Antibacterial and angiogenic potential of iron oxide nanoparticles-stabilized acrylate-based scaffolds for bone tissue engineering applications.

Pickering emulsion polymerization, stabilized by inorganic nanoparticles such as iron oxide nanoparticles (IONPs), can be used to fabricate scaffolds with the desired porosity and pore size. These nanoparticles create stable emulsions that can be processed under harsh polymerization conditions. IONPs, apart from serving as an emulsifier, impart beneficial bioactivities such as antibacterial and pro-angiogenic activity. Here, we coated IONPs with three different weights of oleic acid (5.0 g, 7.5 g, and 10.0 g) to synthesize oleic acid-IONPs (OA-IONPs) that possess the desired hydrophobicity (contact angle > 100°). Next, glycidyl methacrylate and trimethylolpropane triacrylate were polymerized using the Pickering emulsion polymerization technique stabilized by the OA-IONPs. The physicochemical properties of the resulting porous scaffolds were thoroughly characterized using scanning electron microscopy (SEM), thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FT-IR), vibrating sample magnetometry (VSM), and a universal testing machine (UTM). The SEM images confirmed the formation of a porous scaffold. The IONPs content, measured using inductively coupled plasma mass spectrometry (ICP-MS), was in the range of 22-26 µg/mg of the scaffold. The mechanical strengths of the scaffolds were in the range of cancellous bone. The degradation profile of the scaffolds varied between 29% and 41% degradation over 30 days. In vitro cytotoxicity studies conducted using the fibroblast (L929) and osteosarcoma (MG-63) cell lines proved that these scaffolds were non-toxic. SEM images showed that the MG-63 cells adhered firmly to the scaffolds and exhibited a well-spread morphology. The antibacterial activity was confirmed by percentage inhibition studies, SEM analysis of bacterial membrane distortion, and reactive oxygen species (ROS) generation in the bacteria. Chick chorioallantoic membrane assay showed that the total vessel length and branch points were significantly increased in the presence of the scaffolds. These results confirm the pro-angiogenic potential of the fabricated scaffolds. The physicochemical, mechanical, and biological properties of the material suggest that the developed scaffolds would be suitable for bone tissue engineering applications.

Biomimetic ion substituted and Co-substituted hydroxyapatite nanoparticle synthesis using Serratia Marcescens.

Biomimicry is becoming deep-rooted as part of bioceramics owing to its numerous functional advantages. Naturally occurring hydroxyapatite (HA) apart from primary nano structures are also characterised by various ionic substitutions. The ease of accommodating such key elements into the HA lattice is known to enhance bone healing properties of bioceramics. In this work, hydroxyapatite synthesized via biomimetic approach was substituted with individual as well as multiple cations for potential applications in bone repair. Ion substitutions of Sr, Mg and Zn was carried out on HA for the first time by using Serratia grown in a defined biomineralization medium. The individual ions of varying concentration substituted in Serratia HA (SHA) (Sr SHA, Mg SHA and Zn SHA) were analysed for crystallinity, functional groups, morphology and crystal size. All three showed decreased crystallinity, phase purity, large agglomerated aggregates and needle-shaped morphologies. Fourier transform infrared spectroscopy (FTIR) spectra indicated increased carbonate content of 5.8% resembling that of natural bone. Additionally, the reduced O-H intensities clearly portrayed disruption of HA lattice and subsequent ion-substitution. The novelty of this study lies primarily in investigating the co-substitution of a combination of 1% Sr, Zn and Mg in SHA and establishing the associated change in bone parameters. Scanning electron microscope (SEM) and transmission electron microscope (TEM) images clearly illustrated uniform nano-sized agglomerates of average dimensions of 20-50 nm length and 8-15 nm width for Sr SHA; 10-40 nm length and 8-10 nm width for both Zn SHA and Mg SHA and 40-70 nm length and 4-10 nm width in the case of 1% Sr, Zn, Mg SHA. In both individual as well as co-substitutions, significant peak shifts were not observed possibly due to the lower concentrations. However, cell volumes increased in both cases due to presence of Sr2+ validating its dominant integration into the SHA lattice. Rich trace ion deposition was presented by energy dispersive X-ray spectroscopy (EDS) and quantified using inductively coupled plasma optical emission spectrometer (ICP-OES). In vitro cytotoxicity studies in three cell lines viz. NIH/3T3 fibroblast cells, MG-63 osteosarcoma cells and RAW 264.7 macrophages showed more than 90% cell viability proving the biocompatible nature of 1% Sr, Zn and Mg in SHA. Microbial biomineralization by Serratia produced nanocrystals of HA that mimicked "bone-like apatite" as evidenced by pure phase, carbonated groups, reduced crystallinity, nano agglomerates, variations in cell parameters, rich ion deposition and non-toxic nature. Therefore ion-substituted and co-substituted biomineralized nano SHA appears to be a suitable candidate for applications in biomedicine addressing bone injuries and aiding regeneration as a result of its characteristics close to that of the human bone.

