Improving the cell-membrane-penetrating activity of globins by introducing positive charges on protein surface: A case study of sperm whale myoglobin.

Globins are heme proteins such as hemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb), playing important roles in biological system. In addition to normal functions, zebrafish Ngb was able to penetrate cell membranes, whereas less was known for other globin members. In this study, to improve the cell-membrane-penetrating activity of globins, we used sperm whale Mb as a model protein and constructed a quadruple mutant of G5K/Q8K/A19K/V21K Mb (termed 4K Mb), by introduction of four positive charges on the protein surface, which was designed according to the amino acid alignment with that of zebrafish Ngb. Spectroscopic and crystallographic studies showed that the four positively charged Lys residues did not affect the protein structure. Cell-membrane-penetrating essay further showed that 4K Mb exhibited enhanced activity compared to that of native Mb. This study provides valuable information for the effect of distribution of charged residues on the protein structure and the cell-membrane-penetrating activity of globins. Therefore, it will guide the design of protein-based biomaterials for biological applications.

Polymeric Micelles Loading Proteins through Concurrent Ion Complexation and pH-Cleavable Covalent Bonding for In Vivo Delivery.

Protein drugs have great potential as targeted therapies, yet their application suffers from several drawbacks, such as instability, short half-life, and adverse immune responses. Thus, protein delivery approaches based on stimuli-responsive nanocarriers can provide effective strategies for selectively enhancing the availability and activation of proteins in targeted tissues. Herein, polymeric micelles with the ability of encapsulating proteins are developed via concurrent ion complexation and pH-cleavable covalent bonding between proteins and block copolymers directed to pH-triggered release of the protein payload. Carboxydimethylmaleic anhydride (CDM) is selected as the pH-sensitive moiety, since the CDMamide bond is stable at physiological pH (pH 7.4), while it cleaves at pH 6.5, that is, the pathophysiological pH of tumors and inflammatory tissues. By using poly(ethylene glycol)-poly(l-lysine) block copolymers having 45% CDM addition, different proteins with various sizes and isoelectric points are loaded successfully. By using myoglobin-loaded micelles (myo/m) as a model, the stability of the micelles in physiological conditions and the dissociation and release of functional myoglobin at pH 6.5 are successfully confirmed. Moreover, myo/m shows extended half-life in blood compared to free myoglobin and micelles assembled solely by polyion complex, indicating the potential of this system for in vivo delivery of proteins.

Novel preparation method for sustained-release PLGA microspheres using water-in-oil-in-hydrophilic-oil-in-water emulsion.

An increasing number of drugs are needing improved formulations to optimize patient compliance because of their short half-lives in blood. Sustained-release formulations of drugs are often required for long-term efficacy, and microspheres are among the most popular ones. When drugs are encapsulated into microsphere formulations, different methods of preparation need to be used according to specific clinical requirements and the differing physicochemical characteristics of individual drugs. In this work, we developed a novel method for sustained-release drug delivery using a water-in-oil-in-hydrophilic oil-in-water (w/o/oh/w) emulsion to encapsulate a drug into poly(lactic-co-glycolic acid) (PLGA) microspheres. Different effects were achieved by varying the proportions and concentrations of hydrophilic oil and PLGA. Scanning electron and optical microscopic images showed the surfaces of the microspheres to be smooth and that their morphology was spherical. Microspheres prepared using the w/o/oh/w emulsion were able to load protein efficiently and had sustained-release properties. These results indicate that the above-mentioned method might be useful for developing sustained-release microsphere formulations in the future.

A novel cell encapsulation method using photosensitive poly(allylamine alpha-cyanocinnamylideneacetate).

A photosensitive alpha-cyanocinnamylideneacetyl group was coupled to poly(allylamine) to obtain a photosensitive polymer. This photosensitive poly(allylamine alpha-cyanocinnamylideneacetate) can cross-link upon light exposure. Microcapsules were fabricated from alginate in contact with Ca+2 ion, followed by coating with the photosensitive poly(allylamine alpha-cyanocinnamylideneacetate). The microcapsules, thus formed, can be strengthened significantly by the light-induced cross-linking of poly(allylamine alpha-cyanocinnamylideneacetate). Only 16 capsules (out of 50) prepared from the photosensitive poly(allylamine alpha-cyanocinnamylideneacetate) fractured after 48 h of agitation. For microcapsules prepared from the unmodified poly(allylamine), 32 capsules fractured. The photo cross-linked capsular membrane was permeable to cytochrome C, moderately permeable to myoglobin, and least permeable to serum albumin. IW32 (a mouse leukaemia cell line) cells were entrapped and cultured within these microcapsules. The cells proliferated to a density of about 9 x 10(6) cells/ml in the capsules after 7 days of cultivation.

Different time courses of cardiac contractile proteins after acute myocardial infarction.

For the first time we have compared time courses of cardiac myosin light chain-1 (MLC-1), beta-type myosin heavy chain (MHC), troponin T (TnT), myoglobin, creatine kinase (CK) and CKMB in the same patients with acute myocardial infarction (AMI). Blood samples were serially collected in 23 patients with first-time AMI. All but 3 patients received intravenous thrombolytic treatment. TnT and MLC-1 time courses were biphasic in most patients and showed two distinct peaks in 13 and 8 patients, respectively. MHC time courses were usually monophasic. Only 1 patient showed a biphasic MHC time course with two distinct peak values. Although MHC and MLC were lower by about the fourth day after onset of AMI in early reperfused patients, reperfusion did not qualitatively alter MLC and MHC release (no significant influence on the first appearance in blood or on time to peak). MLC and MHC peaks correlated closely (r = 0.75, P = 0.0001), whereas TnT peaks were correlated less closely with MLC or MHC peaks (r = 0.58 each, P < 0.007). Peak values of all cardiac contractile proteins correlated closely and significantly with CKMB peaks (0.75 < or = r < or = 0.81, P < or = 0.0006). Myoglobin was the first marker to increase in blood after AMI and showed the earliest peaks, whereas MHC increased latest showing the latest peaks. TnT increased significantly (P = 0.0001) earlier than MLC and MHC. These results can be explained by the impact of the intracellular compartmentation of a cardiac protein on the rapidity with which it is released after AMI.