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Functionalized hydrogel microparticles prepared by microfluidics and their interaction with tumour marker carbonic anhydrase IX

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Abstract

Microfluidics allows to accurately control the synthesis of microparticles for specific applications, where size and morphology play an important role. In this work, we introduce microfluidic chip design with dedicated extraction and gelation sections allowing to prepare hydrogel particles in size range of a red blood cell. The influence of the extractive channel size, alginate concentration and type of storage media on the final size of the prepared alginate microparticles are discussed. The second part of the work is dedicated to surface modification of prepared particles using chitosan, pHPMA and monoclonal antibody molecule IgG M75. The specific interaction of the antibody molecule to an antigen domain of Carbonic Anhydrase IX, the transmembrane tumour protein associated with several types of cancer, is demonstrated by fluorescence imaging and compared to an isotypic antibody molecule.

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... This enables a considerable reduction of the ecological impact of research and development, and potentially increases the rate of production of new formulations. Drop microreactors have been successfully used for activities including the measurement of kinetic constants of chemical and biochemical reactions [2][3][4], drug-protein association constants [5], cell screening [6][7][8], synthesis of nanoparticles [9][10][11] and hydrogel particles [12][13][14]. ...
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The reversible, temperature-dependent change in the permeability of a phospholipid bi-layer has been used for controlling the diffusion rate of encapsulated molecular payload from liposomes. Liposomes were pre-loaded with a fluorescent dye and immobilized in calcium alginate hydrogel microparticles that also contained iron oxide nanoparticles. The composite microparticles were produced by a drop-on-demand ink-jet method. The ability of iron oxide nanoparticles to locally dissipate heat upon exposure to a radio-frequency (RF) alternating magnetic field was used to control the local temperature and therefore diffusion from the liposomes in a contactless way using RF coil. Several different release patterns were realised, including repeated on-demand release. The internal structure of the composite alginate-liposome-magnetite microparticles was investigated and the influence of microparticle concentration on the heating rate determined. In order to achieve a temperature rise required for the liposome membrane melting, the concentration of alginate beads should be at least 25% of their maximum packing density for the nanoparticle concentration and specific absorption rate used.
Article
A novel method for the fabrication of cell-like microparticles consisting of a hydrogel core and a mesoporous silica shell has been developed. It consists of two steps: (i) inkjet fabrication of calcium alginate cores in the size range of 40–90 μm that can contain immobilised nanoparticles and liposome-based internal compartments; (ii) a sol–gel process for depositing a thin mesoporous silica layer on the microparticle surface using alkoxysilane precursors. The structure of the silica coating has been investigated and its effect on the diffusion rates of proteins with increasing molecular weight (lysozyme, myoglobin, ovalbumin and BSA) has been determined. The silica shell was shown to act as an efficient semi-permeable barrier that allows the diffusion of smaller proteins but rejects larger ones.
Article
Composite microparticles consisting of a calcium alginate gel matrix with embedded liposomes made from cholesterol:DPPC (dipalmitoylphosphatidylcholine) mixtures were considered. Factors affecting the encapsulation stability of liposomes during the gel formation by ionic cross-linking - namely temperature and the cholesterol:DPPC ratio - were systematically investigated. The liposomes were found to be tolerant to Ca(2+) ions during cross-linking of the gel and stable in the hydrogel matrix for extended periods of time when cholesterol was present in the phospholipid bilayer and temperature was kept sufficiently below the phase transition. The temperature-controlled release rate of encapsulated fluorescent dye was quantified. It is shown that a defined quantity of encapsulated substance can be repeatedly released from the embedded liposomes "on-demand" by short temperature pulses of suitably chosen duration and amplitude. This makes the hydrogel-liposome composites potential candidates for applications such as controlled drug delivery or study of reaction-diffusion phenomena in compartmentalised systems.
Article
Stimuli-responsive capsules are of interest in drug delivery, fragrance release, food preservation, and self-healing materials. Many methods are used to trigger the release of encapsulated contents. Here we highlight mechanisms for the controlled release of encapsulated cargo that utilize chemical reactions occurring in solid polymeric shell walls. Triggering mechanisms responsible for covalent bond cleavage that result in the release of capsule contents include chemical, biological, light, thermal, magnetic, and electrical stimuli. We present methods for encapsulation and release, triggering methods, and mechanisms and conclude with our opinions on interesting obstacles for chemically induced activation with relevance for controlled release.
