A crisantaspase é uma enzima do tipo asparaginase (ASNase) produzida pela bactéria Erwinia chrysanthemi e utilizada como biofármaco no tratamento da leucemia linfoblástica aguda (LLA) em casos de hipersensibilidade à ASNase de E. coli. As principais desvantagens deste biofármaco são a curta meia-vida (10 horas) e imunogenicidade. Nesse sentido, sua forma peguilada (PEG-crisantaspase) não só reduziria o efeito imunogênico, como também melhoraria a meia-vida plasmática. Atualmente, somente a ASNase de E. coli está disponível comercialmente na forma peguilada e essa, por ter sido uma das primeiras proteínas a serem peguiladas, é resultado de um processo de peguilação aleatória em resíduos de lisina. Portanto, apresenta alto grau de polidispersão em relação à quantidade de cadeias de PEG ligadas à enzima. Nesse trabalho desenvolvemos um processo de obtenção de crisantaspase peguilada de maneira sítio-específica, no grupamento N-terminal (PEG-crisantaspase). A crisantaspase foi obtida de forma recombinante na cepa E. coli BL21, cultivada em agitador metabólico e biorreator, em meio Luria Bertani. A produtividade volumétrica no biorreator aumentou 37% em comparação com o agitador metabólico (460 e 335 U·L-1·h-1 respectivamente). A crisantaspase foi recuperada por choque osmótico e purificada por cromatografia de troca catiônica (coluna HiTrap SP FF, 5 mL, eluição em pH 7,5), apresentando atividade específica de 694 U·mg-1, fator de purificação de 31 e rendimento de 69%. A crisantaspase purificada foi peguilada com mPEG-NHS 10 kDa, em tampão fosfato 100 mM, 22 °C, razão molar enzima:PEG 1:50 durante 30 min e sob diferentes valores de pH (6,5-9,0). O melhor rendimento de peguilação N-terminal (50%) foi em pH 7,5 com menor formação de estruturas poli-peguiladas (7%). A PEG-crisantaspase foi isolada por cromatografia de exclusão molecular, retendo 50% da atividade específica (357 U·mg-1) com valor de kM três vezes maior do que o da crisantaspase (150 e 48,5 µM respectivamente). Entretanto, apresentou maior estabilidade em altas temperaturas. Em duas semanas, a crisantaspase perdeu 93% de sua atividade específica enquanto que a PEG-crisantaspase foi estável por 20 dias. Portanto, a enzima PEG-crisantaspase desenvolvida representa uma alternativa promissora para o tratamento da LLA. / Crisantaspase is an asparaginase enzyme (ASNase) produced by Erwinia chrysanthemi bacterium and used as biopharmaceutical in the treatment of acute lymphoblastic leukemia (ALL) in case of hypersensivity to E. coli ASNase. The main disadvantages of this biopharmaceutical are the short half-life (10 hours) and immunogenicity. In this sense, its PEGylated form (PEG-crisantaspase) could not only reduce the immunogenic effect but also improve plasma half-life. Currently, only E. coli ASNase is commercially available in its pegylated form. Since ASNase was one of the first proteins to be pegylated, it corresponds to a random PEGylation process on lysine residues and consequently preparations are highly polydisperse. In this work we developed a process to obtain a site-specific N-terminal PEGylated crisantaspase (PEG-crisantaspase). Crisantaspase was recombinantly expressed in E. coli BL21 strain, grown in shaker and bioreactor, in Luria Bertani medium. Volumetric productivity in bioreactor increased 37% compared to shaker conditions (460 and 335 U·L-1·h-1 respectively). Crisantaspase was extracted by osmotic shock and purified by cation exchange chromatography (HiTrap SP FF column, 5 mL, elution at pH 7.5), presenting specific activity of 694 U·mg-1,31 purification fold and an yield of 69%. Purified crisantaspase was PEGylated with 10 kDa mPEG-NHS in 100mM phosphate buffer, 22°C, enzyme:PEG molar ratio of 1:50 for 30 min, and at different pH values (6.5-9.0). The highest N-terminal pegylation yield (50%) was at pH 7.5 with less poly-PEGylated forms (7%). PEG-crisantaspase was purified by size-exclusion chromatography, retaining 50% of specific activity (357 U·mg-1) with a kM value 3 times higher than crisantaspase (150 and 48,5 µM respectively). Nonetheless, PEG-crisantaspase was found to be more stable at high temperatures and over the time. In two weeks, crisantaspase lost 93% of its specific activity, while PEG-crisantaspase was stable for 20 days. Therefore, the novel PEG-crisantaspase enzyme developed represents a promising alternative for the treatment of ALL.