Cancer nanomedicine: a review of nano-therapeutics and challenges ahead.

Cancer is known as the most dangerous disease in the world in terms of mortality and lack of effective treatment. Research on cancer treatment is still active and of great social importance. Since 1930, chemotherapeutics have been used to treat cancer. However, such conventional treatments are associated with pain, side effects, and a lack of targeting. Nanomedicines are an emerging alternative due to their targeting, bioavailability, and low toxicity. Nanoparticles target cancer cells via active and passive mechanisms. Since FDA approval for Doxil®, several nano-therapeutics have been developed, and a few have received approval for use in cancer treatment. Along with liposomes, solid lipid nanoparticles, polymeric nanoparticles, and nanoemulsions, even newer techniques involving extracellular vesicles (EVs) and thermal nanomaterials are now being researched and implemented in practice. This review highlights the evolution and current status of cancer therapy, with a focus on clinical/pre-clinical nanomedicine cancer studies. Insight is also provided into the prospects in this regard.

Mussel-Inspired Adhesive Hydrogels Based on Laponite-Confined Dopamine Polymerization as a Transdermal Patch.

Transdermal patch for local drug delivery has attained huge attention as an attractive alternative to existing drug delivery techniques as it is painless and user-friendly. However, most adhesive hydrogels either do not have adequate adhesion with the skin or cause discomfort while being removed from the skin surface due to excessive adhesion. To address this challenge, we developed an adhesive hydrogel based on laponite-confined dopamine polymerization as a transdermal patch. Laponite RDS nanoclay was used to control the hydrogel's viscous behavior and dopamine polymerization. The laponite polymerized polydopamine (l-PDA) was incorporated into poly(vinyl alcohol) (PVA) to make the PVA-l-PDA hydrogel. The laponite-confined polymerization improved the hydrogels' water contact angle and adhesion strength. The adhesion strength of the PVA-l-PDA hydrogel was adequate to adhere to the evaluated goat skin, glass, and polypropylene surfaces. Notably, the PVA-l-PDA hydrogel was easy to peel off from the skin. Further, we evaluated the drug release profile in goat skin using lidocaine as a model drug. We observed the controlled release of lidocaine from the PVA-l-PDA hydrogel compared to the PVA-PDA hydrogel. In addition, the nanoclay-confined adhesive hydrogel did not show any cytotoxic effect in fibroblasts. Altogether, PVA-l-PDA hydrogels offer appropriate adhesive strength, toughness, and biocompatibility. Thus, the PVA-l-PDA hydrogel has the potential to be an efficient transdermal patch.

Modular amphiphilic poly(aryl ether)-based supramolecular nanomicelles: an efficient endocytic drug carrier.

A rationally designed amphiphilic poly(aryl ether)-based dendrimer self-assembles into nanomicelles and exhibits tunable morphology upon varying the hydrophilic chain length. The 30 nm-sized dendrimer nanomicelles successfully entrapped Doxorubicin, demonstrated the sustained release of Doxorubicin and can successfully penetrate cancer cells through caveolae-dependent endocytosis, compared to the free drug.

Gelatin grafted poly(D,L-lactide) as an inhibitor of protein aggregation: An in vitro case study.

