Release Of Macromolecules Research Articles

Drug Delivery Using Stimuli-Responsive Polymer Gel Spheres

Polymeric gels that undergo deformation upon appropriate changes in pH or temperature have considerable promise as drug delivery vehicles. Uptake of drug macromolecules into swelling and nonswelling gel spheres and release of drug macromolecules from deswelling and nondeforming gel spheres into a target fluid are investigated here. A mathematical model for gel–solution composite, a composite of a distributed parameter system (gel spheres) and a lumped parameter system (surrounding solution), is developed. The polymer network displacement in swelling/deswelling gels is described by a stress diffusion coupling model. The analytical solution for network displacement is used to predict solvent intake by swelling gels, solvent efflux from deswelling gels, and changes in pressure, porosity, and effective drug diffusivity resulting from network displacement. These in turn influence drug uptake during and after gel swelling and drug release from gel during and after gel deswelling. Numerical results illustrate benefits of gel swelling for drug loading and merits of different modes of drug release. Comparisons are made, as concerns drug uptake and drug release, with gels not subject to deformation.

Ordered Mesoporous Polymer−Silica Hybrid Nanoparticles as Vehicles for the Intracellular Controlled Release of Macromolecules

A two-dimensional hexagonal ordered mesoporous polymer-silica hybrid nanoparticle (PSN) material was synthesized by polymerization of acrylate monomers on the surface of SBA-15 mesoporous silica nanoparticles. The structure of the PSN material was analyzed using a series of different techniques, including transmission electron microscopy, powder X-ray diffraction, and N(2) sorption analysis. These structurally ordered mesoporous polymer-silica hybrid nanoparticles were used for the controlled release of membrane-impermeable macromolecules inside eukaryotic cells. The cellular uptake efficiency and biocompatibility of PSN with human cervical cancer cells (HeLa) were investigated. Our results show that the inhibitory concentration (IC(50)) of PSN is very high (>100 μg/mL per million cells), while the median effective concentration for the uptake (EC(50)) of PSN is low (EC(50) = 4.4 μg/mL), indicating that PSNs are fairly biocompatible and easily up-taken in vitro. A membrane-impermeable macromolecule, 40 kDa FITC-Dextran, was loaded into the mesopores of PSNs at low pH. We demonstrated that the PSN material could indeed serve as a transmembrane carrier for the controlled release of FITC-Dextran at the pH level inside live HeLa cells. We believe that further developments of this PSN material will lead to a new generation of nanodevices for intracellular controlled delivery applications.

Impacts of nanowhisker on formation kinetics and properties of all-cellulose composite gels

Composite gels, with cellulose nanowhiskers as the reinforcing phase and regenerated cellulose as the matrix, were firstly developed by a rapid thermal-induced phase separation followed by a regenerating process. The gelation behavior, regeneration kinetics, morphology and properties of the all-cellulose composite gel system were investigated by advanced rheometer, potentiometric titration, scanning electron microscopy, Fourier-transform infrared spectroscopy, texture analyzer and swelling testing in the presence of different amount of cellulose nanowhiskers. The results revealed that cellulose nanowhiskers could act as the “bridge” to facilitate the crosslinking of cellulose chains during gel formation process. Moreover, they can significantly improve the dimensional stability and mechanical strength of the regenerated gels. In vitro experiment demonstrated that these gels, featuring membrane–matrix hybrid structures, could be used for controlled release of macromolecules in the simulated body fluid.

Generation of Endosomolytic Reagents by Branching of Cell-Penetrating Peptides: Tools for the Delivery of Bioactive Compounds to Live Cells in Cis or Trans

We describe the synthesis and cellular delivery properties of multivalent and branched delivery systems consisting of cell-penetrating peptides assembled onto a peptide scaffold using native chemical ligation. A trimeric delivery system presenting three copies of the prototypical cell-penetrating peptide TAT shows an endosomolytic activity much higher than its monomeric and dimeric counterparts. This novel reagent promotes the endosomal release of macromolecules internalized into cells by endocytosis, and as a result, it can be used to achieve cytosolic delivery of bioactive but cell-impermeable macromolecules in either cis (covalent conjugation) or trans (simple coincubation).

