European Journal of Pharmaceutics and Biopharmaceutics (v.68, #1)

APV Diary (S1-S3).

pH-sensitive film coatings: Towards a better understanding and facilitated optimization by F. Siepmann; C. Wahle; B. Leclercq; B. Carlin; J. Siepmann (2-10).
The major aims of this study were: (i) to prepare and characterize polymeric film coatings with pH-dependent properties for oral administration; and (ii) to better understand the underlying mass transport mechanisms upon exposure to simulated gastric and intestinal fluids. Propylene glycol alginate (containing free carboxylic groups) was chosen as a pH-sensitive film former, which was blended with different amounts of ethylcellulose (being water-insoluble throughout the gastro-intestinal tract). The water uptake kinetics of thin free films in 0.1 M HCl and phosphate buffer pH 7.4 were monitored gravimetrically and quantitatively described using an appropriate analytical solution of Fick’s law of diffusion. Interestingly, the addition of only a low percentage (2.5–10%) of propylene glycol alginate to ethylcellulose significantly increased both, the rate and extent of the films’ water uptake, irrespective of the pH of the release medium. Importantly, diffusion was found to be the pre-dominant mass transport mechanism for all system compositions and types of release media. The apparent water diffusivity in the polymeric films could quantitatively be determined as a function of the polymer blend ratio. It significantly increased with increasing pH of the release medium, due to the presence of the free carboxylic groups in propylene glycol alginate. Also the dry mass loss of the polymer networks was much more pronounced at high compared to low pH. The differences in both water uptake as well as dry mass loss resulted in a clear pH-dependence of the drug release kinetics from coated pellets. Importantly, desired pH-sensitive release rates can easily be adjusted by varying the propylene glycol alginate content.
Keywords: Solid dosage form; Polymeric drug delivery systems; Coating; Pellets; Controlled release; pH-sensitivity; Ethylcellulose; Release mechanism; Diffusion;

Oral pulsatile delivery systems based on swellable hydrophilic polymers by Andrea Gazzaniga; Luca Palugan; Anastasia Foppoli; Maria Edvige Sangalli (11-18).
Upon contact with aqueous fluids, swellable hydrophilic polymers undergo typical chain relaxation phenomena that coincide with a glassy–rubbery transition. In the rubbery phase, these polymers may be subject to swelling, dissolution and erosion processes or, alternatively, form an enduring gel barrier when cross-linked networks (hydrogels) are dealt with. Because of the peculiar hydration and biocompatibility properties, such materials are widely exploited in the pharmaceutical field, particularly as far as hydrophilic cellulose derivatives are concerned. In oral delivery, they have for long been employed in the manufacturing of prolonged release matrices and, more recently, for pulsatile (delayed) release devices as well. Pulsatile delivery, which is meant as the liberation of drugs following programmed lag phases, has drawn increasing interest especially in view of emerging chronotherapeutic approaches. In pursuit of pulsatile release, various design strategies have been proposed, chiefly including reservoir, capsular and osmotic formulations. In most cases, water-swellable polymers play a key role in the overall delivery mechanism after being activated by physiological media. Based on these premises, the aim of the present review is to survey the main oral pulsatile delivery systems, for which swelling, dissolution and/or erosion of hydrophilic polymers are primarily involved in the control of release.
Keywords: Swellable hydrophilic polymers; Dissolution; Erosion; Swelling; Hydrophilic cellulose derivatives; Oral pulsatile/delayed release;

