UV-Triggered Hydrogel Coating of the Double Network Polyelectrolytes for Enhanced Endothelialization.

The left atrial appendage (LAA) occluder is an important medical device for closing the LAA and preventing stroke. The device-related thrombus (DRT) prevents the implantation of the occluder in exerting the desired therapeutic effect, which is primarily caused by the delayed endothelialization of the occluder. Functional coatings are an effective strategy for accelerating the endothelialization of occluders. However, the occluder surface area is particularly large and structurally complex, and the device is subjected to a large shear friction in the sheath during implantation, which poses a significant challenge to the coating. Herein, a hydrogel coating by the in situ UV-triggered polymerization of double-network polyelectrolytes is reported. The findings reveal that the double network and electrostatic interactions between the networks resulted in excellent mechanical properties of the hydrogel coating. The sulfonate and Arg-Gly-Asp (RGD) groups in the coating promoted hemocompatibility and endothelial growth of the occluder, respectively. The coating significantly accelerated the endothelialization of the LAA occluder in a canine model is further demonstrated. This study has potential clinical benefits in reducing both the incidence of DRT and the postoperative anticoagulant course for LAA closure.

Adventitial delivery of miR-145 to treat intimal hyperplasia post vascular injuries through injectable and in-situ self-assembling peptide hydrogels.

Intimal hyperplasia is a common lesion that can be observed in diverse vascular diseases. Drug-eluting stents and drug-coated balloons, which can release anti-proliferative agents to inhibit smooth muscle cell (SMC) proliferation, are developed to prevent intimal hyperplasia. However, these intervention devices still cannot achieve satisfactory clinical outcomes. In contrast to endovascular drug delivery, vascular adventitial drug delivery is a new strategy. To develop a vascular adventitial drug delivery system to treat intimal hyperplasia post vascular injuries, we loaded miR-145-5p-agomir (miR-145) into an injectable and in-situ self-assembling RAD peptide hydrogel. In vitro data showed that the miR-145 could be well incorporated into the RAD peptide hydrogels and released in a slow and controlled manner. The released miR-145 could transfect SMCs successfully, and the transfected SMCs exhibited a reduced migration capacity and higher expressions of SMC contractile biomarkers as compared to the non-transfected SMCs. In vivo data showed that the retention of the miR-145 was greatly elongated by the RAD peptide hydrogels. In addition, the application of the miR-145-loaded RAD peptide hydrogels surrounding injured arteries decreased the proliferative SMCs, promoted the regeneration of endothelium, reduced the macrophage infiltration, inhibited the neointimal formation and prevented adverse ECM remodeling via downregulation of KLF4 expression. The RAD peptide hydrogels loaded with miR-145 can successfully inhibit intimal hyperplasia after vascular injuries and thus hold great potential as an innovative extravascular drug delivery approach to treat vascular diseases. STATEMENT OF SIGNIFICANCE: Intimal hyperplasia is a common lesion that can be observed in diverse vascular diseases. Drug-eluting stents and drug-coated balloons, which can release anti-proliferative agents to inhibit smooth muscle cell (SMC) proliferation, are developed to prevent intimal hyperplasia. However, these intervention devices still cannot achieve satisfactory clinical outcomes. In contrast to endovascular drug delivery, vascular adventitial drug delivery is a new strategy. Our work here demonstrates that the RAD peptide hydrogels loaded with miR-145-5p-agomir (miR-145) can successfully reverse intimal hyperplasia after vascular injuries and thus hold great potential as an innovative vascular adventitial drug delivery approach to treat vascular diseases. Our work proposes a possible paradigm shift from endovascular drug delivery to extravascular drug delivery for vascular disorder treatment.

A pDNA/rapamycin nanocomposite coating on interventional balloons for inhibiting neointimal hyperplasia.

