Nonclinical Evaluation of Single-Mutant E. coli Asparaginases Obtained by Double-Mutant Deconvolution: Improving Toxicological, Immune and Inflammatory Responses.

Acute lymphoblastic leukaemia is currently treated with bacterial L-asparaginase; however, its side effects raise the need for the development of improved and efficient novel enzymes. Previously, we obtained low anti-asparaginase antibody production and high serum enzyme half-life in mice treated with the P40S/S206C mutant; however, its specific activity was significantly reduced. Thus, our aim was to test single mutants, S206C and P40S, through in vitro and in vivo assays. Our results showed that the drop in specific activity was caused by P40S substitution. In addition, our single mutants were highly stable in biological environment simulation, unlike the double-mutant P40S/S206C. The in vitro cell viability assay demonstrated that mutant enzymes have a higher cytotoxic effect than WT on T-cell-derived ALL and on some solid cancer cell lines. The in vivo assays were performed in mice to identify toxicological effects, to evoke immunological responses and to study the enzymes' pharmacokinetics. From these tests, none of the enzymes was toxic; however, S206C elicited lower physiological changes and immune/allergenic responses. In relation to the pharmacokinetic profile, S206C exhibited twofold higher activity than WT and P40S two hours after injection. In conclusion, we present bioengineered E. coli asparaginases with high specific enzyme activity and fewer side effects.

Recombinant production of a highly efficient photolyase from Thermus thermophilus.

Ultraviolet (UV) radiation from sunlight can damage DNA, inducing mutagenesis and eventually leading to skin cancer. Topical sunscreens are used to avoid the effect of UV irradiation, but the topical application of DNA repair enzymes, such as photolyase, can provide active photoprotection by DNA recovery. Here we produced a recombinant Thermus thermophilus photolyase expressed in Escherichia coli, evaluated the kinetic parameters of bacterial growth and the kinetics and stability of the enzyme. The maximum biomass (𝑋𝑚𝑎𝑥 ) of 2.0 g L-1 was reached after 5 h of cultivation, corresponding to 𝑃X  = 0.4 g L-1 h. The µð‘šð‘Žð‘¥ corresponded to 1.0 h-1 . Photolyase was purified by affinity chromatography and high amounts of pure enzyme were obtained (3.25 mg L-1 of cultivation). Two different methods demonstrated the enzyme activity on DNA samples and very low enzyme concentrations, such as 15 µg mL-1 , already resulted in 90% of CPD photodamage removal. We also determined photolyase kM of 9.5 nM, confirming the potential of the enzyme at very low concentrations, and demonstrated conservation of enzyme activity after freezing (-20°C) and lyophilization. Therefore, we demonstrate T. thermophilus photolyase capacity of CPD damage repair and its potential as an active ingredient to be incorporated in dermatological products.

Enzymes for dermatological use.

Skin is the ultimate barrier between body and environment and prevents water loss and penetration of pathogens and toxins. Internal and external stressors, such as ultraviolet radiation (UVR), can damage skin integrity and lead to disorders. Therefore, skin health and skin ageing are important concerns and increased research from cosmetic and pharmaceutical sectors aims to improve skin conditions and provide new anti-ageing treatments. Biomolecules, compared to low molecular weight drugs and cosmetic ingredients, can offer high levels of specificity. Topically applied enzymes have been investigated to treat the adverse effects of sunlight, pollution and other external agents. Enzymes, with a diverse range of targets, present potential for dermatological use such as antioxidant enzymes, proteases and repairing enzymes. In this review, we discuss enzymes for dermatological applications and the challenges associated in this growing field.

Challenges in estimating the encapsulation efficiency of proteins in polymersomes - Which is the best method?

