Kam W. Leong

CHITOSAN-DNA NANOPARTICLES AS GENE CARRIERS: SYNTHESIS, CHARACTERIZATION AND TRANSFECTION EFFICIENCY

Chitosan-DNA nanoparticles were prepared using a complex coacervation process. The important parameters for the nanoparticle synthesis were investigated, including the concentrations of DNA, chitosan and sodium sulfate, temperature of the solutions, pH of the buffer, and molecular weights of chitosan and DNA. At an amino group to phosphate group ratio (N/P ratio) between 3 and 8 and a chitosan concentration of 100 microg/ml, the size of particles was optimized to approximately 100--250 nm with a narrow distribution, with a composition of 35.6 and 64.4% by weight for DNA and chitosan, respectively. The surface charge of these particles was slightly positive with a zeta potential of +12 to +18 mV at pH lower than 6.0, and became nearly neutral at pH 7.2. The chitosan-DNA nanoparticles could partially protect the encapsulated plasmid DNA from nuclease degradation as shown by electrophoretic mobility analysis. The transfection efficiency of chitosan-DNA nanoparticles was cell-type dependent. Typically, it was three to four orders of magnitude, in relative light units, higher than background level in HEK293 cells, and two to ten times lower than that achieved by LipofectAMINE-DNA complexes. The presence of 10% fetal bovine serum did not interfere with their transfection ability. Chloroquine could be co-encapsulated in the nanoparticles at 5.2%, but with negligible enhancement effect despite the fact that chitosan only showed limited buffering capacity compared with PEI. The present study also developed three different schemes to conjugate transferrin or KNOB protein to the nanoparticle surface. The transferrin conjugation only yielded a maximum of four-fold increase in their transfection efficiency in HEK293 cells and HeLa cells, whereas KNOB conjugated nanoparticles could improve gene expression level in HeLa cells by 130-fold. Conjugation of PEG on the nanoparticles allowed lyophilization without aggregation, and without loss of bioactivity for at least 1 month in storage. The clearance of the PEGylated nanoparticles in mice following intravenous administration was slower than unmodified nanoparticles at 15 min, and with higher depositions in kidney and liver. However, no difference was observed at the 1-h time point.

BIOMEDICAL APPLICATIONS OF POLYMER-COMPOSITE MATERIALS: A REVIEW

An overview of various biomedical applications of polymer-composite materials reported in the literature over the last 30 years is presented in this paper. For the benefit of the readers, general information regarding structure and function of tissues, types and purpose of implants/medical devices, and various other materials used, are also briefly presented. Different types of polymer composite that are already in use or are investigated for various biomedical applications are presented. Specific advantages of using polymer-composite biomaterials in selected applications are also highlighted. The paper also examines the critical issues and scientific challenges that require further research and development of polymer composite materials for their increased acceptance in the biomedical industry.

RNA-guided gene activation by CRISPR-Cas9–based transcription factors

Technologies for engineering synthetic transcription factors have enabled many advances in medical and scientific research. In contrast to existing methods based on engineering of DNA-binding proteins, we created a Cas9-based transactivator that is targeted to DNA sequences by guide RNA molecules. Coexpression of this transactivator and combinations of guide RNAs in human cells induced specific expression of endogenous target genes, demonstrating a simple and versatile approach for RNA-guided gene activation.

DNA-polycation nanospheres as non-viral gene delivery vehicles

Nanospheres synthesized by salt-induced complex coacervation of cDNA and polycations such as gelatin and chitosan were evaluated as gene delivery vehicles. DNA-nanospheres in the size range of 200-750 nm could transfect a variety of cell lines. Although the transfection efficiency of the nanospheres was typically lower than that of lipofectamine and calcium phosphate controls in cell culture, the beta-gal expression in muscle of BALB/c mice was higher and more sustained than that achieved by naked DNA and lipofectamine complexes. This gene delivery system has several attractive features: (1) ligands can be conjugated to the nanosphere for targeting or stimulating receptor-mediated endocytosis; (2) lysosomolytic agents can be incorporated to reduce degradation of the DNA in the endosomal and lysosomal compartments; (3) other bioactive agents or multiple plasmids can be co-encapsulated; (4) bioavailability of the DNA can be improved because of protection from serum nuclease degradation by the polymeric matrix; (5) the nanosphere can be lyophilized for storage without loss of bioactivity.

Nanotopography-induced changes in focal adhesions, cytoskeletal organization, and mechanical properties of human mesenchymal stem cells.

The growth of stem cells can be modulated by physical factors such as extracellular matrix nanotopography. We hypothesize that nanotopography modulates cell behavior by changing the integrin clustering and focal adhesion (FA) assembly, leading to changes in cytoskeletal organization and cell mechanical properties. Human mesenchymal stem cells (hMSCs) cultured on 350 nm gratings of tissue-culture polystyrene (TCPS) and polydimethylsiloxane (PDMS) showed decreased expression of integrin subunits alpha2, alpha , alpha V, beta2, beta 3 and beta 4 compared to the unpatterned controls. On gratings, the elongated hMSCs exhibited an aligned actin cytoskeleton, while on unpatterned controls, spreading cells showed a random but denser actin cytoskeleton network. Expression of cytoskeleton and FA components was also altered by the nanotopography as reflected in the mechanical properties measured by atomic force microscopy (AFM) indentation. On the rigid TCPS, hMSCs on gratings exhibited lower instantaneous and equilibrium Youngs moduli and apparent viscosity. On the softer PDMS, the effects of nanotopography were not significant. However, hMSCs cultured on PDMS showed lower cell mechanical properties than those on TCPS, regardless of topography. These suggest that both nanotopography and substrate stiffness could be important in determining mechanical properties, while nanotopography may be more dominant in determining the organization of the cytoskeleton and FAs.