Liver injury caused by drugs: an update

Although severe idiosyncratic drug-induced liver injury (DILI) is a rare event, it has a large impact on the fate of affected patients and the incriminated drug. Hepatic metabolism of drugs, which occurs in the generation of chemically reactive metabolites in critical amounts, seems to underlie most instances of DILI. Genetic polymorphisms in activating and detoxifying enzymes determine, in part, the extent of cellular stress. A cascade of events, where the pathogenetic relevance of single steps is likely to vary from drug to drug, leads to the disturbance of cellular homeostasis, to mitochondrial dysfunction, to the activation of cell death promoting pathways and the release of drug-modified macromolecules and/or danger signals that initiate an innate and/or adaptive immune response. The patient's response to the initial drug-induced cellular dysfunction determines whether adaptation to the drug-induced cellular stress or DILI in one of its many forms of clinical presentation occurs. Although risk factors for developing DILI have been identified and many pathogenetic mechanisms have been elucidated in model systems, idiosyncratic drug reactions remain unpredictable.

Modeling of the Endosomolytic Activity of HA2-TAT Peptides with Red Blood Cells and Ghosts

HA2-TAT is a peptide-based delivery agent that combines the pH-sensitive HA2 fusion peptide from influenza and the cell-penetrating peptide TAT from HIV. This chimeric peptide is engineered to induce the cellular uptake of macromolecules into endosomes via the TAT moiety and to respond to the acidifying lumen of endosomes to cause membrane leakage and release of macromolecules into cells via the HA2 moiety. The question of how HA2 and TAT affect the properties of one another remains, however, unanswered, and the behavior of the peptide inside endosomes is mostly uncharacterized. To address these issues, the binding and membrane leakage activity of a glutamic acid-enriched analogue E5-TAT was assessed with red blood cells and giant unilamellar vesicles as membrane models for endosomes. Hemolysis and microscopy assays reveal that E5-TAT binds to membranes in a pH-dependent manner and causes membrane leakage by inducing the formation of pores through which macromolecules can escape. The TAT moiety contributes to this activity by causing a shift in the pH response of E5 and by binding to negatively charged phospholipids. On the other hand, TAT binding to glycosaminoglycans reduces the lytic activity of E5-TAT. Addition of TAT to the C-terminus of E5 can therefore either increase or inhibit the activity of E5 depending on the cellular components present at the membrane. Taken together, these results suggest a model for the endosomolytic activity of the peptide and provide the basis for the molecular design of future delivery agents.

Designing Bioactive Delivery Systems for Tissue Regeneration

The direct infusion of macromolecules into defect sites generally does not impart adequate physiological responses. Without the protection of delivery systems, inductive molecules may likely redistribute away from their desired locale and are vulnerable to degradation. In order to achieve efficacy, large doses supplied at interval time periods are necessary, often at great expense and ensuing detrimental side effects. The selection of a delivery system plays an important role in the rate of re-growth and functionality of regenerating tissue: not only do the release kinetics of inductive molecules and their consequent bioactivities need to be considered, but also how the delivery system interacts and integrates with its surrounding host environment. In the current review, we describe the means of release of macromolecules from hydrogels, polymeric microspheres, and porous scaffolds along with the selection and utilization of bioactive delivery systems in a variety of tissue-engineering strategies.