Abstract Polymersomes are nanometric vesicles that can encapsulate large and hydrophilic biomolecules, such as proteins, in the aqueous core. Data in literature show large variation in encapsulation efficiency (%EE) values depending on the method used for calculation. We investigated different approaches (direct and indirect) to quantify the %EE of different proteins (catalase, bovine serum albumin-BSA, L-asparaginase and lysozyme) in Pluronic L-121 polymersomes. Direct methods allow quantification of the actual payload of the polymersomes and indirect methods are based on the quantification of the remaining non-encapsulated protein. The protein-loaded polymersomes produced presented approximately 152 nm of diameter (PDI ~ 0.4). Higher %EE values were obtained with the indirect method (up to 25%), attributed to partial entanglement of free protein in the polymersomes poly(Ethylene Glycol) corona. For the direct methods, vesicles were disrupted with chloroform or proteins precipitated with solvents. Reasonable agreement was found between the two protocols, with values up to 8%, 6%, 17.6% and 0.9% for catalase, BSA, L-asparaginase and lysozyme, respectively. We believe direct determination is the best alternative to quantify the %EE and the combination of both protocols would make results more reliable. Finally, no clear correlation was observed between protein size and encapsulation efficiency.

Peg-Grafted Liposomes for L-Asparaginase Encapsulation.

L-asparaginase (ASNase) is an important biological drug used to treat Acute Lymphoblastic Leukemia (ALL). It catalyzes the hydrolysis of L-asparagine (Asn) in the bloodstream and, since ALL cells cannot synthesize Asn, protein synthesis is impaired leading to apoptosis. Despite its therapeutic importance, ASNase treatment is associated to side effects, mainly hypersensitivity and immunogenicity. Furthermore, degradation by plasma proteases and immunogenicity shortens the enzyme half-life. Encapsulation of ASNase in liposomes, nanostructures formed by the self-aggregation of phospholipids, is an attractive alternative to protect the enzyme from plasma proteases and enhance pharmacokinetics profile. In addition, PEGylation might prolong the in vivo circulation of liposomes owing to the spherical shielding conferred by the polyethylene (PEG) corona around the nanostructures. In this paper, ASNase was encapsulated in liposomal formulations composed by 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) containing or not different concentrations of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N [methoxy (polyethylene glycol)-2000] (DSPE-PEG). Nanostructures of approximately 142-202 nm of diameter and polydispersity index (PDI) of 0.069 to 0.190 were obtained and the vesicular shape confirmed by Transmission Electron Microscopy (TEM and cryo-TEM). The encapsulation efficiency (%EE) varied from 10% to 16%. All formulations presented activity in contact with ASNase substrate, indicating the liposomes permeability to Asn and/or enzyme adsorption at the nanostructures' surface; the highest activity was observed for DMPC/DSPE-PEG 10%. Finally, we investigated the activity against the Molt 4 leukemic cell line and found a lower IC50 for the DMPC/DSPE-PEG 10% formulation in comparison to the free enzyme, indicating our system could provide in vivo activity while protecting the enzyme from immune system recognition and proteases degradation.

Correction: Novel site-specific PEGylated L-asparaginase.

[This corrects the article DOI: 10.1371/journal.pone.0211951.].

Polymeric micelles of pluronic F127 reduce hemolytic potential of amphiphilic drugs.

One of the main toxicities associated to intravenous administration of amphiphilic drugs is pronounced hemolytic activity. To overcome this limitation, we investigated the anti-hemolytic properties of polymeric micelles of Pluronics, triblock copolymers of poly(ethylene oxide) and poly(propylene oxide). We studied the encapsulation of the amphiphilic compound miltefosine (HePC) into polymeric micelles of Pluronics F108, F68, F127, L44, and L64. In vitro hemolysis indicated that, among the five copolymers studied, only F127 completely inhibited hemolytic effect of HePC at 50 µg/mL, this effect was also observed for other two amphiphilic molecules (cetyltrimethylammonium bromide and cethylpyridinium chloride). To better understand this interaction, we analyzed the HC50 (concentration causing 50% of hemolysis) for HePC free and loaded into F127 micelles. Copolymer concentration influenced the hemolytic profile of encapsulated HePC; for F127 the HC50 increased relative to free HePC (40 µg/mL) up to 184, 441, 736 and 964 µg/mL, for 1, 3, 6 and 9% F127, respectively. Interestingly, a linear relationship was found between HC50-HePC and F127 concentration. At 3% of F127, it is possible to load up to 300 µg/mL of HePC with no hemolytic effect. By achieving this level of hemolysis protection, a promising application is on the view, bringing the parenteral use of HePC and other amphiphilic drugs. Additionally, small-angle X-ray scattering (SAXS) was used to asses structural information on the interactions between HePC and F127 micelles.

