Mechanistic details of the membrane perforation and passive translocation of TAT peptides

The trans-activator of transcription (TAT) peptide is regarded as the “gold standard” for cell-penetrating peptides, capable of traversing a mammalian membrane passively into the cytosolic space. This characteristic has been exploited through conjugation of TAT for applications such as drug delivery. However, the process by which TAT achieves membrane penetration remains ambiguous and unresolved. Mechanistic details of TAT peptide action are revealed herein by using three complementary methods: quartz crystal microbalance with dissipation (QCM-D), scanning electrochemical microscopy (SECM) and atomic force microscopy (AFM). When combined, these three scales of measurement define that the membrane uptake of the TAT peptide is by trans-membrane insertion using a “worm-hole” pore that leads to ion permeability across the membrane layer. AFM data provided nanometre-scale visualisation of TAT punctuation using a mammalian-mimetic membrane bilayer. The TAT peptide does not show the same specificity towards a bacterial mimetic membrane and QCM-D and SECM showed that the TAT peptide demonstrates a disruptive action towards these membranes. This investigation supports the energy-independent uptake of the cationic TAT peptide and provides empirical data that clarify the mechanism by which the TAT peptide achieves its membrane activity. The novel use of these three biophysical techniques provides valuable insight into the mechanism for TAT peptide translocation, which is essential for improvements in the cellular delivery of TAT-conjugated cargoes including therapeutic agents required to target specific intracellular locations.

In vitro microenvironments to study breast cancer bone colonisation

Bone metastasis occurs frequently in patients with advanced breast cancer and is a major cause of morbidity and mortality in these patients. In order to advance current therapies, the mechanisms leading to the formation of bone metastases and their pathophysiology have to be better understood. Several in vitro models have been developed for systematic studies of interactions between breast cancer cells and the bone microenvironment. Such models can provide insights into the molecular basis of bone metastatic colonisation and also may provide a useful platform to design more physiologically relevant drug testing assays. This review describes different in vitro approaches and discusses their advantages and disadvantages.

In vitro modeling of the prostate cancer microenvironment

Prostate cancer is the most commonly diagnosed malignancy in men and advanced disease is incurable. Model systems are a fundamental tool for research and many in vitro models of prostate cancer use cancer cell lines in monoculture. Although these have yielded significant insight they are inherently limited by virtue of their two-dimensional (2D) growth and inability to include the influence of tumour microenvironment. These major limitations can be overcome with the development of newer systems that more faithfully recreate and mimic the complex in vivo multi-cellular, three-dimensional (3D) microenvironment. This article presents the current state of in vitro models for prostate cancer, with particular emphasis on 3D systems and the challenges that remain before their potential to advance our understanding of prostate disease and aid in the development and testing of new therapeutic agents can be realised.

The potential for production of freeze-dried oral vaccines using alginate hydrogel microspheres as protein carriers

Oral administration of dry vaccine formulations is acknowledged to offer major clinical and logistical benefits by eliminating the cold chain required for liquid preparations. A model antigen, bovine serum albumin (BSA) was encapsulated in alginate microspheres using aerosolisation. Hydrated microspheres 25 to 65 μm in size with protein loading of 3.3 % w/w were obtained. Environmental scanning electron microscopy indicated a stabilizing effect of encapsulated protein on alginate hydrogels revealed by an increase in dehydration resistance. Freeze drying of alginate microspheres without use of a cryoprotectant resulted in fragmentation and subsequent rapid loss of the majority of the protein load in simulated intestinal fluid in 2 h, whereas intact microspheres were observed following freeze-drying of BSA-loaded microspheres in the presence of maltodextrin. BSA release from freeze-dried preparations was limited to less than 7 % in simulated gastric fluid over 2 h, while 90 % of the protein load was gradually released in simulated intestinal fluid over 10 h. SDS-PAGE analysis indicated that released BSA largely preserved its molecular weight. These findings demonstrate the potential for manufacturing freeze-dried oral vaccines using alginate microspheres.

Single molecule microscopy in 3D cell cultures and tissues

From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy.

Borax Mediated Layer-by-Layer Self-Assembly of Neutral Poly(vinyl alcohol) and Chitosan

We report a multilayer film of poly(vinyl alcohol) (PVA)-borate complex and chitosan by using a layer-by-layer approach. PVA is an uncharged polymer, but hydroxyl functional groups of PVA can be crosslinked by using borax as a cross-linking agent. As a result electrostatic charges and intra- and interchain cross-links are introduced in the PVA chain and provide physically cross-linked networks. The PVA-borate was then deposited on a flat Substrate as well as on colloidal particles with chitosan as an oppositely charged polyelectrolyte. Quartz crystal microbalance. scanning electron microscopy, and atomic force microscopy were used to follow the growth of thin film oil flat substrate. Analogous experiments were performed on melamine formaldehyde colloidal particles (3-3.5 mu m) to quantify the process for the preparation of hollow rnicrocapsules. Removal of the core in 0.1 N HCI results in hollow microcapsules. Characterization of microcapsules by transmission electron microscopy revealed formation of stable microcapsules. Further, self-assembly of PVA-borate/chitosan was loaded with the anticancer drug doxorubicin, and release rates were determined at different pH Values to highlight the drug delivery potential of this system.

