High-throughput screening and rational design of biofunctionalized surfaces with optimized biocompatibility and antimicrobial activity.

Peptides are widely used for surface modification to develop improved implants, such as cell adhesion RGD peptide and antimicrobial peptide (AMP). However, it is a daunting challenge to identify an optimized condition with the two peptides showing their intended activities and the parameters for reaching such a condition. Herein, we develop a high-throughput strategy, preparing titanium (Ti) surfaces with a gradient in peptide density by click reaction as a platform, to screen the positions with desired functions. Such positions are corresponding to optimized molecular parameters (peptide densities/ratios) and associated preparation parameters (reaction times/reactant concentrations). These parameters are then extracted to prepare nongradient mono- and dual-peptide functionalized Ti surfaces with desired biocompatibility or/and antimicrobial activity in vitro and in vivo. We also demonstrate this strategy could be extended to other materials. Here, we show that the high-throughput versatile strategy holds great promise for rational design and preparation of functional biomaterial surfaces.

Fusion peptide engineered "statically-versatile" titanium implant simultaneously enhancing anti-infection, vascularization and osseointegration.

Although antimicrobial titanium implants can prevent biomaterial-associated infection (BAI) in orthopedics, they display cytotoxicity and delayed osseointegration. Therefore, versatile implants are desirable for simultaneously inhibiting BAI and promoting osseointegration, especially "statically-versatile" ones with nonessential external stimulations for facilitating applications. Herein, we develop a "statically-versatile" titanium implant by immobilizing an innovative fusion peptide (FP) containing HHC36 antimicrobial sequence and QK angiogenic sequence via sodium borohydride reduction promoted Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC-SB), which shows higher immobilization efficiency than traditional CuAAC with sodium ascorbate reduction (CuAAC-SA). The FP-engineered implant exhibits over 96.8% antimicrobial activity against four types of clinical bacteria (S. aureus, E. coli, P. aeruginosa and methicillin-resistant S. aureus), being stronger than that modified with mixed peptides. This can be mechanistically attributed to the larger bacterial accessible surface area of HHC36 sequence. Notably, the implant can simultaneously enhance cellular proliferation, up-regulate expressions of angiogenesis-related genes/proteins (VEGF and VEGFR-2) of HUVECs and osteogenesis-related genes/proteins (ALP, COL-1, RUNX-2, OPN and OCN) of hBMSCs. In vivo assay with infection and non-infection bone-defect model reveals that the FP-engineered implant can kill 99.63% of S. aureus, and simultaneously promote vascularization and osseointegration. It is believed that this study presents an excellent strategy for developing "statically-versatile" orthopedic implants.

Antimicrobial Titanium Surface via Click-Immobilization of Peptide and Its in Vitro/Vivo Activity.

The use of antimicrobial peptides (AMPs)-functionalized titanium implants is an efficient method for preventing bacterial infection. However, the attachment of AMPs to the surface of titanium implants remains a challenge. In this study, a "clickable" titanium surface was developed by using a silane coupling agent with an alkynyl group. The antimicrobial titanium implant was then constructed through the reaction between the "clickable" surface and azido-AMPs (PEG-HHC36:N3-PEG12-KRWWKWWRR) via click chemistry of Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Such an antimicrobial titanium implant, with an AMP density of 897.4 ± 67.3 ng/cm2 (2.5 ± 0.2 molecules per nm2) on the surface, exhibited good and stable antimicrobial activity, inhibited 90.2% of Staphylococcus aureus and 88.1% of Escherichia coli after 2.5 h of incubation, and even inhibited 69.5% of Staphylococcus aureus after 4 days of degradation. The CCK-8 assay indicated that the antimicrobial titanium implant exhibited negligible cytotoxicity to mouse bone mesenchymal stem cells. In vivo assay illustrated that this implant could kill 78.8% of Staphylococcus aureus after 7 days. This method has great potential for the preparation of antimicrobial titanium implants and the prevention of infections in the clinic.

Immobilization of an antimicrobial peptide on silicon surface with stable activity by click chemistry.

Infections associated with biomedical implants and devices pose a serious clinical challenge in hospitals worldwide. Antimicrobial peptides (AMPs) have become a great prospect to inhibit this type of infection due to their broad-spectrum antimicrobial activity and low cytotoxicity. However, it is still a challenge to apply AMPs on the biomaterial surface as the activity of AMPs is sensitive to salt or enzyme. In the present study, we prepared a spacer molecule, poly[2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (polySBMA), on a model silicon surface via surface-initiated atom transfer radical polymerization (SI-ATRP). We then modified the antimicrobial peptide HHC36 (KRWWKWWRR) with l-propargylglycine (PraAMP) to improve its salt-tolerant activity and integrated PraAMP onto the spacer molecule using click chemistry. We employed X-ray photoelectron spectroscopy (XPS), contact angle goniometry, and atomic force microscopy (AFM) to confirm the success of the immobilization process. We also characterized the antimicrobial activity and stability of the surface with an antimicrobial assay. The results reveal that the modified surface exhibits good antimicrobial activity to inhibit 98.26% of E. coli, 83.72% of S. aureus, and 81.59% of P. aeruginosa. Furthermore, as compared to the control group without the polySBMA spacer, the modified surface improved its resistance to enzymolysis. An in vitro CCK-8 assay also illustrated that this surface showed negligible cytotoxicity to mouse bone mesenchymal stem cells.

Synthesis and anionophoric activities of dimeric polyamine-sterol conjugates: the impact of rigid vs. flexible linkers.

Two dimeric spermine-choloyl conjugates were synthesized and found to be capable of promoting the transport of anions across egg-yolk L-α-phosphatidylcholine-based liposomal membranes, via an anion-exchange mechanism and with moderate selectivity with respect to monoanionic ions. A Hill analysis indicated that these two conjugates exhibited similar aggregation behaviors. However, the conjugate bearing a rigid p-bis(aminomethyl)benzene moiety functioned more efficiently than the analogue having a flexible putrescine linker.