A novel thermoresponsive hydrogel based on chitosan by Yannic B. Schuetz; Robert Gurny; Olivier Jordan (19-25).
Injectable thermosetting chitosan hydrogels are attractive systems for drug delivery and tissue engineering that combine biodegradability, biocompatibility and the ability to form in situ gel-like implants. Thermally-induced gelation relies advantageously on biopolymer secondary interactions, avoiding potentially toxic polymerization reactions that may occur with in situ polymerizing formulations. In view of a biomedical use, such formulations have to be sterilizable and storable on extended periods without losing their thermosetting properties. These two key features have been studied in the present paper. Chitosans from two different sources were added with several phosphate-free polyols or polyoses as gelling agents. Despite a reduction in chitosan molecular weight following autoclaving, the hydrogels prepared with autoclaved chitosan showed the desired thermosetting properties. Hence, chitosan steam sterilization combined with aseptic preparation of the hydrogel allows a sterile formulation to be obtained. Whereas thermosetting hydrogels were shown to be unstable when refrigerated, freezing was shown to be conceivable as a storage method. When trehalose or mannitol was used as stabilizing agent, the formulation reconstituted from a lyophilizate displayed thermosetting properties and was still injectable, paving the way to the development of a clinically utilizable, novel chitosan thermosetting hydrogel.
Keywords: Hydrogels; Drug delivery; Chitosan; Thermosetting; Thermoresponsive; Lyophilization; In situ gelling;

Characterization of thermosensitive chitosan-based hydrogels by rheology and electron paramagnetic resonance spectroscopy by Sabine Kempe; Hendrik Metz; Martin Bastrop; Annette Hvilsom; Renata Vidor Contri; Karsten Mäder (26-33).
Chitosan, an amino-polysaccharide, has been proposed as a promising biopolymer for tissue repair and drug delivery. Chitosan solutions containing glycerol-2-phosphate (β-GP) have been described as injectable in situ gelling thermosensitive formulations, which undergo sol–gel transition at physiological pH and temperatures. This feature makes them suitable for the parenteral administration of drugs, especially for peptides and proteins. The aim of the present study was to get a deeper insight into the macro- and microstructure of chitosan/β-GP systems. In addition to oscillating rheology, electron paramagnetic resonance (EPR) spectroscopy was applied to examine the microviscosity and pH inside the gels depending on the β-GP concentration and to follow the loading and release of spin-labelled Insulin. All chitosan/β-GP solutions showed a physiological pH ranging from 6.6 to 6.8 that did not change during gelation, irrespective of the proportion of β-GP. The dynamics of the spin-labelled Insulin and its microviscosity inside the gels and during release were monitored by EPR spectroscopy. The results indicate that the Insulin was incorporated into the aqueous environment of the gel and was released in its native form. The in vitro drug release from the gels was governed by diffusion of drug from the gel matrix. A sustained release of Insulin was observed over a period of 2 weeks. Increasing the proportion of β-GP increased the amount of released Insulin and the velocity thereof.
Keywords: Chitosan; Hydrogel; Thermogelling; Glycerol phosphate; Rheology; EPR; ESR;

Thermoresponsive hydrogels in biomedical applications by Leda Klouda; Antonios G. Mikos (34-45).
Environmentally responsive hydrogels have the ability to turn from solution to gel when a specific stimulus is applied. Thermoresponsive hydrogels utilize temperature change as the trigger that determines their gelling behavior without any additional external factor. These hydrogels have been interesting for biomedical uses as they can swell in situ under physiological conditions and provide the advantage of convenient administration. The scope of this paper is to review the aqueous polymer solutions that exhibit transition to gel upon temperature change. Typically, aqueous solutions of hydrogels used in biomedical applications are liquid at ambient temperature and gel at physiological temperature. The review focuses mainly on hydrogels based on natural polymers, N-isopropylacrylamide polymers, poly(ethylene oxide)–b-poly(propylene oxide)–b-poly(ethylene oxide) polymers as well as poly(ethylene glycol)-biodegradable polyester copolymers.
Keywords: Thermosensitive hydrogels; In situ gelation; Drug delivery; Tissue engineering; Lower critical solution temperature;

