Advanced Bioactive Inorganic Materials for Bone Regeneration and Drug Delivery

Bioceramics play an important role in repairing and regenerating bone defects. Annually, more than 500,000 bone graft procedures are performed in the United states and approximately 2.2 million are conducted worldwide. The estimated cost of these procedures approaches $2.5billion per year. Around 60% of the bone graft substitutes available on the market involve bioceramics. It is reported that bioceramics in the world market increase by 9% per year. For this reason, the research of bioceramics has been one of the most active areas during, the past several years. Considering the significant importance of bioceramics, our goal was to compile this book to review the latest research advances in the field of bioceramics. The text also summarizes our work during the past 10 years in an effort to share innovative concepts, design of bioceramisc, and methods for material synthesis and drug delivery. We anticipate that this text will provide some useful information and guidance in the bioceramics field for biomedical engineering researchers and material scientists. Information on novel mesoporous bioactive glasses and silicate-based ceramics for bone regeneration and drug delivery are presented. Mesoporous bioactive glasses have shown multifunctional characteristics of bone regeneration and drug delivery due to their special mesopore structures,whereas silicated-based bioceramics, as typical third-generation biomaterials,possess significant osteostimulation properties. Silica nanospheres with a core-shell structure and specific properties for controllable drug delivery have been carefully reviewed-a variety of advanced synthetic strategies have been developed to construct functional mesoporous silica nanoparticles with a core-shell structure, including hollow, magnetic, or luminescent, and other multifunctional core-shell mesoporous silica nanoparticles. In addition, multifunctional drug delivery systems based on these nanoparticles have been designed and optimized to deliver the drugs into the targeted organs or cells,with a controllable release fashioned by virtue of various internal and external triggers. The novel 3D-printing technique to prepare advanced bioceramic scaffolds for bone tissue engineering applications has been highlighted, including the preparation, mechanical strength, and biological properties of 3D-printed porous scaffolds of calcium phosphate cement and silicate bioceramics. Three-dimensional printing techniques offer improved large-pore structure and mechanical strength. In addition , biomimetic preparation and controllable crystal growth as well as biomineralization of bioceramics are summarized, showing the latest research progress in this area. Finally, inorganic and organic composite materials are reviewed for bone regeneration and gene delivery. Bioactive inorganic and organic composite materials offer unique biological, electrical, and mechanical properties for designing excellent bone regeneration or gene delivery systems. It is our sincere hope that this book will updated the reader as to the research progress of bioceramics and their applications in bone repair and regeneration. It will be the best reward to all the contributors of this book if their efforts herein in some way help reader in any part of their study, research, and career development.

Gene electrotransfer into murine skeletal muscle: A systematic analysis of parameters for long-term gene expression

Skeletal muscle is an attractive target tissue for delivery of therapeutic genes, since it is well vascularized, easily accessible, and has a high capacity for protein synthesis. For efficient transfection in skeletal muscle, several protocols have been described, including delivery of low voltage electric pulses and a combination of high and low voltage electric pulses. The aim of this study was to determine the influence of different parameters of electrotransfection on short-term and long-term transfection efficiency in murine skeletal muscle, and to evaluate histological changes in the treated tissue. Different parameters of electric pulses, different time lags between plasmid DNA injection and application of electric pulses, and different doses of plasmid DNA were tested for electrotransfection of tibialis cranialis muscle of C57BI/6 mice using DNA plasmid encoding green fluorescent protein (GFP). Transfection efficiency was assessed on frozen tissue sections one week after electrotransfection using a fluorescence microscope and also noninvasively, followed by an in vivo imaging system using a fluorescence stereo microscope over a period of several months. Histological changes in muscle were evaluated immediately or several months after electrotransfection by determining infiltration of inflammatory mononuclear cells and presence of necrotic muscle fibers. The most efficient electrotransfection into skeletal muscle of C57BI/6 mice in our experiments was achieved when one high voltage (HV) and four low voltage (LV) electric pulses were applied 5 seconds after the injection of 30 μg of plasmid DNA. This protocol resulted in the highest short-term as well as long-term transfection. The fluorescence intensity of the transfected area declined after 2-3 weeks, but GFP fluorescence was still detectable 18 months after electrotransfection. Extensive inflammatory mononuclear cell infiltration was observed immediately after the electrotransfection procedure using the described parameters, but no necrosis or late tissue damage was observed. This study showed that electric pulse parameters, time lag between the injection of DNA and application of electric pulses, and dose of plasmid DNA affected the duration of transgene expression in murine skeletal muscle. Therefore, transgene expression in muscle can be controlled by appropriate selection of electrotransfection protocol.

