PHAs are linear biopolyesters produced as energy- and carbon-storage materials by many bacteria. The medical applications of PHA have been extensively explored in recent years for implant biomedical applications including sutures, nerve conduits, patches, slings, cardiovascular patches, stents, guided tissue repair/regeneration devices, bone marrow scaffolds and so on. PHA nanoparticles have also been developed as controlled drug-release vectors owing to their biocompatibility and biodegradability.
The surface of PHA-based nanoparticles can be modified by many different types of signaling molecules and thus can be used for purposes other than targeted delivery. This can also be achieved by engineering proteins other than PhaP such as other PHA surface associated proteins including PhaC, PhaR, and PhaZ. These proteins provide targets for displaying various molecules on polymer nanoparticle surfaces for protein immobilization and display. The phasin PhaP is a small amphiphilic protein that can associate with most hydrophobic polymer granule surfaces via strong hydrophobic interactions. PhaP is abundant on the surface of natural PHA granules, and can reach approximately 5% (wt/wt) of the total cell protein in PHA-accumulating bacteria. Thus, multifunctional polymer nanoparticles can be obtained through protein engineering of PhaP and applied to a variety of versatile situations. The functional fusion proteins were able to produce easily by recombinant bacteria cultivation. This method provides a simple and convenient way to immobilize and present proteins on the surface of hydrophobic biomaterials without complicated procedures to modify both proteins and materials.
Human B7-2 is a 329-amino-acid (aa) protein containing a putative 23-aa signal peptide, a 224-aa extracellular domain, a 21-aa transmembrane domain, and a 61-aa cytoplasmic domain. The absence of the signal peptide, the transmembrane domain, and the cytoplasmic domain did not affect the costimulatory activity of B7 molecules. In this study, the 224-aa extracellular domain was subcloned and fused with PhaP and immobilized on the surface of PHBHHx nanoparticles through the hydrophobic interactions between PhaP and polymer. As measured by western blot, 400 μg of GST-B7-2-PhaP fusion proteins could be immobilized on the surface of 1 mg PHBHHx nanoparticles, which by extrapolation implies 240 μg (3.2 pMol) B7-2-PhaP fusion protein was present on the surface of 1 mg of nanoparticles. When 1 μg ml-1 immobilized B7-2-PhaP proteins was applied to cultured cells, the amount of nanoparticles used was approximately 4.2 μg ml-1. Previously we demonstrated that 5 μg ml-1 PHBHHx nanoparticles could enhance the cell viability of rodent lymphocytes from spleen. Interestingly, the main degradation product of the PHBHHx polymer, 3-hydroxybutyrate, can promote interleukin-2 and interferon-γ secretion by rodent T lymphocytes (data not published). This may explain the slight T cell proliferation resulting from the non-coated PHBHHx nanoparticle treatment in this study.
Phytohemagglutinin is a lectin found in plants, and is used as a mitogen to trigger T lymphocyte cell division. Isolated lymphocytes were treated with 5 μg ml-1 phytohemagglutinin to increase cell numbers prior to the proliferation assay. Thus, the expanded cells were mainly T cells, although small numbers of B cells and other monocytes remained in the culture. This may explain why non-immobilized B7-2-PhaP fusion proteins also resulted in cell proliferation. PhaP is a bacterial protein that might be recognized by B cells, thus triggering an immune response resulting in lymphocyte proliferation. However, whether the B7-2-PhaP fusion protein was immobilized or not, the combined treatment of B7-2-PhaP protein and anti-CD3 antibody achieved the highest proliferation ratio and number of activated T cells. Compared with the non-immobilized free B7-2-PhaP protein, the B7-2-PhaP protein loaded nanoparticles showed a higher activity for cell proliferation stimulation. This can be explained by the advantage of the immobilization system, which can be used to provide several signal molecules that interact concurrently with ligands on cell surfaces.