Enhanced resistance of polyelectrolyte multilayer microcapsules to pepsin erosion and release properties of encapsulated indomethacin

Polyelectrolyte multilayer films were prepared through layer-by-layer (LbL) self-assembly using polysaccharide sodium alginate (ALG) and chitosan (CHI). After incubation in an enzyme pepsin solution, the multilayer film was partially destroyed as detected by the decrease in fluorescent intensity because of the enzymatic degradation of CHI. The enzymatic desorption was also observed from the microcapsule wall made of the ALG/CHI multilayer film directly deposited on indomethacin (IDM) microcrystals through LbL self-assembly. After pepsin erosion, the IDM release from the microcapsules monitored by UV absorbance was obviously accelerated because of desorption. To enhance the stability of the ALG/CHI multilayer film to the enzymatic erosion, some physical and chemical methods were established to increase film thickness or to cross-link the polysaccharides within the film. Increasing the layer number and raising the deposition temperature effectively slowed down the enzymatic desorption and release rate. Especially, increasing deposition temperature was more effective because of producing a more perfect structure in the ALG/CHI multilayer film. Cross-linking the neighboring layers of ALG and CHI with 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide in the ALG/CHI multilayer film significantly reduced the enzymatic desorption and release rate. Therefore, increasing deposition temperature and cross-linking neighboring layers are effective methods to protect the multilayer film fabricated using LbL assembly from the enzymatic erosion and to prolong the release of the encapsulated drug.     

Tunable DNA release from cross-linked ultrathin DNA/PLL multilayered films

A novel ultrathin enzymatically degradable multilayered film using DNA as building blocks was fabricated by the layer-by-layer (LbL) technique. The UV-vis spectrometry and AFM experiments showed that the buildup of DNA and poly-L-lysine (PLL) was a kind of "exponentially growing films". The dye adsorption experiment suggested that the DNA molecules in the multilayered films were effectively protected by PLL. The films were further cross-linked by glutaraldehyde (GA). The cross-linking density of the films was modulated through the simple controlling of the time of the GA incubation process. An in vitro enzymatic degradation was carried out to investigate the DNA release profiles. The UV-vis spectrometry and fluorescence measurements indicated that the DNA release profiles were accordingly changed with the cross-linking density of the films. The nanoscale, easily processed enzymatically biodegradable PLL/DNA film with the ability to precisely control DNA release profiles may serve as a novel DNA delivery system, which may have great potential for gene therapy applications in implantable materials and biomedical devices.     

Multifunctional polyelectrolyte multilayer films: combining mechanical resistance, biodegradability, and bioactivity

Cross-linked polyelectrolyte multilayer films (CL PEM) have an increased rigidity and are mechanically more resistant than native (e.g., uncrosslinked) films. However, they are still biodegradable, which make them interesting candidates for biomedical applications. In this study, CL PEM films have been explored for their multifunctional properties as (i) mechanically resistant, (ii) biodegradable, and (iii) bioactive films. Toward this end, we investigated drug loading into CL chitosan/hyaluronan (CHI/HA) and poly(L-lysine)/hyaluronan (PLL/HA) films by simple diffusion of the drugs. Sodium diclofenac and paclitaxel were chosen as model drugs and were successfully loaded into the films. The effect of varying the number of layers in the (CHI/HA) films as well as the cross-linker concentration on diclofenac loading were studied. Diclofenac was released from the film in about 10 h. Paclitaxel was also found to diffuse within CL films. Its activity was maintained after loading in the CL films, and cellular viability could be reduced by about 55% over 3 days. Such a simple approach may be applied to other types of cross-linked films and to other drugs. These results prove that it is possible to design multifunctional multilayer films that combine mechanical resistance, biodegradability, and bioactivity properties into a single PEM architecture.     

Biodegradation and in vivo biocompatibility of rosin: a natural film-forming polymer

The specific aim of the present study was to investigate the biodegradation and biocompatibility characteristics of rosin, a natural film-forming polymer. Both in vitro as well as in vivo methods were used for assessment of the same. The in vitro degradation of rosin films was followed in pH 7.4 phosphate buffered saline at 37°C and in vivo by subdermal implantation in rats for up to 90 days. Initial biocompatibility was followed on postoperative days 7, 14, 21, and 28 by histological observations of the surrounding tissues around the implanted films. Poly (DL-lactic-co-glycolic acid) (PLGA) (50∶50) was used as reference material for biocompatibility. Rate and extent of degradation were followed in terms of dry film weight loss, molecular weight (MW) decline, and surface morphological changes. Although the rate of in vitro degradation was slow, rosin-free films showed complete degradation between 60 and 90 days following subdermal implantation in rats. The films degraded following different rates, in vitro and in vivo, but the mechanism followed was primarily bulk degradation. Rosin films demonstrated inflammatory reactions similar to PLGA, indicative of good biocompatibility. Good biocompatibility comparable to PLGA is demonstrated by the absence of necrosis or abscess formation in the surrounding tissues. The study provides valuable insight, which may lead to new applications of rosin in the field of drug delivery.     