2-Methacryloyloxyethyl phosphorylcholine polymer (MPC)-coating improves the transfection activity of GALA-modified lipid nanoparticles by assisting the cellular uptake and intracellular dissociation of plasmid DNA in primary hepatocytes

We previously reported that modification of GALA peptide on the surface of liposomes enhanced fusion with endosomal membrane, and cytoplasmic release of encapsulated macromolecules. We report herein that an additional coating of GALA-modified liposomes with 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer resulted in a two order of magnitude enhancement in the transfection activity of encapsulating plasmid DNA (pDNA). Quantification of the delivered gene copies in whole cells and isolated nuclei revealed that the increase of transfection activity can be attributed to improved efficiencies in cellular uptake and post-nuclear delivery processes. Imaging studies revealed that the intracellular dissociation of pDNA from the lipid envelope is enhanced by GALA modification and further coating with MPC polymer in a stepwise manner. The MPC polymer-coating decreased the ζ-potential of GALA-modified liposomes, suggesting that it assisted in the functional display of negatively charged GALA on the cationic liposomes by providing shielding from mutual electrostatic interactions. Collectively, these data indicate that MPC polymer-coating induced the fusogenic activity of the GALA-modified envelope with endosomes, leading to a more effective cytoplasmic release pDNA. The extensive fusion of the lipid envelope may also reduce electrostatic interactions between mRNA and cationic lipid components, thereby resulting in an enhancement in the translation process.

Engineering gas-foamed large porous particles for efficient local delivery of macromolecules to the lung

Gas-foamed large porous particles (gfLPP) based on poly(lactic-co-glycolic) acid (PLGA) have been recently suggested as potential carriers for pulmonary drug delivery. In this work, we attempt to engineer gfLPP for efficient local delivery of macromolecules in the lungs. Particles were fabricated by the double emulsion-solvent evaporation technique using ammonium bicarbonate as porogen. To improve particle technological properties, two lipid aid excipients, namely dipalmitoylphosphatidylcholine (DPPC) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), were tested. Preliminary technological studies performed on unloaded gfLPP showed that the addition of an appropriate amount of NH4(HCO3), which spontaneously produces CO2 and NH3 during solvent evaporation, is essential to achieve a homogeneous population of highly porous particles with optimal aerodynamic properties. Then, the effect of the presence of DPPC or DOTAP upon the properties of gfLPP containing a model hydrophilic macromolecule, rhodamine B isothiocyanate–dextran (Rhod-dex), was assessed. We found that in the case of hydrophilic macromolecules unable to interact with PLGA end-groups, such as Rhod-dex, excipient addition is essential to increase the amount of drug entrapped within gfLPP, being as high as 80% only for DPPC- or DOTAP-engineered gfLPP. Also Rhod-dex release profile from gfLPP was strongly affected by excipient addition in the initial formulation, with lipid-engineered gfLPP allowing for a more prolonged release of Rhod-dex as compared to excipient-free gfLPP. A further modulation of Rhod-dex initial release rate could be achieved when DOTAP was used, likely due to the electrostatic interactions occurring between macromolecule and cationic phospholipid. Conceiving the developed gfLPP for drug inhalation, DPPC- and DOTAP-engineered gfLPP displayed optimal MMADexp values falling within the range 6.1–7.6μm and very low geometric standard deviations (GSD) varying between 1.2 and 1.3. In vivo deposition studies performed after intra-tracheal administration of gfLPP in rats confirmed the ability of the developed dry powders to deposit along bronchia and bronchioles. In perspective, lipid-engineered gfLPP represent a viable alternative to LPP developed so far to achieve local and prolonged release of hydrophilic macromolecules, such as nucleic acids, in the lungs.

Applications of Micro- and Nano-technology to Study Cell Adhesion to Material Surfaces

