Current Topics in Medicinal Chemistry - Volume 8, Issue 4, 2008

The fields of engineering, basic sciences, and medicine are rapidly converging when it comes to biomedical engineering. It was not too long ago that biomedical engineers were almost exclusively involved with designing imaging and other medical instrumentations, mainly from an engineering perspective; today scientists in this field come from various backgrounds and have added expertise such as cell/tissue culture and protein synthesis to their engineering armamentarium. A multidisciplinary approach has become imperative to solve the medical challenges ahead. The use of biomaterials as implants in the human body has become a commonplace, with implants ranging from dental prosthetics to artificial knees and coronary stents. Even as the quality and complexity of these implants have increased, there has been a growing movement in the field of biomaterials to regenerate various diseased tissues instead of replacing them with man-made implants. Over the past 30 years, this effort has evolved into a field know as tissue engineering or regenerative medicine. Thus, the role of biomaterials in this field has changed from restoration of function to assisting in the re-growth of functional tissues. Consequently, some of the biggest challenges in the area of biomaterials lie at the interface of biology and materials. This special issue of the journal contains articles on tissue engineering from some of the foremost laboratories in the world and provides a glimpse of the cutting edge work in this very complex field. Another significant challenge in the area of biomaterials is site-specific or targeted delivery of therapeutic materials such as drugs and genes. Systemic delivery often dilutes the amount of therapeutics needed at the required site while increasing the risk of adverse side effects. On the other hand, targeted systems can deliver high concentrations exclusively to the diseased site, thus increasing the efficacy and reducing the overall quantity of drug that has to be administered. Such systems are especially valuable in the fields of cardiology and oncology. Once again, we are fortunate that some of the leaders in the field have agreed to publish their latest research in this special issue. The papers in this issue represent but a small cross-section of the kind of exciting and excellent research ongoing in the area of biomaterials. They also show the multidisciplinary nature of the work and we hope that the readers will be encouraged to seek collaborations and share their expertise with those in the field.

Implantable medical devices are increasingly important in the practice of modern medicine. Unfortunately, almost all medical devices suffer to a different extent from adverse reactions, including inflammation, fibrosis, thrombosis and infection. To improve the safety and function of many types of medical implants, a major need exists for development of materials that evoked desired tissue responses. Because implant-associated protein adsorption and conformational changes thereafter have been shown to promote immune reactions, rigorous research efforts have been emphasized on the engineering of surface property (physical and chemical characteristics) to reduce protein adsorption and cell interactions and subsequently improve implant biocompatibility. This brief review is aimed to summarize the past efforts and our recent knowledge about the influence of surface functionality on protein:cell:biomaterial interactions. It is our belief that detailed understandings of bioactivity of surface functionality provide an easy, economic, and specific approach for the future rational design of implantable medical devices with desired tissue reactivity and, hopefully, wound healing capability.

Delivery of therapeutic agents from self-assembled monolayers (SAMs) on 316L stainless steel (SS) has been demonstrated as a viable method to deliver drugs for localized coronary artery stent application. SAMs are highlyordered, nano-sized molecular coatings, adding 1-10 nm thickness to a surface. Hydroxyl terminated alkanethiol SAMs of 11-mercapto-1-undecanol (-OH SAM) were formed on 316L SS with 48 hr immersion in ethanolic solutions. Attachment of ibuprofen (a model drug) to the functional SAMs was carried out in toluene for 5 hrs at 60 C using Novozume-435 as a biocatalyst. SAM formation and subsequent attachment of ibuprofen was characterized collectively using X-ray photoelectron spectroscopy (XPS), Fourier transform infrared spectroscopy (FTIR), and contact angle (CA) measurements. The quantitative in vitro release of ibuprofen into a “physiological” buffer solution was characterized using reverse phase HPLC. Drug release kinetics showed that 14.1 μg of ibuprofen eluted over a period of 35 days with 2.7μg being eluted in the first day and the remaining being eluted over a period of 35 days. The drug release kinetics showed an increase in ibuprofen elution that occurred during first 14 days (2.7μg in 1 day to 9.5 μg in 14 days), following which there was a decrease in the rate of elution. Thus, functional SAMs on 316L SS could be used as tethers for drug attachment and could serve as a drug delivery mechanism from stainless steel implants such as coronary artery stents.