Enzymatic desorption of layer-by-layer assembled multilayer films and effects on the release of encapsulated indomethacin microcrystals

Polyelectrolyte multilayer films were prepared through layer-by-layer (LbL) self-assembly of chitosan (CHI) and pyrene labeled poly(2-acrylamido-2-methylpropanesulfonic acid) (APy). After incubation in an enzyme pepsin solution, multilayer films were partially destroyed as detected by a decrease in fluorescence intensity due to enzymatic degradation of CHI and desorption of APy. The multilayer desorption rate was the highest at pH 4.0. Increasing temperature from 20 degrees C to 60 degrees C accelerated desorption. The enzymatic desorption was also observed from microcapsule walls made of CHI/alginate (ALG) multilayer films directly deposited on indomethacin (IDM) microcrystals by LbL self-assembly. After pepsin erosion, the IDM release from the microcapsule monitored by UV absorbance was obviously accelerated due to desorption. The influence of incubation time, pH, and temperature of the pepsin solution on the IDM release was investigated. The release rate was the fastest after incubation in the pepsin solution at pH 4.0 due to the highest activity of pepsin. Increasing incubation temperature from 20 degrees C to 60 degrees C, however, slowed down the release rate, which was considered to be due to the formation of more perfect and compact multilayer films through the chain rearrangement at higher temperatures. The CHI/ALG multilayer film was found to maintain its barrier function to the IDM diffusion even after 6-h incubation in the pepsin solution.     

Elasticity of native and cross-linked polyelectrolyte multilayer films

Mechanical properties of polyelectrolyte multilayer films were studied by nanoindentation using the atomic force microscope (AFM). Force-distance measurements using colloidal probe tips were systematically obtained for supported films of poly(L-lysine) and hyaluronan that are suited to bio-application. Both native and covalently cross-linked films were studied as a function of increasing layer number, which increases film thickness. The effective Young's modulus perpendicular to the film, Eperpendicular, was determined to be a function of film thickness, cross-linking, and sample age. Thick PEM films exhibited a lower Eperpendicular than thinner PEM, whereas the Young's modulus of cross-linked films was more than 10-fold larger than native films. Moduli range from approximately 20 kPa for native films up to approximately 800 kPa for cross-linked ones. Young's moduli increased slightly with sample age, plateauing after approximately 4 weeks. Spreading of smooth muscle cells on these substrates with pre-attached collagen proved to be highly dependent on film rigidity with stiffer films giving greater cell spreading.     

Chitosan film as rhBMP2 carrier: delivery properties for bone tissue application

Tissue engineering approaches need biomaterials with suitable properties to provide an appropriate environment for cell attachment and growth. The performance of these biomaterials can be greatly enhanced through the incorporation of bioactive agents. For this reason, we developed chitosan films with cell-attachment ability, rhBMP-2 carrier capacity, and good in vivo performance, and we employ them as covering for implantable materials. In this work, we have tried to explain how the rh-BMP2 is delivered to the surroundings from the development chitosan films. Protein diffusion from film, film stability versus in vitro dissolution, and biodegradation were evaluated to study rhBMP-2 delivery. Our results show that chitosan film has sufficiently good features to be used as an rhBMP-2 carrier. A low diffusion rate was observed, which was sufficient to quickly induce an in vitro differentiation stimulus, although heavily activated films retain more than 80-85% of the protein on the film. On the other hand, we estimated that chitosan film dissolution due to initial acidification in the wound environment is no more than 15-20%. We also estimated chitosan film response to lysozyme and concluded that degradation via this process proceeded at a slow kinetic rate. In addition, rhBMP-2 in vitro activity after film processing, as well as in vivo film behavior, were studied. We confirm that rhBMP-2 remains active on the film and after release, both in vitro and in vivo. These results support the conclusion that the developed chitosan film allows sustained release of the rhBMP-2 osteoinductive protein and could be used as an activated coat for implant and surgical prosthesis.     