Poly (lactic-co-glycolic acid) nanospheres allow for high l-asparaginase encapsulation yield and activity.

l-Asparaginase (ASNase) is an amidohydrolase used as a chemotherapeutic agent for the treatment of acute lymphoblastic leukemia (ALL). The nanoencapsulation of this enzyme is strategic to avoid its immediate immunogenic effects that lead to a decrease in the enzyme half-life. In this work, ASNase-containing nanoparticles (NPs) were prepared by double emulsification, through an ultrasonic sonicator or an Ultra-Turrax, using two copolymers of 50:50 (w/w) poly (lactic-co-glycolic acid) (PLGA) with different ranges of molecular weight (24-38 kDa and 30-60 kDa) and varying the concentration of polyvinyl alcohol (PVA) as a stabilizer (0.5, 1.0, 1.5 and 2.0%) as well as the emulsification time (30 and 60 s). Using 24-38 kDa PLGA and 1.0% PVA, we obtained by cavitation NPs with hydrodynamic diameter of 384 nm, polydispersity index of 0.143 and Zeta potential of -16.4 mV, whose ASNase encapsulation efficiency was as high as 87 ±â€¯2%. The encapsulated enzyme showed an activity 22% higher than that of the free enzyme, and no conformational changes were detected by circular dichroism. The enzyme release from NPs entrapped in dialysis bags (500 kDa molecular weight cut-off) allowed selecting a controlled system able to release about 60% of the enzyme within 14 days, for which the Korsmeyer-Peppas model provided the best correlation (R2 = 0.966).

Biased Agonist TRV027 Determinants in AT1R by Molecular Dynamics Simulations.

Functional selectivity is a phenomenon observed in G protein-coupled receptors in which intermediate active-state conformations are stabilized by mutations or ligand binding, resulting in different sets of signaling pathways. Peptides capable of selectively activating ß-arrestin, known as biased agonists, have already been characterized in vivo and could correspond to a new therapeutic approach for treatment of cardiovascular diseases. Despite the potential of biased agonism, the mechanism involved in selective signaling remains unclear. In this work, molecular dynamics simulations were employed to compare the conformational profile of the angiotensin II type 1 receptor (AT1R) crystal bound to angiotensin II, bound to the biased ligand TRV027, and in the apo form. Our results show that both ligands induce changes near the NPxxY motif in transmembrane domain 7 that are related to receptor activation. However, the biased ligand does not cause the rotamer toggle alternative positioning and displays an exclusive hydrogen-bonding pattern. Our work sheds light on the biased agonism mechanism and will help in the future design of novel biased agonists for AT1R.

From Synthesis to Characterization of Site-Selective PEGylated Proteins.

Covalent attachment of therapeutic proteins to polyethylene glycol (PEG) is widely used for the improvement of its pharmacokinetic and pharmacological properties, as well as the reduction in reactogenicity and related side effects. This technique named PEGylation has been successfully employed in several approved drugs to treat various diseases, even cancer. Some methods have been developed to obtain PEGylated proteins, both in multiple protein sites or in a selected amino acid residue. This review focuses mainly on traditional and novel examples of chemical and enzymatic methods for site-selective PEGylation, emphasizing in N-terminal PEGylation, that make it possible to obtain products with a high degree of homogeneity and preserve bioactivity. In addition, the main assay methods that can be applied for the characterization of PEGylated molecules in complex biological samples are also summarized in this paper.