DNA molecules and human therapeutics

Nucleic acid molecules are championing a new generation of reverse engineered biopharmaceuticals. In terms of potential application in gene medicine, plasmid DNA (pDNA) vectors have exceptional therapeutic and immunological profiles as they are free from safety concerns associated with viral vectors, display non-toxicity and are simpler to develop. This review addresses the potential applications of pDNA molecules in vaccine design/development and gene therapy via recombinant DNA technology as well as a staged delivery mechanism for the introduction of plasmid-borne gene to target cells via the nasal route.

Adenoviral gene therapy for non-small cell lung cancer

Adenoviral gene therapy is an experimental approach to cancer refractory to standard cancer therapies. Adenoviruses can be utilized as vectors to deliver therapeutic transgenes into cancer cells, while gene therapy with oncolytic adenoviruses exploits the lytic potential of viruses to kill tumor cells. Although adenoviruses demonstrate several advantages over other vectors - such as the unparalleled transduction efficacy and natural tropism to a wide range of tissues - the gene transfer efficacy to cancer cells has been limited, consequently restricting the therapeutic effect. There are, however, several approaches to circumvent this problem. We utilized different modified adenoviruses to obtain information on adenovirus tropism towards non-small cell lung cancer (NSCLC) cells. To enhance therapeutic outcome, oncolytic adenoviruses were evaluated. Further, to enhance gene delivery to tumors, we used mesenchymal stem cells (MSCs) as carriers. To improve adenovirus specificity, we investigated whether widely used cyclooxygenase 2 (Cox-2) promoter is induced by adenovirus infection in nontarget cells and whether selectivity can be retained by the 3 untranslated region (UTR) AU-rich elements. In addition, we investigated whether switching adenovirus fiber can retain gene delivery in the presence of neutralizing antibodies. Our results show that adenoviruses, whose capsids were modified with arginine-glycine-aspartatic acid (RGD-4C), the serotype 3 knob, or polylysins displayed enhanced gene transfer into NSCLC cell lines and fresh clinical specimens from patients. The therapeutic efficacy was further improved by using respective oncolytic adenoviruses with isogenic 24bp deletion in the E1A gene. Cox-2 promoter was also shown to be induced in normal and tumor cells following adenovirus infection, but utilization of 3 UTR elements can increase the tumor specificity of the promoter. Further, the results suggested that use of MSCs could enhance the bioavailability and delivery of adenoviruses into human tumors, although cells had no tumor tropism per se. Finally, we demonstrated that changing adenovirus fiber can allow virus to escape from existing neutralizing antibodies when delivered systemically. In conclusion, these results reveal that adenovirus gene transfer and specificity can be increased by using modified adenoviruses and MSCs as carriers, and fiber modifications simultaneously decrease the effect of neutralizing antibodies. This promising data suggest that these approaches could translate into clinical testing in patients with NSCLC refractory to current modalities.

