Tailoring Drug Release Kinetics by the Design and Optimization of Substituents in Azobenzene Molecules in the Photosensitive Liposomal System.

A light-sensitive moiety, e.g., azobenzene, for the light-sensitive liposomal drug carrier has shown advantages as an advanced drug delivery system in site-specific smart therapy due to its reversible photoisomerization characteristics. In this work, a series of 4-position-cholesterol-functionalized azobenzene derivatives with 4'-position substituted pyridine, quinoline, isoquinoline, triethylamine, or ethylenediamine were synthesized, and the relationship between the molecular structure and drug release behaviors was clarified. We found that the charge and electrophilicity of substituents were two important factors (expressed as the characteristic time) that can precisely regulate the isomerization ratio in the liposomal system. There was an approximately linear correlation between the characteristic time of photoisomerization and the fitted first-order constant of photoinduced drug release rate. The photoinduced drug release could be achieved at the desired time and in an appropriate amount by tailoring the substituents at the 4'-position of azobenzene-cholesterol derivatives.

Prolongation of the degradation period and improvement of the angiogenesis of zein porous scaffolds in vivo.

Zein porous scaffolds modified with fatty acids have shown great improvement in mechanical properties and good cell compatibility in vitro, indicating the potential application as a bone tissue engineering substitute. The present study was conducted to systematically investigate whether the addition of fatty acids affects the short-term (up to 12 weeks) and long-term (up to 1 year) behaviors of scaffolds in vivo, mainly focusing on changes in the degradation period and inflammatory responses. Throughout the implantation period, no abnormal signs occurred and zein porous scaffolds modified with oleic acid showed good tolerance in rabbits, characterized by the growth of relatively more blood vessels in the scaffolds and only a slight degree of fibrosis histology. Moreover, the degradation period was prolonged from 8 months to 1 year as compared to the control. These results affirmed further that zein could be used as a new kind of natural biomaterial suitable for bone tissue engineering.

Nanostructured Polyaniline Coating on ITO Glass Promotes the Neurite Outgrowth of PC 12 Cells by Electrical Stimulation.

A conducting polymer polyaniline (PANI) with nanostructure was synthesized on indium tin oxide (ITO) glass. The effect of electrical stimulation on the proliferation and the length of neurites of PC 12 cells was investigated. The dynamic protein adsorption on PANI and ITO surfaces in a cell culture medium was also compared with and without electrical stimulation. The adsorbed proteins were characterized using SDS-PAGE. A PANI coating on ITO surface was shown with 30-50 nm spherical nanostructure. The number of PC 12 cells was significantly greater on the PANI/ITO surface than on ITO and plate surfaces after cell seeding for 24 and 36 h. This result confirmed that the PANI coating is nontoxic to PC 12 cells. The electrical stimulation for 1, 2, and 4 h significantly enhanced the cell numbers for both PANI and ITO conducting surfaces. Moreover, the application of electrical stimulation also improved the neurite outgrowth of PC 12 cells, and the number of PC 12 cells with longer neurite lengths increased obviously under electrical stimulation for the PANI surface. From the mechanism, the adsorption of DMEM proteins was found to be enhanced by electrical stimulation for both PANI/ITO and ITO surfaces. A new band 2 (around 37 kDa) was observed from the collected adsorbed proteins when PC 12 cells were cultured on these surfaces, and culturing PC 12 cells also seemed to increase the amount of band 1 (around 90 kDa). When immersing PANI/ITO and ITO surfaces in a DMEM medium without a cell culture, the number of band 3 (around 70 kDa) and band 4 (around 45 kDa) proteins decreased compared to that of PC 12 cell cultured surfaces. These results are valuable for the design and improvement of the material performance for neural regeneration.

Two cholesterol derivative-based PEGylated liposomes as drug delivery system, study on pharmacokinetics and drug delivery to retina.