Challenges for the Self-Assembly of Poly(Ethylene Glycol)⁻Poly(Lactic Acid) (PEG-PLA) into Polymersomes: Beyond the Theoretical Paradigms.

Polymersomes (PL), vesicles formed by self-assembly of amphiphilic block copolymers, have been described as promising nanosystems for drug delivery, especially of biomolecules. The film hydration method (FH) is widely used for PL preparation, however, it often requires long hydration times and commonly results in broad size distribution. In this work, we describe the challenges of the self-assembly of poly (ethylene glycol)-poly(lactic acid) (PEG-PLA) into PL by FH exploring different hydrophilic volume fraction (f) values of this copolymer, stirring times, temperatures and post-FH steps in an attempt to reduce broad size distribution of the nanostructures. We demonstrate that, alongside f value, the methods employed for hydration and post-film steps influence the PEG-PLA self-assembly into PL. With initial FH, we found high PDI values (>0.4). However, post-hydration centrifugation significantly reduced PDI to 0.280. Moreover, extrusion at higher concentrations resulted in further improvement of the monodispersity of the samples and narrow size distribution. For PL prepared at concentration of 0.1% (m/v), extrusion resulted in the narrower size distributions corresponding to PDI values of 0.345, 0.144 and 0.081 for PEG45-PLA69, PEG114-PLA153 and PEG114-PLA180, respectively. Additionally, we demonstrated that copolymers with smaller f resulted in larger PL and, therefore, higher encapsulation efficiency (EE%) for proteins, since larger vesicles enclose larger aqueous volumes.

Development of L-Asparaginase Biobetters: Current Research Status and Review of the Desirable Quality Profiles.

L-Asparaginase (ASNase) is a vital component of the first line treatment of acute lymphoblastic leukemia (ALL), an aggressive type of blood cancer expected to afflict over 53,000 people worldwide by 2020. More recently, ASNase has also been shown to have potential for preventing metastasis from solid tumors. The ASNase treatment is, however, characterized by a plethora of potential side effects, ranging from immune reactions to severe toxicity. Consequently, in accordance with Quality-by-Design (QbD) principles, ingenious new products tailored to minimize adverse reactions while increasing patient survival have been devised. In the following pages, the reader is invited for a brief discussion on the most recent developments in this field. Firstly, the review presents an outline of the recent improvements on the manufacturing and formulation processes, which can severely influence important aspects of the product quality profile, such as contamination, aggregation and enzymatic activity. Following, the most recent advances in protein engineering applied to the development of biobetter ASNases (i.e., with reduced glutaminase activity, proteolysis resistant and less immunogenic) using techniques such as site-directed mutagenesis, molecular dynamics, PEGylation, PASylation and bioconjugation are discussed. Afterwards, the attention is shifted toward nanomedicine including technologies such as encapsulation and immobilization, which aim at improving ASNase pharmacokinetics. Besides discussing the results of the most innovative and representative academic research, the review provides an overview of the products already available on the market or in the latest stages of development. With this, the review is intended to provide a solid background for the current product development and underpin the discussions on the target quality profile of future ASNase-based pharmaceuticals.

Therapeutic l-asparaginase: upstream, downstream and beyond.

l-asparaginase (l-asparagine amino hydrolase, E.C.3.5.1.1) is an enzyme clinically accepted as an antitumor agent to treat acute lymphoblastic leukemia and lymphosarcoma. It catalyzes l-asparagine (Asn) hydrolysis to l-aspartate and ammonia, and Asn effective depletion results in cytotoxicity to leukemic cells. Microbial l-asparaginase (ASNase) production has attracted considerable attention owing to its cost effectiveness and eco-friendliness. The focus of this review is to provide a thorough review on microbial ASNase production, with special emphasis to microbial producers, conditions of enzyme production, protein engineering, downstream processes, biochemical characteristics, enzyme stability, bioavailability, toxicity and allergy potential. Some issues are also highlighted that will have to be addressed to achieve better therapeutic results and less side effects of ASNase use in cancer treatment: (a) search for new sources of this enzyme to increase its availability as a drug; (b) production of new ASNases with improved pharmacodynamics, pharmacokinetics and toxicological profiles, and (c) improvement of ASNase production by recombinant microorganisms. In this regard, rational protein engineering, directed mutagenesis, metabolic flux analysis and optimization of purification protocols are expected to play a paramount role in the near future.