Metallomicelles as potent catalysts for the ester hydrolysis reactions in water

Synthetic amphiphiles have been employed for the investigation of diverse topics, e.g. membrane mimetics, drug delivery, ion sensing and even in certain separation processes. Metal-complexing amphiphiles comprise an interesting class of compounds possessing multiple utilities. Upon solubilization in water they form metallomicelles. For achieving specific catalysis of a variety of reactions, metallomicelles were utilized by applying the principles of coordination chemistry and self-organizing systems. Because of their certain similarities with the natural enzymes, metallomicelles were synthesized as catalysts for many reactions. In particular the metallomicelles play a catalytic role in reactions involving the hydrolysis of activated carboxylate esters, phosphate esters and amides at ambient conditions near neutral pH. Apart from the hydrolysis reactions, these were exploited to play pertinent role as Lewis acid catalysts in cycloaddition reactions, and in other reactions such as phenolic oxidation in presence of hydrogen peroxide. In this review we emphasize with the help of assorted examples, the design, synthesis of metal-complexing amphiphiles and their aggregation behavior leading to catalytic hydrolysis reactions in aqueous media.

Organized Nanostructures of Thermoresponsive Poly(N-isopropylacrylamide) Block Copolymers Obtained Through Controlled RAFT Polymerization

Kontrolloidut radikaalipolymerointimenetelmät, kuten RAFT-polymerointi, ovat moderni tapa valmistaa polymeerejä säädellysti. RAFT-polymeroinnilla polymeerien ketjunpituutta, moolimassajakaumaa, mikrorakennetta (taktisuus, järjestys), koostumusta ja funktionaalisuutta kyetään hallitsemaan. Siten menetelmällä voidaan valmistaa uudenlaisia polymeeriarkkitektuureja, kuten blokki- ja tähtipolymeerejä, sekä hybridimateriaaleja ja biokonjugaatteja. Polymeeristen rakennuspalikoiden itsejärjestyminen, missä huolellisesti syntetisoidut polymeerit järjestyvät halutulla tavalla nanoskaalassa, on suosittu tutkimuskohde materiaalitieteessä. On huomattava, että blokkipolymeerien itsejärjestyminen on vielä suhteellisen nuori tutkimusaihe. Tämän hetkiset polymeeriset nanomateriaalit ovat suhteellisen yksinkertaisia luonnon luomuksiin verrattuina, tarjoten jatkuvasti uusia mahdollisuuksia seuraavan sukupolven polymeereille. Tässä työssä RAFT-polymeroinnilla syntetisoitiin amfifiilisiä di- ja triblokkikopolymeerejä sekä tutkittiin niiden järjestymistä nanorakenteiksi. Kaikissa blokkikopolymeereissä käytettiin lämpöherkkää poly(N-isopropyyliakryyliamidia). Siten polymeerit ja tutkitut materiaalit reagoivat lämpötilanmuutokseen ympäristössä eli ovat ns. ympäristöherkkiä. Työssä tutkittiin taktisuuden kontrollointia N-isopropyyliakryyliamidin RAFT-polymeroinnissa. Polymeerin taktisuutta sekä ketjunpituutta ja blokkijärjestystä säätämällä voitiin hallita polymeerin itsejärjestymistä vesiliuoksessa. Amfifiiliset polymeerit järjestyivät laimeissa vesiliuoksissa erilaisiksi misellirakenteiksi, muodostaen ns. mikrosäiliöitä. Tällaisilla polymeereillä odotetaan olevan sovelluksia esim. lääkeainevapautuksessa. Amfifiilejä käytetään myös esimerkiksi apuaineina pinnoitteissa ja kosmetiikassa. Kiinteässä tilassa tutkitut triblokkikopolymeerit muodostivat teoreettisesti ennustettuja morfologioita. Lämpöherkän materiaalin hydrogeelit toimivat suodatinmembraanina nanokokoluokassa. RAFT-polymeroinnilla syntetisoituja polymeereja voidaan sellaisenaan käyttää kultananopartikkeleiden päällystämiseen. Kultananopartikkelit ovat erittäin kiinostavia mm. niiden stabiilisuuden ja ainutlaatuisten pintaominaisuuksien vuoksi. Kun amfifiilisiä polymeerejä kiinnitettiin kultapartikkelin pinnalle, sen liuos- ja optisia ominaisuuksia voitiin säädellä pH:n ja lämpötilan avulla. Tällaisilla kultananopartikkeleilla on sovelluksia mm. diagnostiikassa, sensoreina ja solukuvauksessa.

