Glutamate acts on acid-sensing ion channels to worsen ischaemic brain injury.

Glutamate is traditionally viewed as the first messenger to activate NMDAR (N-methyl-D-aspartate receptor)-dependent cell death pathways in stroke1,2, but unsuccessful clinical trials with NMDAR antagonists implicate the engagement of other mechanisms3-7. Here we show that glutamate and its structural analogues, including NMDAR antagonist L-AP5 (also known as APV), robustly potentiate currents mediated by acid-sensing ion channels (ASICs) associated with acidosis-induced neurotoxicity in stroke4. Glutamate increases the affinity of ASICs for protons and their open probability, aggravating ischaemic neurotoxicity in both in vitro and in vivo models. Site-directed mutagenesis, structure-based modelling and functional assays reveal a bona fide glutamate-binding cavity in the extracellular domain of ASIC1a. Computational drug screening identified a small molecule, LK-2, that binds to this cavity and abolishes glutamate-dependent potentiation of ASIC currents but spares NMDARs. LK-2 reduces the infarct volume and improves sensorimotor recovery in a mouse model of ischaemic stroke, reminiscent of that seen in mice with Asic1a knockout or knockout of other cation channels4-7. We conclude that glutamate functions as a positive allosteric modulator for ASICs to exacerbate neurotoxicity, and preferential targeting of the glutamate-binding site on ASICs over that on NMDARs may be strategized for developing stroke therapeutics lacking the psychotic side effects of NMDAR antagonists.

mPEG-b-P(Glu-co-Phe) nanoparticles increase gastric retention time and gastric ulcer treatment efficacy of 20(S)-ginsenoside Rg3.

BACKGROUND: Gastric ulcer (GU) belongs to gastric mucosal irritation and damage. 20(S)-ginsenoside Rg3 (Rg3) has shown anti-oxidant, antiinflammation, and tissue repair effects which are essential for GU treatment. However, the solubility of Rg3 is poor and low gastrointestinal absorption may limit its anti-ulcer effects. As a result, we aim to increase the gastric retention time and gastric absorption of Rg3 to achieve better GU treatment efficacy. METHODS: The mPEG-b-P(Glu-co-Phe) nanoparticles loaded with Rg3 (Rg3-NPs) were developed. The characteristics of Rg3-NPs, including the morphology, diameter, and stability were analyzed. The Rg3 release profiles, gastric retention of Rg3, in vitro cytotoxicity, and pharmacokinetics of Rg3 were assessed. An alcohol-induced rats GU model was performed, and the rats were randomly separated into five treatment groups. Biochemical analysis, gross evaluation, histopathology, and immunohistochemical analysis were applied to further analyze the anti-ulcer effects of Rg3-NPs. RESULTS: Rg3-NPs were successfully prepared and the Rg3 release was pH sensitive. The gastric retention time of Rg3 is longer in Rg3-NPs group than that in Rg3 group. By slightly increasing nitric oxide (NO), obviously increasing epidermal growth factor (EGF), EGF receptor (EGFR), and superoxide dismutase (SOD), and decreasing endothelin-1 (ET-1) and nitric oxide synthase (NOS2), Rg3-NPs possess better GU treatment efficacy than Rg3. CONCLUSIONS: Rg3-NPs can increase gastric retention time and gastric absorption of Rg3 and promote its GU treatment efficacy.

Computational identification of 4-carboxyglutamate sites to supplement physiological studies using deep learning.

In biological systems, Glutamic acid is a crucial amino acid which is used in protein biosynthesis. Carboxylation of glutamic acid is a significant post-translational modification which plays important role in blood coagulation by activating prothrombin to thrombin. Contrariwise, 4-carboxy-glutamate is also found to be involved in diseases including plaque atherosclerosis, osteoporosis, mineralized heart valves, bone resorption and serves as biomarker for onset of these diseases. Owing to the pathophysiological significance of 4-carboxyglutamate, its identification is important to better understand pathophysiological systems. The wet lab identification of prospective 4-carboxyglutamate sites is costly, laborious and time consuming due to inherent difficulties of in-vivo, ex-vivo and in vitro experiments. To supplement these experiments, we proposed, implemented, and evaluated a different approach to develop 4-carboxyglutamate site predictors using pseudo amino acid compositions (PseAAC) and deep neural networks (DNNs). Our approach does not require any feature extraction and employs deep neural networks to learn feature representation of peptide sequences and performing classification thereof. Proposed approach is validated using standard performance evaluation metrics. Among different deep neural networks, convolutional neural network-based predictor achieved best scores on independent dataset with accuracy of 94.7%, AuC score of 0.91 and F1-score of 0.874 which shows the promise of proposed approach. The iCarboxE-Deep server is deployed at https://share.streamlit.io/sheraz-n/carboxyglutamate/app.py .

