MPEG

Uchegbu and coworkers have studied the complexation and delivery of DNA using a unique poly(amino acid)-based polymer vesicle. A polymer of either poly (L-lysine) or poly(L-omithine) was functionalized with methoxy-poly(ethylene glycol) (mPEG) and hydrophobic palmitic acid chains to synthesize an amphiphilic triblock of either mPEG-6-poly(L-lysine)-6-palmitoyl or mPEG-Z>-poly(L-omithine)-6-palmitoyl. Vesicles formed from these polymers were complexed with DNA and showed improved transfection in vitro over poly(amino acid) complexed with DNA or DNA alone [82]. 

Originally, for preparation of such conjugates the hydroxyl groups of monomethoxy-PEG (mPEG) were activated with cyanuric chloride, and the resulting compound then coupled with proteins (10). This approach suffers from disadvantages, such as the toxicity of cyanuric chloride and its limited applicability for modification of proteins having essential cysteine or tyrosine residues, as manifested by their loss of activity. 

Currently, a common form of activated mPEG used for preparation of therapeutic enzymes is mPEG-succinate-N-hydroxysuccinimide ester (SS-PEG) (11). It reacts with proteins in short periods of time under mild conditions, producing extensively modified conjugates with well preserved biological activity. However, the ester linkage between the polymer and the succinic acid residue has limited stability in aqueous media (5,12). 

Determination of Optimal pH for SC-PEG Reactivity. To triethanolamine-borate buffer (0.3 M, 1 mL) at the appropriate pH, a stock solution of N,a-acetyl-lysine (NAL) in water (50 mM, 0.1 mL) was added followed by a stock solution of SC-activated mPEG-5000 in CH3CN (50 mM active acyl, 0.1 mL). The resultant solution was vortexed and incubated at 28 °C for 1 h. A mixture of the same components but leaving out SC-PEG was used as a control. The TNBS assay version of Snyder and Sobocinski (18) was used to determine the unreacted NAL. 

Scheme 10.11. Domino Ugi/Diels-Alder process on a MPEG-0-CH2-platform.

Figure 7.12 Amine-containing dendrimers can be modified using a number of common reactive modification agents. Excess amine groups can be blocked using acetic anhydride, glycidol, or an NHS-mPEG compound. Amines also can be converted into carboxylates using succinic anhydride.

Figure 14.5 A method of making particles biocompatible includes the use of PEG-based spacers. A lawn of mPEG molecules in interspersed with some longer PEG chains that terminate in carboxylate groups for coupling amine-containing molecules. The result is an extremely hydrophilic surface with low nonspecific binding.

The low nonspecificity of PEG layers also was used to eliminate biomolecule binding to certain areas of an array. Kidambi et al. (2004) patterned an mPEG-carboxylate molecule onto polyelectrolyte multilayers to mask portions of the surface. The extremely low binding character of PEG provides advantages for creating patterned surfaces that other modifiers using aliphatic alkyl linkers do not provide. 

In addition, branched chain compounds have been developed consisting of a functional group or a reactive group followed by a PEG4 chain, which then leads to three branches each having an mPEG arm on them. Such compounds are expected to provide large exclusion volumes in aqueous solution to surround, protect, and solubilize modified molecules. 

Figure 18.26 The branched PEGylation compound NHS-dPEG fmPEG h contains three mPEG arms, which provide an increased sphere of hydration around modified molecules compared to straight-chain PEGylation compounds.

Figure 25.1 mPEG polymers may be activated by trichloro-s-tnazinc for the modification of amine-containing... 

The following protocol for mPEG activation using TsT and its coupling to proteins is based on the protocols of Abuchowski et al. (1977b) and Gotoh et al. (1993). 

Add to the TsT solution, 50 g of mPEG-5000 (monomethoxypolyethylene glycol having a molecular weight of 5000). Mix well to dissolve. 

Remove excess solvents by rotary evaporation. The TsT-mPEG should be used immediately or stored in anhydrous conditions at 4°C. 

Dissolve the protein to be modified with TsT-mPEG in ice-cold 0.1 sodium borate, pH 9.4, at a concentration of 2-10 mg/ml. Other buffers at lower pH values (down to pH 7.2) can be used and still obtain modification, but the yield will be less. Avoid amine-containing buffers such as Tris or the presence of sulfhydryl-containing compounds, such as disulfide reductants. 

Slowly add TsT-mPEG to the protein solution at a level of at least a 5-fold molar excess over the desired modification level. For example, Gotoh et al. (1993) added 100 lmg of TsT-mPEG-5000 to 19 mg of protein dissolved in 6 ml of buffer. Add the polymer over a period of about 15 minutes with stirring at 4°C. 

Remove excess TsT-mPEG by dialysis or gel filtration using a column of Sephacryl S-300. 

PEG contains no carboxylate groups in its native state, but can be modified to possess them by reaction with anhydride compounds. Either PEG or mPEG may be acylated with anhydrides... 

Figure 25.2 mPEG may be derivatized with succinic anhydride to produce a carboxylate end. A reactive NHS ester can be formed from this derivative by use of a carbodiimide-mediated reaction under nonaqueous conditions. The succinimidyl succinate-mPEG is highly reactive toward amine nucleophiles. 

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