Vivian Saez

PEGylated Interferon-α2b: A Branched 40K Polyethylene Glycol Derivative

PurposeThe conjugation of interferon-α2b (IFN-α2b) to a branched-chain (40,000) polyethylene glycol (PEG2,40K) was studied.MethodsWe studied the conjugation of IFN-α2b at different pH values (6.5, 7, and 8), using the PEG2,40K reagent in either solution or solid state. MonoPEGylated interferon was isolated by ion-exchange chromatography and characterized using (1) sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (2) cation exchange high-performance liquid chromatography, (3) bicinchoninic acid protein assay, (4) enzyme-linked immunosorbent assay, (5) cell-based bioassays, (6) thermal stability (at 60°C), (7) tryptic digestion, and (8) pharmacokinetics in rats.ResultsPEGylation reaction gave 30–55% PEG2,40K-IFN-α2b, 1–10% polyPEGylated interferon, and 35–70% unmodified IFN-α2b. Compared to the polyPEGylated IFN-α2b species, the pure (96%) monoPEGylated conjugate retained a significantly higher bioactivity (IU/mg): 1.7 × 104 ± 8.5 × 103vs. 2.8 × 106 ± 1.4 × 106 for antiviral and 1.9 × 104 ± 9.5 × 103vs. 3.1 × 106 ± 1.6 × 106 for antiproliferative activity. Immunorecognition against IFN was reduced by the PEG2,40K moiety in the conjugate. This monoPEGylated IFN-α2b, which migrated as a single band in gel electrophoresis, was found to be a heterogeneous, complex mixture of different positional isomers. PEGylation markedly enhanced both the resistance to tryptic degradation and the thermal stability of IFN-α2b. The serum half-life of 40K PEG–IFN was 330-fold longer, while plasma residence time was increased 708 times compared to native IFN.ConclusionThe PEG2,40K conjugate of IFN-α2b has increased in vitroand in vivo stability as compared to the native cytokine.

Microencapsulation of Alpha Interferons in Biodegradable Microspheres

The immunomodulatory, antiproliferative, and antiviral properties of interferons alpha (IFN-α) have made these cytokines attractive for numerous clinical applications. However, most of the current available IFN pharmaceutical dosage forms need to be injected frequently and may provoke adverse reactions in patients. This problem might be overcome by using biodegradable microspheres loaded with IFN. The encapsulation of IFN-α in microspheres and the current status of this technology are the main subjects reviewed here. To this end, we describe (i) the main methods and experimental parameters used to obtain IFN-loaded microspheres and (ii) characterization of these microspheres in terms of morphology, particle size, loading/encapsulation efficiency, residual water content, residual solvent content, release profile, and sterility. Also, we discuss both the characterization of the encapsulated IFN and the stabilization/protection of IFN during microencapsulation. Finally, a brief overview of preclinical and clinical studies using IFN-containing microspheres is given.

Extraction of PLGA-Microencapsulated Proteins Using a Two-Immiscible Liquid Phases System Containing Surfactants

PurposeThe extraction of proteins from PLGA/PLA microspheres by a two-immiscible liquid phases system with the addition of surfactants was investigated.MethodsFirst, the extraction without surfactants and the interaction between proteins (IFN-α2b and EGF) and empty microspheres (PLGA or PLA) was studied. Next, proteins stability in presence of different surfactants was evaluated by: (1) bicinchoninic acid protein assay, (2) reversed phase-high performance liquid chromatography, and (3) enzyme-linked immunosorbent assay. Then, proteins were extracted with PBS/dichloromethane including selected surfactants and characterized by the above mentioned techniques, biological activity tests, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrospray ionization mass spectrometry.ResultsWithout surfactants, protein recovery was only 27–43% for IFN-α2b and 58–73% for EGF. Protein content in solutions incubated with blank microspheres decreased to 66% for IFN-α2b and 86% for EGF. It was only possible to quantify the EGF and IFN-α2b in the same manner as in PBS alone when the surfactant added was Pluronic F-68 and SDS, respectively. Addition of these surfactants allowed the complete isolation of both biomolecules from the microspheres. The extraction procedure did not affect the encapsulated proteins.ConclusionProteins can be quantitatively extracted, without changes, from PLGA/PLA microspheres using PBS/dichloromethane system that include an appropriate surfactant.