Drug-coated balloon (DCB) is a therapeutic method that can effectively deliver antiproliferative drugs such as paclitaxel and rapamycin (RAPA) with no permanent implants left behind. However, delayed reendothelialization due to the toxicity of the delivered drugs leads to poor therapeutic effects. Here, we propose a new design of DCB coating, which incorporates both vascular endothelial growth factor (VEGF)-encoding plasmid DNA (pDNA) that can promote endothelial repair and RAPA into protamine sulfate (PrS). We demonstrate that the PrS/pDNA/RAPA coating had stability and good anticoagulation properties in vitro. We further show that the coating exhibited excellent transfer capacity from balloon substrates to vessel walls both in vitro and in vivo. Furthermore, the PrS/pDNA/RAPA coating effectively inhibited neointimal hyperplasia after balloon-induced vascular injuries through the down-regulation of the mammalian target of Rapamycin (mTOR) and promoted endothelium regeneration through increased expression of VEGF in vivo. These data indicate that our nanocomposite coating has great potential for use as a novel coating of DCB to treat neointimal hyperplasia after vascular injuries.

Photothermal-assisted surface-mediated gene delivery for enhancing transfection efficiency.

The development of gene therapy puts forward the requirements for efficient delivery of genetic information into diverse cells. However, in some cases of transfection, especially those for transfecting some primary cells and for delivering large size plasmid DNA (pDNA), the existing conventional transfection methods show poor efficiency. How to further improve transfection efficiency in these hard-to-achieve issues remains a crucial challenge. Here, we report a photothermal-assisted surface-mediated gene delivery based on a polydopamine-polyethylenimine (PDA-PEI) surface. The PDA-PEI surface was prepared through PEI-accelerated dopamine polymerization, which showed efficiency in the immobilization of PEI/pDNA polyplexes and remarkable photothermal properties. Upon IR irradiation, we observed improved transfection efficiencies of two important hard-to-achieve transfection issues, namely the transfection of primary endothelial cells, which are kinds of typical hard-to-transfect cells, and the transfection of cells with large-size pDNA. We demonstrate that the increases of transfection efficiency were due to the hyperthermia-induced pDNA release, the local cell membrane disturbance, and the polyplex internalization. This work highlights the importance of local immobilization and release of pDNA to gene deliveries, showing great potential applications in medical devices in the field of gene therapy.

Surface-Mediated Stimuli-Responsive Gene Delivery Based on Breath Figure Film Combined with Matrix Metalloproteinase-Sensitive Hydrogel.

Surface-mediated gene delivery appears to be potential gene delivery modes for various applications. Still, controlled and smart delivery manners are required especially considering the need for gene therapy to deliver gene with selectivity. A surface that can effectively payload DNA, promote cell adhesion, and stimuli response is an important prerequisite. Here, we report a matrix metalloproteinase (MMP)-responsive surface-mediated gene delivery system by combining MMP-degradable hydrogel with a breath figure (BF) porous film. The MMP-degradable hydrogel containing plasmid DNA was loaded into the surface pores of the BF film as DNA reservoirs. The upper surface without hydrogel on the BF film served as footholds of integrin adhesions. MMP is one of the important endogenous signals in tumor-related pathologic changes, and MMP expressions in cancer cells are significantly higher than those in normal cells. Consequently, our surface-mediated gene delivery locally and rapidly released the payload DNA in response to cancer cells and transfected them. This work highlights the importance of the combination of stimuli-response and surface-mediated gene delivery to functional materials, showing good potential applications in the field of gene therapy.

Thermo-triggered ultrafast self-healing of microporous coating for on-demand encapsulation of biomacromolecules.

Medical coatings cooperated with biomacromolecules can regulate biological events and tissue responses, thus increasing medical implant longevity and providing improved and/or new therapeutic functions. In particular, medical coatings, which can load the correct species and doses of biomacromolecules according to individual diagnoses, will significantly optimize treatment effects and satisfy the rising clinical need of "precision medicine". Herein, we report on a dynamic microporous coating with an ultrafast self-healing property to fulfill the "load-and-play" concept for "precision medicine". A structure-switchable coating based on poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) triblock copolymer network is constructed. The coating can be switched to microporous morphology via a water swelling and freeze-drying process. Then, through a mild thermo-trigger as low as 40 °C, this spongy coating can undergo self-healing to switch back to a pore-free structure within minutes to even 5 s. Based on this dynamic coating, we suggest a simple and versatile method to encapsulate biomacromolecules for surface-mediated delivery. The ultrafast self-healing of the microporous structure enables uniform incorporation of biomacromolecules with an easily achieved high loading of albumin of 16.3 µg/cm2 within 1 min. More importantly, controllable encapsulation can be realized by simple control of the concentration of the loading solution. We further demonstrate that the encapsulated biomacromolecules retained their bioactivity. This work may benefit clinicians with flexibility to provide personalized medical coatings for individual patients during treatment.

