PEGylation versus glycosylation: effect on the thermodynamics and thermostability of crisantaspase.

Thermostability is an important and desired feature of therapeutic proteins and is critical for the success or failure of protein drugs development. It can be increased by PEGylation-binding of poly(ethylene glycol) moieties-or glycosylation-post-translational modification to add glycans. Here, the thermostability and thermodynamic parameters of native, PEGylated, and glycosylated versions of the antileukemic enzyme crisantaspase were investigated. First-order kinetics was found to describe the irreversible deactivation process. Activation energy of the enzyme-catalyzed reaction (E*) was estimated for native, PEGylated, and glycosylated enzyme (10.2, 14.8, and 18.8 kJ mol-1 respectively). Half-life decreased progressively with increasing temperature, and longer half-life was observed for PEG-crisantaspase (87.74 min) at 50 °C compared to the native form (9.79 min). The activation energy of denaturation of PEG-crisantaspase (307.1 kJ mol-1) was higher than for crisantaspase (218.1 kJ mol-1) and Glyco-crisantaspase (120.0 kJ mol-1), which means that more energy is required to overcome the energy barrier of the unfolding process. According to our results, PEG-crisantaspase is more thermostable than its native form, while Glyco-crisantaspase is more thermosensitive.

Doxorubicin nanoformulations on therapy against cancer: An overview from the last 10 years.

Doxorubicin (DOX) is a natural antibiotic with antineoplastic activity. It has been used for over 40 years and remains one of the most used drugs in chemotherapy for a variety of cancers. However, cardiotoxicity limits its use for long periods. To overcome this limitation, encapsulation in smart drug delivery systems (DDS) brings advantages in comparison with free drug administration (i.e., conventional anticancer drug therapy). In this review, we present the most relevant nanostructures used for DOX encapsulation over the last 10 years, such as liposomes, micelles and polymeric vesicles (i.e., polymersomes), micro/nanoemulsions, different types of polymeric nanoparticles and hydrogel nanoparticles, as well as novel approaches for DOX encapsulation. The studies highlighted here show these nanoformulations achieved higher solubility, improved tumor cytotoxicity, prolonged DOX release, as well as reduced side effects, among other interesting advantages.

Recent advances in the design of antimicrobial peptide conjugates.

Antimicrobial peptides (AMPs) are ubiquitous host defense peptides characterized by their antibiotic activity and lower propensity for developing resistance compared to classic antibiotics. While several AMPs have shown activity against antibiotic-sensitive and even multi-drug resistant strains, some bottlenecks to further development and clinical applications are still present, for instance, low antimicrobial activity, instability under physiological conditions, systemic toxicity and the potential for compromising the innate host defense immunity. Conjugation to molecules such as proteins, synthetic polymers, small molecules and nanoparticles are strategies under investigation to boost the therapeutic efficacy of AMPs. This review focuses on the design and application of AMPs' conjugates. In silico tools for creating new AMPs and AMPs' conjugates and their clinical development are also discussed. Furthermore, key future considerations regarding the major achievements and challenges of AMPs' conjugates in the antimicrobial resistance context are presented as a take-home message.

Lysine-PEGylated Cytochrome C with Enhanced Shelf-Life Stability.

Cytochrome c (Cyt-c), a small mitochondrial electron transport heme protein, has been employed in bioelectrochemical and therapeutic applications. However, its potential as both a biosensor and anticancer drug is significantly impaired due to poor long-term and thermal stability. To overcome these drawbacks, we developed a site-specific PEGylation protocol for Cyt-c. The PEG derivative used was a 5 kDa mPEG-NHS, and a site-directed PEGylation at the lysine amino-acids was performed. The effects of the pH of the reaction media, molar ratio (Cyt-c:mPEG-NHS) and reaction time were evaluated. The best conditions were defined as pH 7, 1:25 Cyt-c:mPEG-NHS and 15 min reaction time, resulting in PEGylation yield of 45% for Cyt-c-PEG-4 and 34% for Cyt-c-PEG-8 (PEGylated cytochrome c with 4 and 8 PEG molecules, respectively). Circular dichroism spectra demonstrated that PEGylation did not cause significant changes to the secondary and tertiary structures of the Cyt-c. The long-term stability of native and PEGylated Cyt-c forms was also investigated in terms of peroxidative activity. The results demonstrated that both Cyt-c-PEG-4 and Cyt-c-PEG-8 were more stable, presenting higher half-life than unPEGylated protein. In particular, Cyt-c-PEG-8 presented great potential for biomedical applications, since it retained 30-40% more residual activity than Cyt-c over 60-days of storage, at both studied temperatures of 4 °C and 25 °C.

Building better biobetters: From fundamentals to industrial application.