Article
New method of synthesis of water-soluble polymer-drug conjugates, exhibiting remarkable anticancer activity in mice models, has been developed. In the conjugates, an anticancer drug doxorubicin (DOX) is attached to a polymer carrier based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer via a hydrolytically labile hydrazone bond. New methacrylamide-type comonomers, containing either hydrazide group or hydrazon of DOX, were used for copolymerization with HPMA. In contrast to the synthetic procedure described earlier the new method is simpler, cheaper, and results in a better-defined conjugate structure. The conjugates are fairly stable in buffer at pH 7.4 (model of blood stream) but release DOX under mild acid conditions modeling the tumor microenvironment. The conjugates showed significant in vivo antitumor activity in treatment of T-cell lymphoma EL-4 bearing mice with up to 100% long-term survivors. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008
Article
This article describes the simulation of flow velocity distribution and the process of formation of monodisperse calcium alginate microparticles (Ca-alg MPs) by using microfluidic T-junction geometries. Based on sheath stresses, water-in-oil (w/o) emulsions of an aqueous solution of a biopolymer (e.g. sodium alginate, Na-alg) are emulsified in an oil phase. The emulsions are collected into a bath containing calcium-ions to form the Ca-alg MPs. Experimental data show that Ca-alg MPs with diameters ranging from 70 μm to 220 μm with a variation less than 10% are precisely generated. The size of the emulsions can be tunable by adjusting the relative sheath/sample flow rate ratio. In addition, the encapsulation of silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) is studied. The approach in manipulation of microparticles will provide many potential usages for pharmaceutical application. The proposed method has advantages of being readily controlled, cost-effective and easy to operate, together with its ability to produce a uniform size.
Article
Microfluidic devices are finding increasing application as analytical systems, biomedical devices, tools for chemistry and biochemistry, and systems for fundamental research. Conventional methods of fabricating microfluidic devices have centered on etching in glass and silicon. Fabrication of microfluidic devices in poly(dimethylsiloxane) (PDMS) by soft lithography provides faster, less expensive routes than these conventional methods to devices that handle aqueous solutions. These soft-lithographic methods are based on rapid prototyping and replica molding and are more accessible to chemists and biologists working under benchtop conditions than are the microelectronics-derived methods because, in soft lithography, devices do not need to be fabricated in a cleanroom. This paper describes devices fabricated in PDMS for separations, patterning of biological and nonbiological material, and components for integrated systems.
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This review presents the most outstanding contributions in the field of biodegradable polymeric nanoparticles used as drug delivery systems. Methods of preparation, drug loading and drug release are covered. The most important findings on surface modification methods as well as surface characterization are covered from 1990 through mid-2000.
Article
The interactions between alginate and polycations have been studied by using different labelling techniques. Binding of poly-L-lysine (PLL) to alginate in the gel state is mainly governed by the amount of dissociable negative charges on the bead surface. PLL was found to bind more rapidly to gel beads made from alginate with a high content of mannuronic acid. The binding was enhanced by increasing the alginate concentration on the surface by making inhomogeneous beads. When the capsules were stored in the presence of cations with high affinity for alginate (Ca2+, Sr2+), PLL was washed off. Less PLL is bound to strontium alginate than to calcium alginate beads. Two mechanisms appear to be responsible for the binding of sodium alginate to alginate PLL capsules (coating): (i) an electrostatic interaction between the soluble coating material and excess positive charges on PLL on the surface; (ii) the formation of a calcium alginate gel on the surface owing to leaching of calcium ions from the core. The stability and efficiency of the coating as a function of molecular size and sequential structure of the coating polymer have also been investigated.