Synthesis and biological evaluation of a cross-linked hyaluronan-mitomycin C hydrogel

A cross-linked hyaluronan (HA) hydrogel that contained a covalently bound derivative of the anti-proliferative drug mitomycin C (MMC) was synthesized and evaluated in vitro and in vivo. The HA-MMC hydrogel was prepared by coupling MMC-aziridinyl-N-acrylate with thiol-modified HA followed by cross-linking with poly(ethylene glycol) diacrylate (PEGDA). MMC was released from 0.5% and 2.0% MMC films by hydrolysis in proportion to the MMC loading. When incubated in vitro with human T31 tracheal scar fibroblasts, 0.5% MMC films inhibited proliferation, whereas 2.0% MMC films were cytotoxic. When implanted in vivo into a rat peritoneal cavity, neither 0.5% nor 2.0% HA-MMC films elicited a severe peritoneal fluid leukocyte response. Importantly, MMC reduced the thickness of fibrous tissue formed surrounding the implanted films. Thus, cross-linked HA-MMC films have strong potential as anti-fibrotic barriers for the prevention of post-surgical adhesions.     

Drug release from and hydrolytic degradation of a poly(ethylene glycol) grafted poly(3-hydroxyoctanoate)

Monoacrylate-poly(ethylene glycol)-grafted poly(3-hydroxyoctanoate) (PEGMA-g-PHO) copolymers were synthesized to develop a swelling-controlled release delivery system for ibuprofen as a model drug. The in vitro hydrolytic degradation of and the drug release from a film made of the PEGMA-g-PHO copolymer were carried out in a phosphate buffer saline (pH 7.4) medium. The hydrolytic degradation of the copolymer was strongly dependent on the degree of grafting (DG) of the PEGMA group. The degradation rate of the copolymer films in vitro increased with increasing DG of the PEGMA group on the PHO chain. The copolymer films showed a controlled delivery of ibuprofen to the medium in periods of time that depend on the composition, hydrophilic/hydrophobic characteristics, initial drug loading amount and film thickness of the graft copolymer support. The drug release rate from the grafted copolymer films was faster than the rate of weight loss of the films themselves. In particular, a combination of the low DG of the PEGMA group in the PHO chains with the low ibuprofen solubility in water led to long-term constant release from these matrices in vitro.     

Micromatricial metronidazole benzoate film as a local mucoadhesive delivery system for treatment of periodontal diseases

The main objective of this study was to develop a local, oral mucoadhesive metronidazole benzoate (MET) delivery system that can be applied and removed by the patient for the treatment of periodontal diseases. Mucoadhesive micromatricial chitosan/poly(ε-caprolactone) (CH/PCL) films and chitosan films were prepared. thermal behavior, morphology, and particle size measurements were used to evaluate the prepared films. The effect of different molar masses of CH and different ratios of medium Mwt molar mass chitosan (MCH):PCL on water absorption, in vitro bioadhesion, mechanical properties, and in vitro drug release was examined. In vivo performance of the selected formulation was also evaluated. Differential scanning calorimetry examination revealed that MET existed mainly in amorphous form. Under microscopic examination, PCL microparticles were homogeneously dispersed in the films. The use of different molar masses of CH and different ratios of (MCH):PCL affected the size of the entrapped particles. Addition of PCL significantly decreased percentage water uptake and bioadhesion force compared with pure CH film. With regard to mechanical properties, the 2-layered film containing 1∶0.625 MCH:PCL had the best tensile properties. At fixed CH:PCL ratio (1∶1.25), the slowest drug release was obtained from films containing high molar mass CH. On the other hand, the 2-layered film that consisted of 1∶0.625 MCH:PCL had the slowest MET release. In vivo evaluation of the selected film revealed that metronidazole concentration in saliva over 6 hours ranged from 5 to 15 μg/mL, which was within and higher than the reported range of minimum inhibitory concentration for metronidazole. A significant in vitro/in vivo correlation under the adopted experimental conditions was obtained. Published: September 14, 2007     

In vitro processing of the artificial analog of beta-lipotropin

The processing of the recombinant analogue of beta-lipotropin (beta-LHP) having 11 additional N-terminal amino acid residues and separated from the hormone by the processing signal, was investigated using rat adrenal secretory granule lysate as a test system of processing "in vitro". It was found that incubation of the beta-LPH analogue with secretory granule enzymes leads to its limited specific degradation with a release of native beta-endorphin. It is concluded that the additional N-terminal amino acids induced no qualitative changes in beta-LPH processing.     

