Proteins loading/release

Bound proteins are released by boiling for 2 min at 95° in the 4 x sample loading buffer and separated by SDS-PAGE for immunoblot analyses as described for Ni affinity chromatography in Section 2.4. 

For other, larger proteins, the release was very slow - in 100 hours no more than 5% of loaded catalyse, urease, glucose oxidase or hemoglobin were released. 

Theoretical and model analysis based on a nanofluidic approach is needed for this situation. One may ask, is it possible to release proteins loaded in nanotubules We have found that the addition of the polycation PEI in the release solvent resulted in much quicker protein release, as demonstrated in Figure 14.9. In this case, most of the insulin was released in 1 hour instead of 100 hours. 10-40% of glucose oxidase, catalyse, and hemoglobin were released within 4 hours through complexation with PEI. It is unclear, whether the proteins were replaced by the polycation or released in a complex with PEI. 

Jiang and Zhu (2000) and Qiu and Zhu (2001) have reported the fabrication of multilayered devices composed of stacks of compression-molded disks of alternating compositions. One type of disk is either P(SA-EG) or P[SA-co-TMAgly)-Z>-EG] and the other is a pH-sensitive, protein-loaded blend of, for example, poly(methacrylic acid) and polyethoxazoline. The release of model proteins, myoglobin, bovine serum albumin, and FITC-dextran, and compounds such as brilliant blue have been studied and pulsatile release profiles have been demonstrated (Jiang and Zhu, 2000 Qiu and Zhu, 2001). 

Amidi M, Romeijn SG, Borchard G, Junginger HE, Hennink WE, Jiskoot W (2006) Preparation and characterization of protein-loaded N-trimethyl chi-tosan nanoparticles as nasal delivery system. J Control Release 111(1-2) 107-116. 

The matrix polyelectrolyte capsules have high protein-loading capacity, and both the loading and, in principle, the release are driven by electrostatic interaction with polyelectrolytes [111]. Moreover, the loading and release can be controlled by the number of polyelectrolyte adsorption steps [112] as well as by the pore size of the CaCC>3 cores [116],... 

The dwell time of a transferrin molecule with the reticulocyte may be only a minute or two (52, 80) or possibly as long as 10 min (86), after which the protein is released for another cycle of iron transport. The affinity of reticulocyte receptors for apotransferrin appears less than for iron-loaded molecules (80). This property may facilitate the release of the protein from the receptor after it has donated its iron and ensures that the apoprotein will not impede the delivery of iron to reticulocytes by competing with iron-bearing molecules for available receptors on the cell surface. 

One phase contains the polysaccharide chitosan (CS) and a diblock copolymer of ethylene oxide and the polyanion sodium tripolyphosphate (TPP). It was stated that the size (200-1000 nm) and zeta potential (between + 20 mV and + 60 mV) of nanoparticles can be conventionally modulated by varying the ratio of CS/PEO to PPO. Furthermore, using BSA as a model protein, it was shown that these new nanoparticles have a high protein loading capacity (entrapment efficiency up to 80 % of the protein) and provide a continuous release of the entrapped protein for up to 1 week [56]. 

Preparation of Nanoparticles Loaded with Model Proteins for Release Experiments... 

Khan, M. A., Healy, M. S., and Bernstein, H. (1992), Low temperature fabrication of protein loaded microspheres, Proc. Int. Symp. Controlled Release Bioactive Mater., 19, 518-519. 

Park, T. G., Lee, H. Y., and Nam, Y. S. (1998), A new preparation method for protein loaded poly(D,L-lactic-co-glycolic acid) microspheres and protein release mechanism study, J. Controlled Release, 55,181-191. 

Whitaker, M.J., Hao, J., Davies, O.R., Serhatkulu, G., Stolnik-Trenkic, S., Howdle, S.M., and Shakesheff, K.M. (2005). The production of protein-loaded microparticles by supercritical fluid enhanced mixing and spraying. J. Controlled Release 101, 85-92. 

Yan, C. Resau, J.H. Hewetson, J. Mest, M. Rill, W.L. Kende, M. Characterization and morphological analysis of protein-loaded poly(lactide-co-glycolide) microparticles prepared by water-in-oil-in-water emulsion technique. J. Controlled Release 1994, 32, 231-241. 

Figure 9.10 Proteins are released from matrices by diffusion through constricted pores. Schematie view of a protein/hydrophobic polymer matrix system, (a) Cross-sectional views of matrices at two different loadings. At higher loadings connected clusters of pores are formed, providing continuous paths for diffusion to the surface.

Modeling Release of Proteins from Matrices. Consider a protein-loaded matrix that is constructed as a thin slab. Release of the protein occurs essentially through the top and bottom faces of this slab since the thickness of the slab is small compared to the other dimensions. The desorption of protein from this slab can be described by Fick s second law of diffusion. Equation (9-14), in which an effective diffusion coefficient, Dgff, is used in place of Di-p. For example ... 

Figure 9.11 Release of bovine serum albumin from EVAc matrices. Controlled release of BSA from an EVAc matrix. Solid particles of BSA either (a) 45 to 75 /rm or (b) 150 to 250 /rm in diameter were dispersed within EVAc by solvent evaporation to achieve final protein loadings from 10, 20, 30, 40, or 50%. In each case five identical slabs, each 70 mg and 1 mm thick, were incubated in cacodylate-buffered water containing 0.02% gentamicin. Periodically, the buffered water was replaced, and the amount of protein released from the matrix was determined by measuring the concentration of protein in the solution that was removed. Each symbol represents the average cumulative fraction of protein released (cumulative mass of protein released/initial mass of protein within the matrix) for the five samples error bars indicate the standard deviation, which in some cases are smaller than the symbols. Data from [16].

The total tortuosity is listed for protein release from EVAc matrices prepared with different molecular weight fractions of EVAc. y-Globulin matrices were prepared with 119-180 nm particles and 40% protein loading [31]. BSA matrices were prepared with 106-150particles and 25% protein loading [38]. 

To understand how microscopic properties of the material influence these phenomena, it is necessary to develop more complex models of protein release. When detailed information on the microgeometry of the porous network in the polymers is available (as it is for protein-loaded EVAc matrices [37]) or can be estimated accurately, detailed models of protein release can be developed. For example, percolation models of pore network topology (such as those described in Chapter 4, see Figure 4.20) were coupled with analytical models of pore-to-pore diffusion rates to predict the rate of diffusion of proteins from EVAc matrices [16]. Effective diffusion coefficients predicted using this approach agree with those estimated by measuring rates of protein release from the matrix (Figure 9.13). 

TMC is supposed to possess the property of permeation enhancement, and therefore has been studied as a delivery vector for proteins and genes. For example, TMC nanoparticles were prepared by ionic crosslinking with tripolyphosphate (TPP) and used as a delivery system for ovalbumin [30]. The loading efficiency and capacity of the protein were up to 95% and 50%, respectively, with an improved integrity. Most importantly, transportation of the protein-loaded particles across nasal mucosa was confirmed by an in vivo uptake test. In another study, the influence of DQ on the property of protein-loaded TMC/TPP nanoparticles was investigated [31]. A lower DQ leads to an increased particle size and a slower release rate of protein. 

---