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.

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.

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.

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.

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.

Mechanistic insights expatiating the biological role and regulatory implications of estrogen and HER2 in breast cancer metastasis.

Breast cancer (BCa) has become the leading cause of death in women worldwide. Irrespective of advancement in cancer treatments, e.g., surgery, radiation, chemotherapy, hormonal therapy, immunotherapy, and targeted therapy, recurrence leading to metastasis poses the greatest threat in BCa management. BCa receptors estrogen (ER), progesterone (PR), and human epidermal growth factor receptor-2 (HER2) hold significant reputations as prognostic and predictive biomarkers in therapeutic decision-making. Under normal physiological conditions, these receptors modulate critical biological functions, e.g., cell migration, proliferation, and apoptosis events, etc. However, aberrant expression causes deviations, triggering signaling course to adapt permanent switching "ON" mode. The later events induce rapid and unrestrained proliferation leading to cancer. As conventional ways of cancer management ultimately lead to resistance; therefore, recently targeted therapies have been extensively studied to conquer resistance. Targeting various small molecules in downstream signaling has become an area of interest in scientific society. The severity of cancer converts many folds soon after it takes on a migratory approach that eventually commences metastasis. Cancer migration comprises protrusion of cytoplasm at the leading edge of the migration forward-facing, establishing adhesions with the basic cell-matrix, disassembly of the adhesions at the back end of the cell, and actin-myosin fiber contractions to pull the bulk of the cytoplasm forward. On the other hand, metastatic progression comprises a cascade of events, including invasion, migration, and establishment of tumor microenvironment. The progression of BCa from early stage to metastatic development causes remarkable heterogeneity. Interference at any explicit level could hamper the process, and it has thus become an area of interest for scientists. Metastasis is the ultimate cause of spreading tumor cells to invade distant organs. Recently small molecule inhibitors of protein tyrosine kinases, which can cross the blood-brain barrier, have become a center point of research for investigators in developing novel treatment strategies against BCa management.

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.

Tyrosine kinase inhibitors and their unique therapeutic potentialities to combat cancer.

Cancer is one of the leading causes of death with a mortality rate of 12%. Although significant progress has been achieved in cancer research, the effective treatment of cancer remains the greatest global challenge in medicine. Dysregulation of tyrosine kinases (TK) is one of the characteristics of several types of cancers. Thus, drugs that target and inhibit these enzymes, known as TK inhibitors (TKIs), are considered vital chemotherapeutics to combat various types of cancer. The oral bioavailability of available TKIs and their targeted therapy are their potential benefits. Based on these characteristics, most TKIs are included in first/second-line therapy for the treatment of different cancers. This review aims to shed light on orally-active TKIs (natural and synthetic molecules) and their promising implication in the therapy of numerous types of tumors along with their mechanisms of action. Further, recent progress in the development of synthetic and isolation of natural TKIs is reviewed. A significant growth in research regarding the development of new-generation TKIs is made with time (23 FDA-approved TKIs from 2018) due to their better therapeutic response. Oral bioavailability should be considered as an important parameter while developing of new-generation TKIs; however, drug delivery systems can also be used to address issue of poor bioavailability to a certain extent. Moreover, clinical trials should be designed in consideration of the development of resistance and tumor heterogeneity.

Microneedles in Smart Drug Delivery.

Significance: In biomedical setup, at large, and drug delivery, in particular, transdermal patches, hypodermal needles, and/or dermatological creams with the topical appliance are among the most widely practiced routes for transdermal drug delivery. Owing to the stratum corneum layer of the skin, traditional drug delivery methods are inefficient, and the effect of the administered therapeutic cues is limited. Recent Advances: The current advancement at the microlevel and nanolevel has revolutionized the drug delivery sector. Particularly, various types of microneedles (MNs) are becoming popular for drug delivery applications because of safety, patient compliance, and smart action. Critical Issues: Herein, we reviewed state-of-the-art MNs as a smart and sophisticated drug delivery approach. Following a brief introduction, the drug delivery mechanism of MNs is discussed. Different types of MNs, that is, solid, hollow, coated, dissolving, and hydrogel forming, are discussed with suitable examples. The latter half of the work is focused on the applied perspective and clinical translation of MNs. Furthermore, a detailed overview of clinical applications and future perspectives is also included in this review. Future Directions: Regardless of ongoing technological and clinical advancement, the focus should be diverted to enhance the efficacy and strength of MNs. Besides, the possible immune response or interference should also be avoided for successful clinical translation of MNs as an efficient drug delivery system.