Co-liposomes comprising a lipidated multivalent RGD-peptide and a cationic gemini cholesterol induce selective gene transfection in alpha v beta 3 and alpha v beta 5 integrin receptor-rich cancer cells

The alpha v beta 3 and alpha v beta 5 integrins, transmembrane glycoprotein receptors, are over-expressed in numerous tumors and in endothelial cells that constitute tumor blood vessels. As this protein selectively binds to the Arg-Gly-Asp (RGD) sequence containing peptides, it is an attractive way to target tumors. Herein we have developed novel formulations for integrin mediated selective gene delivery. These formulations are composed of a novel palmitoylated tetrameric RGD containing scaffold (named RAFT-RGD), cationic gemini cholesterol (GL5) and a natural helper lipid 1,2-dioleoyl-L-alpha-glycero-3-phosphatidylethanolamine (DOPE). We have optimized a co-liposomal formulation to introduce the multivalent RGD-containing macromolecule in GL5: DOPE (GL5D) mixture to produce GL5D-RGD. We have unambiguously shown the selectivity of these formulations towards cancer cells that over express alpha v beta 3 and alpha v beta 5 integrins. Two reporter plasmids, pEGFP-C3 and PGL-3, were employed for the transfection experiments and it was shown that GL5D-RGD Liposomes increased exclusively the transfection in alpha v beta 3 and alpha v beta 5 overexpressing HeLa cells.

Design,optimization and in vivo evaluation of non-viral vectors for gene therapy. Applications in ocular disorders

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The effect of nocodazole on the transfection efficiency of lipid-bilayer coated gold nanoparticles

Nonviral vectors are safer than viral systems for gene therapy applications. However, the limited efficacy always prevents their being widely used in clinical practice. Aside from searching new gene nonviral vectors, many researchers focus on finding out new substances to improve the transfection efficiency of existent vectors. In this work, we found a transfection enhancer, nocodazole (NCZ), for dimethyldioctadecylammonium (DODAB, a cationic lipid) bilayer coated gold nanoparticles (AuNPs) mediated gene delivery. It was found that NCZ produces 3-fold transfection enhancement to HEK 293T cells assessed by flow cytometry (FCM). The result was further confirmed by luciferase assay, in which NCZ induced more than 5 times improvement in transfection efficiency after 48 h of transfection. The results from the inductively coupled plasma mass spectrometry (ICP-MS) and FCM showed that NCZ did not affect the internalization of DODAB-AuNPs/DNA complexes. The trafficking of the complexes by transmission electron microscopy (TEM) indicated that the interrupted transportation of the complexes to the lysosomes contributed greatly to the transfection enhancement.

A Novel Biodegradable and Light-Breakable Diblock Copolymer Micelle for Drug Delivery

A facile approach to the preparation of light-responsive copolymer micelles is developed. This approach is based on the attachment of hydrophobic groups to one block of a diblock copolymer via a light-sensitive linkage. The micelles can be dissociated under light irradiation and release the encapsulated pyrene. The obtained polymeric micelles are expected to be of use as drug-delivery vehicles.

Prostate cancer cell-specific VEGF siRNA delivery system using cell targeting peptide conjugated polyplexes

A polymeric gene carrier was developed to deliver vascular endothelial growth factor (VEGF) small interfering RNA (siRNA) for prostate cancer cells in a target-specific manner. Prostate cancer-binding peptide (PCP) was conjugated with polyethylenimine (PEI) via a poly(ethylene glycol) (PEG) linker (PEI-PEG-PCP). The PEI-PEG-PCP conjugate could effectively condense siRNA to form stable polyelectrolyte complexes (polyplexes) with an average diameter of approximately 150 nm in an aqueous solution. VEGF siRNA/PEI-PEG-PCP polyplexes exhibited significantly higher VEGF inhibition efficiency than PCP-unmodified polycationic carriers (PEI-PEG or PEI) in human prostate carcinoma cells (PC-3 cells). The enhanced gene silencing activity of VEGF siRNA/PEI-PEG-PCP was maintained even under serum conditions, owing to the steric stabilization of the polyplexes with hydrophilic PEG grafts. Confocal microscopic studies revealed that the siRNA/PEI-PEG-PCP polyplexes were delivered into PC-3 cells in a PCP ligand-specific manner.