Biopharmaceuticals from microorganisms: from production to purification

ABSTRACT The use of biopharmaceuticals dates from the 19th century and within 5-10 years, up to 50% of all drugs in development will be biopharmaceuticals. In the 1980s, the biopharmaceutical industry experienced a significant growth in the production and approval of recombinant proteins such as interferons (IFN α, β, and γ) and growth hormones. The production of biopharmaceuticals, known as bioprocess, involves a wide range of techniques. In this review, we discuss the technology involved in the bioprocess and describe the available strategies and main advances in microbial fermentation and purification process to obtain biopharmaceuticals.

Biopharmaceuticals from microorganisms: from production to purification.

The use of biopharmaceuticals dates from the 19th century and within 5-10 years, up to 50% of all drugs in development will be biopharmaceuticals. In the 1980s, the biopharmaceutical industry experienced a significant growth in the production and approval of recombinant proteins such as interferons (IFN α, ß, and γ) and growth hormones. The production of biopharmaceuticals, known as bioprocess, involves a wide range of techniques. In this review, we discuss the technology involved in the bioprocess and describe the available strategies and main advances in microbial fermentation and purification process to obtain biopharmaceuticals.

Nanostructures for protein drug delivery.

Use of nanoscale devices as carriers for drugs and imaging agents has been extensively investigated and successful examples can already be found in therapy. In parallel, recombinant DNA technology together with molecular biology has opened up numerous possibilities for the large-scale production of many proteins of pharmaceutical interest, reflecting in the exponentially growing number of drugs of biotechnological origin. When we consider protein drugs, however, there are specific criteria to take into account to select adequate nanostructured systems as drug carriers. In this review, we highlight the main features, advantages, drawbacks and recent developments of nanostructures for protein encapsulation, such as nanoemulsions, liposomes, polymersomes, single-protein nanocapsules and hydrogel nanoparticles. We also discuss the importance of nanoparticle stabilization, as well as future opportunities and challenges in nanostructures for protein drug delivery.

Biopharmaceuticals from microorganisms: from production to purification

ABSTRACT The use of biopharmaceuticals dates from the 19th century and within 5-10 years, up to 50% of all drugs in development will be biopharmaceuticals. In the 1980s, the biopharmaceutical industry experienced a significant growth in the production and approval of recombinant proteins such as interferons (IFN , , and ) and growth hormones. The production of biopharmaceuticals, known as bioprocess, involves a wide range of techniques. In this review, we discuss the technology involved in the bioprocess and describe the available strategies and main advances in microbial fermentation and purification process to obtain biopharmaceuticals.

Poly(lactic-co-glycolic acid) matrix incorporated with nisin as a novel antimicrobial biomaterial.

The use of poly(lactic-co-glycolic acid) (PLGA) matrix as a biomolecule carrier has been receiving great attention due to its potential therapeutic application. In this context, we investigated the PLGA matrix capacity to incorporate nisin, an antimicrobial peptide capable of inhibiting the growth of Gram-positive bacteria and bacterial spores germination. Nisin-incorporated PLGA matrices were evaluated based on the inhibitory effect against the nisin-bioindicator Lactobacillus sakei. Additionally, the PLGA-nisin matrix stability over an 8-months period was investigated, as well as the nisin release profile. For the incorporation conditions, we observed that a 5 h incorporation time, at 30 °C, with 250 µg/mL nisin solution in PBS buffer pH 4.5, resulted in the highest inhibitory activity of 2.70 logAU/mL. The PLGA-nisin matrix was found to be relatively stable and showed sustained drug delivery, with continuous release of nisin for 2 weeks. Therefore, PLGA-nisin matrix is could be used as a novel antimicrobial delivery system and an alternative to antibiotics incorporated into PLGA matrices.