Co-delivery of gemcitabine and cisplatin via Poly (L-glutamic acid)-g-methoxy poly (ethylene glycol) micelle to improve the in vivo stability and antitumor effect.

PURPOSE: The intention of the study was to co-delivery gemcitabine and cisplatin with totally different nature by prodrug and micelle strategy to improve its in vivo stability and antitumor effect. METHODS: A prodrug of gemcitabine (mPEG-PLG-GEM) was synthesized through the covalent conjugation between the primary amino group of gemcitabine and the carboxylic group of poly (L-glutamic acid)-g-methoxy poly (ethylene glycol) (mPEG-PLG). It was prepared into micelles by a solvent diffusion method, and then combined with cisplatin through chelation to prepare gemcitabine and cisplatin co-loaded mPEG-PLG micelles (mPEG-PLG-GEM@CDDP micelles). RESULTS: Gemcitabine and cisplatin in each micelle group were released more slowly than in solutions. In addition, pharmacokinetics behaviors of them were improved after encapsulated in prodrug micelles. T1/2z of gemcitabine and cisplatin encapsulated in micelles were prolonged to 6.357 h (mPEG-PLG-GEM), 10.490 h (mPEG-PLG@CDDP), 5.463 h and 12.540 h (mPEG-PLG-GEM@CDDP) compared with GEM@CDDP solutions (T1/2z = 1.445 h and 7.740 h). The ratio of synergy between gemcitabine and cisplatin (3:1 ~ 1:1(n/n)) was guaranteed in the systemic circulation, thus improving its antitumor effect. The results of biochemical analysis showed that GEM@CDDP-Sol was more toxic to kidneys and marrow compared with mPEG-PLG-GEM@CDDP micelles. CONCLUSIONS: By prodrug strategy, gemcitabine and cisplatin with totally different nature were prepared into micelles and obtained a better pharmacokinetic behavior. And the dual drug delivery system performed a better in vivo stability and antitumor effect compared with each single drug delivery system in the experiment. Scheme. Schematic of mPEG-PLG-GEM@CDDP micelles' formation and action process.

Thermodynamic Solution Properties of a Biodegradable Chelant (L-glutamic-N,N-diacetic Acid, L-GLDA) and Its Sequestering Ability toward Cd2.

The thermodynamics of the interaction of L-glutamic-N,N-diacetic acid (GLDA) with protons was studied potentiometrically at different temperatures, ionic strengths and ionic media. Four protonation constants and corresponding enthalpy changes occurred at infinite dilution together with temperature and ionic strength coefficients. The medium effect was also interpreted in terms of the formation of weak complexes between the ligand and the cations of supporting electrolytes, resulting in a greater tendency of GLDA to chemically interact with Na+ rather than K+ and, in turn, (CH3)4N+. Formation constants of GLDA with Cd2+ were determined in NaCl(aq) at different ionic strength values. Five complex species were found, namely CdL2-, CdHL-, CdH2L0(aq), Cd2L0(aq), and Cd(OH)L3-, whose formation constant values at infinite dilution were log ß = 12.68, 17.61, 20.76, 17.52, and 1.77, respectively. All the species results were relevant in the pH range of natural waters, although the Cd2L0(aq) was observed only for CCd ≥ CGLDA and concentrations of >0.1 mmol dm-3. The sequestering ability of GLDA toward Cd2+, evaluated by means of pL0.5, was maximum at pH~10, whereas the presence of a chloride containing a supporting electrolyte exerted a negative effect. Among new generation biodegradable ligands, GLDA was the most efficient in Cd2+ sequestration.

Self-assembled PEGylated amphiphilic polypeptides for gene transfection.