Ultrathin κ-Carrageenan/Chitosan Hydrogel Films with High Toughness and Antiadhesion Property.

Designing tough biopolymer-based hydrogels as structural biomaterials has both scientific and practical significances. We report a facile approach to prepare polysaccharide-based hydrogel films with remarkable mechanical performances and antiadhesion property. The hydrogel films with a thickness of 40-60 µm were prepared by mixing aqueous solutions of κ-carrageenan (κ-CG) and protonated chitosan (CS), evaporating the solvent, and then swelling the casted film in water to achieve the equilibrium state. The obtained κ-CG/CS gel films with a water content of 48-88 wt % possessed excellent mechanical properties with a breaking stress of 2-6.7 MPa and a breaking strain of 80-120%, superior to the most existing biopolymer-based hydrogels. The extraordinary mechanical properties of gel films obtained over a wide range of mass ratio of κ-CG to CS should be rooted in the synergistic effect of ionic and hydrogen bonds between the κ-CG and CS molecules. In addition, the tough gel films showed good self-recovery ability, biocompatibility, and cell antiadhesion property, making them promising as an artificial dura mater and diaphragm materials in the surgery. The design principle by incorporating multiple noncovalent bonds to toughen the biopolymer-based hydrogels should be applicable to other systems toward structural biomaterials with versatile properties.

Stiffness of polyelectrolyte multilayer film influences endothelial function of endothelial cell monolayer.

Endothelialization has proved to be critical for maintaining long-term success of implantable vascular devices. The formation of monolayer of endothelial cells (ECs) on the implant surfaces is one of the most important factors for the endothelialization. However, endothelial function of regenerated EC monolayer, which plays a much more important role in preventing the complications of post-implantation, has not received enough attention. Here, a vascular endothelial growth factor (VEGF)-incorporated poly(l-lysine)/hyaluronan (PLL/HA) polyelectrolyte multilayer film was fabricated. Through varying the crosslinking degree, stiffness of the film was manipulated, offering either soft or stiff film. We demonstrated that ECs were able to adhere and proliferate on both soft and stiff films, subsequently forming an integrated EC monolayer. Furthermore, endothelial functions were evaluated by characterizing EC monolayer integrity, expression of genes correlated with the endothelial functions, and nitric oxide production. It demonstrated that EC monolayer on the soft film displayed higher endothelial function compared to that on the stiff film. Our study highlights the influence of substrate stiffness on endothelial function, which offers a new criterion for surface design of vascular implants.

Improved Endothelial Function of Endothelial Cell Monolayer on the Soft Polyelectrolyte Multilayer Film with Matrix-Bound Vascular Endothelial Growth Factor.

Endothelialization on the vascular implants is of great importance for prevention of undesired postimplantation symptoms. However, endothelial dysfunction of regenerated endothelial cell (EC) monolayer has been frequently observed, leading to severe complications, such as neointimal hyperplasia, late thrombosis, and neoatherosclerosis. It has significantly impeded long-term success of the therapy. So far, very little attention has been paid on endothelial function of EC monolayer. Bioinspired by the microenvironment of the endothelium in a blood vessel, this study described a soft polyelectrolyte multilayer film (PEM) through layer-by-layer assembly of poly(l-lysine) (PLL) and hyaluronan (HA). The (PLL/HA) PEM was chemically cross-linked and further incorporated with vascular endothelial growth factor. It demonstrated that this approach could promote EC adhesion and proliferation, further inducing formation of EC monolayer. Further, improved endothelial function of the EC monolayer was achieved as shown with the tighter integrity, higher production of nitric oxide, and expression level of endothelial function related genes, compared to EC monolayers on traditional substrates with high stiffness (e.g., glass, tissue culture polystyrene, and stainless steel). Our findings highlighted the influence of substrate stiffness on endothelial function of EC monolayer, giving a new strategy in the surface design of vascular implants.