Biological drugs or biopharmaceuticals off patent open a large market for biosimilars and biobetters, follow-on biologics. Biobetters, in particular, are new drugs designed from existing ones with improved properties such as higher selectivity, stability, half-life and/or lower toxicity/immunogenicity. Glycosylation is one of the most used strategies to improve biological drugs, nonetheless bioconjugation is an additional alternative and refers to the covalent attachment of polymers to biological drugs. Extensive research on novel polymers is underway, nonetheless PEGylation is still the best alternative with the longest clinical track record. Innovative trends based on genetic engineering techniques such as fusion proteins and PASylation are also promising. In this review, all these alternatives wereexplored as well as current market trends, legislation and future perspectives.

Pegylated catalase as a potential alternative to treat vitiligo and UV induced skin damage.

The metabolic function of catalase (CAT) is to prevent oxidative damage to tissues through the hydrolysis of hydrogen peroxide, which is a strong oxidizing agent. It has been suggested as an alternative to treat skin diseases related to oxidative stress, such as vitiligo. Owing to the instability associated to the protein nature, topical use of CAT is challenging and, in this sense, PEGylation can be an interesting alternative. Here, we conjugated CAT to methoxy-poly(ethylene oxide) (mPEG) of 10, 20 and 40 kDa, by means of a nucleophilic attack of ε-amino groups to an electron-deficient carbonyl group of the reactive PEG, resulting in site specifically PEGylated bioconjugates. PEGylation yields ranged from 31% ± 2% for CAT-PEG40 to 59% ± 4% for CAT-PEG20 and were strongly affected by the reaction pH owing to the protonation/deprotonation state of primary amines of lysine and N-terminal residues. PEGylated conjugates were purified by size-exclusion chromatography (purity > 95%) and characterized by circular dichroism. Irrespectively of MW, PEG did not affected CAT secondary and tertiary structure, but a decrease in specific activity was observed, more pronounced when PEGs of higher MWs were used. However, this loss of activity is compensated by the increased long-term stability, with a gain of >5 times in t1/2. In vitro antioxidant activity of CAT-PEG20 showed complete elimination of lipid peroxidation at the skin upper layer (stratum corneum) suitable for a topical use to treat vitiligo, as well as other skin conditions related to oxidative stress.

Influence of lysosomal protease sensitivity in the immunogenicity of the antitumor biopharmaceutical asparaginase.

L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.

l-Asparaginase Encapsulation into Asymmetric Permeable Polymersomes.

This work reports, for the encapsulation of l-asparaginase, an anticancer enzyme into hybrid PMPC25-PDPA70/PEO16-PBO22 asymmetric polymersomes previously developed by our group, with loading capacities with over 800 molecules per vesicle. Enzyme-loaded polymersomes show permeability and capacity to hydrolyze l-asparagine, which is essential to cancer cells. The nanoreactors proposed in this work can be potentially used in further studies to develop novel therapeutic alternatives based on l-asparaginase.

Development and characterization of miltefosine-loaded polymeric micelles for cancer treatment.

Miltefosine presents antineoplastic activity but high hemolytic potential. Its use in cancer has been limited to treating cutaneous metastasis of breast cancer. To decrease hemolytic potential, we developed a formulation of miltefosine-loaded polymeric micelles (PM) of the copolymer Pluronic-F127. A central composite design was applied and the analysis of variance showed that the optimum level of hydrodynamic diameter and polydispersity index predicted by the model and experimentally confirmed were 29nm and 0.105, respectively. Thermal analyses confirmed that miltefosine was molecularly dispersed within PM. Pluronic-F127 PM with miltefosine 80µM presented a significant reduction of hemolytic effect (80%, p<0.05) in comparison to free drug. In vitro assays against HeLa carcinoma cells demonstrated similar cytotoxicity to free miltefosine and PM. Our results suggest that, by lowering hemolytic potential, miltefosine-loaded Pluronic-F127 PM a promising alternative to broaden this drug use in cancer therapy, as well as of other alkylphosphocholines.

Molecular Determinants for the Binding Mode of Alkylphosphocholines in the C2 Domain of PKCα.

Alkylphosphocholines (APCs) and alkyl-lysophosphocholines (ALPs) are antineoplastic agents that interfere with cellular membranes and signaling proteins. Protein kinase Cα (PKCα) is a signaling protein composed by catalytic (C3, C4) and regulatory domains (C1, C2). The C2 needs calcium (Ca(2+) ) and phosphatidylserine (PS) for activation. Miltefosine inhibits PKCα competitively with regard to PS and non-competitively with regard to Ca(2+) , however, the mechanism of action is unknown. We employed molecular docking, molecular dynamics and chemometric methods to verify how 7 APCs and ALPs derivatives and PS interact with the C2 domain. All ligands except PS were grouped in 2 clusters according to their interactions inside the enzyme. The findings showed that PS's phosphoryl oxygens interact with Ca(2+) , the serine moiety interacts with Asn189, and the carbonyl oxygen of the alkylic chain interacts with Arg249 and Thr251. On the other hand, ligands' phosphoryl oxygens interact with Asn189, Arg249, Thr250, and one water molecule instead of Ca(2+) . Because of the different binding mode, we hypothesize that the ligands cause conformational changes in the calcium binding region. Moreover, the packing mismatch between bilayer-forming lipids and ALP/APC chain impedes the C2 domain from docking to the internal leaflet of cellular membranes, interrupting PKCα activation.