Micro- and nano-technologies provide new tools to study and control cell adhesion. These technologies are well suited to reconstitute the cell's natural environment and mimic its biochemical, morphological and mechanical peculiarities. 'Smart' material surfaces and coatings, which allow controlling the binding and release of specific macromolecules and/or cells, are being developed at a high pace. Moreover, the application of geometrical constraints at the micro- or nano-scale reveals some of the physico-chemical principles underlying molecular and cellular organization. These technologies, combined with the growing knowledge in molecular biology and the spatial and temporal resolutions given by the various microscopy techniques, are going to boost our understanding of cell physiology. The availability of well-defined multicellular assemblies opens new ways to test and analyze cells, either in a cluster, or in assemblies mimicking tissue organization. These techniques help bridging the gap between molecular biology and tissue or organism physiology. First, we list the main chemical and physical parameters in the cell micro-environment that influence its survival, proliferation, differentiation or migration. Then we review some examples where micro- and nano-technologies are used to control cell spreading and adhesion in different ways: (1) via the distance between adhesive molecules, (2) via the geometry of adhesive zones, (3) via surfaces with switchable adhesiveness, or (4) via three-dimensional coatings used as reservoir of active molecules.

Hydrogel-based drug delivery systems: Comparison of drug diffusivity and release kinetics

Hydrogels are extensively studied as matrices for the controlled release of macromolecules. To evaluate the mobility of embedded molecules, these drug delivery systems are usually characterized by release studies. However, these experiments are time-consuming and their reliability is often poor. In this study, gels were prepared by step-growth polymerization of poly(ethylene glycol) (PEG) and loaded with fluoresceine isothiocyanate (FITC) labeled dextrans. Mechanical testing and swelling studies allowed prediction of the expected FITC-dextran diffusivity. The translational diffusion coefficients ( D) of the incorporated FITC-dextrans were measured by fluorescence recovery after photobleaching (FRAP) and pulsed field gradient NMR spectroscopy. Because the determined values of D agreed well with those obtained from release studies, mechanical testing, FRAP, and pulsed field gradient NMR spectroscopy are proposed as alternatives to release experiments. The applied methods complemented each other and represented the relative differences between the tested samples correctly. Measuring D can therefore be used to rapidly evaluate the potential of newly developed drug delivery systems.

Size-dependent release of fluorescent macromolecules and nanoparticles from radically cross-linked hydrogels

Hydrogels play an important role in drug delivery and tissue engineering applications due to their excellent biocompatibility and their variable mechanical and physical properties, which allow their optimization for many different aspects of the intended use. In this study, we examined the suitability of poly(ethylene glycol) (PEG)-based hydrogels as release systems for nanometer-sized drugs or drug carriers, like nanoparticles, using the radically cross-linkable oligo(poly(ethylene glycol)fumarate) (OPF) together with two cross-linking agents. Different fluorescent nanoparticulate probes with respect to size and physical structure were incorporated in the cross-linked hydrogels, and the obtained release profiles were correlated with the physical properties and the chemical structure of the gels, indicating a strong dependence of the release on the chosen PEG prepolymers. The prepared hydrogels were characterized by oscillatory rheometry and swelling experiments. Release experiments as well as diffusion measurements using fluorescence recovery after photobleaching showed the great potential of this type of hydrogels for the preparation of adjustable release systems by altering the molecular weights of the used PEG molecules.

Development of new chitosan/carrageenan nanoparticles for drug delivery applications

The use of polymeric nanoparticles, especially those composed of natural polymers, has become a very interesting approach in drug delivery, mainly because of the advantages offered by their small dimensions. The aim of this work was to develop a novel formulation of nanoparticles comprised of two natural marine-derived polymers, namely chitosan and carrageenan, and to evaluate their potential for the association and controlled release of macromolecules. Nanoparticles were obtained in a hydrophilic environment, under very mild conditions, avoiding the use of organic solvents or other aggressive technologies for their preparation. The developed nanocarriers presented sizes within 350-650 nm and positive zeta potentials of 50-60 mV. Polymeric interactions between nanoparticles' components were evaluated by Fourier transform infrared spectroscopy. Using ovalbumin as model protein, nanoparticles evidenced loading capacity varying from 4% to 17% and demonstrated excellent capacity to provide a controlled release for up to 3 weeks. Furthermore, nanoparticles have demonstrated to exhibit a noncytotoxic behavior in biological in vitro tests performed using L929 fibroblasts, which is critical regarding the biocompatibility of those carriers. In summary, the developed chitosan-carrageenan nanoparticles have shown promising properties to be used as carriers of therapeutic macromolecules, with potential application not only strictly in drug delivery, but also in broader areas, such as tissue engineering and regenerative medicine.