Selective enzymatic degradation of poly(ε-caprolactone) containing multiblock copolymers by A. Kulkarni; J. Reiche; J. Hartmann; K. Kratz; A. Lendlein (46-56).
The hydrolytic and Pseudomonas lipase catalysed enzymatic degradation was studied for PDC multiblock copolymers consisting of poly(ε-caprolactone) (PCL) segments and poly(p-dioxanone) (PPDO) segments with variable composition. The enzymatic degradation of these multiblock copolymers is significantly accelerated by Pseudomonas lipase in contrast to the hydrolytic degradation where the degradation behaviour is determined by the PPDO segments. Degradation time intervals up to 200 h are selected, where the PPDO segments remain stable and do not contribute to the degradation process. A linear correlation between weight loss and increasing PCL content of the multiblock copolymers was found. X-ray diffraction data confirm that both crystalline and amorphous PCL are attacked by the enzymes. SEM cross-section images reveal that Pseudomonas lipase penetrates into the PDC polymers. The present study impressively demonstrates that selective enzymatic degradation of PCL containing multifunctional polymers is a beneficial tool for controlling their degradation properties.
Keywords: Enzymatic degradation; Hydrolytic degradation; Multiblock copolymer; Biodegradation; Shape-memory polymer; Segmented polyester urethanes;

Polymers in the prevention of peritoneal adhesions by Yoon Yeo; Daniel S. Kohane (57-66).
Peritoneal adhesions are serious complications of surgery, and can result in pain, infertility, and potentially lethal bowel obstruction. Pharmacotherapy and barrier devices have reduced adhesion formation to varying degrees in preclinical studies or clinical trials; however, complete prevention of adhesions remains to be accomplished. We and others have hypothesized that the limitations of the two approaches could be overcome by combining their strengths in the context of controlled drug delivery. Here we review the role of polymeric systems in the prevention of peritoneal adhesions, with an emphasis on our recent work in developing and applying polymeric drug delivery systems such as nano- or microparticles, hydrogels, and hybrid systems for peritoneal use.
Keywords: Peritoneal adhesions; Adhesion barrier; Controlled drug delivery; Particulate drug delivery system; Hydrogels;

Synthesis and characterization of cyclic acetal based degradable hydrogels by Sachiko Kaihara; Shuichi Matsumura; John P. Fisher (67-73).
While many synthetic, hydrolytically degradable hydrogels have been developed for biomedical applications, there are only a few examples whose polymer backbone does not form acidic products upon degradation. In order to address this concern, we proposed to develop a hydrogel based on a cyclic acetal unit that produces diols and propanals upon hydrolytic degradation. In particular, we proposed the fabrication of hydrogels formed by the free radical polymerization of two diacrylate monomers, 5-ethyl-5-(hydroxymethyl)-β,β-dimethyl-1,3-dioxane-2-ethanol diacrylate (EHD), a cyclic acetal having two acryl groups, and poly(ethylene glycol)diacrylate (PEGDA). However, the hydrophobicity of the EHD monomer inhibits hydrogel fabrication. Therefore this work develops a strategy to form hydrogels with a co-monomer system, one of which is hydrophobic, and subsequently describes the properties of the resulting hydrogel. Using benzoyl peroxide as an initiator and N,N-dimethyl-p-toluidine as an accelerator, the EHD and PEGDA monomers were reacted in an acetone/water co-solvent system. The chemical structure of the resulting EH–PEG [5-ethyl-5-(hydroxymethyl)-β,β-dimethyl-1,3-dioxane-2-ethanol-co-PEG] hydrogel was then characterized by FT-IR. Physicochemical properties of the EH–PEG hydrogel, including swelling degree, sol fraction, and contact angle, were determined so as to characterize the properties of these materials and ultimately investigate their use in drug delivery and tissue engineering applications. Results showed that EH–PEG hydrogel may be formed using the co-solvent system. Further results indicated that swelling degree is dependent upon initiator concentration, monomer concentration, and molar ratios of monomers, while sol fraction significantly depended on initiator concentration and monomer concentration, only. These results demonstrate the ability to fabricate hydrogels using EHD and PEGDA system as well as to control the properties of the resulting hydrophilic networks.
Keywords: Cyclic acetal; Hydrogel; Biomaterial; Swelling; Hydrolytic degradation;