Cell penetrating peptide-based polyplexes shelled with polysaccharide to improve stability and gene transfection.

Cell-penetrating peptides (CPP) have been widely developed as a strategy to enhance cell penetrating ability and transfection. In this work, octa-arginine modified dextran gene vector with pH-sensitivity was developed via host-guest interactions. α-Cyclodextrin was modified with octa-arginine (CDR), which had excellent cell penetrating ability. Dextran was selected as a backbone and modified with azobenzene as guest units by acid-labile imine bonds (Az-I-Dex). The supramolecular polymer CDR/Az-I-Dex with high a C/A molar ratio (molar ratio of CD on CDR to Az on Az-I-Dex) was unfavorable for DNA condensation. The dextran shell of CDR/Az-I-Dex/DNA polyplexes improved the stability under physiological conditions. However, once treated with acetate buffer (pH 5.4) for 3 h, large aggregates formed rapidly due to the cleavage of the dextran shell. As expected, the vector had cell viability of 80% even when the CDR concentration increased to 100 µg mL(-1). Moreover, due to the effective cellular uptake efficiency, CDR/Az-I-Dex/DNA polyplexes had 6-300 times higher transfection efficiency than CDR/DNA polyplexes. It was even higher than high molecular weight PLL-based polyplexes of HEK293 T cells. Importantly, chloroquine as an endosomal escape agent could not improve the transfection of CDR/Az-I-Dex/DNA polyplexes, which indicated that the CDR/Az-I-Dex supramolecular polymer had its own ability for endosomal escape. These results suggested that the CPP-based polyplexes shelled with polysaccharide can be promising non-viral gene delivery carriers.

pH-sensitive controlled release of doxorubicin from polyelectrolyte multilayers.

Controlled and stimulated release of small drug molecules from polyelectrolyte multilayers is still a challenge due to the limitations to incorporate and control the interactions between small molecules and multilayers. Herein we reported a facile method to fabricate polyelectrolyte multilayers with pH-sensitive controlled release of doxorubicin (DOX). A pH-responsive polyelectrolyte was prepared by conjugating DOX onto hyaluronan (HA) via pH-responsive hydrazone bond with an 8% grafting degree. The HA-DOX was then incorporated into the multilayers via the layer-by-layer assembly with poly-L-lysine (PLL). The growth of the multilayers was tracked by spectroscopic ellipsometry. The morphology and structure of the films were characterized by scanning electron microscopy and UV-vis spectroscopy, respectively. The in vitro drug release experiments indicated that the release of DOX was pH-dependent: there was almost no release at pH 7.4, while the releases were significantly promoted at pH 6.0 and 5.0. Furthermore, human hepatoma (HepG2) cells were remarkably inhibited under the conditions at pH 5.0 when they were cultured with the (HA-DOX/PLL) multilayers. The multilayers with the properties of pH-sensitive DOX release would be potentially applied to the biomedical devices for tumor treatments.

"Mixed-charge self-assembled monolayers" as a facile method to design pH-induced aggregation of large gold nanoparticles for near-infrared photothermal cancer therapy.

The acidic microenvironment of tumor tissues has proven to be one of the major differences from other normal tissues. The near-infrared (NIR) light irradiation of aggregated gold nanoparticles in a tumor acidic pH-induced manner could then provide an effect approach to treat solid tumors with the advantage of minimizing the undesired damage to normal tissues. Although it is well-known the aggregation of larger nanoparticles will result in a better NIR photothermal effect, the preparation of pH-sensitive gold nanoparticles in large sizes remains a big challenge because of their worse dispersive stability. In this paper, we introduce a facile way to endow large gold nanoparticles with tunable pH-aggregation behaviors by modifying the nanoparticle surface with mixed-charge self-assembly monolayers compromising positively and negatively charged thiol ligands. Four different size nanoparticles were used to study the general principle of tailoring the pH-induced aggregation behaviors of mixed-charge gold nanoparticles (MC-GNPs) by adjusting the surface ligand composition. With proper surface ligand composition, the MC-GNPs in four different sizes that all exhibited aggregation at tumor acidic pH were obtained. The biggest MC-GNPs showed the most encouraging aggregation-enhanced photothermal efficacy in vitro when they formed aggregates. The mixed-charge self-assembled monolayers were then proved as a facile method to design pH-induced aggregation of large gold nanoparticles for better NIR photothermal cancer therapy.