In the present study, three biodegradable block copolymers composed of a poly(ethylene glycol) block and a copolypeptide block with varying compositions of cationic L-lysine (L-Lys) and hydrophobic benzyl-L-glutamate (Bzl-L-Glu) were designed for gene delivery applications. The polypeptides were synthesized by ring opening polymerization (ROP) and after orthogonal deprotection of Boc-L-Lys side chains, the polymer exhibited an amphiphilic character. To bind or encapsulate plasmid DNA (pDNA), different formulations were investigated: a nanoprecipitation and an emulsion technique using various organic solvents as well as an aqueous pH-controlled formulation method. The complex and nanoparticle (NP) formations were monitored by dynamic light scattering (DLS), and pDNA interaction was shown by gel electrophoresis and subsequent controlled release with heparin. The polypeptides were further tested for their cytotoxicity as well as biodegradability. The complexes and NPs presenting the most promising size distributions and pDNA binding ability were subsequently evaluated for their transfection efficiency in HEK293T cells. The highest transfection efficiencies were obtained with an aqueous formulation of the polypeptide containing the highest L-Lys content and lowest proportion of hydrophobic, helical structures (P1*), which is therefore a promising candidate for efficient gene delivery by biodegradable gene delivery vectors.

VGLUT substrates and inhibitors: A computational viewpoint.

The vesicular glutamate transporters (VGLUTs) bind and move glutamate (Glu) from the cytosol into the lumen of synaptic vesicles using a H+-electrochemical gradient (ΔpH and Δψ) generated by the vesicular H+-ATPase. VGLUTs show very low Glu binding and to date, no three-dimensional structure has been elucidated. Prior studies have attempted to identify the key residues involved in binding VGLUT substrates and inhibitors using homology models and docking experiments. Recently, the inward and outward oriented crystal structures of d-galactonate transporter (DgoT) emerged as possible structure templates for VGLUT. In this review, a new homology model for VGLUT2 based on DgoT has been developed and used to conduct docking experiments to identify and differentiate residues and binding orientations involved in ligand interactions. This review describes small molecule-ligand interactions including docking using a VGLUT2 homology model derived from DgoT.

The mitochondrial carrier Citrin plays a role in regulating cellular energy during carcinogenesis.

Citrin, encoded by SLC25A13 gene, is an inner mitochondrial transporter that is part of the malate-aspartate shuttle, which regulates the NAD+/NADH ratio between the cytosol and mitochondria. Citrullinemia type II (CTLN-II) is an inherited disorder caused by germline mutations in SLC25A13, manifesting clinically in growth failure that can be alleviated by dietary restriction of carbohydrates. The association of citrin with glycolysis and NAD+/NADH ratio led us to hypothesize that it may play a role in carcinogenesis. Indeed, we find that citrin is upregulated in multiple cancer types and is essential for supplementing NAD+ for glycolysis and NADH for oxidative phosphorylation. Consequently, citrin deficiency associates with autophagy, whereas its overexpression in cancer cells increases energy production and cancer invasion. Furthermore, based on the human deleterious mutations in citrin, we found a potential inhibitor of citrin that restricts cancerous phenotypes in cells. Collectively, our findings suggest that targeting citrin may be of benefit for cancer therapy.

Combretastatin A4 Nanodrug-Induced MMP9 Amplification Boosts Tumor-Selective Release of Doxorubicin Prodrug.

Tumor-associated enzyme-activated prodrugs can potentially improve the selectivity of chemotherapeutics. However, the paucity of tumor-associated enzymes which are essential for prodrug activation usually limits the antitumor potency. A cooperative strategy that utilizes combretastatin A4 nanodrug (CA4-NPs) and matrix metalloproteinase 9 (MMP9)-activated doxorubicin prodrug (MMP9-DOX-NPs) is developed. CA4 is a typical vascular disrupting agent that can selectively disrupt immature tumor blood vessels and exacerbate the tumor hypoxia state. After treatment with CA4-NPs, MMP9 expression can be significantly enhanced by 5.6-fold in treated tumors, which further boosts tumor-selective active drug release of MMP9-DOX-NPs by 3.7-fold in an orthotopic 4T1 mammary adenocarcinoma mouse model. The sequential delivery of CA4-NPs and MMP9-DOX-NPs exhibits enhanced antitumor efficacy with reduced systemic toxicity compared with the noncooperative controls.

A Versatile Boc Solid Phase Synthesis of Daptomycin and Analogues Using Site Specific, On-Resin Ozonolysis to Install the Kynurenine Residue.