O Monometoxi-polietilenoglicol succinimidil ácido propiônico (mPEG-SPA 5 e 20 kDa) foi analisado como adjuvante e inibidor da atividade neurotóxica da toxina tetânica (TxT) adsorvida ou não em Al(OH)3, à qual o polímero foi conjugado. Avaliou-se a toxicidade das amostras por DL50, demonstrando que a atividade neurotóxica da TxT foi inibida. A via subcutânea foi mais efetiva na indução de resposta à TxT tratada pelo mPEG-SPA e o efeito adjuvante do Al(OH)3 se deu pela intramuscular. Trinta cavalos foram submetidos a esquema de imunização seletiva, dividindo-se os dezoito escolhidos em grupos para imunização com TxT conjugada ao mPEG-SPA 5.000 e 5.000(2X) e TxT adsorvida ou não. Os soros dos cavalos foram analisados por ToBI Teste, que avaliou a evolução da resposta imune. Os soros também foram analisados por imunodifusão, eletroforese e immunoblotting, tendo este indicado uma provável superioridade antigênica da TxT Fluida relativamente aos adjuvantes. A conjugação mPEG-SPA provou ser efetiva na produção do soro antitetânico terapêutico para uso humano. / Monometoxypolyethylene glycol succinimidyl propionic acid (SPA-mPEG 5 and 20 kDa) was analyzed as adjuvant and inhibitor of tetanus toxin neurotoxic activity (TxT) adsorbed or not by Al(OH)3, to which the polymer was conjugated. The samples toxicity was evaluated by DL50, disclosing that TxT neurotoxic activity was inhibited. The subcutaneous inoculation was more effective in induction of response to TxT treated with SPA-mPEG and the adjuvant effect of Al(OH)3 was evidenced by the intramuscular. Thirty horses were submitted to a selective scheme of immunization and eighteen were divided in groups to be immunized with TxT conjugated to SPA-mPEG 5,000 and 5,000(2X) and TxT adsorbed or not. The horses sera were analysed by ToBI Test, which evaluated the immune response development. The sera were also analysed through immunodifusion, electrophoresis and immunoblotting and the last one indicates a probable antigenic superiority of TxT fluid relatively to the adjuvants. The SPA-mPEG conjugation proved to be effective for anti-tetanus human therapeutic serum production.

While a number of PEGylated proteins have been studied for injectable applications and reviewers have used this data to speculate possible oral delivery improvements, a detailed investigation of PEGylated insulin for oral delivery and the development of an optimized pH-sensitive carrier for PEGylated insulin conjugates had yet to be accomplished. In order to proceed with oral delivery study, improvements in yield, with respect to previous PEGylation methods were necessary to enable the completion of high throughput drug delivery studies. Subsequently, a reaction scheme for the covalent attachment of PEG to insulin using nitrophenyl carbonate-PEG was developed. It was demonstrated that this reaction occurred at a 1:1 ratio and was site specific at the B29Lys position. A P(MAA-g-EG) hydrogel carrier was developed to optimize loading and release behavior for PEGylated insulin. It was demonstrated that the density and length of polymer grafts affected both loading and release behavior of PEGylated insulin. The best performing grafted polymers had a 3:1 methacrylic acid: ethylene glycol (MAA:EG) ratio and achieved loading efficiencies from 96% to nearly 100%. With respect to release, polymer particles containing fewer, but longer grafts shown to release faster than polymers with shorter grafts with the same MAA:EG ratio. Finally, the effects of PEGylation on intestinal absorption was investigated using an intestinal epithelial model as well as a rat model. It was demonstrated that PEGylated insulin in the presence of P(MAA-g-EG) microparticles did not significantly alter the tight junctions over unmodified insulin. However, the conjugate permeabilities across the membrane were reduced. The pharmacological availability (PA) was then verified by injecting the insulin conjugates subcutaneously in fasted Sprague-Dawley rats. It was determined that PEG 1000 insulin (1KPI) had a PA roughly equivalent to insulin, while it was reduced by 59% for 2KPI and by 81% for 5KPI. The effectiveness of utilizing PEGylated insulin as an oral drug delivery candidate was evaluated with a closed loop intestinal study, in which PEGylated insulin or insulin in solution was delivered directly to the jejunum. It was shown that 1KPI and insulin performed identically; with a pharmacological availability of 0.56%. 2KPI, however improved the pharmacological availability of insulin by 2.8 times. These results demonstrate that PEGylation holds promise for improving the oral delivery of proteins. / text