Alkylphosphocholines as Promising Antitumor Agents: Exploring the Role of Structural Features on the Hemolytic Potential.

Alkylphosphocholines (APC) are promising antitumor agents, which have the cellular membrane as primary target; however, red blood cell damage limits their wide therapeutic use. A variety of APC analogs has been synthesized and tested showing less hemolytic effect than the class prototype, Miltefosine (HePC). In this work, chemometric methods were applied to a set of 34 APC derivatives to identify the most relevant structural and molecular features of hemolytic activity. The APC derivatives were divided into three groups: (i) N-methylpiperidine and N-methylmorpholine derivatives with a long alkyl chain or flexible cyclopentadecyl rings, displaying a hemolytic rate of 17 %; (ii) adamantyl and cyclopentadecyl derivatives, showing an average hemolysis of 39 %; and, N,N,N-trimethylammonium, trans-N,N,N-trimethylcyclohexanamine, and trans-N,N,N-trimethylcyclopentanamine derivatives, whose average hemolysis was 41 %. The findings suggested that the presence of either bulky cationic head groups, or rings such as adamantyl and cyclohexyl, primarily increases the hemolysis of compounds with eleven atoms in the alkyl chain. Moreover, the macrocyclic cyclopentadecyl seems to be important to the hemolytic potential especially of compounds with five carbon atoms in the alkyl chain. Regarding linear carbon chain derivatives with no ring substitution, less bulky cationic head groups seem to favor hemolysis. Thus, in order to design more potent and less toxic APC antitumors, the reported structural/molecular patterns should not be included in their structure.

LPS removal from an E. coli fermentation broth using aqueous two-phase micellar system.

In biotechnology, endotoxin (LPS) removal from recombinant proteins is a critical and challenging step in the preparation of injectable therapeutics, as endotoxin is a natural component of bacterial expression systems widely used to manufacture therapeutic proteins. The viability of large-scale industrial production of recombinant biomolecules of pharmaceutical interest significantly depends on the separation and purification techniques used. The aim of this work was to evaluate the use of aqueous two-phase micellar system (ATPMS) for endotoxin removal from preparations containing recombinant proteins of pharmaceutical interest, such as green fluorescent protein (GFPuv). Partition assays were carried out initially using pure LPS, and afterwards in the presence of E. coli cell lysate. The ATPMS technology proved to be effective in GFPuv recovery, preferentially into the micelle-poor phase (K(GFPuv) < 1.00), and LPS removal into the micelle-rich phase (%REM(LPS) > 98.00%). Therefore, this system can be exploited as the first step for purification in biotechnology processes for removal of higher LPS concentrations.

Novel potential drug against T. cruzi and its interaction with surfactant micelles.

The interaction of 5-nitro-2-furfurilylidene benzhydrazide (5NFB), potential anti-trypanosomal compound, with micellar solutions was studied. The results indicated that 50 mug of 5NFB completely kills 20 million T. cruzi epimastigote cells within 3 days, whereas the same amount of benznidazole kills 30% of the cells after 4 days. 5NFB solubility in surfactants solutions (SDS, DTAB, C12EO8) increased linearly with surfactant concentration. According to small angle X-ray scattering (SAXS), 5NFB does not affect micellar structural features. A comparison between C12EO8 effects on T. cruzi epimastigote cells and on erythrocytes showed that surfactant lytic effect is stronger in parasite cells, enlightening the potential of 5NFB micellar formulations.

Experimental and theoretical investigation of the micellar-assisted solubilization of ibuprofen in aqueous media.

