments.

Optical Resolution of Two Pharmaceutical Bases with Various Uses of Tartaric Acid Derivatives and Their Sodium Salts: Racemic Ephedrine and Chloramphenicol Base.

The optically active dibenzoyltartaric acid, tartaric acid, and its sodium salts were successfully applied to the optical resolution of (1R,2S)(1S,2R)-2-(methylamino)-1-phenylpropan-1-ol (EPH) and (1R,2R)(1S,2S)-2-amino-1-(4-nitrophenyl)propane-1,3-diol (AD) as resolving agents. It was observed that both compounds' resolution using a mixture of salts of quasi-racemic resolving agents showed a change in chiral recognition under the same conditions compared to the result of the use of the single enantiomeric resolving agent. The changes are followed by detailed analytical (XRD, FTIR, and DSC) studies. Meanwhile, the DASH indexing software package was also tested on powder XRD patterns of pure initial materials and intermediate salt samples of high diastereomeric excess.

Catalytic Syn-Selective Nitroaldol Approach to Amphenicol Antibiotics: Evolution of a Unified Asymmetric Synthesis of (-)-Chloramphenicol, (-)-Azidamphenicol, (+)-Thiamphenicol, and (+)-Florfenicol.

A unified strategy for an efficient and high diastereo- and enantioselective synthesis of (-)-chloramphenicol, (-)-azidamphenicol, (+)-thiamphenicol, and (+)-florfenicol based on a key catalytic syn-selective Henry reaction is reported. The stereochemistry of the ligand-enabled copper(II)-catalyzed aryl aldehyde Henry reaction of nitroethanol was first explored to forge a challenging syn-2-amino-1,3-diol structure unit with vicinal stereocenters with excellent stereocontrol. Multistep continuous flow manipulations were carried out to achieve the efficient asymmetric synthesis of this family of amphenicol antibiotics.

Unified Strategy to Amphenicol Antibiotics: Asymmetric Synthesis of (-)-Chloramphenicol, (-)-Azidamphenicol, and (+)-Thiamphenicol and Its (+)-3-Floride.

The asymmetric synthesis of (-)-chloramphenicol, (-)-azidamphenicol, and (+)-thiamphenicol and its (+)-3-floride, (+)-florfenicol, is reported. This approach toward the amphenicol antibiotic family features two key steps: (1) a cinchona alkaloid derived urea-catalyzed aldol reaction allows highly enantioselective access to oxazolidinone gem-diesters and (2) a continuous flow diastereoselective decarboxylation of thermally stable oxazolidinone gem-diesters to form the desired trans-oxazolidinone monoesters with two adjacent stereocenters that provide the desired privileged scaffolds of syn-vicinal amino alcohols in the amphenicol family.

CmlI N-Oxygenase Catalyzes the Final Three Steps in Chloramphenicol Biosynthesis without Dissociation of Intermediates.

CmlI catalyzes the six-electron oxidation of an aryl-amine precursor (NH2-CAM) to the aryl-nitro group of chloramphenicol (CAM). The active site of CmlI contains a (hydr)oxo- and carboxylate-bridged dinuclear iron cluster. During catalysis, a novel diferric-peroxo intermediate P is formed and is thought to directly effect oxygenase chemistry. Peroxo intermediates can facilitate at most two-electron oxidations, so the biosynthetic pathway of CmlI must involve at least three steps. Here, kinetic techniques are used to characterize the rate and/or dissociation constants for each step by taking advantage of the remarkable stability of P in the absence of substrates (decay t1/2 = 3 h at 4 °C) and the visible chromophore of the diiron cluster. It is found that diferrous CmlI (CmlIred) can react with NH2-CAM and O2 in either order to form a P-NH2-CAM intermediate. P-NH2-CAM undergoes rapid oxygen transfer to form a diferric CmlI (CmlIox) complex with the aryl-hydroxylamine [NH(OH)-CAM] pathway intermediate. CmlIox-NH(OH)-CAM undergoes a rapid internal redox reaction to form a CmlIred-nitroso-CAM (NO-CAM) complex. O2 binding results in formation of P-NO-CAM that converts to CmlIox-CAM by enzyme-mediated oxygen atom transfer. The kinetic analysis indicates that there is little dissociation of pathway intermediates as the reaction progresses. Reactions initiated by adding pathway intermediates from solution occur much more slowly than those in which the intermediate is generated in the active site as part of the catalytic process. Thus, CmlI is able to preserve efficiency and specificity while avoiding adventitious chemistry by performing the entire six-electron oxidation in one active site.