Gene transfection of hyperbranched PEI grafted by hydrophobic amino acid segment PBLG

The complex copolymer of hyperbranched polyethylenimine (PEI) with hydrophobic poly(gamma-benzyl L-glutamate) segment (PBLG) at their chain ends was synthesized. This water-soluble copolymer PEI-PBLG (PP) was characterized for DNA complexation (gel retardation assay, particle size, DNA release and DNase I protection), cell viability and in vitro transfection efficiency. The experiments showed that PP can effectively condense pDNA into particles. Size measurement of the complexes particles indicated that PP/DNA tended to form smaller nanoparticles than those of PEI/DNA, which was caused by the hydrophobic PBLG segments compressing the PP/DNA complex particles in aqueous solution. The representative average size of PP/DNA complex prepared using plasmid DNA (pEGFP-N1, pDNA) was about 96 nm. The condensed pDNA in the PP/pDNA complexes was significantly protected from enzymatic degradation by DNase1. Cytotoxicity studies by MTT colorimetric assays suggested that the PP had much lower toxicity than PEI. The in vitro transfection efficiency of PP/pDNA complexes improved a lot in HeLa cells, Vero cells and 293T cells as compared to that of PEI25K by the expression of Green Fluorescent Protein (GFP) as determined by flow cytometry. Thus, the water-soluble PP copolymer showed considerable potential as carriers for gene delivery.

Poly(L-lysine)-graft-chitosan copolymers: Synthesis, characterization, and gene transfection effect

Polypeptide/polysaccharide graft copolymers poly(L-lysine)-graft-chitosan (PLL-g-Chi) were prepared by ring-opening polymerization (ROP) of epsilon-benzoxycarbonyl L-lysine N-carboxyanhydrides (Z-L-lysine NCA) in the presence of 6-O-triphenylmethyl chitosan. The PLL-g-Chi copolymers were thoroughly characterized by H-1 NMR, C-13 NMR, Fourier transform infrared (FT-IR), and gel permeation chromatography (GPC). The number-average degree of polymerization of PLL grafted onto the chitosan backbone could be adjusted by controlling the feed ratio of NCA to 6-O-triphenylmethyl chitosan. The particle size of the complexes formed from the copolymer and calf thymus DNA was measured by dynamic light scattering (DLS). It was found in the range of 120 similar to 340 nm. The gel retardation electrophoresis showed that the PLL-g-Chi copolymers possessed better plasmid DNA-binding ability than chitosan. The gene transfection effect in HEK 293T cells of the copolymers was evaluated, and the results showed that the gene transfection ability of the copolymer was better than that of chitosan and was dependent on the PLL grafting ratio. The PLL-g-Chi copolymers could be used as effective gene delivery vectors.

Intracoronary adenovirus-mediated delivery and overexpression of the beta(2)-adrenergic receptor in the heart : prospects for molecular ventricular assistance.

BACKGROUND: Genetic modulation of ventricular function may offer a novel therapeutic strategy for patients with congestive heart failure. Myocardial overexpression of beta(2)-adrenergic receptors (beta(2)ARs) has been shown to enhance contractility in transgenic mice and reverse signaling abnormalities found in failing cardiomyocytes in culture. In this study, we sought to determine the feasibility and in vivo consequences of delivering an adenovirus containing the human beta(2)AR cDNA to ventricular myocardium via catheter-mediated subselective intracoronary delivery. METHODS AND RESULTS: Rabbits underwent percutaneous subselective catheterization of either the left or right coronary artery and infusion of adenoviral vectors containing either a marker transgene (Adeno-betaGal) or the beta(2)AR (Adeno-beta(2)AR). Ventricular function was assessed before catheterization and 3 to 6 days after gene delivery. Both left circumflex- and right coronary artery-mediated delivery of Adeno-beta(2)AR resulted in approximately 10-fold overexpression in a chamber-specific manner. Delivery of Adeno-betaGal did not alter in vivo left ventricular (LV) systolic function, whereas overexpression of beta(2)ARs in the LV improved global LV contractility, as measured by dP/dt(max), at baseline and in response to isoproterenol at both 3 and 6 days after gene delivery. CONCLUSIONS: Percutaneous adenovirus-mediated intracoronary delivery of a potentially therapeutic transgene is feasible, and acute global LV function can be enhanced by LV-specific overexpression of the beta(2)AR. Thus, genetic modulation to enhance the function of the heart may represent a novel therapeutic strategy for congestive heart failure and can be viewed as molecular ventricular assistance.

