Optimizing protein adsorption and antimicrobial properties

The biological response varies considerably across the in vivoapplications resulting in different requirements of biomaterials design. However, all undesirable biological reactions are associated with a series events know an “biofouling” that started with protein adsorption following by other biomolecules and cell adhesion. Therefore, creating a non-biofouling surface with minimal non-specific protein adsorption is of great importance to avoid undesirable biological response. There are two main conditions in which form non-adhesive surfaces as suggested: strongly hydrophilic or strongly hydrophobic[4]. Construction of hydrophilic surfaces of biomaterials is more favored in biomedical applications. Various polymers have been studied to generate a hydrophilic and protein-resistance surfaces. Among them, PEG has been the most widely used polymers for antifouling applications. PEG is a water-soluble amphiphilic polyether and termed as the “gold standard” of antifouling polymers[5].
PEG was coupled to a titanium oxide (TiO2) surface by a 3,4-dihydroxyphenylalanine (DOPA) derivative as cross-linker[6]. The DOPA was used to construct a hydroxylated surface to graft PEG via an amination reaction. The antiadhesive property of PEG functionalized TiO2 surface was assessed through the protein adsorption of BSA. Results revealed a reduction of BSA adsorption by a factor of 4 on PEG-surface compared to bare surface. Similarly, PEG was grafted to PVDF porous membranes via an amination reaction with reactive graphene oxide (GO) additives[7]. The antifouling ability and hydrophilicity of PEGylated PVDF/GO surface were significantly enhanced. The flux recovery rate (FRR) of PEGylated surface was 90.2% with a total fouling rate (Rt) of 20.7%, whereas the FRR of original PVDF/GO surface was 86% with a Rt of 26.7%. In one demonstration, a click reaction was conducted on silicon surface to create amine terminated layer for coating PEG with improved grafting density and uniformity[8]. The PEGylated silicon surface showed no fouling of human serum albumin and relatively lower adsorption of lysozyme. Silicon based biomaterials have been widely used for the development of ophthalmic devices such as contact lenses and intraocular lenses[9]. However, silicon-based contact lenses are always associated with limited wettability and excessive protein adsorption leading to ocular discomfort[10]. PEG coating was applied to intraocular lenses for improving hydrophilicity and antifouling property[11]. There is a commercial PEG based contact lens coating technology, Tangible™ Hydra-PEG, to improve the lubricity and antifouling ability of contact lens.
In addition to macroscopic surfaces, PEG can be incorporated to nanoparticle system to confer protein-repellent properties. NPs are widely used in nanomedicine and drug delivery applications. Whereas the protein corona formed on the surface of nanoparticle can induce fast uptake by macrophages, the reduced targeting efficiency and lower cancer cell uptake specifically for anticancer drug delivery[12]. PEG can be grafted on a wide range of NPs such as inorganic NPs, magnetic metallic NPs, polymeric NPs and nanoscale metal-organic frameworks. Mesoporous silica NPs was first surface modified with PEI-coated carbon dots for effective transepithelial transport and then coated with PEG for better mucus permeability and oral bioavailability[13]. PEG was coated on biodegradable PLGA NPs to improve the mucus permeability and retention of NPs as well[14]. The in vivoanimal studies indicated a considerably improved colorectal retention of PEG-modified PLGA NPs compared to pristine PLGA NPs. The retention of PEG-modified PLGA NPs reached 2-hours post-administration in contrast to 15 min of bare PLGA NPs. A sequential antifouling surface can be constructed on porous silica NPs by grafting PEG via a photo-triggered system. PEG was conjugated to PEI surface with biotin conjugates as targeting molecules via a photo-cleavable ortho-nitrobenzyl linker; and PEI was conjugated to the surface of silica NPs. PEGylation afforded the antifouling property of NPs and avoided the clearance by macrophages. Upon light irradiation, the outer PEG layer would be detached from PEI surface leaving the negatively charged carboxylic acids. Together with positive charged amine groups on the surface, a zwitterionic surface was generated that preserves targeting efficiency of biotin and offers further antifouling property.
Zeolitic imidazolate framework (ZIF-8) NPs with encapsulated doxorubincin (DOX) was modified by PEG in one-pot[15]. PEGylation endowed improved colloidal stability of ZIF-8 NPs in both water and cell culture medium. There was a pH-sensitive drug releasing behavior of DOX@ZIF-8/PEG NPs and higher cytotoxicity to hepatocellular carcinoma cells than free DOX suggesting an enhanced cancer cell targeting ability. Drugs can be conjugated to PEG directly prior to decorate the surface of drug carrier. Curcumin was coupled to the hydroxyl groups of PEG to form drug conjugates; then the PEGylated curcumin was physically attached to magnetic Fe3O4 NPs. Such PEG modified drug delivery system possessed higher drug loading efficiency and a pH-sensitive drug releasing profile.