Dual effect of chloramphenicol peptides on ribosome inhibition.

Chloramphenicol peptides were recently established as useful tools for probing nascent polypeptide chain interaction with the ribosome, either biochemically, or structurally. Here, we present a new 10mer chloramphenicol peptide, which exerts a dual inhibition effect on the ribosome function affecting two distinct areas of the ribosome, namely the peptidyl transferase center and the polypeptide exit tunnel. According to our data, the chloramphenicol peptide bound on the chloramphenicol binding site inhibits the formation of both acetyl-phenylalanine-puromycin and acetyl-lysine-puromycin, showing, however, a decreased peptidyl transferase inhibition compared to chloramphenicol-mediated inhibition per se. Additionally, we found that the same compound is a strong inhibitor of green fluorescent protein synthesis in a coupled in vitro transcription-translation assay as well as a potent inhibitor of lysine polymerization in a poly(A)-programmed ribosome, showing that an additional inhibitory effect may exist. Since chemical protection data supported the interaction of the antibiotic with bases A2058 and A2059 near the entrance of the tunnel, we concluded that the extra inhibition effect on the synthesis of longer peptides is coming from interactions of the peptide moiety of the drug with residues comprising the ribosomal tunnel, and by filling up the tunnel and blocking nascent chain progression through the restricted tunnel. Therefore, the dual interaction of the chloramphenicol peptide with the ribosome increases its inhibitory effect and opens a new window for improving the antimicrobial potency of classical antibiotics or designing new ones.

Mechanism for Six-Electron Aryl-N-Oxygenation by the Non-Heme Diiron Enzyme CmlI.

The ultimate step in chloramphenicol (CAM) biosynthesis is a six-electron oxidation of an aryl-amine precursor (NH2-CAM) to the aryl-nitro group of CAM catalyzed by the non-heme diiron cluster-containing oxygenase CmlI. Upon exposure of the diferrous cluster to O2, CmlI forms a long-lived peroxo intermediate, P, which reacts with NH2-CAM to form CAM. Since P is capable of at most a two-electron oxidation, the overall reaction must occur in several steps. It is unknown whether P is the oxidant in each step or whether another oxidizing species participates in the reaction. Mass spectrometry product analysis of reactions under (18)O2 show that both oxygen atoms in the nitro function of CAM derive from O2. However, when the single-turnover reaction between (18)O2-P and NH2-CAM is carried out in an (16)O2 atmosphere, CAM nitro groups contain both (18)O and (16)O, suggesting that P can be reformed during the reaction sequence. Such reformation would require reduction by a pathway intermediate, shown here to be NH(OH)-CAM. Accordingly, the aerobic reaction of NH(OH)-CAM with diferric CmlI yields P and then CAM without an external reductant. A catalytic cycle is proposed in which NH2-CAM reacts with P to form NH(OH)-CAM and diferric CmlI. Then the NH(OH)-CAM rereduces the enzyme diiron cluster, allowing P to reform upon O2 binding, while itself being oxidized to NO-CAM. Finally, the reformed P oxidizes NO-CAM to CAM with incorporation of a second O2-derived oxygen atom. The complete six-electron oxidation requires only two exogenous electrons and could occur in one active site.

Photocatalytic degradation of the antibiotic chloramphenicol and effluent toxicity effects.

Chloramphenicol sodium succinate (CAP, C15H15Cl2N2 Na2O8) is a broad-spectrum antibiotic exhibiting activity against both Gram-positive and Gram-negative bacteria as well as other groups of microorganisms only partially removed by conventional activated sludge wastewater treatment plants. Thus, CAP and its metabolites can be found in effluents. The present work deals with the photocatalytic degradation of CAP using TiO2 as photocatalyst. We investigated the optimization of reaction contact time and concentration of TiO2 considering CAP and its by-products removal as well as effluent ecotoxicity elimination. Considering a CAP real concentration of 25mgL(-1), kinetic degradation curves were determined at 0.1, 0.2, 0.4, 0.8, 1.6 and 3.2gL(-1) TiO2 after 5, 10, 30, 60 and 120min reaction time. Treated samples were checked for the presence of by-products and residual toxicity (V. fischeri, P. subcapitata, L. sativum and D. magna). Results evidenced that the best combination for CAP and its by-products removal could be set at 1.6gL(-1) of TiO2 for 120min with an average residual toxicity of approximately 10%, that is the threshold set for negative controls in most toxicity tests for blank and general toxicity test acceptability.