Amyloids are a group of proteins that are capable of forming aggregated amyloid fibrils, which is responsible for many neurodegenerative diseases including Alzheimer's disease (AD). In our previous study, synthesis and characterization of star-shaped poly(D,L-lactide)-b-gelatin (ss-pLG) have been reported. In the present work, we have extended our work to study ss-pLG against protein aggregation. To the best of our knowledge, this is the first report on the inhibition of amyloid fibrillation by protein grafted poly(D,L-lactide). Bovine serum albumin (BSA) was chosen as the model protein, which readily forms fibril under high temperature. We found that ss-pLG efficiently suppressed the fibril formation of BSA compared with gelatin (Gel), which was supported by Thioflavin T assay, circular dichroism (CD) spectroscopy and atomic force microscopy (AFM). In addition, ss-pLG significantly curtailed amyloid-induced hemolysis. We also found that incubation of ss-pLG with neuroblastoma cells (MC65) protected the cells from fibril-induced toxicity. The rescuing efficiency of ss-pLG was better than Gel, which could be attributed to the reduced lamella thickness in branched ss-pLG. These results suggest the significance of gelatin grafting, which probably allows gelatin to interact with the key residues of the amyloidogenic core of BSA effectively.

Design and evaluation of Konjac glucomannan-based bioactive interpenetrating network (IPN) scaffolds for engineering vascularized bone tissues.

Synthetic bone grafts are being developed to overcome the limitations of conventional treatments for bone defects. In this study, we have fabricated bioactive binary and novel ternary interpenetrating polymer network (IPN) scaffolds using a combination of natural and synthetic polymers. The binary IPN scaffolds were prepared using Konjac glucomannan (KGM) and polyvinyl alcohol (PVA). In the novel ternary IPN scaffolds, polycaprolactone (PCL) was added to PVA and KGM. SEM images showed that these scaffolds were microporous with good interconnectivity. Compression testing confirmed that both the scaffolds are mechanically strong, with the ternary scaffolds having moduli comparable to the natural bone. In vitro cytocompatibility studies performed with NIH/3T3 fibroblasts cells and MG-63 osteosarcoma cells demonstrated the non-toxic and osseointegrating nature of the scaffolds. Confocal images confirmed that the cells migrated into the interconnected pores of the scaffolds. RT-PCR analysis showed that both binary and ternary scaffolds enhanced the expression of the major bone marker genes, viz., ALP, BMP-2, COLLAGEN-1, and OSTEOCALCIN. However, the expression of these osteogenic markers was significantly enhanced in the ternary scaffolds compared to the binary scaffolds. In vivo chick chorioallantoic membrane (CAM) assay shows that these scaffolds possess excellent pro-angiogenic properties. Hence, these desirable biological properties, coupled with the suitable physicochemical properties, make these IPN scaffolds ideal for treating bone defects.

Cysteine immobilisation on the polyethylene terephthalate surfaces and its effect on the haemocompatibility.

Nitric oxide (NO) is an important signalling molecule involved in haemostasis. NO, present as endogenous S-nitrosothiols, is released by cysteine through a transnitrosation reaction. To exploit this mechanism, cysteine was immobilised onto the different carboxylated polyethylene terephthalate (PET) surfaces using 1-step EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) crosslinking mechanism. Immobilised cysteine concentration and NO release were dependent on the surface carboxyl density. Stability studies showed that the immobilised cysteine concentration and NO release reduced within 6 h. Immobilisation of cysteine derivatives eliminated the possibility of formation of polycysteine and its electrostatic interaction with the carboxylated PET. The immobilised cysteine concentration did not recover after DTT treatment, eliminating the possibility of disulphide bond formation. Further, cysteine was immobilised using a 2-step EDC crosslinking mechanism. Although the cysteine concentration reduced during stability studies, it recovered upon DTT treatment, indicating that cysteine forms amide bonds with the carboxylated PET and the observed decrease in cysteine concentration is probably due to the formation of disulphide bonds. The haemocompatibility of the cysteine immobilised PET surfaces showed similar results compared to the carboxylated PET. The loss of thiol groups due to the disulphide bond restricts the transnitrosation reaction. Hence, these materials can be used primarily in short-term applications.

Kinetic study of NTPDase immobilization and its effect of haemocompatibility on polyethylene terephthalate.