Gene transfer and protein release of fission yeast by application of a high voltage electric pulse

A high voltage electric pulse can be applied to induce the uptake of DNA into cells and the release of protein from cells. In transformation procedures, electroporation is widely used since the technique is simple, rapid, reproducible, and highly efficient. In extraction of protein, on the other hand, electroextraction has many advantages over other conventional extractions. We have developed a highly efficient method for the electroporation of fission yeast. In particular, application of a high voltage electric pulse to fission yeast improves the cellular uptake and release of macromolecules controlled by both osmotic conditions and electric field strength.

Organized Shells on Clay Nanotubes for Controlled Release of Macromolecules

The use of tubular halloysite clay as a nanotemplate for layer-by-layer (LbL) shell assembly and its utilization for controlled release of drug macromolecules are studied. The LbL nanoshell allowed additional control for the sustained release of drug loaded halloysite tubes. The number of polymeric layers in the shell and molecular weight of the assembled polymers influences the drug release rate. Three bilayer shells of chitosan and gelatin of 15 nm thicknesses gave the best encapsulation and retardation in the release rate of dexamethasone. An encapsulation of the macromolecules inside the lumen of the biocompatible clay nanotubes coupled with the polyelectrolyte shell formation provides a novel formulation for the controlled release of bioactive agents.

NSP-Interacting GTPase

Despite the significant progress in the identification of essential components of the nuclear transport machinery, some events of this process are still unclear. Particularly, functional information about the release of nuclear-exported macromolecules at the cytoplasmic side of the nuclear pore complex and their subsequent trans-cytoplasmic movement is lacking. Recently, we identified a cytoplasmic GTPase, designated NIG (NSP-interacting GTPase), which may play a relevant role in these processes. NIG interacts in vivo with the geminivirus NSP and promotes the translocation of the viral protein from the nucleus to the cytoplasm where it is redirected to the cell surface to interact with the viral movement protein, MP. Here we position the NIG function into the mechanistic model for the intracellular trafficking of viral DNA and discuss the putative role of NIG in general cellular nucleocytoplasmic transport of nucleic acid-protein complexes.Addendum to: Carvalho CM, Fontenelle MR, Florentino LH, Santos AA, Zerbini FM, Fontes EPB. A novel nucleocytoplasmic traffic GTPase identified as a functional target of the bipartite geminivirus nuclear shuttle protein. Plant J 2008; DOI: 10.1111/j.1365-313X.2008.03556.x.

Drug-delivery study and estimation of polymer–solvent interaction parameter for bisacrylate ester-modified Pluronic hydrogels

In this study, Pluronic F127 hydrogels were characterised as an injectable system for the controlled release of drugs with variable molecular weights (FITC-Dextran at 70 and 40 kDa). In addition, the polymer–solvent interaction parameter ( χ) was successfully estimated. Pluronic hydrogels (10–25 wt.%) were redox cured and their swelling behaviour investigated in PBS (pH 7.45) at 37 °C. After swelling to equilibrium, the hydrogels were compressed and the rubber-elasticity theory was applied to evaluate χ. Tensile tests proved the hydrogels were elastic and their χ values ranged between 0.50 and 0.53. The full drug load could be delivered over a period of ∼15 h suggesting that redox cured Pluronic F127 hydrogels can function as injectable systems for controlled and sustained release of macromolecules.