In this study, two cholesterol derivatives, (4-cholesterocarbonyl-4'-(N,N,N-triethylamine butyloxyl bromide) azobenzene (CAB) and 4-cholesterocarbonyl-4'-(N,N-diethylamine butyloxyl) azobenzene (ACB), one of which is positively charged while the other is neutral, were synthesized and incorporated with phospholipids and cholesterol to form doxorubicin (DOX)-loaded liposomes. PEGylation was achieved by including 1,2-distearoyl-sn-glycero-3-phosphatiylethanol-amine-N-[methoxy-(polyethylene glycol)-2000 (DSPE-PEG2000). Our results showed that PEGylated liposomes displayed significantly improved stability and the drug leakage was decreased compared to the non-PEGylated ones in vitro. The in vivo study with rats also revealed that the pharmacokinetics and circulation half-life of DOX were significantly improved when liposomes were PEGylated (p < 0.05). In particular, the neutral cholesterol derivative ACB played some role in improving liposomes' stability in systemic circulation compared to the conventional PC liposome and the positively charged CAB liposome, with or without PEGylation. In addition, in the case of local drug delivery, the positively charged PEG-liposome not only delivered much more of the drug into the rats' retinas (p < 0.001), but also maintained much longer drug retention time compared to the neutral PEGylated liposomes.

Meningoencephalitis as an initial manifestation of neuromyelitis optica spectrum disorder.

Two patients presented with initial symptoms of headache and fever, and two weeks later had disturbance of consciousness. Cerebrospinal fluid (CSF) showed pleocytosis >500×10²/L. Magnetic resonance imaging (MRI) showed multiple brain lesions at sites of high aquaporin-4 (AQP-4) expression. Case 1 presented optic neuritis four years after the first attack and case 2 had symptoms of myelitis three weeks after headache. Serum AQP-4 antibody was positive in both cases, and the diagnosis of neuromyelitis optica spectrum disorder (NMOSD) was made. Accordingly, NMOSD can initially present as meningoencephalitis mimicking intracranial infection, and the characteristic MRI imaging is quite critical for differentiation.

In vitro/in vivoidentification of zein degraded peptides using HPLC-MS/MS and their safety evaluation.

The identification of degraded products of implanted scaffolds is desirable to avoid regulatory concerns.In vivoidentification of products produced by the degradation of natural protein-based scaffolds is complex and demands the establishment of a routine analytical method. In this study, we developed a method for the identification of peptides produced by the degradation of zein bothin vitroandin vivousing high performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS). Forin vitroexperiments, zein was degraded enzymatically and analyzed produced peptides.In vitrostudy showed cytocompatibility of peptides present in the hydrolysate of zein with no induction of apoptosis and cell senescence. Forin vivoexperiment, zein gels were prepared and subcutaneously implanted in rats. Peptides produced by the degradation of zein were identified and few were selected as targeted (unique peptides) and two peptides were synthesized as the reference sequence of these peptides. Further, peptide analysis using HPLC-MS/MS of different organs was performed after 2 and 8 weeks of implantation of zein gel in rats. It was found that zein-originated peptides were accumulated in different organs. QQHIIGGALF or peptides with same fractions were identified as unique peptides. These peptides were also found in control rats with regular rat feed, which means the degradation of implanted zein biomaterial produced food related peptides of non-toxic nature. Furthermore, hemotoxylin and eosin (H&E) staining exhibited normal features. Overall, zein degraded products showed cytocompatibility and did not induce organ toxicity, and QQHIIGGALF can act as a standard peptide for tracing and determining zein degradation. The study also provides the feasibility of complex analysis on identification and quantification of degradation products of protein-based scaffolds.

Water-responsive 4D printing based on self-assembly of hydrophobic protein "Zein" for the control of degradation rate and drug release.

Four-dimensional (4D) printing is a promising technology that provides solutions for compelling needs in various fields. Most of the reported 4D printed systems are based on the temporal shape transformation of printed subjects. Induction of temporal heterogenicity in functions in addition to shape may extend the scope of 4D printing. Herein, we report a 4D printing approach using plant protein (zein) gel inspired by the amyloid fibrils formation mechanism. The printing of zein gel in a specialized layered-Carbopol supporting bath with different water concentrations in an ethanol-water mixture modulates hydrophobic and hydrogen bonding that causes temporal changes in functions. The part of the construct printed in a supporting bath with higher water content exhibits higher drug loading, faster drug release and degradation than those printed in the supporting bath with lower water content. Tri-segment conduit and butterfly-shaped construct with two asymmetrical wings are printed using this system to evaluate biomedical function as nerve conduit and drug delivery system. 4D printed conduits are also effective as a drug-eluting urethral stent in the porcine model. Overall, this study extends the concept of 4D printing beyond shape transformation and presents an approach of fabricating specialized baths for 4D printing that can also be extended to other materials to obtain 4D printed medical devices with translational potential.