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.

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.

Endogenous and Exogenous Stimuli-Responsive Drug Delivery Systems for Programmed Site-Specific Release.

In this study, we reviewed state-of-the-art endogenous-based and exogenous-based stimuli-responsive drug delivery systems (DDS) for programmed site-specific release to overcome the drawbacks of conventional therapeutic modalities. This particular work focuses on the smart chemistry and mechanism of action aspects of several types of stimuli-responsive polymeric carriers that play a crucial role in extracellular and intracellular sections of diseased tissues or cells. With ever increasing scientific knowledge and awareness, research is underway around the globe to design new types of stimuli (external/internal) responsive polymeric carriers for biotechnological applications at large and biomedical and/or pharmaceutical applications, in particular. Both external/internal and even dual/multi-responsive behavior of polymeric carriers is considered an essential element of engineering so-called 'smart' DDS, which controls the effective and efficient dose loading, sustained release, individual variability, and targeted permeability in a sophisticated manner. So far, an array of DDS has been proposed, developed, and implemented. For instance, redox, pH, temperature, photo/light, magnetic, ultrasound, and electrical responsive DDS and/or all in all dual/dual/multi-responsive DDS (combination or two or more from any of the above). Despite the massive advancement in DDS arena, there are still many challenging concerns that remain to be addressed to cover the research gap. In this context, herein, an effort has been made to highlight those concerning issues to cover up the literature gap. Thus, the emphasis was given to the drug release mechanism and applications of endogenous and exogenous based stimuli-responsive DDS in the clinical settings.

Redox-responsive nano-carriers as tumor-targeted drug delivery systems.

With ever increasing scientific knowledge and awareness, research is underway around the globe to design new types of stimuli (external/internal) responsive nano-carriers for biotechnological applications at large and biomedical/pharmaceutical in particular. Based on literature evidence, stimuli-responsive carriers have been classified into four major categories, i.e. (1) physical, (2) chemical, (3) biological, and (4) dual (combination of any of the first three classes). Among various types, redox-responsive nano-carriers are of supreme interests and discussed here in this review. The difference in redox potential in tumor and normal tissue is considered as a potential target for tumor targeting leading to the development of redox-responsive drug delivery systems (DDS). In this regard, a high concentration of glutathione in tumor/intracellular environment has extensively been exploited. Disulfide bonds were found as a promising tool for designing redox-responsive which tend to cleave in a reductive environment forming sulfhydryl groups. Many nano-carriers have been explored widely to control tumor growth. These systems were used against the tumor xenograft animal model and showed improved tumor targeting with tumor growth inhibition. Herein, an effort has been made to summarize various aspects from design to development of numerous types of redox-responsive DDS including liposomes, micelles, nanoparticles, nanogel and prodrug based nanomedicines. An emphasis is also given on various types of nano-carriers with special reference to the tumor-targeted drug delivery applications. Also, dual responsive nano-carriers (in addition to redox-responsive) have also been briefly discussed. Towards the end of the chapter, the information is also given on their future perspectives.

Spectrophotometric method for the determination of Gemifloxacin mesylate in pure and tablet dosage form.

A spectrophotometric method for the determination of Gemifloxacin mesylate (GFX) is developed and validated according to ICH guidelines. GFX is a fluoroquinolone that is used in the treatment of pneumonia. The analysis of the pure drug was carried out at its λmax 270 nm. The method was linear from 0.5-5µg/mL, r(2) 0.999 and equation is 0.102-0.000. The % RSD for inter-day (0.969%) and intra-day (0.714%) assuring a good precision and accuracy was close to 100%. Limit of detection and Limit of quantification were 0.197 and 0.599µg/mL, respectively. The validation results and statistical data demonstrate that the method is accurate, sensitive, cost effective and reproducible and has an importance in quality assurance of GFX analysis. The developed method was proved suitable for analysis of GFX in the pure and tablet dosage forms without interference of excepients.