Macromolecule release and smoothness of semi-interpenetrating PVP–pHEMA networks for comfortable soft contact lenses

Knowledge about the microstructure and the release rate of hydrophilic macromolecules is required for a rational development of comfortable and safe contact lenses. Semi-interpenetrating networks of poly(hydroxyethyl methacrylate) (pHEMA) with poly(vinyl pyrrolidone) (PVP) were prepared by free radical polymerization of HEMA in the presence of PVP K30 or K90F, under anhydrous conditions or after addition of water, and evaluated in terms of swelling, porosity, PVP release rate, air–water surface tension, and friction coefficient. The greater water content was during polymerization, the higher was the swelling degree and porosity. Microphase separation above a certain volume of water resulted in hydrogels with bumpy surface and interconnected pores. All hydrogels showed a high optical clarity and slowly released PVP (20% after 9 days). In general, the greater the content of PVP or the higher its molecular weight was, the lower the friction coefficients were. In the case of hydrogels prepared with water, the friction was influenced by the balance between the ability to hold water in the network (which contributes to the sliding and PVP release) and the deleterious effect of an irregular surface. Controlled delivery of PVP revealed as a critical factor for improving the frictional behavior of pHEMA contact lenses.

Photochemical Internalization: A New Tool for Drug Delivery

The utilisation of macromolecules in the therapy of cancer and other diseases is becoming increasingly important. Recent advances in molecular biology and biotechnology have made it possible to improve targeting and design of cytotoxic agents, DNA complexes and other macromolecules for clinical applications. In many cases the targets of macromolecular therapeutics are intracellular. However, degradation of macromolecules in endocytic vesicles after uptake by endocytosis is a major intracellular barrier for the therapeutic application of macromolecules having intracellular targets of action. Photochemical internalisation (PCI) is a novel technology for the release of endocytosed macromolecules into the cytosol. The technology is based on the activation by light of photosensitizers located in endocytic vesicles to induce the release of macromolecules from the endocytic vesicles. Thereby, endocytosed molecules can be released to reach their target of action before being degraded in lysosomes. PCI has been shown to stimulate intracellular delivery of a large variety of macromolecules and other molecules that do not readily penetrate the plasma membrane, including type I ribosome-inactivating proteins (RIPs), DNA delivered as gene-encoding plasmids or by means of adenovirus or adeno-associated virus, peptide nucleic acids (PNAs) and chemotherapeutic agents such as bleomycin and in some cases doxorubicin. PCI of PNA may be of particular importance due to the low therapeutic efficacy of PNA in the absence of an efficient delivery technology and the 10-100-fold increased efficacy in combination with PCI. The efficacy and specificity of PCI of macromolecular therapeutics has been improved by combining the macromolecules with targeting moieties, such as the epidermal growth factor. In general, PCI can induce efficient light-directed delivery of macromolecules into the cytosol, indicating that it may have a variety of useful applications for site-specific drug delivery as for example in gene therapy, vaccination and cancer treatment.

Macromolecule Release from Monodisperse PLG Microspheres: Control of Release Rates and Investigation of Release Mechanism

Novel macromolecular therapeutics such as peptides, proteins, and DNA are advancing rapidly toward the clinic. Because of typically low oral bioavailability, macromolecule delivery requires invasive methods such as frequently repeated injections. Parenteral depots including biodegradable polymer microspheres offer the possibility of reduced dosing frequency but are limited by the inability to adequately control delivery rates. To control release and investigate release mechanisms, we have encapsulated model macromolecules in monodisperse poly(D,L-lactide-co-glycolide) (PLG) microspheres using a double-emulsion method in combination with the precision particle fabrication technique. We encapsulated fluorescein-dextran (F-Dex) and sulforhodamine B-labeled bovine serum albumin (R-BSA) into PLG microspheres of three different sizes: 31, 44, and 80 microm and 34, 47, and 85 microm diameter for F-Dex and R-BSA, respectively. The in vitro release profiles of both compounds showed negligible initial burst. During degradation and release, the microspheres hollowed and swelled at critical time points dependant upon microsphere size. The rate of these events increased with microsphere size resulting in the largest microspheres exhibiting the fastest overall release rate. Monodisperse microspheres may represent a new delivery system for therapeutic proteins and DNA and provide enhanced control of delivery rates using simple injectable depot formulations.