Advanced materials and processing for drug delivery: the past and the future.

Design and synthesis of efficient drug delivery systems are of vital importance for medicine and healthcare. Materials innovation and nanotechnology have synergistically fueled the advancement of drug delivery. Innovation in material chemistry allows the generation of biodegradable, biocompatible, environment-responsive, and targeted delivery systems. Nanotechnology enables control over size, shape and multi-functionality of particulate drug delivery systems. In this review, we focus on the materials innovation and processing of drug delivery systems and how these advances have shaped the past and may influence the future of drug delivery.

Electrohydrodynamics: A facile technique to fabricate drug delivery systems

Electrospinning and electrospraying are facile electrohydrodynamic fabrication methods that can generate drug delivery systems (DDS) through a one-step process. The nanostructured fiber and particle morphologies produced by these techniques offer tunable release kinetics applicable to diverse biomedical applications. Coaxial electrospinning/electrospraying, a relatively new technique of fabricating core-shell fibers/particles have added to the versatility of these DDS by affording a near zero-order drug release kinetics, dampening of burst release, and applicability to a wider range of bioactive agents. Controllable electrospinning/spraying of fibers and particles and subsequent drug release from these chiefly polymeric vehicles depends on well-defined solution and process parameters. The additional drug delivery capability from electrospun fibers can further enhance the materials functionality in tissue engineering applications. This review discusses the state-of-the-art of using electrohydrodynamic technique to generate nanofiber/particles as drug delivery devices.

Fabrication of controlled release biodegradable foams by phase separation.

Highly porous biodegradable foams with controlled release function were fabricated by a phase separation technique. This technique involved inducing phase changes in a homogeneous solution of polymers with naphthalene or phenol used as solvents. A variety of foams with pore sizes ranging from 20 to 500 microm were made of poly(L-lactic acid) (PLLA), poly(bisphenol A-phenylphosphonate (BPA/PP), and its copolymer with poly[bis(2-ethoxy)- hydrophosphonic terephthalate] (PP/PPET). Controlled delivery capability was demonstrated by studying the release of sulforhodamine B and alkaline phosphatase (AP) from these highly porous structures. After an initial burst, AP was released from BPA/PP and PLLA foams at a near steady rate of 0.32 +/- 0.04 and 0.49 +/- 0.13 mg/day/g foam, respectively. These foams were intended for use as cell transplantation devices and tissue grafts such as synthetic bone grafts. Hydroxyapatite (HA) was added into the foams in an attempt to enhance interaction of these foams with bone. This composite was analyzed by energy dispersive spectroscopy, differential scanning calorimetry, and thermomechanical analysis. Since phosphates are known to have good affinity to calcium, poly(phosphoester) foams were treated with 1M calcium chloride solution in an attempt to study the possible interaction of the degrading poly(phosphoester) with calcium. After three weeks in 1 M calcium chloride solution, the complex modulus of the poly(phosphoester) foams changed from 40 to 1948 kPa, with a concurrent decrease in loss tangent from 0.349 to 0.170.

Polyphosphoesters in drug and gene delivery

Polymers with repeating phosphoester bonds in the backbone are structurally versatile, and biodegradable through hydrolysis, and possibly enzymatic digestion at the phosphoester linkages under physiological conditions. These biodegradable polyphosphoesters are appealing for biological and pharmaceutical applications because of their potential biocompatibility and similarity to bio-macromolecules such as nucleic acids. In the first part of this review, we will focus on one particular structure synthesized by extending oligomeric lactide prepolymers with ethylphosphate groups. This amorphous to semi-crystalline polymer is promising in delivering anti-cancer therapeutics in the form of microspheres. In the second half, we will discuss the conjugation of charged groups to the side chain of the phosphate, constituting one of the few biodegradable cationic polymers in the field for non-viral gene delivery. Capable of delivering exogenous genes to a cell nucleus or providing an extracellular sustained release of DNA, these cationic polyphosphoesters also serve as a valuable model to understand the important characteristics that render a polymer an effective gene carrier.

Poly(α-hydroxy acids): carriers for bone morphogenetic proteins

A broad spectrum of cells and cell products is associated with bone homeostasis and the renewal of bone following injury. The coupled interactions among cells provide the power behind sculpting of bone, sustaining form, and ensuring functionality. Local and systemic regulatory molecules (e.g. growth factors, hormones) direct cellular interactions through autocrine, paracrine, and hormonal pathways. Recently, genes for a class of osteogenic regulatory molecules have been cloned, and gene product expression has enabled investigators to assess safety and efficacy in animal studies. The molecules are known as bone morphogenetic proteins (BMPs). Therapeutic applications of BMPs depend on a carrier system. A carrier could spatially and temporally localize BMP for regional needs and be custom-tailored for acute craniofacial applications or for recalcitrant extremity non-unions. The poly(alpha-hydroxy acids) (PHAs) may be suitable for these applications. Therefore, the purposes of this paper are (i) to mention, briefly, basic concepts of the bone wound continuum and the possible therapeutic roles of BMPs; (ii) to outline several properties of selected PHAs relevant to bone regeneration dynamics; and (iii) to review selected preclinical studies with PHAs.