A novel gene, optrA, that confers transferable resistance to oxazolidinones and phenicols and its presence in Enterococcus faecalis and Enterococcus faecium of human and animal origin.

OBJECTIVES: The oxazolidinone-resistant Enterococcus faecalis E349 from a human patient tested negative for the cfr gene and 23S rRNA mutations. Here we report the identification of a novel oxazolidinone resistance gene, optrA, and a first investigation of the extent to which this gene was present in E. faecalis and Enterococcus faecium from humans and food-producing animals. METHODS: The resistance gene optrA was identified by whole-plasmid sequencing and subsequent cloning and expression in a susceptible Enterococcus host. Transformation and conjugation assays served to investigate the transferability of optrA. All optrA-positive E. faecalis and E. faecium isolates of human and animal origin were analysed for their MICs and their genotype, as well as the location of optrA. RESULTS: The novel plasmid-borne ABC transporter gene optrA from E. faecalis E349 conferred combined resistance or elevated MICs (when no clinical breakpoints were available) to oxazolidinones (linezolid and tedizolid) and phenicols (chloramphenicol and florfenicol). The corresponding conjugative plasmid pE349, on which optrA was located, had a size of 36 331 bp and also carried the phenicol exporter gene fexA. The optrA gene was functionally expressed in E. faecalis, E. faecium and Staphylococcus aureus. It was detected more frequently in E. faecalis and E. faecium from food-producing animals (20.3% and 5.7%, respectively) than from humans (4.2% and 0.6%, respectively). CONCLUSIONS: Enterococci with elevated MICs of linezolid and tedizolid should be tested not only for 23S rRNA mutations and the gene cfr, but also for the novel resistance gene optrA.

Pharmacokinetics of chloramphenicol following administration of intravenous and subcutaneous chloramphenicol sodium succinate, and subcutaneous chloramphenicol, to koalas (Phascolarctos cinereus).

Clinically normal koalas (n = 19) received a single dose of intravenous (i.v.) chloramphenicol sodium succinate (SS) (25 mg/kg; n = 6), subcutaneous (s.c.) chloramphenicol SS (60 mg/kg; n = 7) or s.c. chloramphenicol base (60 mg/kg; n = 6). Serial plasma samples were collected over 24-48 h, and chloramphenicol concentrations were determined using a validated high-performance liquid chromatography assay. The median (range) apparent clearance (CL/F) and elimination half-life (t(1/2)) of chloramphenicol after i.v. chloramphenicol SS administration were 0.52 (0.35-0.99) L/h/kg and 1.13 (0.76-1.40) h, respectively. Although the area under the concentration-time curve was comparable for the two s.c. formulations, the absorption rate-limited disposition of chloramphenicol base resulted in a lower median C(max) (2.52; range 0.75-6.80 µg/mL) and longer median tmax (8.00; range 4.00-12.00 h) than chloramphenicol SS (C(max) 20.37, range 13.88-25.15 µg/mL; t(max) 1.25, range 1.00-2.00 h). When these results were compared with susceptibility data for human Chlamydia isolates, the expected efficacy of the current chloramphenicol dosing regimen used in koalas to treat chlamydiosis remains uncertain and at odds with clinical observations.

Aptasensing of chloramphenicol in the presence of its analogues: reaching the maximum residue limit.

