Dhirendra S. Katti

Biodegradable polyphosphazenes for drug delivery applications

Biodegradable polymers such as poly(alpha-hydroxy acids), poly(anhydrides), poly(ortho esters), poly(amino acids) and polyphosphazenes have raised considerable interest as short-term medical implants due to their transient nature. Among these, polyphosphazenes are a relatively new class of polymers, quite distinct from all the biodegradable polymers synthesized so far, due to their synthetic flexibility and versatile adaptability for applications. These are high molecular weight, essentially linear polymers with an inorganic backbone of alternating phosphorous and nitrogen atoms bearing two side groups attached to each phosphorous atom. Controlled tuning of physico-chemical properties, including biodegradability, can be achieved in this class of polymers via macromolecular substitutions. Biodegradable polyphosphazenes, due to their hydrolytic instability, nontoxic degradation products, ease of fabrication and matrix permeability, are an excellent platform for controlled drug delivery applications. This review discusses the mode of degradation and drug delivery applications of biodegradable polyphosphazenes.

Toxicity, biodegradation and elimination of polyanhydrides

Although originally developed for the textile industry, polyanhydrides have found extensive use in biomedical applications due to their biodegradability and excellent biocompatibility. Polyanhydrides are most commonly synthesized from diacid monomers by polycondensation. Efficient control over various physicochemical properties, such as biodegradability and biocompatibility, can be achieved for this class of polymers, due to the availability of a wide variety of diacid monomers as well as by copolymerization of these monomers. Biodegradation of these polymers takes place by the hydrolysis of the anhydride bonds and the polymer undergoes predominantly surface erosion, a desired property to attain near zero-order drug release profile. This review examines the mode of degradation and elimination of these polyanhydrides in vivo as well as the biocompatibility and toxicological aspects of various polyanhydrides.

Improved Biomaterials for Tissue Engineering Applications: Surface Modification of Polymers

Tissue engineering approaches that combine biomaterial-based scaffolds with protein delivery systems have provided a potential strategy for improved regeneration of damaged tissue. The success of polymeric scaffolds is determined by the response it elicits from the surrounding biological environment. This response is governed, to a large extent, by the surface properties of the scaffold. Surfaces of polymeric scaffolds have a significant effect on protein and cell attachment. Multiple approaches have been developed to provide micrometer to nanometer scale alterations in surface architecture of scaffolds to enable improved protein and cell interactions. Chemical modification of polymeric scaffold surfaces is one of the upcoming approaches that enables enhanced biocompatibility while providing a delivery vehicle for proteins. Similarly, physical adsorption, radiation mediated modifications, grafting, and protein modifications are other methods that have been employed successfully for alterations of surface properties of polymeric scaffolds. The goal of this review is to provide an overview of the role of surface properties /chemistry in tissue engineering and briefly discuss some of the methods of surface modification that can provide improved cell and protein interactions. It is hoped that these improved polymeric scaffolds will lead to accelerated and functional tissue regeneration.

Electrospraying: a facile technique for synthesis of chitosan-based micro/nanospheres for drug delivery applications.

Electrospraying is a novel technique for the generation of micro/nanospheres for biomedical applications. Apart from being a high yield technique; electrospraying has an added advantage of not making use of an external dispersion/emulsion phase which often involves ingredients that are undesirable for biomedical applications. In this study, we report the use of electrospraying for the synthesis of chitosan micro/nanospheres. The focus was to optimize the fabrication parameters involved in electrospraying for reproducible synthesis of chitosan based micro/nanospheres and to study their potential as delivery vehicles for bioactive agents. The influence of the following was studied (i) electrospraying voltage, (ii) needle gauge, (iii) concentration of chitosan solution, (iv) concentration of acetic acid solution, and (v) electrospraying distance. SEM analysis demonstrated that microspheres of less than 1 mum were obtained when chitosan concentration was 2% dissolved in 90% acetic acid. The working distance and needle gauge that yielded favorable results were 7 cm and 26 g, respectively. Average particle size of ampicillin loaded chitosan micro/nanospheres was 520 nm with zeta potential of +28.2 mV and encapsulation efficiency of 80.4%. The particles were characterized for drug release kinetics and results demonstrated an initial burst release followed by a sustained release over a period of 120 h. Further, antibacterial activity of drug loaded micro/nanospheres demonstrated that the encapsulated drug was in its active form postexposure to high voltage during electrospraying. This study indicates that electrospraying is a facile technique for the synthesis of chitosan micro/nanospheres for drug delivery applications.