SMS 201-995: a very potent and selective octapeptide analogue of somatostatin with prolonged action

Stepwise modification of a conformationally stabilised analogue of the fragment of somatostatin which had been thought to be essential biologically active moiety has enabled us to synthesise the analogue H-(D) Phe-Cys-Phe-(D) Trp-Lys-Thr-Cys-Thr(ol) code-named SMS 201-995, which in vitro is three times more potent than the native hormone in inhibiting the secretion of growth hormone, which is highly resistant to degradation by pure enzymes and by tissue homogenates, which in vivo in rat and rhesus monkey is (depending on test system) at least 20 times more active than somatostatin, which is much longer acting, and which moreover in both species is much more selective in inhibiting the secretion of growth hormone than that of insulin. The compound is active by several routes of administration including the oral, is well tolerated both in laboratory animals and in man, and is currently undergoing preliminary clinical trial.     

Biodegradable chitosan matrix for the controlled release of steroids.

Chitosan, a polysaccharide, having structural characteristics similar to glycosaminoglycans, seems to be nontoxic and bioabsorbable. This study highlights the use of chitosan matrix for controlled drug delivery systems. The steroid drugs, namely testosterone, progesterone and beta-oestradiol were mixed with chitosan and the films were prepared by evaporation technique. The in vitro release profile of these steroids from the film matrix was monitored, as a function of time, in phosphate buffered saline (PBS, pH 7.4) at 37 degree C using a U-V-spectrophotometer. The degradation, of these chitosan and drug loaded chitosan films, was also investigated by weight loss and tensile strength studies. The steroid release from chitosan films was compared with the release of these drugs from their microbeads. It appears, the films and the microbeads stayed intact during the dissolution study of 90 days and the possibility of using these systems in contraceptive applications and novel drug delivery systems are discussed.     

Effect of pectin charge density on formation of multilayer films with chitosan

The effect of pectin charge density on the formation of multilayer films with chitosan (PEC/CHI) is studied by means of electro-optics. Pectins of low (21%) and high (71%) degrees of esterification, which are inversely proportional to the pectin charge density, are used to form films on colloidal beta-FeOOH particles at pH 4.0 when the CHI is fully ionized. We find that, after deposition of the first 3-4 layers, the film thickness increases linearly with the number of adsorbed layers. However, the increase in the film thickness is larger when the film is terminated with CHI. Irregular increase of the film thickness is more marked for the PEC with higher density of charge. Oscillation in the electrical polarizability of the film-coated particles with the number of deposited layers is also registered in the PEC/CHI films. The charge balance of the multilayers, calculated from electrical polarizability of the film-coated particles, is positive, with larger excess of positive charge within the film constructed from CHI and less charged PEC. This is attributed to the ability of CHI to diffuse into the film at each deposition step. Despite the CHI diffusion, the film thickness increases linearly due to the dissolution of unstable PEC/CHI complexes from the film surface.     

Controlled release of proteins from their poly(ethylene glycol) conjugates: drug delivery systems employing 1,6-elimination

Several tripartate releasable PEG linkers (rPEG) that can provide anchimeric assistance to hydrolysis (cyclization prodrugs) were prepared and, after conjugation to lysozyme demonstrated rapid cleavage in rat plasma compared to nonassisted, permanently bound PEG. By varying the chemical structure and adding steric hindrance, the half-life of the protein conjugates can be adjusted from slow to very fast. The pharmacokinetics (PK) of regeneration of native protein, from various rPEG conjugates can, for the first time, be easily followed in the rat using green fluorescent protein. The PK in mice was also determined for rPEG-Interleukin 2 (rPEG-IL-2) conjugates in vivo using an ELISA assay. Thus, a systematic study of rPEGylated proteins, either in vivo or in vitro during processing, has been investigated based on regeneration of native protein. The employment of releasable PEG polymers substantially broadens the applications of PEGylation drug delivery technology by introducing the benefits of controlled release of native protein therapeutics.     

