Biomaterials (v.31, #17)
In vivo study of an injectable poly(acrylonitrile)-based hydrogel paste as a bulking agent for the treatment of urinary incontinence
by Pascal Ramseyer; Lionel A. Micol; Eva-Maria Engelhardt; Maria-Chiara Osterheld; Jeffrey A. Hubbell; Peter Frey (pp. 4613-4619).
Urinary incontinence can be treated by endoscopic injection of bulking agents, however, no optimal therapeutic effect has been achieved upon this treatment yet. In the present study, the development of a injectable poly(acrylonitrile) hydrogel paste is described, and its efficacy and histological behavior, once injected into the submucosal space of the minipig bladder, are evaluated. A device was developed to mix poly(acrylonitrile) hydrogel powder with glycerin, used as carrier, prior to injection into the submucosal space of the bladder. Several paste deposits, depending on the size of the bladder, were injected per animal. The implants were harvested at days 7, 14, 21, 28, 84 and 168 and analyzed morphologically and by histology. The persistence of the implants was demonstrated. However, at later time points the implants were split up and surrounded by granulomatous tissue, which was gradually replaced by histiocytes and adipocytes. Transitory focal urothelial metaplasia was observed only at day 7 and moderate foreign body reaction was detected predominantly between the second and fifth week. This study demonstrated the feasibility to develop an injectable paste of poly(acrylonitrile) hydrogel thought to provide the expected bulking effect, necessary for the treatment of urinary incontinence.
Keywords: Polyacrylonitrile; Hydrogel; Swelling; Bladder; Urinary tract
Hydroxyapatite nucleation and growth mechanism on electrospun fibers functionalized with different chemical groups and their combinations
by Wenguo Cui; Xiaohong Li; Chengying Xie; Huihui Zhuang; Shaobing Zhou; Jie Weng (pp. 4620-4629).
Controlled nucleation and growth of hydroxyapatite (HA) crystals on electrospun fibers should play important roles in fabrication of composite scaffolds for bone tissue engineering, but no attempt has been made to clarify the effects of chemical group densities and the cooperation of two and more groups on the biomineralization process. The aim of the current study was to investigate into HA nucleation and growth on electrospun poly(dl-lactide) fibers functionalized with carboxyl, hydroxyl and amino groups and their combinations. Electrospun fibers with higher densities of carboxyl groups, combination of hydroxyl and carboxyl groups with the ratio of 3/7, and combination of amino, hydroxyl and carboxyl groups with the ratio of 2/3/5 were favorable for HA nucleation and growth, resulting in higher content and lower crystal size of formed HA. Carboxyl groups were initially combined with calcium ions through electrostatic attraction, and the introduction of hydroxyl groups could modulate the distance between carboxyl groups. The introduction of amino groups may lead to the inner ionic bonding with carboxyl groups, but can accelerate phosphate ions to form HA through a chelate ring with the calcium ion and carbonyl oxygen. The biological evaluation indicated that the mineralized scaffolds acted as an excellent cell support to maintain desirable cell–substrate interactions, to provide favorable conditions for cell proliferation and to stimulate the osteogenic differentiation.
Keywords: Electrospun fibers; Functional groups; Hydroxyapatite nucleation; Growth mechanism; Biological evaluation
Stimulation of in vivo angiogenesis by in situ crosslinked, dual growth factor-loaded, glycosaminoglycan hydrogels
by Roberto Elia; Peter W. Fuegy; Aaron VanDelden; Matthew A. Firpo; Glenn D. Prestwich; Robert A. Peattie (pp. 4630-4638).
As part of a study of elicited angiogenesis, hyaluronan (HA)-based hydrogels crosslinked by polyethylene glycol diacrylate (PEGDA) were loaded with combinations of the cytokine growth factors vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), keratinocyte growth factor (KGF) and platelet-derived growth factor (PDGF). GF release in vivo was controlled by covalent incorporation of thiol-modified heparin into thiolated HA hydrogels, which were injected into the ear pinnae of mice and allowed to crosslink in situ. GF release in vivo was controlled by covalent incorporation of thiol-modified heparin in the gels. The ears were harvested at 7 or 14 days post-implantation, and vascularization evaluated via a Neovascularization Index ( NI). The study demonstrates that in situ gelling implants produced no gross inflammation, redness or swelling, and an improved tolerance compared to HA-based dry film implants. All treatments showed significantly more vascularization than either contralateral ears or ears receiving a sham surgery. The maximum response was observed after 14 days in the ears receiving 0.3% Hp, gelatin-containing gels loaded with VEGF + KGF ( NI = 3.91). The study revealed injected growth factor-loaded HA-based hydrogels can successfully produce localized controllable vascularization, while minimizing tissue necrosis, polymorphonuclear leukocytes and inflammation. The ability to target and controllably release growth factors can prove a useful tool in specific diseased tissue/organ angiogenesis.