Pre-clinical and clinical trials suggest that pre-treatment with dexamethasone (Dex) may facilitate enhanced uptake of subsequently administered chemotherapeutic agents. To reduce the side effects associated with systemic administration of Dex, solid lipid nanoparticles (SLNs) containing dexamethasone palmitate (Dex-P) were prepared as a means of achieving tumor-targeted drug delivery. These studies were aimed at evaluating the physicochemical properties and both the physiological and storage stability of the SLNs. SLNs were prepared using nanotemplate engineering technology. Stearyl alcohol (SA) was used as the lipid phase with Brij® 78 and Polysorbate 60 as surfactants and PEG6000 monostearate as a long-chain PEGylating agent. Both formulations exhibited a small particle size, ellipsoidal shape, and low polydispersity. 1H-NMR spectroscopy confirmed that SLNs have the expected solid core and PEGylated surface. Analysis of the bulk materials indicated that a number of complex interactions are present among the SLN components, including a eutectic between SA and Brij® 78. Dex-P could be incorporated in SLNs at 10-30% w/w SA with encapsulation efficiencies >85%. A preferential interaction with the SA-Brij® 78 eutectic was identified, indicating a possible interfacial localization. For comparison, SLNs were also prepared with ascorbyl palmitate (AP) and curcumin. Higher drug loads were achieved with both palmitate-containing prodrugs than curcumin, though all appeared to align differently within the SLNs. SLNs undergo a concentration-dependent particle size growth when incubated at physiological temperature. However, they appear to remain intact with over 85% of the added Dex-P retained at 24 h in conditions mimicking human plasma. In the presence of carboxylesterase, SLNs became turbid and showed a reduction in particle size as compared to controls. This instability was shown to be a result of the hydrolysis of PEG6000 monostearate and Polysorbate 60. To enhance storage stability, a lyophilization protocol designed to minimize changes in the physicochemical properties of SLNs was developed. During a 3 month period, lyophilized SLNs stored at 4°C demonstrated the greatest stability, showing a consistent particle size and an encapsulation efficiency >80%. Overall, these results indicate that Dex-P loaded SLNs possess the physicochemical properties and stability desirable for development as a tumor-targeted drug delivery system.