Article
To explore the feasibility of alginate gel as a vehicle for liposomes, we investigated the effects of various factors associated with the loading of drug-containing liposomes into the gel beads. The loading process includes (I) mixing of liposomes and alginate solution, (2) calcium induced gelation of alginates, (3) the time-dependent contraction of a gel body squeezing out interior water, and (4) possible leakage or release of a drug entrapped in liposomes in a series of each of theses processes. These effects were examined in terms of the leakage of a marker 5(6)-carboxyfluorescein (CF) from liposomes of egg phosphatidylcholine (EPC) and EPC/cholesterol (EPC/Cho) and liposome (phosphorus) release from curing and fully-cured gel beads whose initial polymer concentrations were 4 and 2%. Major findings were: (1) Alginate induced the leakage of a water-soluble drug incorporated in the liposomes as a function of the polymer concentration and the mixing time. (2) Calcium ions also stimulated the leakage of the drug. EPC/Cho liposomes were several times more resistant to the leakage of CF than were EPC liposomes. (3) The liposomes were well loaded without any loss in the gel bead despite the squeezing outflow of water and the bead contraction during gel curing. (4) Such curing caused leakage of the drug from the EPC liposomes in the very early stage while no effect was observed in the EPC/Cho liposomes. (5) In the gel-eroding medium (pH 7.4 Tris-HCl, 37 degrees C), the total drug release was controlled by the erosion rate of the bead body. Immediately after the bead erosion, EPC liposomes retained about 60% of the drug in the 2% bead and only about 20% in the 4% bead, whereas EPC/Cho liposomes retained more than 85% regardless of the initial alginate concentration. The results provide valuable information for the design and applicability of the gel-loaded liposome delivery system.
Article
MN/CA IX is a cell surface protein, strongly associated with several types of human carcinomas. It exerts activity of carbonic anhydrase and capacity of binding to cell surface receptors. In the present work, we used affinity purified MN/CA IX protein to demonstrate that the cells adhere to immobilized MN/CA IX and that the monoclonal antibody M75 abrogates cell attachment to MN/CA IX. Using synthetic oligopeptides, we identified M75 epitope and located it in the proteoglycan domain, which contains a sixfold tandem repeat of six amino acids GEEDLP. From phage display library of random heptapeptides we identified and chemically synthesized those which compete for the epitope with M75 and inhibit adhesion of cells to MN/CA IX. These heptapeptides might serve as lead compounds for drug design.
Article
In a very short time, since their emergence, the field of controlled delivery of proteins has grown immensely. Because of their relatively large size, they have low transdermal bioavailabilities. Oral bioavailability is generally poor since they are poorly absorbed and easily degraded by proteolytic enzymes in the gastrointestinal tract. Ocular and nasal delivery is also unfavorable due to degradation by enzymes present in eye tissues and nasal mucosa. Thus parenteral delivery is currently most demanding and suitable for delivery of such molecules. In systemic delivery of proteins, biodegradable microspheres as parenteral depot formulation occupy an important place because of several aspects like protection of sensitive proteins from degradation, prolonged or modified release, pulsatile release patterns. The main objective in developing controlled release protein injectables is avoidance of regular invasive doses which in turn provide patient compliance, comfort as well as control over blood levels. This review presents the outstanding contributions in field of biodegradable microspheres as protein delivery systems, their methods of preparation, drug release, stability, interaction with immune system and regulatory considerations.
Article
Biocompatibility of biomaterials and biomaterial-based medical devices is a critical issue for the long-term function on multiple therapeutic systems. In the past few years, there has been an increasing interest in producing more biocompatible biomaterials and in developing novel assays to analyze the quality of the products. In this study, a battery of in vitro techniques to assess the biocompatibility of alginates with different compositions and purities and alginate-based microcapsules is presented. Study of the protein and polyphenol content of the alginates revealed clear differences between the nonpurified and the purified alginates. A similar behavior was observed when the mitogenic activity and the tumor necrosis factor-alphasecretion induced by the alginates were assessed. Interestingly, when the latter two techniques were adapted to evaluate the different alginate microcapsules, a correlation with the results obtained for the alginate samples was observed. These results reinforce the idea of using the full battery of assays here reported to screen alginates and alginate-based microcapsules before implantation.