Nitric oxide synthase gene therapy enhances the toxicity of cisplatin in cancer cells

BACKGROUND AIMS: Cell-based gene therapy is an alternative to viral and non-viral gene therapy. Emerging evidence suggests that mesenchymal stem cells (MSC) are able to migrate to sites of tissue injury and have immunosuppressive properties that may be useful in targeted gene therapy for sustained specific tissue engraftment. METHODS: In this study, we injected intravenously (i.v.) 1x10(6) MSC, isolated from green fluorescent protein (GFP) transgenic rats, into Rif-1 fibrosarcoma-bearing C3H/HeN mice. The MSC had been infected using a lentiviral vector to express stably the luciferase reporter gene (MSC-GFP-luci). An in vivo imaging system (IVIS 200) and Western blotting techniques were used to detect the distribution of MSC-GFP-luci in tumor-bearing animals. RESULTS: We observed that xenogenic MSC selectively migrated to the tumor site, proliferated and expressed the exogenous gene in subcutaneous fibrosarcoma transplants. No MSC distribution was detected in other organs, such as the liver, spleen, colon and kidney. We further showed that the FGF2/FGFR pathways may play a role in the directional movement of MSC to the Rif-1 fibrosarcoma. We performed in vitro co-culture and in vivo tumor growth analysis, showing that MSC did not affect the proliferation of Rif-1 cells and fibrosarcoma growth compared with an untreated control group. Finally, we demonstrated that the xenogenic MSC stably expressing inducible nitric oxide synthase (iNOS) protein transferred by a lentivirus-based system had a significant inhibitory effect on the growth of Rif-1 tumors compared with MSC alone and the non-treatment control group. CONCLUSIONS: iNOS delivered by genetically modified iNOS-MSC showed a significant anti-tumor effect both in vitro and in vivo. MSC may be used as a target gene delivery vehicle for the treatment of fibrosarcoma and other tumors

Novel macrocyclic and acyclic cationic lipids for gene transfer: Synthesis and in vitro evaluation

The synthesis and in vitro evaluation of four cationic lipid gene delivery vectors, characterized by acyclic or macrocyclic, and saturated or unsaturated hydrophobic regions, is described. The synthesis employed standard protocols, including ring-closing metathesis for macrocyclic lipid construction. All lipoplexes studied, formulated from plasmid DNA and a liposome composed of a synthesized lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol as co-lipid, exhibited plasmid DNA binding and protection from DNase I degradation, and concentration dependent cytotoxicity using Chinese hamster ovary-K1 cells. The transfection efficiency of formulations with cholesterol outperformed those with DOPE, and in many cases the EPC/cholesterol control, and formulations with a macrocyclic lipid (+/- 10:1) outperformed their acyclic counterparts (+/- 3:1).

Microneedles for intradermal and transdermal drug delivery

The formidable barrier properties of the uppermost layer of the skin, the stratum corneum, impose significant limitations for successful systemic delivery of broad range of therapeutic molecules particularly macromolecules and genetic material. Microneedle (MN) has been proposed as a strategy to breach the stratum corneum barrier function in order to facilitate effective transport of molecules across the skin. This strategy involves use of micron sized needles fabricated of different materials and geometries to create transient aqueous conduits across the skin. MN, alone or with other enhancing strategies, has been demonstrated to dramatically enhance the skin permeability of numerous therapeutic molecules including biopharmaceuticals either in vitro, ex vivo or in vivo experiments. This suggested the promising use of MN technology for various possible clinical applications such as insulin delivery, transcutaneous immunisations and cutaneous gene delivery. MN has been proved as minimally invasive and painless in human subjects. This review article focuses on recent and future developments for MN technology including the latest type of MN design, challenges and strategies in MNs development as well as potential safety aspects based on comprehensive literature review pertaining to MN studies to date. (C) 2013 Elsevier B.V. All rights reserved.