Keywords: Angiogenesis; Controlled drug release; Glycosaminoglycans; Growth factors; Cytokine; Vascular endothelial growth factor
Bioactive modification of poly(ethylene glycol) hydrogels for tissue engineering
by Junmin Zhu (pp. 4639-4656).
In this review, we explore different approaches for introducing bioactivity into poly(ethylene glycol) (PEG) hydrogels. Hydrogels are excellent scaffolding materials for repairing and regenerating a variety of tissues because they can provide a highly swollen three-dimensional (3D) environment similar to soft tissues. Synthetic hydrogels like PEG-based hydrogels have advantages over natural hydrogels, such as the ability for photopolymerization, adjustable mechanical properties, and easy control of scaffold architecture and chemical compositions. However, PEG hydrogels alone cannot provide an ideal environment to support cell adhesion and tissue formation due to their bio-inert nature. The natural extracellular matrix (ECM) has been an attractive model for the design and fabrication of bioactive scaffolds for tissue engineering. ECM-mimetic modification of PEG hydrogels has emerged as an important strategy to modulate specific cellular responses. To tether ECM-derived bioactive molecules (BMs) to PEG hydrogels, various strategies have been developed for the incorporation of key ECM biofunctions, such as specific cell adhesion, proteolytic degradation, and signal molecule-binding. A number of cell types have been immobilized on bioactive PEG hydrogels to provide fundamental knowledge of cell/scaffold interactions. This review addresses the recent progress in material designs and fabrication approaches leading to the development of bioactive hydrogels as tissue engineering scaffolds.
Keywords: Poly(ethylene glycol) (PEG); Hydrogel; Bioactive modification; Tissue engineering; Biomimetic scaffold; Extracellular matrix (ECM)
Synergistic effects of tethered growth factors and adhesion ligands on DNA synthesis and function of primary hepatocytes cultured on soft synthetic hydrogels
by Geeta Mehta; Courtney M. Williams; Luis Alvarez; Martha Lesniewski; Roger D. Kamm; Linda G. Griffith (pp. 4657-4671).
The composition, presentation, and spatial orientation of extracellular matrix molecules and growth factors are key regulators of cell behavior. Here, we used self-assembling peptide nanofiber gels as a modular scaffold to investigate how fibronectin-derived adhesion ligands and different modes of epidermal growth factor (EGF) presentation synergistically regulate multiple facets of primary rat hepatocyte behavior in the context of a soft gel. In the presence of soluble EGF, inclusion of dimeric RGD and the heparin binding domain from fibronectin (HB) increased hepatocyte aggregation, spreading, and metabolic function compared to unmodified gels or gels modified with a single motif, but unlike rigid substrates, gels failed to induce DNA synthesis. Tethered EGF dramatically stimulated cell aggregation and spreading under all adhesive ligand conditions and also preserved metabolic function. Surprisingly, tethered EGF elicited DNA synthesis on gels with RGD and HB. Phenotypic differences between soluble and tethered EGF stimulation of cells on peptide gels are correlated with differences in expression and phosphorylation the EGF receptor and its heterodimerization partner ErbB2, and activation of the downstream signaling node ERK1/2. These modular matrices reveal new facets of hepatocellular biology in culture and may be more broadly useful in culture of other soft tissues.
Keywords: Self assembled peptide gel; Extracellular matrix; RGD cell binding domain; Heparin binding domain; Tethered growth factor
The synergistic effects of 3-D porous silk fibroin matrix scaffold properties and hydrodynamic environment in cartilage tissue regeneration
by Yun Wang; Erika Bella; Christopher S.D. Lee; Claudio Migliaresi; Linda Pelcastre; Zvi Schwartz; Barbara D. Boyan; Antonella Motta (pp. 4672-4681).