Layer-by-layer self-assembly of modified hyaluronic acid/chitosan based on hydrogen bonding

The fabrication of hydrogen bonded polymer self-assembly for drug delivery has been accomplished via layer-by-layer sequential assembly from aqueous solution. In this study, the self-assembly was constructed based on hydrogen bonding between DNA base (adenine and thymine) pairs substituted on the backbone of chitosan and hyaluronic acid. Chitosan was modified with adenine, whereas hyaluronic acid was modified with thymine. Subsequently, these two polymers were sequentially absorbed on flat substrate by taking advantage of interactions of DNA base pairs via hydrogen bonding. Interlayer hydrogen bonding of these two polymers produces stable multilayer film without using any cross-linking agent. Thin film formation on quartz substrate has been monitored with UV-vis spectra and an AFM study. Formation of multilayer hydrogen-bonded thin film has been further confirmed with SEM. Encapsulation and release behavior of the therapeutic drug from the multilayer thin film at different conditions has been illustrated using UV-vis spectra. Cell viability of modified polymers using MTT assay confirmed no cytotoxic effect.

Benefits and challenges of antivirulence antimicrobials at the dawn of the post-antibiotic era

In April 2014, the World Health Organization announced the beginning of a post-antibiotic era and declared antimicrobial resistance (AMR) a public health priority demanding global action. If no action is taken, by 2050 AMR will kill more people each year than cancer, with 10 million estimated annual deaths at a cost of $100 trillion to the global economy. New therapies to tackle multidrug resistant bacterial pathogens are urgently needed. Unlike traditional antibiotics, antivirulence drugs inhibit bacterial virulence instead of growth promising to offer a new class of superior therapeutics that will be ‘evolution-proof’ and ‘tailored-spectrum’. This mini-review discusses the latest emerging evidence on the promised benefits of antivirulence drugs over conventional antibiotics, also highlighting the challenges in evaluating these properties for each of the diverse virulence targets that are currently under investigation. The author argues that overcoming such challenges early in the development process constitutes an important step towards successfully progressing each of the expanding number of antivirulence strategies into next-generation therapies for common human and animal infections that are becoming increasingly refractory to all available antibiotics.

Platinum dendritic nanoparticles with magnetic behavior

Magnetic nanoparticles have attracted increasing attention for biomedical applications in magnetic resonance imaging, high frequency magnetic field hyperthermia therapies, and magnetic-field-gradient-targeted drug delivery. In this study, three-dimensional (3D) platinum nanostructures with large surface area that features magnetic behavior have been demonstrated. The well-developed 3D nanodendrites consist of plentiful interconnected nano-arms ∼4 nm in size. The magnetic behavior of the 3D dendritic Pt nanoparticles is contributed by the localization of surface electrons due to strongly bonded oxygen/Pluronic F127 and the local magnetic moment induced by oxygen vacancies on the neighboring Pt and O atoms. The magnetization of the nanoparticles exhibits a mixed paramagnetic and ferromagnetic state, originating from the core and surface, respectively. The 3D nanodendrite structure is suitable for surface modification and high amounts of drug loading if the transition temperature was enhanced to room temperature properly.

Effect of Polymer Grafting on the Bilayer Gel to Liquid-Crystalline Transition

Grafted polymers oil the surface of lipid membranes have potential applications in liposome-based drug delivery and Supported membrane systems. The effect of polymer grafting on the phase behavior of bilayers made up of single-tail lipids is investigated using dissipative particle dynamics. The bilayer is maintained in a tensionless state using a barostat. Simulations are carried Out by varying the grafting fraction, G(f), defined as the ratio of the number of polymer molecules to the number of lipid molecules, and the length of the lipid tails. At low G(f), the bilayer shows I sharp transition from the gel (L-beta) to the liquid-crystalline (L-alpha) phase. This main melting transition temperature is lowered as G(f) is increased, and above a critical value of G(f), the interdigitated L-beta I phase is observed prior to the main transition. The temperature range over which the intermediate phases are observed is a function of the lipid tail length and G(f). At higher grafting fractions, the presence of the L-beta I, phase is attributed to the increase in the area per head group due to the lateral pressure exerted by the polymer brush. The areal expansion and decrease in the melting temperatures as a function of G(f) were found to follow the scalings predicted by the self-consistent mean field theories for grafted polymer membranes. Our study shows that the grafted polymer density can be used to effectively control the temperature range and occurrence of a given bilayer phase.