Water-based nanoparticulate polymeric system for protein delivery: permeability control and vaccine application

The idea of using polymeric nanoparticles as drug carriers is receiving an increasing amount of attention both in academia and industry, Nanoparticles have a number of potential applications in protein, drug and vaccine delivery, as well as gene therapy applications. In this article, we focus on this unique drug delivery technology as a method to control the release rate of substances, not only for protein delivery but also for delivering an experimental vaccine immunogen. Nanoparticles were assembled on the basis of ionic interaction between water-soluble polymers so that the resulting particles were stable in physiologic media. Among the typical polymers used to assemble nanoparticles, different polysaccharides, natural amines, and poly-amines were investigated. The entrapped substances tested included a protein and antigens. Polydextran aldehyde was incorporated into the particle core, to enable physiologic cross-linking as a method to control permeability. This resulted in long-term retention of substances that would otherwise rapidly leak out of the nanoparticles. Results of cross-linking experiments clearly demonstrated that the release rate could be substantially reduced, depending on the degree of cross-linking. For vaccine antigen delivery tests, we measured an antibody production after subcutaneous and oral administration. The data indicated that only the cross-linked antigen was immunogenic when the oral route of administration was used. The data presented in this article address primarily the utility of nanoparticulates for oral delivery of vaccine antigen.     

Biomedical applications of electrostatic layer-by-layer nano-assembly of polymers,enzymes, and nanoparticles

The introduction of electrostatic layer-by-layer (LbL) self-assembly has shown broad biomedical applications in thin film coating, micropatterning, nanobioreactors, artificial cells, and drug delivery systems. Multiple assembly polyelectrolytes and proteins are based on electrostatic interaction between oppositely charged layers. The film architecture is precisely designed and can be controlled to 1-nm precision with a range from 5 to 1000 nm. Thin films can be deposited on any surface including many widely used biomaterials. Microencapsulation of micro/nanotemplates with multilayers enabled cell surface modification, controlled drug release, hollow shell formation, and nanobioreactors. Both in vitro and in vivo studies indicate potential applications in biology, pharmaceutics, medicine, and other biomedical areas.     

Entrapment of 5‐aminolevulinic acid under edible composite film of konjac glucomannan and chitosan

5‐Aminolevulinic acid (5‐ALA) is a known plant regulator and growth promoter. It is a very sensitive and highly unstable compound that is easy to deteriorate. Here we propose a novel approach to stabilize 5‐ALA into a film. Films from konjac glucomannan (KGM), KGM treated with alkali solution (KGOH), chitosan (CHI) as well as blends between KGOH and CHI were fabricated for 5‐ALA entrapment. It was found that the efficiency of KGM film, KGOH film and CHI film for 5‐ALA entrapment was 55.7 ± 0.73%, 58.3 ± 0.36% and 60.3 ± 0.18 %, respectively. A 25:75 (%w/w) blended film (KGOH/CHI) showed the highest entrapment efficiency of 5‐ALA (65.9 ± 0.37%) versus other films. The possible mechanism for entrapment of 5‐ALA in blended film was postulated under two mechanisms. A secondary amide that leads to the interaction between the amino group of CHI and carboxyl group of 5‐ALA is proposed as the first mechanism. The fact that the 5‐ALA molecule was entrapped within the complexity of KGOH structure is proposed as the second mechanism. Therefore, stabilizing 5‐ALA in a film may be an alternative way to use and preserve 5‐ALA for further applications.     

Photo-cross-linked hybrid polymer networks consisting of poly(propylene fumarate) and poly(caprolactone fumarate): controlled physical properties and regulated bone and nerve cell responses

Aiming to achieve suitable polymeric biomaterials with controlled physical properties for hard and soft tissue replacements, we have developed a series of blends consisting of two photo-cross-linkable polymers: polypropylene fumarate (PPF) and polycaprolactone fumarate (PCLF). Physical properties of both un-cross-linked and UV cross-linked PPF/PCLF blends with PPF composition ranging from 0% to 100% have been investigated extensively. It has been found that the physical properties such as thermal, rheological, and mechanical properties could be modulated efficiently by varying the PPF composition in the blends. Thermal properties including glass transition temperature (T g) and melting temperature (T m) have been correlated with their rheological and mechanical properties. Surface characteristics such as surface morphology, hydrophilicity, and the capability of adsorbing serum protein from culture medium have also been examined for the cross-linked polymer and blend disks. For potential applications in bone and nerve tissue engineering, in vitro cell studies including cytotoxicity, cell adhesion, and proliferation on cross-linked disks with controlled physical properties have been performed using rat bone marrow stromal cells and SPL201 cells, respectively. In addition, the role of mechanical properties such as surface stiffness in modulating cell responses has been emphasized using this model blend system.