Surfactants can be used to increase the solubility of poorly soluble drugs in water and to increase drug bioavailability. In this article, the aqueous solubilization of the nonsteroidal, antiinflammatory drug ibuprofen is studied experimentally and theoretically in micellar solutions of anionic (sodium dodecyl sulfate, SDS), cationic (dodecyltrimethylammonium bromide, DTAB), and nonionic (dodecyl octa(ethylene oxide), C12E8) surfactants possessing the same hydrocarbon "tail" length but differing in their hydrophilic headgroups. We find that, for these three surfactants, the aqueous solubility of ibuprofen increases linearly with increasing surfactant concentration. In particular, we observed a 16-fold increase in the solubility of ibuprofen relative to that in the aqueous buffer upon the addition of 80 mM DTAB and 80 mM C12E8 but only a 5.5-fold solubility increase upon the addition of 80 mM SDS. The highest value of the molar solubilization capacity (chi) was obtained for DTAB (chi = 0.97), followed by C12E8 (chi = 0.72) and finally by SDS (chi = 0.23). A recently developed computer simulation/molecular-thermodynamic modeling approach was extended to predict theoretically the solubilization behavior of the three ibuprofen/surfactant mixtures considered. In this modeling approach, molecular-dynamics (MD) simulations were used to identify which portions of ibuprofen are exposed to water (hydrated) in a micellar environment by simulating a single ibuprofen molecule at an oil/water interface (modeling the micelle core/water interface). On the basis of this input, molecular-thermodynamic modeling was then implemented to predict (i) the micellar composition as a function of surfactant concentration, (ii) the aqueous solubility of ibuprofen as a function of surfactant concentration, and (iii) the molar solubilization capacity (chi). Our theoretical results on the solubility of ibuprofen in aqueous SDS and C12E8 surfactant solutions are in good agreement with the experimental data. The ibuprofen solubility in aqueous DTAB solutions was somewhat overpredicted because of challenges associated with accurately modeling the strong electrostatic interactions between the anionic ibuprofen and the cationic DTAB. Our results indicate that computer simulations of ibuprofen at a flat oil/water interface can be used to obtain accurate information about the hydrated and the unhydrated portions of ibuprofen in a micellar environment. This information can then be used as input to a molecular-thermodynamic model of self-assembly to successfully predict the aqueous solubilization behavior of ibuprofen in the three surfactant systems studied.

Micellar solubilization of ibuprofen - influence of surfactant head groups on the extent of solubilization

An important property of micells with particular significance in pharmacy is their ability to increase the solubility of poorly soluble drugs in water, thus increasing their bioavailability. In this work, the solubilization of ibuprofen (IBU) was studied in micellar solutions of there surfactants possessing the same hydrocarbon tail but different hydrophilic head groups, namely sodium dodecyl sulphate (SDS), dodecyltrimethylammonium bromide (DTAB), and n-dodecyl octa(ethylene oxide) (`C IND. 12´ E`O IND. 8´). The results showed that, irrespective of the surfactant type, the solubility of IBU increased linearly with increasing surfactant concentration, as a consequence of the association between the drug and the micelles...

Glucose-6-phosphate dehydrogenase partitioning in two-phase aqueous mixed (nonionic/cationic) micellar systems.

The enzyme glucose-6-phosphate dehydrogenase (G6PD) plays an important role in maintaining the level of NADPH and in producing pentose phosphates for nucleotide biosynthesis. It is also of great value as an analytical reagent, being used in various quantitative assays. In searching for new strategies to purify this enzyme, the partitioning of G6PD in two-phase aqueous mixed (nonionic/cationic) micellar systems was investigated both experimentally and theoretically. Our results indicate that the use of a two-phase aqueous mixed micellar system composed of the nonionic surfactant C(10)E(4) (n-decyl tetra(ethylene oxide)) and the cationic surfactant C(n)TAB (alkyltrimethylammonium bromide, n = 8, 10, or 12) can improve significantly the partitioning behavior of G6PD relative to that obtained in the two-phase aqueous C(10)E(4) micellar system. This improvement can be attributed to electrostatic attractions between the positively charged mixed (nonionic/cationic) micelles and the net negatively charged enzyme G6PD, resulting in the preferential partitioning of G6PD to the top, mixed micelle-rich phase of the two-phase aqueous mixed micellar systems. The effect of varying the cationic surfactant tail length (n = 8, 10, and 12) on the denaturation and partitioning behavior of G6PD in the C(10)E(4) /C(n)TAB/buffer system was investigated. It was found that C(8)TAB is the least denaturing to G6PD, followed by C(10)TAB and C(12)TAB. However, the C(10)E(4)/C(12)TAB/buffer system generated stronger electrostatic attractions with the net negatively charged enzyme G6PD than the C(10)E(4)/C(10)TAB/buffer and the C(10)E(4)/C(8)TAB/buffer systems, when using the same amount of cationic surfactant. Overall, the two-phase aqueous mixed (C(10)E(4)/C(10)TAB) micellar system yielded the highest G6PD partition coefficient of 7.7, with a G6PD yield in the top phase of 71%, providing the optimal balance between the denaturing effect and the electrostatic attractions for the three cationic surfactants examined. A recently developed theoretical framework to predict protein partition coefficients in two-phase aqueous mixed (nonionic/ionic) micellar systems was implemented, and the theoretically predicted G6PD partition coefficients were found to be in reasonable quantitative agreement with the experimentally measured ones.