Polymer assisted hydroxyapatite microspheres suitable for biomedical application

Hollow Microspheres of hydroxyapatite-polymer composite can be used as carriers in drug delivery and fillers in tissue engineering. Based on the concept of soft chemistry, a battery of technique is available in the literature to synthesize hollow microspheres, however, an economically viable synthesis route, having good control over the microarchitect and easy to be scaled up, is yet to be developed. Polymer matrix mediated synthesis of inorganic nanoparticles is known to synthesize nanoparticles with controlled morphology and dimensions. It is termed as biomimetic synthesis. Integrating the biomimetic synthesis of nano-particles and spray drying techniques, a novel process of producing hydroxyapatite-polymer composite hollow microspheres is briefly discussed here.

Assessment of Nanoscience and Nanotechnology Initiatives in India

Nanotechnology is a new technology which is generating a lot of interest among academicians, practitioners and scientists. Critical research is being carried out in this area all over the world.Governments are creating policy initiatives to promote developments it the nanoscale science and technology developments. Private investment is also seeing a rising trend. Large number of academic institutions and national laboratories has set up research centers that are workingon the multiple applications of nanotechnology. Wide ranges of applications are claimed for nanotechnology. This consists of materials, chemicals, textiles, semiconductors, to wonder drug delivery systems and diagnostics. Nanotechnology is considered to be a next big wave of technology after information technology and biotechnology. In fact, nanotechnology holds the promise of advances that exceed those achieved in recent decades in computers and biotechnology. Much interest in nanotechnology also could be because of the fact that enormous monetary benefits are expected from nanotechnology based products. According to NSF, revenues from nanotechnology could touch $ 1 trillion by 2015. However much of the benefits are projected ones. Realizing claimed benefits require successful development of nanoscience andv nanotechnology research efforts. That is the journey of invention to innovation has to be completed. For this to happen the technology has to flow from laboratory to market. Nanoscience and nanotechnology research efforts have to come out in the form of new products, new processes, and new platforms.India has also started its Nanoscience and Nanotechnology development program in under its 10(th) Five Year Plan and funds worth Rs. One billion have been allocated for Nanoscience and Nanotechnology Research and Development. The aim of the paper is to assess Nanoscience and Nanotechnology initiatives in India. We propose a conceptual model derived from theresource based view of the innovation. We have developed a structured questionnaire to measure the constructs in the conceptual model. Responses have been collected from 115 scientists and engineers working in the field of Nanoscience and Nanotechnology. The responses have been analyzed further by using Principal Component Analysis, Cluster Analysis and Regression Analysis.

Loading of itraconazole into porous silica and silicon microparticles: characterization and dissolution tests

Most new drug molecules discovered today suffer from poor bioavailability. Poor oral bioavailability results mainly from poor dissolution properties of hydrophobic drug molecules, because the drug dissolution is often the rate-limiting event of the drug’s absorption through the intestinal wall into the systemic circulation. During the last few years, the use of mesoporous silica and silicon particles as oral drug delivery vehicles has been widely studied, and there have been promising results of their suitability to enhance the physicochemical properties of poorly soluble drug molecules. Mesoporous silica and silicon particles can be used to enhance the solubility and dissolution rate of a drug by incorporating the drug inside the pores, which are only a few times larger than the drug molecules, and thus, breaking the crystalline structure into a disordered, amorphous form with better dissolution properties. Also, the high surface area of the mesoporous particles improves the dissolution rate of the incorporated drug. In addition, the mesoporous materials can also enhance the permeability of large, hydrophilic drug substances across biological barriers. T he loading process of drugs into silica and silicon mesopores is mainly based on the adsorption of drug molecules from a loading solution into the silica or silicon pore walls. There are several factors that affect the loading process: the surface area, the pore size, the total pore volume, the pore geometry and surface chemistry of the mesoporous material, as well as the chemical nature of the drugs and the solvents. Furthermore, both the pore and the surface structure of the particles also affect the drug release kinetics. In this study, the loading of itraconazole into mesoporous silica (Syloid AL-1 and Syloid 244) and silicon (TOPSi and TCPSi) microparticles was studied, as well as the release of itraconazole from the microparticles and its stability after loading. Itraconazole was selected for this study because of its highly hydrophobic and poorly soluble nature. Different mesoporous materials with different surface structures, pore volumes and surface areas were selected in order to evaluate the structural effect of the particles on the loading degree and dissolution behaviour of the drug using different loading parameters. The loaded particles were characterized with various analytical methods, and the drug release from the particles was assessed by in vitro dissolution tests. The results showed that the loaded drug was apparently in amorphous form after loading, and that the loading process did not alter the chemical structure of the silica or silicon surface. Both the mesoporous silica and silicon microparticles enhanced the solubility and dissolution rate of itraconazole. Moreover, the physicochemical properties of the particles and the loading procedure were shown to have an effect on the drug loading efficiency and drug release kinetics. Finally, the mesoporous silicon particles loaded with itraconazole were found to be unstable under stressed conditions (at 38 qC and 70 % relative humidity).