A vast number of manufacturing techniques have been employed in the last five years to manufacture three dimensional (3D) calcium phosphate (CaP) scaffolds, with the intention to replicate the architecture of native bone as well as to repair and restore bone function. Design features such as architectural control and sintering temperature and their impact on scaffold performance is presented in this review. In vitro cell responses to bioceramic scaffolds and their in vivo performances have been enhanced. Current frontiers of active research on HA scaffolds have included the relationship between fluid flow and mechanotransduction as well as cell signaling pathways that induce endothelial cell recruitment and angiogenesis. Additionally, current research has focused on a better understanding of cell signaling and its environmental cues. The availability of non-invasive and non-destructive quantitative imaging modalities has also become critical in aiding the characterization of scaffolds and predicting scaffold performance. It is thus anticipated that further knowledge gained from this research will allow the overall advancement of scaffolds that can be clinically used to restore large bone defects.

Although there have been extensive research efforts to create functional tissues and organs, most successes in tissue engineering have been limited to avascular or thin tissues. The major hurdle in development of more complex tissues lies in the formation of vascular networks capable of delivering oxygen and nutrients throughout the engineered constructs. Sufficient neovascularization in scaffold materials can be achieved through coordinated application of angiogenic factors with proper cell types in biomaterials. This review present the current research developments in the design of biomaterials and their biochemical and biochemical modifications to produce vascularized tissue constructs.

Gene therapy today is hampered by the need of a safe and efficient gene delivery system that can provide a sustained therapeutic effect without cytotoxicity or unwanted immune responses. Bolus gene delivery in solution results in the loss of delivered factors via lymphatic system and may cause undesired effects by the escape of bioactive molecules to distant sites. Controlled gene delivery systems, acting as localized depot of genes, provide an extended sustained release of genes, giving prolonged maintenance of the therapeutic level of encoded proteins. They also limit the DNA degradation in the nuclease rich extra-cellular environment. While attempts have been made to adapt existing controlled drug delivery technologies, more novel approaches are being investigated for controlled gene delivery. DNA encapsulated in nano/micro spheres of polymers have been administered systemically/orally to be taken up by the targeted tissues and provide sustained release once internalized. Alternatively, DNA entrapped in hydrogels or scaffolds have been injected/implanted in tissues/cavities as platforms for gene delivery. The present review examines these different modalities for sustained delivery of viral and non-viral gene-delivery vectors. Design parameters and release mechanisms of different systems made with synthetic or natural polymers are presented along with their prospective applications and opportunities for continuous development.

Chitosan is a polysaccharide that has generated significant interest as a non-viral gene delivery vehicle due to its cationic and biocompatible characteristics. However, transfection efficiency of chitosan is significantly lower compared to other cationic gene delivery agents, e.g. polyethyleneimine (PEI), dendrimers or cationic lipids. This is primarily attributed to its minimal solubility and low buffering capacity at physiological pH leading to poor endosomal escape of the gene carrier and inefficient cytoplasmic decoupling of the complexed nucleic acid. Here we have developed an imidazole acetic acid (IAA)-modified chitosan to introduce secondary and tertiary amines to the polymer in order to improve its endosomal buffering and solubility. The modified polymer was characterized by ninhydrin and 1H NMR assays for degree of modification, while buffering and solubility were analyzed by acid titration. Nanocomplex formation, studied at various polymer-nucleic acid ratios, showed an increase in particle zeta potential for chitosan-IAA, as well as an increase in the effective diameter. Up to 100-fold increase in transfection efficiency of pDNA was seen for chitosan-IAA as compared to native chitosan, nearly matching that of PEI. In addition, transfection of siRNA by the modified polymers showed efficient gene knockdown equivalent to commercially available siPORT Amines. Collectively, these results demonstrate the potential of the imidazole-grafted chitosan as a biocompatible and effective delivery vehicle for both pDNA and siRNA.

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.

Biodegradable polymeric scaffolds are widely used as a temporary extracellular matrix in tissue engineering and regenerative medicine. By physical adsorption of biomolecules on scaffold surface, physical entrapment of biomolecules in polymer microspheres or hydrogels, and chemical immobilization of oligopeptides or proteins on biomaterials, biologically active biomaterials and scaffolds can be derived. These bioactive systems show great potential in tissue engineering in rendering bioactivity and/or specificity to scaffolds. This review highlights some of the biologically active chitosan systems for tissue engineering application and the associated strategies to develop such bioactive chitosan systems.