Poor haemocompatibility of material surfaces is a serious limitation that can lead to failure of blood-contacting devices and implants. In this work, we have improved the haemocompatibility of polyethylene terephthalate (PET) surfaces by immobilizing apyrase/ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) on to the carboxylated PET. NTPDase immobilized PET surfaces scavenge the ADP released by activated platelets, which prevents further platelet activation and aggregation. The surface properties of the modified PET were characterized by scanning electron microscope (SEM), energy dispersive X-ray spectroscopy (EDAX), and contact angle measurement. The enzyme attachment and stability on the modified PET surfaces were evaluated. The kinetics of free enzyme and immobilized enzyme were studied and fitted using the Michaelis-Menten kinetic model. Both free and immobilized NTPDase followed Michaelis-Menten kinetics with similar Michaelis-Menten constants (Km). This suggests that the activity of NTPDase was unchanged upon immobilization. Protein adsorption and %hemolysis was significantly reduced for carboxylated PET and NTPDase immobilized PET surfaces compared to unmodified PET. Lactate dehydrogenase assay showed that the number of adhered platelets reduced by more than an order of magnitude for the NTPDase immobilized PET surface compared to unmodified PET. These results clearly indicate that NTPDase immobilization significantly enhances the haemocompatibility of PET surfaces.

Mechanistic Study on the Antibacterial Activity of Self-Assembled Poly(aryl ether)-Based Amphiphilic Dendrimers.

The increased threat of bacterial resistance against conventional antibiotics has warranted the need for development of membrane targeting antibacterial agents. Several self-assembled cationic amphiphiles with different supramolecular structures have been reported in recent years for potent antibacterial activity with increased specificity. In this study, we describe the self-assembly and antibacterial activity of four lower generation poly(aryl ether)-based amphiphilic dendrimers (AD-1, AD-2, AD-3, and AD-4) containing terminal amine (PAMAM-based), ester, and hydrazide functional groups with varied hydrophobicity. Among the four dendrimers under study, the amine-terminated dendrimer (AD-1) displayed potent antibacterial activity. The ratio of surface cationic charge to hydrophobicity had a significant effect on the antibacterial activity, where AD-3 dendrimer with increased surface cationic charges exhibited a higher minimum inhibitory concentration (MIC) than AD-1. AD-2 (ester terminated) and AD-4 (hydrazide terminated) dendrimers did not show any bactericidal activity. The amphiphilic dendrimer-bacteria interactions, further validated by binding studies, also showed significant changes in bacterial morphology, effective membrane permeation, and depolarization by AD-1 in comparison with AD-3. Molecular dynamics simulations of AD-1 and AD-3 on bacterial membrane patches further corroborated the experimental findings. The structural conformation of AD-1 dendrimer facilitated increased membrane interaction compared to AD-3 dendrimer. AD-1 also displayed selectivity to bacterial membranes over fibroblast cells (4× MIC), corroborating the significance of an optimal hydrophobicity for potent antibacterial activity with no cytotoxicity. The self-assembled (poly(aryl ether)-PAMAM-based) dendrimer (AD-1) also exhibited potent antibacterial activity in comparison with conventional higher generation dendrimers, establishing the implication of self-assembly for bactericidal activity. Moreover, the detailed mechanistic study reveals that optimal tuning of the hydrophobicity of amphiphilic dendrimers plays a crucial role in membrane disruption of bacteria. We believe that this study will provide valuable insights into the design strategies of amphiphilic dendrimers as antibacterial agents for efficient membrane disruption.

Analysis of functionalized polyethylene terephthalate with immobilized NTPDase and cysteine.

Polyethylene terephthalate (PET) was functionalized to introduce carboxyl groups onto its surface by a carboxylation technique. Surface and bulk properties, such as possible surface deterioration, surface roughness and the mechanical strength of the carboxylated polymers, were studied and compared with those of aminolyzed and hydrolyzed PET. Atomic force microscopy studies showed that unlike aminolysis and hydrolysis, which increased the surface roughness significantly due to cracking and pitting, the surface roughness of unmodified and carboxylated PET were comparable. While hydrolysis and aminolysis of PET resulted in significant loss of strength, tensile testing revealed that unmodified and carboxylated polymers had similar strength. The development of mechanically stable, functionalized PET would vastly improve the biomedical applications of this polymer. To understand the potential for improving biomedical applications, biologically active molecules, namely nucleoside triphosphate diphosphohydrolase (NTPDase) and cysteine, were immobilized on the carboxylated PET using amide bonds. NTPDase was also immobilized to aminolyzed PET using imine bonds, while cysteine was immobilized on aminolyzed PET using both imine and amide bonds. Attachment of NTPDase and cysteine was verified by analyzing the NTPDase activity and the cysteine surface concentration. The stability of these immobilizations was also studied.