Degradable polyphosphazene/poly(α-hydroxyester) blends: degradation studies

Biomaterials based on the polymers of lactic acid and glycolic acid and their copolymers are used or studied extensively as implantable devices for drug delivery, tissue engineering and other biomedical applications. Although these polymers have shown good biocompatibility, concerns have been raised regarding their acidic degradation products, which have important implications for long-term implantable systems. Therefore, we have designed a novel biodegradable polyphosphazene/poly(a-hydroxyester) blend whose degradation products are less acidic than those of the poly(a-hydroxyester) alone. In this study, the degradation characteristics of a blend of poly(lactide-co-glycolide) (50 :50 PLAGA) and poly[(50% ethyl glycinato)(50% p-methylphenoxy) phosphazene] (PPHOS-EG50) were qualitatively and quantitatively determined with comparisons made to the parent polymers. Circular matrices (14 mm diameter) of the PLAGA, PPHOS-EG50 and PLAGA–PPHOS-EG50 blend were degraded in nonbuffered solutions (pH 7.4). The degraded polymers were characterized for percentage mass loss and molecular weight and the degradation medium was characterized for acid released in non-buffered solutions. The amounts of neutralizing base necessary to bring about neutral pH were measured for each polymer or polymer blend during degradation. The poly(phosphazene)/poly(lactideco-glycolide) blend required significantly less neutralizing base in order to bring about neutral solution pH during the degradation period studied. The results indicated that the blend degraded at a rate intermediate to that of the parent polymers and that the degradation products of the polyphosphazene neutralized the acidic degradation products of PLAGA. Thus, results from these in vitro degradation studies suggest that the PLAGA–PPHOS-EG50 blend may provide a viable improvement to biomaterials based on acid-releasing organic polymers. r 2002 Elsevier Science Ltd. All rights reserved.

Electrosprayed inulin microparticles for microbiota triggered targeting of colon.

Inulin, a naturally occurring polysaccharide, was acetylated to make it processable by electrospraying, a facile and single step method for microparticle fabrication. Electrospraying process parameters were optimized for fabrication of spherical and monodisperse indomethacin (IDM) loaded inulin acetate (INA) microparticles. The apparent entrapment efficiency of IDM was determined to be 100%, whereas working encapsulation efficiency was estimated to be 35.39 ± 1.63%. Differential scanning calorimetry and X-ray diffraction analysis confirmed molecular dispersion of IDM in an amorphous state within the INA matrix. Finally, the results from in vitro release study performed in simulated gastro-intestinal fluids demonstrated that IDM was released only in simulated colonic fluid that contained inulinase. Therefore, this study demonstrates that acetylation of inulin does not alter its susceptibility to inulinase and that microparticles fabricated from INA can be developed as a colon targeting drug delivery system.

Pullulan-based composite scaffolds for bone tissue engineering: Improved osteoconductivity by pore wall mineralization

Porous hydrogels have been explored for bone tissue engineering; however their poor mechanical properties make them less suitable as bone graft substitutes. Since incorporation of fillers is a well-accepted method for improving mechanical properties of hydrogels, in this work pullulan hydrogels were reinforced with nano-crystalline hydroxyapatite (nHAp) (5 wt% nHAp in hydrogel) and poly(3-hydroxybutyrate) (PHB) fibers (3 wt% fibers in hydrogel) containing nHAp (3 wt% nHAp in fibers). Addition of these fillers to pullulan hydrogel improved compressive modulus of the scaffold by 10 fold. However, the hydrophilicity of pullulan did not support adhesion and spreading of cells. To overcome this limitation, porous composite scaffolds were modified using a double diffusion method that enabled deposition of hydroxyapatite on pore walls. This method resulted in rapid and uniform coating of HAp throughout the three-dimensional scaffolds which not only rendered them osteoconductive in vitro but also led to an improvement in their compressive modulus. These results demonstrate the potential of mineralized pullulan-based composite scaffolds in non-load bearing bone tissue engineering.

Regional drug delivery with radiation for the treatment of Ewing's sarcoma. In vitro development of a taxol release system.