4D printed tri-segment nerve conduit using zein gel as the ink for repair of rat sciatic nerve large defect.

Zein has enormous potential for application in biomedical field due to biodegradation and biocompatibility, we have recently prepared zein gel as a possible 3D printing ink. Our previous studies found that the pore structure in zein material can reduce early inflammation, promote the polarization of macrophages toward the M2 phenotype, and accelerate nerve regeneration. To further explore the role of zein in nerve repair, we used 4D printing technique to create nerve conduits with zein protein gel, and designed 2 types of tri-segment conduits with different degradation rates. Structural parts printed in support baths with higher water content show faster degradation rates than those printed in support baths with lower water content. The conduits that degraded quickly at both ends and slowly in the middle (CB75-CB40-CB75) and the conduits that degraded slowly at both ends and quickly in the middle (CB40-CB75-CB40) were 4D printed, respectively. Animal experiments suggest that the CB75-CB40-CB75 conduit is better for nerve repair, which may be because its degradation pattern can match to the pattern of nerve regeneration better. Our new strategy through 4D printing indicated that fine modulation in conduit degradation can affect efficacy of nerve repair significantly.

Injectable zein gel with in situ self-assembly as hemostatic material.

Zein is a biocompatible and biodegradable corn protein with promising properties for biomedical applications. It is hydrophobic with the ability to self-assemble in an aqueous medium. It can also form a gel in hydroalcoholic solvents at higher concentrations. Few studies have investigated the biomedical significance of zein gels. Herein, we exploited the injectability and water-responsive increase in stiffness of zein gel to achieve hemostasis by physical blockage of the wound and clot formation. The release of components from the gel further aided blood clotting and gave a higher clot strength than a natural clot, which can prevent rebleeding. Rabbit aortic injury and swine femoral artery injury models were used to evaluate the hemostatic efficacy of the zein gel. Zein gel was effective in both hemostatic models without applying external compression due to an in situ increase in stiffness, while the control (Celox™ Gauze) required external compression at the wound site. The zein gel was easily removed after hemostasis due to hydrophobic self-assembly. Overall, zein gel is proposed as an effective hemostatic product for any wound shape owing to its good shape adaptability and rapid in situ blood-responsive stiffness increase.

Self-assembled zein organogels as in situ forming implant drug delivery system and 3D printing ink.

Recently, biomedical applications of organogels have been increasing; however, there is a demand for bio-based polymers. Here, we report self-assembled zein organogels in N-methyl pyrrolidone (NMP), Dimethyl sulfoxide (DMSO), and glycerol formal (GF). The gel formation was driven by the solvent's polarity and the hydrogen bonding component of Hansen Solubility Parameters was important in promoting gelation. Gels exhibited shear-thinning and thixotropic properties. Furthermore, water-induced self-assembly of zein allows mechanically robust in situ implant formation by solvent exchange. Ciprofloxacin was incorporated as a model drug and sustained release depending upon the solvent exchange rate was observed. In situ implants in agarose gel retained antibacterial efficacy against S. aureus for more than 14 days. Zein-based organogels were further applied as 3D printing ink and it was found that zein gel in DMSO had superior printability than gels prepared in NMP and GF. Using three solvents to prepare organogels can enable the encapsulation of various drugs and facilitate the preparation of composite gels with other biocompatible polymers. These organogel systems can further be used for developing 3D printed drug delivery systems or scaffolds for tissue engineering.

Preparation and evaluation of chitosan/polyvinylpyrrolidone/zein composite hemostatic sponges.

Trauma-related excessive bleeding is one of the leading causes of death. Chitosan (CS) sponges have unique advantages in the treatment of massive bleeding, but their application is limited by poor stability and toxic crosslinking agent. In this work, chitosan/polyvinylpyrrolidone/zein (CS/PVP/Zein) sponges with macroporous structure were prepared, which exhibited rapid water absorption capacity and water-triggered expanding property with low cytotoxicity and low hemolysis ratio. In vitro blood coagulation experiments showed that CS/PVP/Zein sponges could clot blood significantly faster than commercial surgical gauze. Further investigation of the hemostatic mechanism suggested that the CS/PVP/Zein sponges could accelerate coagulation by promoting attachment of erythrocytes, activation of platelets, and rapid plasma protein absorption. Prepared sponges were also found effective in the rat femoral artery transection model to control bleeding. Overall, the CS/PVP/Zein sponges exhibited the potential to control trauma-related hemorrhage.