A novel, label-free folding induced aptamer-based electrochemical biosensor for the detection of chloramphenicol (CAP) in the presence of its analogues has been developed. CAP is a broad-spectrum antibiotic that has lost its favor due to its serious adverse toxic effects on human health. Aptamers are artificial nucleic acid ligands (ssDNA or RNA) able to specifically recognize a target such as CAP. In this article, the aptamers are fixed onto a gold electrode surface by a self-assembly approach. In the presence of CAP, the unfolded ssDNA on the electrode surface changes to a hairpin structure, bringing the target molecules close to the surface and triggering electron transfer. Detection limits were determined to be 1.6 × 10(-9) mol L(-1). In addition, thiamphenicol (TAP) and florfenicol (FF), antibiotics with a structure similar to CAP, did not influence the performance of the aptasensor, suggesting a good selectivity of the CAP-aptasensor. Its simplicity and low detection limit (because of the home-selected aptamers) suggest that the electrochemical aptasensor is suitable for practical use in the detection of CAP in milk samples.

Antibiotics that affect the ribosome.

The ribosome is a major bacterial target for antibiotics. Drugs inhibit ribosome function either by interfering in messenger RNA translation or by blocking the formation of peptide bonds at the peptidyl transferase centre. These effects are the consequence of the binding of drugs to the ribosomal subunits. Various mechanisms, including enzymatic detoxification, target alteration (ribosomal [r]RNAs and ribosomal proteins) and reduced accumulation (impermeability and efflux) are involved in bacterial resistance to protein synthesis inhibitors. The fact that some positions in rRNA participate in the binding of antibiotics belonging to distinct families explains why bacteria have developed mechanisms that can lead to cross-resistance.

ProFlow nano-liquid chromatography with a graphene oxide-functionalized monolithic nano-column for the simultaneous determination of chloramphenicol and chloramphenicol glucuronide in foods.

Chloramphenicol (CAP) is an effective antibiotic with broad spectrum against gram-positive and gram-negative bacteria, while it is used to treat various infections in animals. Although CAP is banned for usage in the livestock products including, milk, honey, seafood, and royal jelly, CAP is still often detected in foods of animal origin, posing a threat to consumer health. The use of CAP is restricted in many countries due to its side effect in human metabolic process according to the Expert Committee on Food Additives (ECFA) recommendation. Chloramphenicol glucuronide (CAPG) is also a metabolic product of CAP, which may be a hazardous chemical for human health. Therefore, the development of sensitive separation and quantification method is an important issue, especially for food safety. Herein, we reported the preparation and application of a monolithic nano-column for CAP and CAPG analyses in foods by ProFlow Nano liquid chromatography (ProFlow Nano LC). The monolithic nano-column was prepared by an in situ polymerization using 3-chloro-2-hydroxypropylmethacrylate (HPMA-Cl) and ethylene dimethacrylate (EDMA) and followed graphene oxide (GO) modification. After characterization, the monolithic nano-column was used for the analysis of CAP and CAPG in honey and milk samples by ProFlow Nano LC. The whole method was validated in terms of linearity, sensitivity, precision, recovery, and repeatability, while it led to obtain high sensitivity with limit of quantification was found as 0.02 µg/kg for CAP. Limit of quantification for CAPG was found as 0.08 µg/kg. The developed method with monolithic nano-column was optimized to achieve very sensitive analyses of CAP and CAPG in the food samples. The applicability of the nano-column was successfully demonstrated by the analysis of CAP and CAPG in milk and honey samples. PRACTICAL APPLICATION: This article describes the preparation and application of a monolithic nano-column for the separation and determination of chloramphenicol and chloramphenicol glucuronide in food samples by ProFlow Nano LC. The use of new and advanced techniques is a crucial issue in the food science and technology. In this sense, this study demonstrated a new food analysis method using advanced instrumental technique with a homemade monolithic nano-column.

Profound cardioprotection with chloramphenicol succinate in the swine model of myocardial ischemia-reperfusion injury.