Physicochemical and cell adhesion properties of chitosan films prepared from sugar and phosphate-containing solutions by Ruggero Bettini; Antonello A. Romani; Marina M. Morganti; Angelo F. Borghetti (74-81).
This work was aimed at investigating a series of chitosan films obtained from chitosan, chitosan-phosphate, chitosan-phosphate-d-(+)raffinose or chitosan-phosphate-d-(+)sucrose solutions to preliminarily select a suitable biomaterial for developing a cell substrate for tissue engineering. The prepared films were characterized in terms of physicochemical properties (FT-IR, XRD, optical microscopy, wettability, water absorption, and tensile stress) and effects on proliferation of different types of human cells (endothelial, HUVEC; fibroblast, WI-38).The obtained results indicated that the presence of sucrose or raffinose at high concentration along with phosphate salts in the chitosan film-forming solution affords smooth, amorphous and highly hydrophilic materials in the form of soft and elastic film with optimal cytocompatibility.Owing to improved physicochemical and mechanical properties as well as affinity for differentiated human cells, these novel chitosan films appear as promising candidate biomaterials for tissue regeneration and repair.The major finding is the possibility to improve the biocompatibility of chitosan films by simply modifying their solid state characteristics.
Keywords: Chitosan film; Sugars; Swelling index; Surface characterization; Mechanical properties; Human cell culture; Cytocompatibility; Cell proliferation;

Controlling protein release from scaffolds using polymer blends and composites by Patrick J. Ginty; John J.A. Barry; Lisa J. White; Steve M. Howdle; Kevin M. Shakesheff (82-89).
We report the development of three protein loaded polymer blend and composite materials that modify the release kinetics of the protein from poly(dl-lactic acid) (P dl LA) scaffolds. P dl LA has been combined with either poly(ethylene glycol) (PEG), poly(caprolactone) (PCL) microparticles or calcium alginate fibres using supercritical CO2 (scCO2) processing to form single and dual protein release scaffolds. P dl LA was blended with the hydrophilic polymer PEG using scCO2 to increase the water uptake of the resultant scaffold and modify the release kinetics of an encapsulated protein. This was demonstrated by the more rapid release of the protein when compared to the release rate from P dl LA only scaffolds. For the P dl LA/alginate scaffolds, the protein loaded alginate fibres were processed into porous protein loaded P dl LA scaffolds using scCO2 to produce dual release kinetics from the scaffolds. Protein release from the hydrophilic alginate fibres was more rapid in the initial stages, complementing the slower release from the slower degrading P dl LA scaffolds. In contrast, when protein loaded PCL particles were loaded into P dl LA scaffolds, the rate of protein release was retarded from the slow degrading PCL phase.
Keywords: Tissue engineering; Scaffolds; Protein delivery; Growth factors; Supercritical fluid;

The recent rapid progress of molecular biology together with the steady progress of genome projects has given us some essential and revolutionary information about DNA and RNA to elucidate various biological phenomena at a genetic level. Under these circumstances, the technology and methodology of gene transfection have become more and more important to enhance the efficacy of gene therapy for several diseases. In addition, gene transfection is a fundamental technology indispensable to the further research development of basic biology and medicine regarding stem cells. Stem cells genetically manipulated will enhance the therapeutic efficacy of cell transplantation. In this paper, the carrier and technology of gene delivery are briefly overviewed while the applications to the basic researches of biology and medicine as well as regenerative medical therapy are introduced. A new non-viral carrier and the cell culture system are described to efficiently manipulate stem cells.
Keywords: Non-viral gene carrier; Genetic engineering; Stem cells; Drug delivery system; Regenerative medical therapy;