LPS-protein aggregation influences protein partitioning in aqueous two-phase micellar systems.

Lipopolysaccharide endotoxins (LPS) are the most common pyrogenic substances in recombinant peptides and proteins purified from Gram-negative bacteria, such as Escherichia coli. In this respect, aqueous two-phase micellar systems (ATPMS) have already proven to be a good strategy to purify recombinant proteins of pharmaceutical interest and remove high LPS concentrations. In this paper, we review our recent experimental work in protein partitioning in Triton X-114 ATPMS altogether with some new results and show that LPS-protein aggregation can influence both protein and LPS partitioning. Green fluorescent protein (GFPuv) was employed as a model protein. The ATPMS technology proved to be effective for high loads of LPS removal into the micelle-rich phase (%REM(LPS) > 98 %) while GFPuv partitioned preferentially to the micelle-poor phase (K GFP(uv) < 1.00) due to the excluded-volume interactions. However, theoretically predicted protein partition coefficient values were compared with experimentally obtained ones, and good agreement was found only in the absence of LPS. Dynamic light scattering measurements showed that protein-LPS interactions were taking place and influenced the partitioning process. We believe that this phenomenon should be considered in LPS removal employing any kind of aqueous two-phase system. Nonetheless, ATPMS can still be considered as an efficient strategy for high loads of LPS removal, but being aware that the excluded-volume partitioning theory available might overestimate partition coefficient values due to the presence of protein-LPS aggregation.

Polymeric micelles and molecular modeling applied to the development of radiopharmaceuticals

Micelles composed of amphiphilic copolymers linked to a radioactive element are used in nuclear medicine predominantly as a diagnostic application. A relevant advantage of polymeric micelles in aqueous solution is their resulting particle size, which can vary from 10 to 100 nm in diameter. In this review, polymeric micelles labeled with radioisotopes including technetium (99mTc) and indium (111In), and their clinical applications for several diagnostic techniques, such as single photon emission computed tomography (SPECT), gamma-scintigraphy, and nuclear magnetic resonance (NMR), were discussed. Also, micelle use primarily for the diagnosis of lymphatic ducts and sentinel lymph nodes received special attention. Notably, the employment of these diagnostic techniques can be considered a significant tool for functionally exploring body systems as well as investigating molecular pathways involved in the disease process. The use of molecular modeling methodologies and computer-aided drug design strategies can also yield valuable information for the rational design and development of novel radiopharmaceuticals.

Micelas poliméricas compostas de copolímeros ligadas a um elemento radioativo são utilizadas em Medicina Nuclear com aplicação predominantemente diagnóstica. A vantagem relevante da utilização de micelas poliméricas em solução aquosa é o tamanho de suas partículas, as quais podem variar de 10 a 100 nm de diâmetro. Neste trabalho de revisão são apresentadas micelas poliméricas marcadas com radioisotopos, como tecnécio-99m (99mTc) e índio-111 (111In), assim como suas aplicações clínicas em técnicas de diagnóstico como Tomografia por emissão de Fóton Único (Single photon Emission Computed Tomography - SPECT), cintilografia, e Ressonância Magnética Nuclear (RMN). Neste contexto, sua aplicação em diagnóstico de sistema linfático e linfonodo sentinela recebe atenção especial. O emprego de técnicas de diagnóstico pode ser considerado uma ferramenta importante para a exploração de sistemas no organismo humano assim como para a investigação de caminhos moleculares envolvidos nos processos de diversas doenças. O uso de metodologias de modelagem molecular e estratégias de desenvolvimento de fármacos assistidas computacionalmente também pode fornecer informações valiosas para o planejamento e o desenvolvimento racional de novos radiofármacos.