Autologous cell-based tissue engineering using three-dimensional porous scaffolds has provided a good option for the repair of cartilage defects. Silk fibroin-based scaffolds are naturally degradable materials with excellent biocompatibility and robust mechanical properties, indicating potential applications in cartilage tissue engineering. In this study, silk fibroin scaffolds prepared by freeze-drying (FD) and salt-leaching (SL300 and SL500) were fully characterized and used to study the effects of silk fibroin scaffold properties on chondrocyte attachment, proliferation and differentiation. The synergistic effects of scaffold properties and hydrodynamic environment generated by in vitro rocking culture were also investigated using static cultures as control. FD scaffolds with small pore size and lower porosity increased cell attachment but inhibited cell penetration and limited cell proliferation and differentiation. In contrast, SL scaffolds displaying a bigger pore size, higher porosity and crystallinity resulted in homogenous cell distribution, increasing cell proliferation and advanced chondrocyte differentiation in terms of their spherical morphology, predominant chondrogenic gene expression and abundant cartilaginous extracellular matrix production. A hydrodynamic environment was beneficial to chondrocyte proliferation, differentiation, and integrin gene expression in a pore size dependent manner with superior cartilage matrix production but limited hypertrophic differentiation obtained using chondrocyte-seeded SL500 scaffolds. Integrin α5β1 might mediate these effects. Chondrocyte/SL500 silk fibroin constructs obtained under in vitro rocking culture might serve as an excellent implant for in vivo cartilage defect reparation.
Keywords: Silk fibroin; Scaffolds; Chondrocyte; Hydrodynamic environment; Cartilage tissue engineering
Neuronal polarity selection by topography-induced focal adhesion control
by Aldo Ferrari; Marco Cecchini; Michela Serresi; Paolo Faraci; Dario Pisignano; Fabio Beltram (pp. 4682-4694).
Interaction between differentiating neurons and the extracellular environment guides the establishment of cell polarity during nervous system development. Developing neurons read the physical properties of the local substrate in a contact-dependent manner and retrieve essential guidance cues. In previous works we demonstrated that PC12 cell interaction with nanogratings (alternating lines of ridges and grooves of submicron size) promotes bipolarity and alignment to the substrate topography. Here, we investigate the role of focal adhesions, cell contractility, and actin dynamics in this process. Exploiting nanoimprint lithography techniques and a cyclic olefin copolymer, we engineered biocompatible nanostructured substrates designed for high-resolution live-cell microscopy. Our results reveal that neuronal polarization and contact guidance are based on a geometrical constraint of focal adhesions resulting in an angular modulation of their maturation and persistence. We report on ROCK1/2-myosin-II pathway activity and demonstrate that ROCK-mediated contractility contributes to polarity selection during neuronal differentiation. Importantly, the selection process confined the generation of actin-supported membrane protrusions and the initiation of new neurites at the poles. Maintenance of the established polarity was independent from NGF stimulation. Altogether our results imply that focal adhesions and cell contractility stably link the topographical configuration of the extracellular environment to a corresponding neuronal polarity state.
Keywords: Cell polarity; Neuron; Focal adhesions; Contact guidance; Topography; Cell contractility
Modulation of gene expression and collagen production of anterior cruciate ligament cells through cell shape changes on polycaprolactone/chitosan blends
by Hung-Jen Shao; Yu-Tsang Lee; Chiang-Sang Chen; Jyh-Horng Wang; Tai-Horng Young (pp. 4695-4705).
Our previous study has illustrated that chitosan could enhance human anterior cruciate ligament (ACL) cells to exhibit a dramatic effect on increasing the gene expression of transforming growth factor β1 (TGF-β1), which is a specific gene for wound healing and collagen synthesis. However, human ACL cells could not adhere and proliferate well on chitosan. In order to overcome this drawback, we introduced polycaprolactone (PCL) into chitosan by the method of blending in this study. It was found that the morphology, viability and gene expression of human ACL cells on the chitosan/PCL blends could be effectively regulated. With the increase of PCL content in blends, human ACL cells presented more flatten shape, well-organized cytoskeleton, and higher proliferated ability. Compared to flatten shape, human ACL cells with round shape exhibited higher levels of mRNA expression of TGF-β1 and collagen type III through 3-day culture period. Furthermore, these blended materials could upregulate protein synthesis of human ACL cells, which corresponded to their gene expressions. Therefore, it is possible to combine the advantages of chitosan and PCL to create a new blended material, which could control cellular morphologies specifically, and further to regulate the gene expression and protein production of cells for specific applications. We expected this concept, controlling the cell shape through biomaterial to modulate the behavior of cells, could provide a new vision for the material selection of ligament tissue engineering.
Keywords: Anterior cruciate ligament (ACL) cells; Blends; Chitosan; Polycaprolactone (PCL); Cell shape; Gene expression
The role of cyclic AMP in normalizing the function of engineered human blood microvessels in microfluidic collagen gels
by Keith H.K. Wong; James G. Truslow; Joe Tien (pp. 4706-4714).