Application of imaging techniques in pancreaticobiliary maljunction.

Imaging techniques are useful tools in the diagnosis and treatment of pancreaticobiliary maljunction (PBM). PBM is a precancerous lesion often relative to the disease of the pancreas and biliary tract, for example, cholecystolithiasis, protein plugs, and pancreatitis. For patients with PBM, early diagnosis and timely treatment are highly important, which is largely dependent on imaging techniques. The continuous development of imaging techniques, including endoscopic retrograde cholangiopancreatography, magnetic resonance cholangiopancreatography, computed tomography, ultrasound, and intraoperative cholangiography, has provided appropriate diagnostic and therapeutic tools for PBM. Imaging techniques, including non-invasive and invasive, have distinct advantages and disadvantages. The purpose of this paper is to review the application of various imaging techniques in the diagnosis and treatment of PBM.

Water-Soluble and Degradable Gelatin/Polyaniline Assemblies with a High Photothermal Conversion Efficiency for pH-Switchable Precise Photothermal Therapy.

Photothermal therapy (PTT) is regarded as one of the potential techniques to replace surgery in the treatment of tumors. Polyaniline (PANI) shows better biocompatibility than inorganic reagents, which has been widely used in tumor photoacoustic (PA) imaging and PTT. However, the poor water solubility and nonspecific aggregation of PANI nanoparticles severely restricted their biomedical application. In addition, it is difficult to control the photothermal effect just on cancer cells. Herein, we develop tumor pH-responsive PANI-Gel/Cu assemblies, which can achieve targeted and precise ablation of tumors. Due to the high hydrophilicity of gelatin, the PANI-Gel/Cu assemblies show excellent dispersion in physiological solutions and long-term stability. By taking advantage of the self-doping effect between the carboxyl groups in gelatin and the imine part of the PANI skeleton, the photothermal characteristics of PANI-Gel/Cu assemblies can be promoted effectively by the acid tumor microenvironment, and the PA imaging of PANI-Gel/Cu assemblies can also be activated by tumor pH. Consequently, both the PTT enhancement and PA signal amplification can be triggered under a tumor microenvironment, and PANI-Gel/Cu assemblies can be targeted to cancer cells with the RGD sequences in their gelatin skeleton. In vivo imaging-guided PTT to A549 cancer shows precise treatment with little harm to normal cells, and PANI-Gel/Cu assemblies can disassemble into tiny particles (<15 nm) under laser irradiation. This work overcomes the intrinsic limitation of PANI materials, i.e., poor water solubility and nonspecific aggregation, meanwhile providing a pH-active PANI-based platform for precise and effective ablation of cancer.

Zein-induced immune response and modulation by size, pore structure and drug-loading: Application for sciatic nerve regeneration.

Zein is a biodegradable material with great potential in biomedical applications. However, as a plant-derived protein material, body's immune response is the key factor to determine its clinical performance. Herein, for the first time, the zein-induced immune response is evaluated systemically and locally, comparing with typical materials including alginate (ALG), poly(lactic-co-glycolic) acid (PLGA) and polystyrene (PS). Zein triggers an early inflammatory response consistent with the non-degradable PS, but this response decreases to the same level of the biosafe ALG and PLGA with zein degradation. Changing sphere sizes, pore structure and encapsulating dexamethasone can effectively modulate the zein-induced immune response, especially the pore structure which also inhibits neutrophil recruitment and promotes macrophages polarizing towards M2 phenotype. Thus, porous zein conduits with high and low porosity are further fabricated for the 15 mm sciatic nerve defect repair in rats. The conduits with high porosity induce more M2 macrophages to accelerate nerve regeneration with shorter degradation period and better nerve repair efficacy. These findings suggest that the pore structure in zein materials can alleviate the zein-induced early inflammation and promote M2 macrophage polarization to accelerate nerve regeneration. STATEMENT OF SIGNIFICANCE: Zein is a biodegradable material with great potential in biomedical applications. However, as a plant protein, its possible immune response in vivo is always the key issue. Until now, the systemic study on the immune responses of zein in vivo is still very limited, especially as an implant. Herein, for the first time, the zein-induced immune response was evaluated systemically and locally, comparing with typical biomaterials including alginate, poly(lactic-co-glycolic) acid and polystyrene. Changing sphere sizes, pore structure and encapsulating dexamethasone could effectively modulate the zein-induced immune response, especially the pore structure which also inhibited neutrophil recruitment and promoted macrophages polarizing towards M2 phenotype. Furthermore, the pore structure in zein nerve conduits was proved to alleviate the early inflammation and promote M2 macrophage polarization to accelerate nerve regeneration.