Detection of PEGylated proteins in polyacrylamide gels by reverse staining with zinc and imidazole salts

The reverse staining, with imidazole–SDS–zinc, of PEG‐linked proteins separated by SDS‐PAGE was studied. Using model conjugates (interferon‐α 2b (IFN‐α2b) reacted with either a branched‐chain (40 000) PEG (PEG2,40) or a linear monomethoxy PEG polymer (Mr of 12 000) and chromatographically purified monoPEG2,40‐IFN‐α2b), conventional small‐format analytical gels (<1 mm thick) showed typical detection patterns (i.e., transparent, colorless bands clearly discernible against a zinc imidazolate‐generated white gel background), in less than 20 min. Nonreacted (free) PEG was almost undetected, as expected. The reverse‐stained PEGylated IFN‐α2b patterns were qualitatively indistinguishable from those of parallel gels stained with iodine (I2). The LOD was estimated in the low nanogram range (e.g., at about 7 ng for mono‐ or bi‐PEG2,40 IFN‐α2b per lane on gradient (4–17%) gels). Also, this stain allowed the visualization of Coomassie blue‐undetected PEG‐IFN bands, and could be restained with I2. PEGylated species of lysozyme, a low‐molecular‐weight peptide, ovalbumin, and chymotrypsin were used to demonstrate the generality of this stain. We also show (i) how to counteract the adverse effect of some parameters (e.g., gel thickness above 1 mm, long gel length, low (e.g., 4–6%) acrylamide concentration) on the reverse staining process and (ii) that the properties of the reverse‐stained PEGylated proteins remain unchanged, as judged by analyzing both the ion exchange chromatography‐based positional isomer separation profile and enzyme‐linked immunosorbent response of PEG‐IFN recovered from gels. Consequently, this technique may be useful for the rapid analysis or the small‐scale preparation of PEGylated proteins.

Influence of PLGA and PLGA-PEG on the dissolution profile of oxaliplatin

Oxaliplatin was inserted into polymeric matrices aiming to study the interaction of this drug with these polymers and its capability to diffuse to the environment. Tested polymers were: (1) polyethylene glycol (PEG), (2) poly(lactic-co-glycolic acid) (PLGA), and (3) a copolymer of them (PLGA-PEG). The latter two were synthesized by us using polycondensation in bulk. Oxaliplatin was included in the matrices by the melt mixing process followed by casting. Fourier tran sform infrared spectroscopy (FTIR), proton nuclear magnetic resonance (1H-NMR) and X-ray diffraction (DRX) studies of the polymers were performed proving the obtaining of the desired materials. In addition, the interaction between drug and matrices and the release profile of the oxaliplatin from these matrices were analyzed. Among them, PEG did not control the oxaliplatin release. In turn, PLGA and PLGA-PEG present drug release profiles quite similar. Oxaliplatin was completely released from PLGA and PLGA-PEG in 5 hours, by a relaxation mechanism. There was no evidence of oxaliplatin interaction with the different polymers. In addition, as the PEG improves the biocompatibility and biomasking, obtained results prove the obtaining of a drug release system, which allowed the total use of the drug improving the cancer treatment and even the welfare of the patients.

A reproducible method for obtaining a 2-arms monofunctional peg with high purity

PEGylation has become a widely applied technique to enhancing in vitro and in vivo stability of therapeutic proteins and to increasing materials biocompatibility. PEG branched structures have proven useful for protein and peptide modification. Furthermore, they may be better than linear structures for many purposes. This paper describes an improved procedure for obtaining 2-arms PEG based on L-lysine. The efficiency of the synthesis was not related to moisture of the raw materials. This procedure does not use hazardous reagents as previous protocols do. It implemented a purification process for obtaining the desired structure with high purity ( > 99%). Finally, the procedure described here allows the obtaining of others PEGylation reagents.