Halopemide, a phospholipase D2 inhibitor identified by high-throughput screening. In January 2007 Monovich and co-workers at Novartis identified halopemide, via a highthroughput screen, as an inhibitor of phospholipase D2 (PLD2) [1]. This was a major feat, as the PLD field suffered from a lack of ligands to probe its biological function; indeed, the gold standard PLD inhibitor is n-butanol. Phospholipase D (PLD) is present in mammalian cells in two distinct isoforms, PLD1 and PLD2 [2,3]. Both PLD isoforms catalyze the hydrolysis of the phosphodiester bond of phosphatidylcholine, thereby forming free choline and phosphatidic acid [2-4]. PLD has been shown to be involved in regulating the actin cytoskeleton, vesicle trafficking, and receptor signaling [3]. PLD itself has been shown to be regulated by phosphatidylinositol-4,5-bisphosphate, protein kinase C, ADP Ribosylation Factor, and the Rho family GTPases [3]. Several disease processes are thought be linked to PLD activity, including cancer and inflammation [5]. A sizeable body of evidence exists that shows elevated PLD expression is often correlated with oncogenesis and metastasis [5]. While a firm mechanistic understanding of PLD’s role in cancer has not yet been elucidated PLD is thought to contribute to Raf recruitment, cell cycle checkpoint override, the suppression of apoptosis, and protease secretion and increased cell motility [5]. In some cases the activity of only one PLD isoform is relevant. For example, only PLD2 activity has been shown to be elevated in renal cell carcinoma [2,6]. Due to the burgeoning body of evidence implicating PLD in cancer and inflammatory diseases, and the frequent observance of elevated PLD expression and activity in various tumors PLD ought to be a favorable molecular target for therapeutic intervention [1,5]. Halopemide was originally reported by Janssen in the 1970's as a potential therapy for autism or other neurological conditions [1, 7]. Recently, Monovich and co-workers from Novartis reported parallel synthesis and relatively modest optimization of halopemide analogs for PLD2 inhibition. Thirteen compounds are disclosed, all of which are actually analogs of deschloro-halopemide. Deschloro-halopemide showed PLD2 potency comparable to halopemide (1.4 μM versus 1.500 μM, respectively) [1]. Monovich and coworkers synthesized 13 analogs in which the amide portion of the molecule was varied [1]. By replacing the pfluorophenyl moiety with a 2-indolyl moiety potency was improved from 1.4 μM to 20.0 nM [1]. The 5-fluoro-2- indolyl compound showed moderate oral bioavailability (18.5%) in rats, and while the compound had a high clearance of 2.18 Lh-1kg-1 it also had a half-life of 5.57 h [1]. Also, a Cmax more than 10 times the IC50 was obtained when the 5-fluoro-2-indolyl compound was dosed at 5 mg/kg p.o. or 1 mg/kg iv [1]. The results from this initial optimization project are encouraging, but a better understanding of the pharmacology of these compounds, and other analogs, would provide more progress toward developing these molecules into a pre-clinical candidate for indications such as cancer and inflammation. Specifically, data on potency in various cell types expressing PLD1 and/or PLD2, cell invasion assay data, and in vivo pharmacology data from animal disease models would be very informative. The work of Monovich and co-workers may lead to progress in understanding the pharmacology of a relatively new molecular target, PLD, in diseases such as cancer and inflammation and represents a significant advance in the PLD field. REFERENCES [1] Monovich, L.; Mugrage, B.; Quadros, E.; Toscano, K.; Tommasi, R.; LaVoie, S.; Liu, E.; Du, Z.; LaSala, D.; Boyar, W.; Steed, P. ‘Optimization of Halopemide for Phospholipase D2 inhibition’ Bioorg. Med. Chem. Lett. 2007, 17(8), 2310-2311. [2] McDermott, M.; Wakelam, M.; Morris, A. ‘Phospholipase D’ Biochem. Cell Biol. 2004, 82, 225-253. [3] Jenkins, G.M.; Frohman, M.A.; ‘Phospholipase D: a lipid centric review’ Cell. Mol. Life Sci. 2005, 62, 2305-2316. [4] Saito M. and Kanfer J. ‘Phosphatidohydrolase activity in a solubilized preparation from rat brain particulate fraction’ Arch. Biochem. Biophys. 1975, 169, 318-323. [5] Foster, D.A.; Xu, L. ‘Phospholipase D in Cell Proliferation and Cancer’ Molecular Cancer Research 2003, 1, 789-800. [6] Zhao, Y., Ehara, H., Akao, Y., Shamoto, M., Nakagawa, Y., Banno, Y., Deguchi, T., Ohishi, N., Yagi, K., and Nozawa, Y. ‘Increased activity and intranuclear expression of phospholipase D2 in renal cancer’ Biochem. Biophys. Res. Commun. 2000, 278, 140-143. [7] Loonen, A.J.M.; Wijngaarden, I.V.; Janssen, P.A.J.; Soundijn, W. ‘Regional localization of halopemide, a new psychotropic agent, in the rat brain’ European Journal of Pharmacology 1978, 50, 403- 408.