Article
Expression of CA IX is normally restricted to the mucosa of alimentary tract, but on the other hand, it takes place in a high percentage of human cancers derived from tissues which are normally CA IX-negative. It is a transmembrane protein with two extracellular domains: carbonic anhydrase (CA) with a high catalytic activity and a proteoglycan-like segment (PG), mediating cell-cell adhesion. Both CA and PG domains interact with the microenvironment and they could play a role in tumorigenesis, but their roles are poorly understood. The present work characterizes some newly recognized properties of the PG. One of them is a prevalently negative charge, caused by a high proportion of dicarboxylic amino acids. This is reflected by easy dissociation of complexes formed by PG either with monoclonal antibody M75 or with the cell surface receptor already at slightly acidic pH. This property might facilitate separation of cells from the primary tumor. Released cells may subsequently attach elsewhere in the organism and eventually start metastatic growth. Another aim of the present study was to identify human tumor cell lines which are expressing the presumed CA IX receptor molecule. The same cell lines were also tested for the presence of CA IX protein; we found that expression of CA IX and of the receptor is independent of each other. In addition, we examined the species specificity of CA IX receptors. The PG domain, which contains the epitope of mAb M75 -PGEEDLP- overlapping with the binding site for putative receptor is relatively conserved in evolution: human and rat CA IX cross-react with M75 antibody on western blots. Consistently with this, human and rat cells can attach to purified human CA IX protein. On the other hand, murine CA IX contains an entirely different equivalent of PG sequence and it does not react with M75 antibody or attach to human CA IX protein. This is suggestive of the co-evolution of CA IX protein together with its receptor.
Article
Among several drug delivery systems, liposomal encapsulated anti-cancer agents represent an advanced and versatile technology. Several formulations of liposomal anthracyclines are approved, e.g. for the treatment of metastatic breast cancer (pegylated and non-pegylated liposomal doxorubicin) or AIDS-related Kaposi's sarcoma (pegylated liposomal doxorubicin and liposomal daunorubicin). Meanwhile, virtually all anti-cancer drugs have been encapsulated in liposomes using different technologies. This review will summarize preclinical and clinical data of approved and exemplary emerging liposomal anti-cancer agents.
Article
This paper describes the preparation and characterization of a novel drug delivery system for protein, liposomes-in-alginate (LIA) of biodegradable polymers, which is conceived from a combination of the polymer and the lipid-based delivery systems. LIA were prepared by first entrapping bovine serum albumin (BSA), a model protein within multivesicular liposomes (MVLs) by double emulsification process, which are then encapsulated within alginate hydrogel microcapsule, with untrapped BSA which are added during preparation of MVLs. Factors impacting encapsulation efficiency of MVLs are investigated and release of protein from the microcapsules in vitro is studied. At the same time, characterization of MVLs, microcapsules encapsulated protein formulation and integrality analyse of BSA in microcapsules are also studied, with the aim of improving the entrapment efficiency and prolonging release time. It is found that encapsulation efficiency and size of MVLs are affected by the composition and fabrication parameters of LIA. The data also show LIA have high encapsulation efficiency (up to 95%), little chemical change in drug caused by the formulation process, narrow particle size distribution and spherical particle morphology. Drug release assays conducted in vitro indicates that these formulations provide sustained release of encapsulated drug over a period, about 2 weeks.
Article
In this paper the manipulation of Ca-alginate microspheres, using a microfluidic chip, for the encapsulation of gold nanoparticles is presented. Our strategy is based on hydrodynamic-focusing on the forming of a series of self-assembling sphere structures, the so-called water-in-oil (w/o) emulsions, in the cross-junction microchannel. These fine emulsions, consisting of aqueous Na-alginates, are then dripped into a solution of 20% calcium salt to accomplish Ca-alginate microspheres in an efficient manner. Experimental data show that microspheres with diameters ranging from 50 microm to 2000 microm with a variation less than 5% were precisely generated. The size and gap of the droplets are tunable by adjusting the relative sheath/sample flow rate ratio. Furthermore, we applied them to encapsulated gold nanoparticles, and this one shot operation performs the 'Lab on a Chip'.
Article
We report a microfluidic approach to generating capsules of biopolymer hydrogels. Droplets of an aqueous solution of a biopolymer were emulsified in an organic phase comprising a cross-linking agent. Polymer gelation was achieved in situ (on a microfluidic chip) by diffusion-controlled ionic cross-linking of the biopolymer, following the transfer of the cross-linking agent from the continuous phase to the droplets. Gelation was quenched by collecting particles in a large pool of cross-linking agent-free liquid. The structure of microgels (from capsules to gradient microgels to particles with a uniform structure) was controlled by varying the time of residence of droplets on the microfluidic chip and the concentration of the cross-linking agent in the continuous phase. We demonstrated the encapsulation of a controlled number of polystyrene beads in the microgel capsules. The described approach was applied to the preparation of capsules of several polysaccharides such as alginate, kappa-carrageenan, and carboxymethylcellulose.