Plasmid DNA was encapsulated within poly(d,l-lactic-co-glycolic acid) (PLGA) nanospheres by using polyethylene glycol (PEG) assisted solubilization technique of plasmid DNA in organic solvents. Plasmid DNA was solubilized in an organic solvent mixture composed of 80% methylene chloride and 20% DMSO by producing PEG/DNA nano-complexes having an average diameter less than 100 nm. DNA could be solubilized in the organic solvent mixture to a greater extent with increasing the weight ratio of PEG/DNA. PLGA nanospheres encapsulating DNA were successfully prepared by the single O/W emulsion method. They exhibited greater loading efficiency and better structural integrity, compared to those prepared by the W/O/W double emulsion method. Plasmid DNA could be successfully delivered to macrophage cells to express an exogenous gene. This new formulation enabled high loading of intact plasmid DNA within PLGA nanospheres useful for DNA vaccines.
Keywords: Plasmid DNA; Nanospheres; Gene delivery; Biodegradable; PLGA;

Polymers and nanoparticles: Intelligent tools for intracellular targeting? by M. Breunig; S. Bauer; A. Goepferich (112-128).
In recent years, a new generation of drugs has entered the pharmaceutical market. Some are more potent, but some are also more toxic and thus, therapeutical efficacy may be hindered, and severe side effects may be observed, unless they are delivered to their assigned place of effect. Those targets are not only certain cell types, moreover, in cancer therapy for example, some drugs even have to be targeted to a specific cell organelle. Those targets in eukaryotic cells include among others endo- and lysosomes, mitochondria, the so-called power plants of the cells, and the biggest compartment with almost all the genetic information, the nucleus. In this review, we describe how the drugs can be directed to specific subcellular organelles and focus especially on synthetic polymers and nanoparticles as their carriers. Furthermore, we portray the progress that has been accomplished in recent years in the field of designing the carriers for efficient delivery into these target structures. Yet, we do not fail to mention the obstacles that still exist and are preventing polymeric and nanoparticular drug carrier systems from their broad application in humans.
Keywords: Drug delivery; Intracellular targeting; Polymers; Nanoparticles; Endosomes; Lysosomes; Mitochondria; Nucleus;

The influence of the composition of the polymer coated polyvinyl alcohol (PVA), vinyl alcohol/vinyl amine copolymer (A-PVA) and polyethylenimine (PEI) coated superparamagnetic iron oxide nanoparticles (SPIONs) on the colloidal stability, cytotoxicity and cellular uptake of these particles in different cell media is reported in this paper. Although all examined polymer coated SPIONs were stable in water and PBS buffer these colloidal systems had different stabilities in DMEM or RPMI media without and supplemented with fetal calf serum (FCS). We found that A-PVA coating onto the surface of the SPIONs decreased the cytotoxicity of the polymer compared to the same concentration of A-PVA alone. As well, polyplexes of PEI-SPIONs with DNA in concentration used for transfection experiments showed no cytotoxicity compared to PEI and PEI-SPIONs. Our data show that the choice of medium largely influences the uptake of these particles by HeLa cells. The optimal medium is different for the different examined polymer coated SPIONs and it should be determined in each case, individually.
Keywords: Superparamagnetism; Iron oxide; Nanoparticles; PVA; PEI; Colloidal stability; Cytotoxicity;

Polymer coating of quantum dots – A powerful tool toward diagnostics and sensorics by A.F.E. Hezinger; J. Teßmar; A. Göpferich (138-152).
The use of quantum dots for biological and biomedical applications is one of the fastest moving fields of nanotechnology today. The unique optical properties of these nanometersized semiconductor crystals make them an exciting fluorescent tool for in-vivo and in-vitro imaging as well as for sensoric applications. To apply them in biological fluids or aqueous environment it is essential to modulate the chemical nature of quantum dot surfaces to alter their solubility and add additional chemical functionalities. By employing different coating technologies they cannot only be rendered water soluble but also functionalized to fulfill different tasks, like receptor targeting or sensing of low molecular weight substances. To achieve this goal different polymeric coatings are applied to provide solubility in water and additional functional groups for attachment. Taken together the versatile modifications described in this review make quantum dots a promising alternative to conventional fluorescent dyes and may offer possibilities for new future developments.
Keywords: Quantum dots; Polymer coating; Biocompatible; Sensorics; Diagnostics; Nanoparticles;