BACKGROUND: Emerging evidence suggests that "adaptive" induction of autophagy (the cellular process responsible for the degradation and recycling of proteins and organelles) may confer a cardioprotective phenotype and represent a novel strategy to limit ischemia-reperfusion injury. Our aim was to test this paradigm in a clinically relevant, large animal model of acute myocardial infarction. METHODS AND RESULTS: Anesthetized pigs underwent 45 minutes of coronary artery occlusion and 3 hours of reperfusion. In the first component of the study, pigs received chloramphenicol succinate (CAPS) (an agent that purportedly upregulates autophagy; 20 mg/kg) or saline at 10 minutes before ischemia. Infarct size was delineated by tetrazolium staining and expressed as a % of the at-risk myocardium. In separate animals, myocardial samples were harvested at baseline and 10 minutes following CAPS treatment and assayed (by immunoblotting) for 2 proteins involved in autophagosome formation: Beclin-1 and microtubule-associated protein light chain 3-II. To investigate whether the efficacy of CAPS was maintained with "delayed" treatment, additional pigs received CAPS (20 mg/kg) at 30 minutes after occlusion. Expression of Beclin-1 and microtubule-associated protein light chain 3-II, as well as infarct size, were assessed at end-reperfusion. CAPS was cardioprotective: infarct size was 25±5 and 41±4%, respectively, in the CAPS-pretreated and CAPS-delayed treatment groups versus 56±5% in saline controls (P<0.01 and P<0.05 versus control). Moreover, administration of CAPS was associated with increased expression of both proteins. CONCLUSIONS: Our results demonstrate attenuation of ischemia-reperfusion injury with CAPS and are consistent with the concept that induction of autophagy may provide a novel strategy to confer cardioprotection.

Synthesis, Analytical Characterization and Spectroscopic Investigation of Chloramphenicol Impurity A for the Quality Control of Chloramphenicol and its Formulation as Per International Compendium.

OBJECTIVE: The main aim of the present work is to synthesize chloramphenicol impurity A (CLRMIMP- A) in the purest form and its subsequent characterization by using a panel of sophisticated analytical techniques (LC-MS, DSC, TGA, NMR, FTIR, HPLC, and CHNS) to provide as a reference standard mentioned in most of the international compendiums, including IP, BP, USP, and EP. The present synthetic procedure has not been disclosed anywhere in the prior art. METHODS: A simple, cheaper, and new synthesis method was described for the preparation of CLRM-IMP-A. It was synthesized and characterized by FTIR, DSC, TGA, NMR (1H and 13C), LC-MS, CHNS, and HPLC. RESULTS: CLRM-IMP-A present in drugs and dosage form can alter the therapeutic effects and adverse reaction of a drug considerably, it is mandatory to have a precise method for the estimation of impurities to safeguard the public health. Under these circumstances, the presence of CLRM-IMP-A in chloramphenicol (CLRM) requires strict quality control to satisfy the specified regulatory limit. The synthetic impurity obtained was in the pure form to provide a certified reference standard or working standard to stakeholders with defined potency. CONCLUSION: The present research describes a novel technique for the synthesis of pharmacopoeial impurity, which can help in checking/controlling the quality of the CLRM in the international markets.

A structural study of the interaction between the Dr haemagglutinin DraE and derivatives of chloramphenicol.

Dr adhesins are expressed on the surface of uropathogenic and diffusely adherent strains of Escherichia coli. The major adhesin subunit (DraE/AfaE) of these organelles mediates attachment of the bacterium to the surface of the host cell and possibly intracellular invasion through its recognition of the complement regulator decay-accelerating factor (DAF) and/or members of the carcinoembryonic antigen (CEA) family. The adhesin subunit of the Dr haemagglutinin, a Dr-family member, additionally binds type IV collagen and is inhibited in all its receptor interactions by the antibiotic chloramphenicol (CLM). In this study, previous structural work is built upon by reporting the X-ray structures of DraE bound to two chloramphenicol derivatives: chloramphenicol succinate (CLS) and bromamphenicol (BRM). The CLS structure demonstrates that acylation of the 3-hydroxyl group of CLM with succinyl does not significantly perturb the mode of binding, while the BRM structure implies that the binding pocket is able to accommodate bulkier substituents on the N-acyl group. It is concluded that modifications of the 3-hydroxyl group would generate a potent Dr haemagglutinin inhibitor that would not cause the toxic side effects that are associated with the normal bacteriostatic activity of CLM.

Status of mitochondria in living human fibroblasts during growth and senescence in vitro: use of the laser dye rhodamine 123.