Protein PEGylation (covalent attachment of poly(ethylene glycol) or PEG to proteins) is an excellent example of a drug delivery system that improves pharmacokinetics and pharmacodynamic properties of therapeutics. However, although PEGylation is clinically proven and attracts both scientific and commercial interest, the technique is associated with many process constraints, in particular related to controlling the number of conjugated PEG chains. A novel, solid-phase PEGylation methodology was attempted to overcome the drawbacks of the commonly used solution-phase methods for preparing PEGylated products. The solid-phase PEGylation methodology involved conjugating protein onto a tethered PEG derivative attached onto a solid matrix, followed by hydrolytic cleavage of the PEG chain from the solid matrix under mild conditions to yield PEGylated protein in free solution. PEGs with molecular weights (MWs) 2000 and 4000 Da were used and a heterobifunctional PEG derivative, α-(β-alanine)-ω-carboxy PEG, with a cleavable β-alanine ester terminal was prepared for surface grafting and protein conjugation. The amine terminal of this PEG derivative was used for grafting PEG onto carboxy functionalized hydrophilic Sephadex and hydrophobic polystyrene derivatives. The free carboxyl terminal was used for protein conjugation via amine coupling. A kinetic study of PEG-surface grafting was performed to understand the influence of a number of parameters on the PEG surface concentration and its conformation, including temperature, reaction time, nature of the matrix, solvent and base, and MW of PEG. PEG grafted matrices were characterized using various surface characterization tools including Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). Higher PEG grafting was observed with polystyrene matrices (up to 0.3 mmol/g) than either of the Sephadex derivatives (less than 0.15 mmol/g) using both molecular weights. Detailed surface characterization using XPS studies showed a layer thickness of 11.87 nm was achieved with polystyrene matrices using 4000 Da PEG derivatives after a grafting period of 72 hours at 40°C, indicating the presence of brush conformations for the grafted PEGs. In contrast, mushroom conformations were observed for PEG molecules grafted on both carboxymethyl and carboxypentyl Sephadex derivatives after the same reaction period, with a layer thickness of 2.62 nm and 4.14 nm respectively. Optimized PEG grafting and hydrolysis conditions were developed for solid-phase protein PEGylation using Cytochrome c as a model protein. The presence of PEGylated species were detected by size exclusion chromatography (SEC) from Sephadex derivatives but were absent when using polystyrene matrices. Both Sephadex derivatives gave mainly multi-PEGylated species with poor yields, in place of the expected mono-PEGylated products. A solution-phase PEGylation using the same PEG derivatives was performed successfully and various PEGylated species were identified and characterized using SEC and gel electrophoresis, based on their viscosity radius. An examination of the surface characteristics of the PEG-grafted was carried out by XPS, showing that protein conjugation was greatly influenced by surface force interactions, which depended on the PEG grafting densities and the nature of the solid matrices. Finally, fluorescent images obtained using confocal microscope with fluorescein isothiocyanate labelled Cytochrome c provided supporting evidence regarding the factors that constrained the solid-phase PEGylation process.

The research presented in this thesis focuses on the molecular design of an oxidation-sensitive nanocarrier and its enzyme conjugate with a view of their application in the field of biomaterials. I have polarised our attention on a specific class of polymers, the polysulfides, for their environmental responsiveness (towards oxidising substances, a condition often associated with inflammatory reactions), interesting physico-chemical properties, ease of the preparation and multiple possibilities for further modifications and bioconjugations, which are perfectly suitable for the development as systems for drug delivery applications. In this work we firstly have focused on the synthesis of amphiphilic poly(propylene sulfide)-poly(ethylene glycol) (PPS-PEG) block copolymers by employing vinyl sulfone as the functional group to link the blocks and modify the end of the PEG. This study was followed by an investigation of the macromolecular interchange and payload exchange of the formed polymeric micelles to understand the 'co-formulation' events, employing fluorophores (dansyl groups) and quenchers (dabsyl groups) either as terminal groups in macroamphiphiles or as encapsulated hydrophobic payloads. In another part of the work, I have developed a micellar system with which simultaneously to two of the most important ROS: superoxide and hydrogen peroxide, for inflammation-responsive drug release. The system is composed of superoxide dismutase (SOD) conjugated to oxidation-sensitive amphiphilic polysulfide/PEG block copolymers; the conjugate combines the SOD reactivity towards superoxide with that of hydrophobic thioethers towards hydrogen peroxide. Specifically, here we have demonstrated how this hybrid system can efficiently convert superoxide into hydrogen peroxide, which is then 'mopped-up' by the polysulfides. This mode of operation is functionally analogous to the SOD/catalase combination, with the advantage of being based on a single and more stable system.