A de novo solid-phase synthesis of the cyclic lipodepsipeptide daptomycin via Boc chemistry was achieved. The challenging ester bond formation between the nonproteinogenic amino acid kynurenine was achieved by esterification of a threonine residue with a protected tryptophan. Subsequent late-stage on-resin ozonolysis, inspired by the biomimetic pathway, afforded the kynurenine residue directly. Synthetic daptomycin possessed potent antimicrobial activity (MIC100 =1.0 µg mL-1 ) against S. aureus, while five other daptomycin analogues containing (2R,3R)-3-methylglutamic acid, (2S,4S)-4-methylglutamic acid or canonical glutamic acid at position twelve prepared using this new methodology were all inactive, clearly establishing that the (2S,3R)-3-methylglutamic acid plays a key role in the antimicrobial activity of daptomycin.

[A randomized, double-blind, placebo-controlled study of the efficacy and safety of ampasse in the treatment of chronic cerebral ischemia]. / Randomizirovannoe dvoinoe slepoe platsebo-kontroliruemoe issledovanie éffektivnosti i bezopasnosti preparata ampasse v terapii khronicheskoi ishemii mozga.

AIM: To study the efficacy and safety of ampasse in the treatment of chronic cerebral ischemia. MATERIAL AND METHODS: A randomized, double-blind, placebo-controlled study of the efficacy and safety of the 5-hydroxy-3-carboxypyridine-L-glutamine acid monocalcium salt (ampasse) was performed in 80 patients, aged from 50 to 75 years, with chronic cerebrovascular accident due to arterial hypertension and/or atherosclerosis of the main arteries of the head. The drug was used in daily doses of 5, 10, or 25 mg intravenously once a day for 15 days. Sodium chloride 0.9% was used as a placebo. RESULTS AND CONCLUSION: It has been established that ampasse improves the state of patients with chronic cerebral ischemia in relation to depression, sleep quality and cognitive functions.

Arsinothricin, an arsenic-containing non-proteinogenic amino acid analog of glutamate, is a broad-spectrum antibiotic.

The emergence and spread of antimicrobial resistance highlights the urgent need for new antibiotics. Organoarsenicals have been used as antimicrobials since Paul Ehrlich's salvarsan. Recently a soil bacterium was shown to produce the organoarsenical arsinothricin. We demonstrate that arsinothricin, a non-proteinogenic analog of glutamate that inhibits glutamine synthetase, is an effective broad-spectrum antibiotic against both Gram-positive and Gram-negative bacteria, suggesting that bacteria have evolved the ability to utilize the pervasive environmental toxic metalloid arsenic to produce a potent antimicrobial. With every new antibiotic, resistance inevitably arises. The arsN1 gene, widely distributed in bacterial arsenic resistance (ars) operons, selectively confers resistance to arsinothricin by acetylation of the α-amino group. Crystal structures of ArsN1 N-acetyltransferase, with or without arsinothricin, shed light on the mechanism of its substrate selectivity. These findings have the potential for development of a new class of organoarsenical antimicrobials and ArsN1 inhibitors.

Recording of Neural Activity With Modulation of Photolysis of Caged Compounds Using Microelectrode Arrays in Rats With Seizures.

OBJECTIVE: In this paper, a new method was established to monitor multichannel neural activity with microelectrode arrays (MEAs) under modulation of caged compounds in a rat model of seizures. METHODS: The 16-channel MEAs were fabricated and implanted into the hippocampus of normal rats and epileptic rats for neural spike and local field potential (LFP) recording. Using optical fibers with drug delivery tubing, two different caged compounds [ruthenium-bipyridine-trimethylphosphine glutamate (RuBi-Glu) and ruthenium-bipyridine-trimethylphosphine gamma aminobutyric acid (RuBi-GABA)] were applied, and blue light (465 nm) was used to modulate neural activity. RESULTS: In normal rats, RuBi-Glu excited neural activity, and RuBi-GABA inhibited neural activity. The amplitude of spikes increased 26% from 154 to 194 µV with RuBi-Glu modulation. During RuBi-GABA modulation, spikes recovered to 142 µV. The firing rate increased from 1.4 to 4.5 Hz with RuBi-Glu modulation and decreased to 0.8 Hz after RuBi-GABA modulation. The power of LFPs increased from 566 to 1128 µW with RuBi-Glu modulation and recovered to 710 µW with RuBi-GABA modulation. In epileptic rats, the neural activity during seizures was significantly inhibited by RuBi-GABA modulation. The amplitude of spikes was 242 µV during seizures and decreased to 112 µV with RuBi-GABA modulation. The firing rate decreased from 20.29 to 1.33 Hz with RuBi-GABA modulation. CONCLUSION: Using MEAs, the modulation of neural activity with caged compound photolysis was observed with high temporal-spatial resolution in normal and epileptic rats. SIGNIFICANCE: This new method is important for monitoring neural activity with photo-switchable modulation.