Construction of an anti-fouling surface using aforementioned techniques can benefit the reduction in bacterial adhesion. However, unlike the “passive” strategy relying on the production of low adhesive surface, an “active” approach involves an antibacterial surface based on the incorporation of antibacterial agents[1].
Metal NPs or ions can be incorporated in antibacterial surface. Silver related antibacterial activity has been widely studied. There are numbers of silver incorporated commercial products in healthcare such as Silverlon® surgical dressing and Palindrome™ Precision SI-silver ion antimicrobial dialysis catheter. Silver has been coated on single-walled carbon nanotubes (SWCNT) to achieve antibacterial activities[16]. Results showed a stronger bactericidal activity against foodborne pathogens of PEGylated silver coating than non-PEGylated silver coating. The in ovoadministration of PEG/Ag-SWCNT indicated undetectable toxic effects on development of chicken embryo. Silver doped HA coating was deposited on NiTi alloys through electrodeposition to obtain an antibacterial and bioactive surface for orthopedic applications[17]. A composite coating composed of nano-HA and silver NPs on Ti6Al4V dental implants was developed for enhanced biocompatibility and additional antimicrobial property[18]. Since the HA coating was a porous layer, the antibacterial ability of silver layer would not be masked. There was some initial release of silver ions in the first 24 h immersion in cell culture media followed by a slow release. The initial release could be clinically beneficial for an early infection control.
Quaternary ammonium compounds (QACs) are antimicrobial materials and effectively against various bacteria. The most accepted mechanism of the antibacterial ability of QACs is the disruption of cell membrane due to the sufficiently long cationic polymer chains[20]. Another explanation is the disruption of divalent cations on cell membranes by the highly charged surface[21]. However, both explanations lead to concern about the potential cytotoxicity to human cells by QACs, which quite limited their progress in clinic[3,22].
Antimicrobial peptides (AMPs) are widely used in antibacterial coatings due to their broad-spectrum antimicrobial activity. Magainin I (Mag) has been bonded to TiO2 surfaces via PEG crosslinker[6]. There was a significant reduction in Listeria ivanovii adhesion to PEG-Mag modified surface compared to bare surface by a factor close to 2. Even though some bacteria were adhered to modified surface, such bacteria exhibited abnormal morphology revealing a detrimental effect from the antimicrobial surface. Another AMP, ε-poly-L-lysine (EPL), was dip-coated to 3D PCL/HA scaffolds to confer antibacterial property[23]. The EPL modification endowed a notable improvement in hydrophilicity and broad-spectrum antibacterial activities of PCL/HA scaffolds. Such antimicrobial activities againstS. aureus , E. coli and S. mutans . can retain for 3 days.
Various antibiotics can be immobilized on the surface of biomaterials to directly achieve bactericidal effects. Triclosan has been encapsulated into multilayer films composed of PEG, PCL, chitosan and PAA[24]. Such films were deposited on PDMS substrates by layer-by-layer self-assembly. Zone of inhibition (ZOI) and bacterial LIVE/DEAD staining assays verified the high efficiency of this antibiotics delivery system. The ZOI against E. coli was 15 mm while ZOI against S. aureus was 3 mm. The multilayer film allowed a sustained release of triclosan up to 7 days enabling long-term antibacterial function. Importantly, the sustained antibiotics release was stimuli-responsive that can be triggered by pH and bacteria stimuli, which may address the problems of resistant bacteria. As implanted the triclosan loaded multilayer coated substrates in rabbit models, the implants related infection was considerably eliminated with an infection rate of 16.7% in comparison with infection rate of 83.3% in multilayer alone modified group.
Polymers with bactericide effects can also be employed as antibacterial coating such as chitosan and PHMB. Acid treated carbon nanotubes were incorporated in PCL fibers to enhance mechanical strength as well as to create negatively charged surface[25]. Chitosan as a positively charged polysaccharide can be strongly immobilized to the surface of PCL fibers through electrostatic attraction. ZOI assay confirmed the acquired antibacterial function of PCL fibrous mats attributed to chitosan with ZOI against E. coli of 11.15 ± 0.21 mm and ZOI against S. aureus of 8.38 ± 0.19 mm. A hemostatic and antibacterial sodium alginate/gelatin sponge was fabricated by surface engineering with PHMB and hyaluronic acid by alternately spraying them on the sponge layer-by-layer. Hyaluronic acid was deposited on top of PHMB to endow the surface with better biocompatibility. When encountered with Gram-positive bacteria such as S. aureus , the bacteria secreted hyaluronidase would degrade the hyaluronic acid layer leading to subsequent exposure of PHMB to perform the bactericide function. This bacteria-stimulated antibacterial sponge showed an on-demand strategy and exhibited excellent in vivo anti-infection performance. However, its antibacterial property against Gram-negative bacteria such as E. coli was quite limited due to the masking effect of hyaluronic acid layer on PHMB.