O Monometoxi-polietilenoglicol succinimidil ácido propiônico (mPEG-SPA 5 e 20 kDa) foi analisado como adjuvante e inibidor da atividade neurotóxica da toxina tetânica (TxT) adsorvida ou não em Al(OH)3, à qual o polímero foi conjugado. Avaliou-se a toxicidade das amostras por DL50, demonstrando que a atividade neurotóxica da TxT foi inibida. A via subcutânea foi mais efetiva na indução de resposta à TxT tratada pelo mPEG-SPA e o efeito adjuvante do Al(OH)3 se deu pela intramuscular. Trinta cavalos foram submetidos a esquema de imunização seletiva, dividindo-se os dezoito escolhidos em grupos para imunização com TxT conjugada ao mPEG-SPA 5.000 e 5.000(2X) e TxT adsorvida ou não. Os soros dos cavalos foram analisados por ToBI Teste, que avaliou a evolução da resposta imune. Os soros também foram analisados por imunodifusão, eletroforese e immunoblotting, tendo este indicado uma provável superioridade antigênica da TxT Fluida relativamente aos adjuvantes. A conjugação mPEG-SPA provou ser efetiva na produção do soro antitetânico terapêutico para uso humano. / Monometoxypolyethylene glycol succinimidyl propionic acid (SPA-mPEG 5 and 20 kDa) was analyzed as adjuvant and inhibitor of tetanus toxin neurotoxic activity (TxT) adsorbed or not by Al(OH)3, to which the polymer was conjugated. The samples toxicity was evaluated by DL50, disclosing that TxT neurotoxic activity was inhibited. The subcutaneous inoculation was more effective in induction of response to TxT treated with SPA-mPEG and the adjuvant effect of Al(OH)3 was evidenced by the intramuscular. Thirty horses were submitted to a selective scheme of immunization and eighteen were divided in groups to be immunized with TxT conjugated to SPA-mPEG 5,000 and 5,000(2X) and TxT adsorbed or not. The horses sera were analysed by ToBI Test, which evaluated the immune response development. The sera were also analysed through immunodifusion, electrophoresis and immunoblotting and the last one indicates a probable antigenic superiority of TxT fluid relatively to the adjuvants. The SPA-mPEG conjugation proved to be effective for anti-tetanus human therapeutic serum production.

PEGylation has been used for decades to enhance the pharmacokinetic properties of protein therapeutics. This method has been effective at increasing the serum half-life of these drugs, but the mechanism of how it does this is unclear. Chapter 1 is an introduction to the methods of PEGylation. In chapter 2 we show that the effect of PEGylation on the conformational stability of the WW domain differs based on amino acid linker and conjugation site. We show that all positions in the WW domain that were tested can be stabilized by at least one amino acid linker. The rate of proteolysis is proportional to the degree of conformational stability. Chapter 3 shows that PEG-based desolvation can increase the strength of the interaction between two salt bridge residues, though the effect of structural context is unclear. A crystal structure shows that PEG occupies the space between the PEGylation site and the salt bridge, displacing water. In Chapter 4 we discuss the effect that PEGylation has on the interaction strength of a solvent exposed hydrophobic patch. When the c Log P of the hydrophobic patch increases, PEG increases the conformational stability of the WW domain more dramatically. Chapter 5 is about the effect of PEG based desolvation on the strength of an NH-π hydrogen bond in the WW domain between Trp11 and Asn26. When Trp11 is mutated to Phe, Tyr and naphthylalanine (Nal), the melting temperatures correlate with the calculated interaction energies between the sidechain arene of the hydrogen bond acceptor and formamide. When Asn26 is PEGylated in the presence of each of these amino acids, the effect that PEG has on the conformational stability of the WW domain correlates with the melting temperature of the nonPEGylated variants, the calculated interaction energies, the arene molecular polarizability, and the arene molar volume.

No description available.

The covalent attachment of poly(ethylene glycol) (PEG) to a protein surface (known as PEGylation), has been demonstrated to increase the serum half-life of therapeutic proteins by reducing kidney clearance and immunogenicity and by protecting against proteolysis. Theses beneficial effects could be further enhanced if PEGylation consistently increased protein conformational stability (i.e. the difference in free energy between the folded and unfolded states). However, the effects of PEGylation on protein conformational stability are unpredictable; PEGylation has been reported to increase, decrease, or have no effect on the conformational stability of medicinal proteins.This thesis details the results of two studies aimed at discovering the structural determinants which influence the thermodynamic impact of PEGylation on the WW domain, a small model protein. Chapter 1 is a brief introduction to protein therapeutics and protein PEGylation. Chapter 2 describes a study which demonstrates that the thermodynamic impact of PEGylation is strongly dependent on the site to which PEG is conjugated. The studies described in Chapter 3 elaborate on this site dependence, and demonstrate that PEG stabilizes the WW domain through interactions with the surface of the folded peptide, and that two factors – the orientation of the PEG chain (relative to the protein surface) and the identity of nearby side chains – play a critical role in determining the thermodynamic impact of PEGylation.