Rhodamine 123, a fluorescent laser dye that is selectively taken up into mitochondria of living cells, was used to examine mitochondrial morphology in early-passage (young), late-passage (old), and progeric human fibroblasts. Mitochondria were readily visualized in all cell types during growth (mid-log) and confluent stages. In all cell strains at confluence, mitochondria became shorter, more randomly aligned, and developed a higher proportion of bead-like forms. Treatment of cells for six days with Tevenel, a chloramphenicol analog that inhibits mitochondrial protein synthesis, brought about a marked depletion of mitochondria and a diffuse background fluorescence. Cyanide produced a rapid release of preloaded mitochondrial fluorescence followed by detachment and killing of cells. Colcemid caused a random coiling and fragmentation of mitochondria particularly in the confluent stage. No gross differences were discernible in mitochondria of the three cell strains in mid-log and confluent states or after these treatments. Butanol-extractable fluorescence after loading with rhodamine 123 was lower in all cell strains in confluent compared to mid-log stages. At confluence all three cell strains had similar rhodamine contents at zero-time and after washout up to 24 h. At the mid-log stage, young cells contained more rhodamine initially and lost it more rapidly than old or progeria cells, in that order. The data indicate no gross derangement in the morphology or number of mitochondria in old and progeria fibroblasts but there is a reduction of protonmotive force evident in these cells at the mid-log stage that may be growth limiting.

Selection of mammalian cells resistant to a chloramphenicol analog.

This study describes the selection and preliminary characterization of mammalian cells resistant to 100 mug Tevenel/ml. Tevenel, the sulfamoyl analog of chloramphenicol, is a specific inhibitor of mitochondrial protein synthesis. After growth in suspension culture for 5 days in 100 mug Tevenel/ml and subsequent plating in 100 mug Tevenel/ml, LMTK- cells yielded resistant clones. As a control, L cells treated identically yielded no clones. Three resistant clones were chosen for study. Each resistant cell line had an identical growth rate in the presence and absence of 100 mug Tevenel/ml. By plating efficiency analysis, the resistant cells were found to be cross-resistant to D-chloramphenicol. The change responsible for resistance was found to be stable for at least 100 generations in the absence of the drug. Protein synthesis by isolated mitochondria of resistant cells was found to be less inhibited by concentrations of both Tevenel and D-chloramphenicol up to 200 mug/ml than the protein synthesis by LMTK- mitochondria. This resistance in vitro was not changed by incubation of the mitochondria in 0.01% Triton X-100.

Chromatographic evaluation of polymers imprinted with analogs of chloramphenicol and application to selective solid-phase extraction.

To obtain a highly selective material for the antibiotic chloramphenicol, which has several harmful side effects in humans, different molecularly imprinted polymers (MIPs) were prepared. In order to avoid a major traditional drawback associated with MIPs of residual template bleeding, molecules that are structurally related to chloramphenicol were used as templates for polymer synthesis. Chromatographic evaluation indicated that the employed template imparted a significant influence on the recognition properties of the corresponding polymer. A strong retention of chloramphenicol under nonpolar elution conditions (k = 68.03, IF = 17.72) and under aqueous elution conditions (k = 92.44, IF = 1.35) was achieved. After chromatographic evaluation, the MIP was utilized as the recognition sorbent in a solid-phase extraction to determine chloramphenicol using either an organic or aqueous washing solvent. Recoveries of nearly 100% from the chloramphenicol standard solution and nearly 90% from honey samples spiked with chloramphenicol were attained. Furthermore, the applicability of the MIP for sample cleanup was demonstrated.

Sustained-release microparticle dry powders of chloramphenicol palmitate or thiamphenicol palmitate prodrugs for lung delivery as aerosols.

The purpose of this study was to design inhalable sustained-release nanoparticle-in-microparticles, i.e. nano-embedded microparticles, for the lung delivery of chloramphenicol or thiamphenicol as aerosols. The palmitate ester prodrugs of the two antibiotics were used to prepare PLGA-based nanoparticles or to form pure prodrug nanoparticles. Prodrug-loaded PLGA nanoparticles or pure prodrug nanoparticles were prepared using the emulsion-solvent evaporation method. Dry microparticle powders for inhalation were then produced by spray-drying the nanoparticle suspensions supplemented with lactose as a bulking agent and L-leucine as a dispersing enhancer. Examined under the scanning electron microscopy, the obtained microparticles appeared to be spherical and shriveled, with no crystal-like structures. Drug loading was satisfactory (14 to 34% (m/m)) and the aerodynamic properties determined with a Next Generation Impactor were appropriate for lung delivery, with mass median aerodynamic diameters close to 3 µm. The in vitro release profiles showed that sustained released was achieved with these formulations, with an almost complete release over 14 days.