In vivo evaluation of a novel dexamethasone-heparin-double-coated stent for inhibition of artery restenosis and thrombosis.

To evaluate the efficacy and safety of dexamethasone-heparin-double-coated stent (DHDCS) on inhibition of artery lumen reduction and neointimal hyperplasia in porcine model we carried out this study. Bare mental stents (BMS, n = 12), protein-coated stents (PCS, n = 12), heparin microballoon-coated stents (HMCS, n = 12), and DHDCS (n = 12), prepared by the spray drying method, were implanted into the selected internal iliac artery, external iliac artery, sacrococcygeal artery, and femoral artery of each of the selected pigs (n = 12), which were randomly divided into four groups on average. Thirty days and ninety days after the implantation, aorta angiography was performed on all the 12 mini-pigs to evaluate the artery lumen reduction. Subsequently, in order to analyze their histological appearance, the pigs were killed, and their arteries with the stents inside were taken out, embedded in plastic for hard histological section and hematoxylin-eosin (H.E.) staining, and examined by light microscopy and scanning electron microscopy (SEM). The artery lumen reduction and average neointimal hyperplasia in the group of DHDCS were significantly lesser than those in the other three groups of BMS, PCS, and HMCS. This study shows that DHDCS is capable of inhibiting the proliferation of intima and lumen area reduction of the target artery within stents, and effectively and safely reducing the incidence of regional thrombosis and restenosis for a short term.

Effects of the micro/nanostructure of electrospun zein fibres on cells in simulated blood flow environment.

In order to prevent thrombosis, reduce intima hyperplasia, and to maintain long-term patency after implantation of an artificial blood vessel, the formation of intact endothelial cells layer on an inner surface of graft is desirable. The present study aimed to improve endothelial cell adhesion by regulating the morphology of the inner surface of artificial blood vessels. Zein fibre membranes with three fibre diameters (small, ~100 nm; medium, ~500 nm; and large, ~1000 nm) were constructed by electrospinning. A flow chamber device was designed to simulate the blood flow environment. The morphology and adhesion of human umbilical vein fusion cells (EA.hy926) on the surface of the fibre membranes were studied under a shear stress of approximately 15 dynes/cm2. The results showed that oriented electrospun zein fibre surfaces with both medium- and large-diameter fibres can regulate the morphology of endothelial cells (EA.hy926), which are aligned by the fibre direction. The three fibre membranes improved the adhesion of endothelial cells significantly compared to that on the flat membrane. When the fibre direction was fixed parallel to the fluid direction, the medium-diameter oriented-fibre membrane could significantly improve the ability endothelial cells to resist shear stress, and there was a significant difference at 1, 2 and 4 h time points compared with the shear stress resistance on the small-diameter and large-diameter oriented-fibre membranes. When the fibre direction was perpendicular to the fluid direction, again the medium-diameter oriented-fibre membrane improved the ability of endothelial cells to resist shear stress significantly at 1 and 2 h time points. It was concluded that by changing the diameter and arrangement of electrospun fibres, cell morphology control and shear stress resistance can be achieved.

Improvement of mechanical properties of zein porous scaffold by quenching/electrospun fiber reinforcement.