Mixed-charge nanoparticles for long circulation, low reticuloendothelial system clearance, and high tumor accumulation.

Mixed-charge zwitterionic surface modification shows great potential as a simple strategy to fabricate nanoparticle (NP) surfaces that are nonfouling. Here, the in vivo fate of 16 nm mixed-charge gold nanoparticles (AuNPs) is investigated, coated with mixed quaternary ammonium and sulfonic groups. The results show that mixed-charge AuNPs have a much longer blood half-life (≈30.6 h) than do poly(ethylene glycol) (PEG, M¯w = 2000) -coated AuNPs (≈6.65 h) and they accumulate in the liver and spleen far less than do the PEGylated AuNPs. Using transmission electron microscopy, it is further confirmed that the mixed-charge AuNPs have much lower uptake and different existing states in liver Kupffer cells and spleen macrophages one month after injection compared with the PEGylated AuNPs. Moreover, these mixed-charge AuNPs do not cause appreciable toxicity at this tested dose to mice in a period of 1 month as evidenced by histological examinations. Importantly, the mixed-charge AuNPs have higher accumulation and slower clearance in tumors than do PEGylated AuNPs for times of 24-72 h. Results from this work show promise for effectively designing tumor-targeting NPs that can minimize reticuloendothelial system clearance and circulate for long periods by using a simple mixed-charge strategy.

Mussel-inspired polydopamine: a biocompatible and ultrastable coating for nanoparticles in vivo.

Bioinspired polydopamine (PDA) has served as a universal coating to nanoparticles (NPs) for various biomedical applications. However, one remaining critical question is whether the PDA shell on NPs is stable in vivo. In this study, we modified gold nanoparticles (GNPs) with finely controlled PDA nanolayers to form uniform core/shell nanostructures (GNP@PDA). In vitro study showed that the PDA-coated GNPs had low cytotoxicity and could smoothly translocate to cancer cells. Transmission electron microscopy (TEM) analysis demonstrated that the PDA nanoshells were intact within cells after 24 h incubation. Notably, we found the GNP@PDA could partially escape from the endosomes/lysosomes to cytosol and locate close to the nucleus. Furthermore, we observed that the PDA-coated NPs have very different uptake behavior in two important organs of the liver and spleen: GNP@PDA in the liver were mainly uptaken by the Kupffer cells, while the GNP@PDA in the spleen were uptaken by a variety of cells. Importantly, we proved the PDA nanoshells were stable within cells of the liver and spleen for at least six weeks, and GNP@PDA did not show notable histological toxicity to main organs of mice in a long time. These results provided the direct evidence to support that the PDA surface modification can serve as an effective strategy to form ultrastable coatings on NPs in vivo, which can improve the intracellular delivery capacity and biocompatibility of NPs for biomedical application.

Multilayers based on cationic nanocomplexes for co-delivery of doxorubicin and DNA.

Co-delivery of drugs and genes has synergistic advantages in many fields such as cancer treatments. In this study, we present a multilayers for co-delivery of doxorubicin (DOX) and DNA. Ferrocene-modified polyethyleneimine (PEI-Fc) is synthesized, and it can form micelles in solution with ferrocene core and PEI shell. DOX and DNA are thus incorporated into the core and shell of the micelles, respectively, to form the PEI-Fc-DOX-DNA nanocomplexes. Such cationic nanocomplexes are used to construct multilayers through layer-by-layer assembly with negatively charged dextran sulfate (DS). We show that the multilayers can release DOX, and the release can be significantly enhanced in a hydrogen peroxide condition. Moreover, the multilayers have the ability to transfect cells through a substrate-mediated mode. The (PEI-Fc-DOX-DNA/DS) multilayers can be potentially applied to the biomedical devices for cancer treatment, regenerative medicine, etc.