Indole-3-acetylaspartate and indole-3-acetylglutamate, the IAA-amide conjugates in the diploid strawberry achene, are hydrolyzed in growing seedlings.

MAIN CONCLUSION: Indole-3-acetylaspartate and indole-3-acetylglutamate are the stored auxin amino acid conjugates of the achene of the diploid strawberry and serve as sources of auxin during seedling growth. The edible part of the strawberry, a pseudocarp, has long been known to enlarge in response to auxin produced by the developing achenes, the botanical true fruit. Auxin homeostasis involves a complex interaction between biosynthesis, conjugate formation and hydrolysis, catabolism and transport. Strawberry tissues are capable of synthesizing auxin conjugates, and transcriptome data support the expression of genes involved in IAA conjugate formation and hydrolysis throughout embryo development and subsequent seedling growth. Using a highly sensitive and selective mass spectrometric method, we identified all the low molecular weight indole-auxin amino acid conjugates in achenes of F. vesca as consisting of indole-3-acetylaspartate (IAasp) and indole-3-acetylglutamate (IAglu). In contrast to what has been proposed to occur in Arabidopsis, we determined that IAasp and IAglu are hydrolyzed by seedlings to provide a source of free IAA for growth.

A-type K+ channels impede supralinear summation of clustered glutamatergic inputs in layer 3 neocortical pyramidal neurons.

A-type K+ channels restrain the spread of incoming signals in tufted and apical dendrites of pyramidal neurons resulting in strong compartmentalization. However, the exact subunit composition and functional significance of K+ channels expressed in small diameter proximal dendrites remain poorly understood. We focus on A-type K+ channels expressed in basal and oblique dendrites of cortical layer 3 pyramidal neurons, in ex vivo brain slices from young adult mice. Blocking putative Kv4 subunits with phrixotoxin-2 enhances depolarizing potentials elicited by uncaging RuBi-glutamate at single dendritic spines. A concentration of 4-aminopyridine reported to block Kv1 has no effect on such responses. 4-aminopyridine and phrixotoxin-2 increase supralinear summation of glutamatergic potentials evoked by synchronous activation of clustered spines. The effect of 4-aminopyridine on glutamate responses is simulated in a computational model where the dendritic A-type conductance is distributed homogeneously or in a linear density gradient. Thus, putative Kv4-containing channels depress excitatory inputs at single synapses. The additional recruitment of Kv1 subunits might require the synchronous activation of multiple inputs to regulate the gain of signal integration.

Reducing the toxicity of amphotericin B by encapsulation using methoxy poly(ethylene glycol)-b-poly(l-glutamic acid-co-l-phenylalanine).

Amphotericin B (AmB) is an antifungal drug used for serious fungal infections and leishmaniosis. However, its clinical application is limited because of its high toxicity. To resolve this problem, herein we loaded AmB into methoxy poly(ethylene glycol)-b-poly(l-glutamic acid-co-l-phenylalanine) (mPEG-b-P(Glu-co-Phe)) nanoparticles (l-AmB) via electrostatic, hydrophobic and π-π interactions. The l-AmB has excellent stability both in PBS and in plasma and shows a remarkably reduced hemolysis (17.1 ± 1.5%, 6 h) compared to the free AmB (94.2 ± 5.3%, 6 h). The nephrotoxicity of l-AmB is significantly lower than that of free AmB. The maximum tolerance dose (MTD) of l-AmB is 3.0 mg kg-1, which is 3.75 fold that of free AmB (MTD = 0.8 mg kg-1). The antimicrobial activity of the conjugate was retained in vivo, with l-AmB proving to be a more protective treatment for Aspergillus fumigatus infections in mice than AmB alone. These indicate that l-AmB is a formulation of AmB with low side effects.

A site-specific branching poly-glutamate tag mediates intracellular protein delivery by cationic lipids.