As a novel bone substitute material, zein-based scaffolds (ZS) should have suitable mechanical properties and porosity. ZS has shown good compressive properties matching cancellous bone, but there is still a demand to improve its mechanical properties, especially tensile and bending properties without adding plasticizers. The present study explored two simple and environment-friendly factors for this purpose: fiber reinforcement and quenching. Addition of electrospun zein fibers enhanced all mechanical properties significantly including compressive, tensile, and bending moduli; compressive and bending strengths of ZS with both higher (70-80%) and lower (50-60%) porosities, no matter whether heating treated or not treated. Especially, all these parameters were further enhanced significantly by addition of heating treated fibers. AFM provided evidence that high temperature modification could significantly alter the micro-elastic properties of zein electrospun fibers, i.e., increased stiffness of fibers. Quenching treatment also enhanced compressive, tensile, and bending strengths significantly. Finally, quenching treated ZS were implanted into critical-sized bone defects (15 mm) of the rabbit model to compare the repair efficacy with a commercial ß-tricalcium phosphate product. The results demonstrated that there were no remarkable differences in bone reconstructions between these two materials.

Preparation of stretchable composite film and its application in skin burn repair.

The poor elasticity of wound dressings often leads to wound healing failure due to rupture and fall off. In this study, the composite films of zein and hydrogel poly (acrylic acid) were developed in order to obtain stretchable wound dressing for skin burn repair. The mechanical test revealed that the maximum elongation of break of composite films could reach 349.76% when the mass ratio of zein to poly (acrylic acid) was 1.5. SEM and FTIR analysis demonstrated the good elasticity of composite films might be due to the formation of a dense structure and the strong interaction between zein and poly (acrylic acid). Interestingly, the composite films exhibited great adhesiveness to human finger skin and stretchable ability under strenuous joint exercise. CCK-8 assay and fluorescence staining showed that the composite films and their extract had good cytocompatibility on human foreskin fibroblasts (L929) cells. The in vivo experiment on rat's skin burning model indicated that the composite films could promote wound healing and collagen synthesis by comparison with commercial gauze. It could be concluded that the stretchable composite films of zein and hydrogel poly (acrylic acid) had the potential as the wound dressing.

Preparation and evaluation of captopril loaded gastro-retentive zein based porous floating tablets.

In this study, gastro-retentive porous floating tablets of captopril based on zein are reported using l-menthol as a porogen. Tablets were prepared by the direct compression method. Removing of l-menthol through sublimation process generated pores in tablets, which decreased the density to promote floating over gastric fluid. Prepared tablets showed no floating lag time and prolong total floating time (>24 h). Drug release was found dependent upon porosity of tablets, an increase in porosity of tablets resulted in increased drug release, so it can be tuned by varying concentration of l-menthol. In addition to floating and sustained release properties, porous tablets showed robust mechanical behavior in wet conditions, which can enable them to withstand real gastric environment stress. In vivo studies using New Zealand rabbits also confirmed the prolonged gastric retention (24 h) and plasma drug concentration-time profile showed sustained release of captopril with higher Tmax and MRT as compared to marketed immediate-release tablets. Overall, it was concluded that effective gastric retention can be achieved using porous zein tablets using l-menthol as a porogen.

Preparation of ciprofloxacin loaded zein conduits with good mechanical properties and antibacterial activity.

Conduit scaffolds have potential applications in tissue engineering as nerve conduits, urological stent and blood vessel graft. Zein is a well-reported biopolymer in tissue engineering and drug delivery systems. Herein, we prepared ciprofloxacin loaded zein conduits using a facile rolling method. Zein conduits (ZCs) were evaluated for physical structure, porosity, bending stiffness, degradation, drug release, in vitro and in vivo antibacterial efficacy and cells toxicity. ZCs showed porous structure with porosity > 60 % and good mechanical strength with bending stiffness of 28.54 N.mm2. Slow enzymatic degradation (87 % in 30 days) was also observed for ZCs. Slow release of ciprofloxacin up to 42 days was observed that could assure prevention of post-implantation infection. In vitro and in vivo antibacterial study verified the short-time and long-time antibacterial efficacy of zein conduits on Gram-positive and Gram-negative bacteria. Live/dead measurement and CCK-8 assay on L929 cells demonstrated good cell compatibility for all zein conduits (>90 % cell viability and cells proliferation in 3 days). Overall, the rolling method could be exploited for preparation of ciprofloxacin loaded zein conduits, which had the potential for tissue engineering applications.