Enhanced retention and cellular uptake of nanoparticles in tumors by controlling their aggregation behavior.

Effective accumulation of nanoparticles (NPs) in tumors is crucial for NP-assisted cancer diagnosis and treatment. With the hypothesis that aggregation of NPs stimulated by tumor microenvironment can be utilized to enhance retention and cellular uptake of NPs in tumors, we designed a smart NP system to evaluate the effect of aggregation on NPs' accumulation in tumor tissue. Gold nanoparticles (AuNPs, ~16 nm) were facilely prepared by surface modification with mixed-charge zwitterionic self-assembled monolayers, which can be stable at the pH of blood and normal tissues but aggregate instantly in response to the acidic extracellular pH of solid tumors. The zwitterionic AuNPs exhibited fast, ultrasensitive, and reversible response to the pH change from pH 7.4 to pH 6.5, which enabled the AuNPs to be well dispersed at pH 7.4 with excellent stealth ability to resist uptake by macrophages, while quickly aggregating at pH 6.5, leading to greatly enhanced uptake by cancer cells. An in vivo study demonstrated that the zwitterionic AuNPs had a considerable blood half-life with much higher tumor accumulation, retention, and cellular internalization than nonsensitive PEGylated AuNPs. A preliminary photothermal tumor ablation evaluation suggested the aggregation of AuNPs can be applied to cancer NIR photothermal therapy. These results suggest that controlled aggregation of NPs sensitive to tumor microenvironment can serve as a universal strategy to enhance the retention and cellular uptake of inorganic NPs in tumors, and modifying NPs with a mixed-charge zwitterionic surface can provide an easy way to obtain stealth properties and pH-sensitivity at the same time.

Zwitterionic polycarboxybetaine coating functionalized with REDV peptide to improve selectivity for endothelial cells.

Surface immobilization of bioactive molecules has been a promising strategy to develop in situ endothelialization for cardiovascular implants. With the aim to construct endothelial cell specific coating with low fouling property, zwitterionic carboxybetaine methacrylate and butyl methacrylate were copolymerized as coating materials, spin-coated onto substrates, and immobilized with endothelial cell selective peptide Arg-Glu-Asp-Val (REDV) through functionalization of carboxy groups in carboxybetaine by NHS/EDC chemistry. Experimental results proved that carboxybetaine-REDV coating maintained desirable antifouling ability and fine hemocompatibility. Separate culture and coculture of HUVECs (human umbilical vein endothelial cells) with HUASMCs (human umbilical artery smooth muscle cells) showed that the coating was able to enhance the competitive growth of endothelial cells while limiting the adhesion, proliferation, and migration of smooth muscle cells. The existence of zwitterionic carboxybetaine helps to screen undesirable adsorption of platelets, and its nonspecific resistance to smooth muscle cells contributes to the realization of endothelial cell selectivity.

Fast and long-acting antibacterial properties of chitosan-Ag/polyvinylpyrrolidone nanocomposite films.

Infection associated with medical devices is one of the most frequent complications of modern medical biomaterials. Preparation of antibacterial films on the medical devices is a great challenge owing to bactericidal efficiency, long acting and biocompatibility. In this study, silver nanoparticles (Ag NPs) doped chitosan/polyvinylpyrrolidone (PVP) films were successfully prepared by dip coating method. The nanocomposite films with spherical Ag NPs (diameters in 10-50 nm) were stable after being immersed in PBS for 35 days. Through regulating the concentration of AgNO3, the nanocomposite films showed good cell compatibility. The nanocomposite films could eliminate 100% Staphylococcus aureus (ATCC 6538) and Escherichia coli (ATCC 8739) in 5 min and had favorable long-acting antibacterial property. The increase of PVP amount obviously enhanced anti-adhesion activity of the nanocomposite film. Such nanocomposite films can be expected to have good potential in biomaterials applications.