Recently, several studies have suggested the radiosensitizing effect of taxol, a microtubular inhibitor. Our overall hypothesis is that a combination of radiation and taxol may demonstrate therapeutic efficacy over doses of either individually. Studies examining taxol use have mostly focused on systemic administration, which can lead to undesired effects. To circumvent these side effects, we propose a locally administered polymeric microsphere delivery system combined with radiation therapy for the treatment of Ewings sarcoma. The present study focuses on the in vitro ability of taxol when present as a microencapsulated drug delivery system, and delivered locally at the site of the sarcoma/tumor, to block cells in the G2/M phase of the cell cycle and potentially enhance the radiation sensitivity of cells. Using the bioresorbable poly(anhydride-co-imide), poly[pyromellityl-imidoalanine-1,6-bis(carboxy-phenoxy)hexane] (PMA-CPH), and the radiosensitizing agent taxol, a microsphere based delivery system was fabricated. A solvent evaporation technique was used to encapsulate taxol at doses of 1%, 5%, and 10% in PMA-CPH microspheres. Release kinetics studies demonstrated that the total amount of taxol released and the release rate were directly dependent on loading percentage. Taxols bioactivity and radiosensitizing ability were measured using flow cytometry. Co-culture of Ewings sarcoma cells with and without taxol-loaded microspheres demonstrated that released taxol retained its bioactivity and effectively blocked cells in the radiosensitive G2/M phase of mitosis. The taxol-radiation delivery system studied achieved an 83% decrease in tumor cell count compared to control. Taxol effectively sensitized Ewings sarcoma cells to radiation with radiosensitivity shown to be independent of radiation dose at levels of dosages studied. This work has demonstrated that taxol can be effectively released from a biodegradable PMA-CPH microsphere delivery system while maintaining potent combined cytotoxic and radiosensitizing abilities.

Pore orientation mediated control of mechanical behavior of scaffolds and its application in cartilage-mimetic scaffold design

Scaffolds with aligned pores are being explored in musculoskeletal tissue engineering due to their inherent structural anisotropy. However, influence of their structure on mechanical behavior remains poorly understood. In this work, we elucidate this dependence using chitosan-gelatin based random and aligned scaffolds. For this, scaffolds with horizontally or vertically aligned pores were fabricated using unidirectional freezing technique. Random, horizontal and vertical scaffolds were characterized for their mechanical behavior under compressive, tensile and shear loading regimes. The results revealed conserved trends in compressive, tensile and shear moduli, with horizontal scaffolds showing the least moduli, vertical showing the highest and random showing intermediate. Further, these scaffolds demonstrated a highly viscoelastic behavior under cyclic compressive loading, with a pore orientation dependent relative energy dissipation. These results established that mechanical behavior of porous scaffolds can be modulated by varying pore orientation alone. This finding paved the way to recreate the structural and consequent mechanical anisotropy of articular cartilage tissue using zonally varied pore orientation in scaffolds. To this end, monolithic multizonal scaffolds were fabricated using a novel sequential unidirectional freezing technique. The superficial zone of this scaffold had horizontally aligned pores while the deep zone consisted of vertically aligned pores, with a transition zone between the two having randomly oriented pores. This depth-dependent pore architecture closely mimicked the collagen alignment of native articular cartilage which translated into similar depth-dependent mechanical anisotropy as well. A facile fabrication technique, biomimetic pore architecture and associated mechanical anisotropy make this multizonal scaffold a promising candidate for cartilage tissue engineering.

Structural and functional characterization of proteins adsorbed on hydrophilized polylactide-co-glycolide microfibers

Background: Hydrophobic biopolymers such as polylactide-co-glycolide (PLGA, 85:15) have been extensively explored as scaffolding materials for tissue engineering applications. More recently, electrospun microfiber-based and nanofiber-based scaffolds of PLGA have received increased attention because they act as physical mimics of the fibrillar extracellular matrix. However, the hydrophobicity of the PLGA microfiber surface can limit its use in biomedical applications. Therefore, in a previous study, we fabricated Pluronic® F-108 (PF-108)-blended PLGA microfibrous scaffolds that alleviated the hydrophobicity associated with PLGA by enriching the surface of microfibers with the ethylene oxide units present in PF-108. Methods: In this study, we report the influence of the extent of surface enrichment of PLGA microfibers on their interaction with two model proteins, ie, bovine serum albumin (BSA) and lysozyme. BSA and lysozyme were adsorbed onto PLGA microfiber meshes (unmodified and modified) and studied for the amount, secondary structure conformation, and bioactivity of released protein. Results: Irrespective of the type of protein, PF-108-blended PLGA microfibers showed significantly greater protein adsorption and release than the unblended PLGA samples. However, in comparison with BSA, lysozyme showed a 7–9-fold increase in release. The Fourier transform infrared spectroscopy studies for secondary structure determination demonstrated that irrespective of type of microfiber surface (unblended or blended), adsorbed BSA and lysozyme did not show any significant change in secondary structure (α-helical content) as compared with BSA and/or lysozyme in the free powder state. Further, the bioactivity assay of lysozyme released from blended PLGA microfiber meshes demonstrated 80%–85% bioactivity, indicating that the process of adsorption did not significantly affect biological activity. Therefore, this study demonstrated that the decreased hydrophobicity of blended PLGA microfibrous meshes not only improved the amount of protein adsorbed (lysozyme and BSA) but also maintained the secondary structure and bioactivity of the adsorbed proteins. Conclusion: Modulating the hydrophobicity of PLGA via blending with PF-108 could be a viable strategy to improve its interaction with proteins and subsequent cell interaction in tissue engineering applications.