Intracellular protein delivery is of significance for cellular protein analysis and therapeutic development, but remains challenging technically. Herein, we report a general and highly potent strategy for intracellular protein delivery based on commercially available cationic lipids. In this strategy, a designed double branching poly-glutamate tag is site-specifically attached onto the C-terminal of protein cargos via expressed protein ligation (EPL), which mediates the entrapment of proteins into cationic liposomes driven by electrostatic interaction. The resultant protein-lipid complexes can enter into cytosol with a high efficiency even at the low protein concentration while maintaining protein's biological activity.

Degradable 3D-Printed Hydrogels Based on Star-Shaped Copolypeptides.

We present a star copolypeptide-based hydrogel ink capable of structural microfabrication using 3D extrusion printing. The material comprises an amphiphilic block copolymer structure of poly(benzyl-l-glutamate)- b-oligo(l-valine), which spontaneously forms hydrogels through hydrophobic interactions. The chemical design allows the bulk phase of the hydrogel to remain intact after application of shear due to its self-recovery behavior. It is demonstrated that the composition of the materials is ideally suited for 3D printing with scaffolds capable of maintaining structural cohesion after extrusion. Post extrusion UV-triggered fixation of the printed structures is carried out, resulting in stable hydrogel constructs. The constructs were found to be degradable, exhibited favorable release of encapsulated molecular cargo, and do not appear to affect the metabolic health of the commonly used fibroblastic cell line Balb/3T3 in the absence of the reactive diluent N, N'-methylenebis(acrylamide). The star copolypeptide inks allow for rapid prototyping enabling the fabrication of defined intricate microstructures, providing a platform for complex scaffold development that would otherwise be unattainable with other processing techniques such as molding or casting.

C,N co-doped porous TiO2 hollow sphere visible light photocatalysts for efficient removal of highly toxic phenolic pollutants.

Herein, C,N co-doped porous TiO2 hollow sphere visible light photocatalysts were fabricated using biocompatible N-lauroyl-l-glutamic acid as a doped precursor and soft-template by a mild and facile self-assembly soft-template method, followed by calcination at 550 °C in air. The structure, morphology, and surface elemental composition were characterized in detail by X-ray diffraction, Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, scanning electron microscopy, and transmission electron microscopy. The results show that the prepared TiO2 photocatalysts have a porous hollow sphere structure and are co-doped with C and N. The visible-light-driven photocatalytic degradation rates of phenol and 2-chlorophenol are ∼92 and 90%, respectively. The photocatalytic reaction rate constants of phenol and dichlorophen on HPT550 porous TiO2 hollow spheres were about ∼4 and ∼2 times higher than those on P25, respectively. This enhancement is because the C,N co-doped porous TiO2 hollow spheres not only extend the photoresponse to the visible light region as C,N co-doping narrows the bandgap (2.7 eV), but also expose a large number of surface active sites that favor visible-light-driven photocatalysis. Moreover, the porous hollow structure favors multiple reflections of photons in the interior, increasing the utilization ratio of light. It is worth to pay more efforts to the development of visible light photocatalysts and further promote their practical application.

Dry-powder formulations of non-covalent protein complexes with linear or miktoarm copolymers for pulmonary delivery.

Pulmonary delivery of protein therapeutics has considerable clinical potential for treating both local and systemic diseases. However, poor protein conformational stability, immunogenicity and protein degradation by proteolytic enzymes in the lung are major challenges to overcome for the development of effective therapeutics. To address these, a family of structurally related copolymers comprising polyethylene glycol, mPEG2k, and poly(glutamic acid) with linear A-B (mPEG2k-lin-GA) and miktoarm A-B3 (mPEG2k-mik-(GA)3) macromolecular architectures was investigated as potential protein stabilisers. These copolymers form non-covalent nanocomplexes with a model protein (lysozyme) which can be formulated into dry powders by spray-drying using common aerosol excipients (mannitol, trehalose and leucine). Powder formulations with excellent aerodynamic properties (fine particle fraction of up to 68%) were obtained with particle size (D50) in the 2.5 µm range, low moisture content (<5%), and high glass transitions temperatures, i.e. formulation attributes all suitable for inhalation application. In aqueous medium, dry powders rapidly disintegrated into the original polymer-protein nanocomplexes which provided protection towards proteolytic degradation. Taken together, the present study shows that dry powders based on (mPEG2k-polyGA)-protein nanocomplexes possess potentials as an inhalation delivery system.