Nearly all engineered tissues must eventually be vascularized to survive. To this end, we and others have recently developed methods to synthesize extracellular matrix-based scaffolds that contain open microfluidic networks. These scaffolds serve as templates for the formation of endothelial tubes that can be perfused; whether such microvascular structures are stable and/or functional is largely unknown. Here, we show that compounds that elevate intracellular concentrations of the second messenger cyclic AMP (cAMP) strongly normalize the phenotype of engineered human microvessels in microfluidic type I collagen gels. Cyclic AMP-elevating agents promoted vascular stability and barrier function, and reduced cellular turnover. Under conditions that induced the highest levels of cAMP, the physiology of engineered microvessels in vitro quantitatively mirrored that of native vessels in vivo. Computational analysis indicated that cAMP stabilized vessels partly via its enhancement of barrier function.
Keywords: Microvascular tissue engineering; Collagen gels; Permeability; Microfluidic channels; Cyclic AMP
The use of decellularized adipose tissue to provide an inductive microenvironment for the adipogenic differentiation of human adipose-derived stem cells
by L.E. Flynn (pp. 4715-4724).
The development of an engineered adipose tissue substitute, capable of supporting reliable, predictable, and complete fat tissue formation, would be of significant value in the fields of plastic and reconstructive surgery. Towards the goal of engineering an optimized microenvironment for adipogenesis, a decellularization strategy was developed for adipose tissue, which yielded 3-D scaffolds with preserved extracellular matrix architecture. A significant volume of scaffolding material could be obtained from a human tissue source that is commonly discarded. Histology, immunohistochemistry, and scanning electron microscopy confirmed the efficacy and reproducibility of the approach, and also indicated that the basement membrane was conserved in the processed matrix, including laminin and collagen type IV. Seeding experiments with human adipose-derived stem cells indicated that the decellularized adipose tissue (DAT) provided an inductive microenvironment for adipogenesis, supporting the expression of the master regulators PPARγ and CEBPα, without the need for exogenous differentiation factors. High levels of adipogenic gene expression and glycerol-3-phosphate dehydrogenase activity were observed in the induced DAT scaffolds, as compared to cells grown in monolayer or cell aggregate culture. The protein data emphasized the importance of the cell donor source in the development of tissue-engineering strategies for large-volume soft tissue regeneration.
Keywords: Adipose tissue engineering; Extracellular matrix; Scaffold; Mesenchymal stem cell; Gene expression
The effect of a laminin-5-derived peptide coated onto chitin microfibers on re-epithelialization in early-stage wound healing
by Seung-Ki Min; Sang-Chul Lee; Seong-Doo Hong; Chong-Pyoung Chung; Won Ho Park; Byung-Moo Min (pp. 4725-4730).
Considerable effort has been directed towards regenerating defective tissues using tissue-engineering methods. Recently, peptides have been recognized as a valuable scientific tool in the field of tissue-engineering. The PPFLMLLKGSTR motif of the human laminin-5 α3 chain has been previously reported to promote keratinocyte survival; however, the in vivo effects of the PPFLMLLKGSTR motif have not yet been studied. These studies raised the hypothesis that a laminin-5-derived peptide can promote wound healing by accelerating re-epithelialization in vivo. To examine this hypothesis, we applied chitin microfibrous matrices coated with the PPFLMLLKGSTR motif in both rat and rabbit full-thickness cutaneous wound models. Compared with vehicle-treated and peptide-treated cutaneous wounds, the application significantly promoted early-stage wound healing by accelerating re-epithelialization, notably reduced inflammatory cell infiltration, and prominently enhanced fibroblast proliferation. These findings support our hypothesis that the PPFLMLLKGSTR motif acts as a very effective wound healing accelerator by enhancing re-epithelialization.
Keywords: Laminin-5-derived peptide; Wound dressing material; Tissue-engineering; Re-epithelialization; Wound healing
Fibrin-polylactide-based tissue-engineered vascular graft in the arterial circulation
by Sabine Koch; Thomas C. Flanagan; Joerg S. Sachweh; Fadwa Tanios; Heike Schnoering; Thorsten Deichmann; Ville Ellä; Minna Kellomäki; Nina Gronloh; Thomas Gries; René Tolba; Thomas Schmitz-Rode; Stefan Jockenhoevel (pp. 4731-4739).
There is a clear clinical requirement for the design and development of living, functional, small-calibre arterial grafts. Here, we investigate the potential use of a small diameter, tissue-engineered artery in a pre-clinical study in the carotid artery position of sheep. Small-calibre (∼5 mm) vascular composite grafts were molded using a fibrin scaffold supported by a poly(L/D)lactide 96/4 (P(L/D)LA 96/4) mesh, and seeded with autologous arterial-derived cells prior to 28 days of dynamic conditioning. Conditioned grafts were subsequently implanted for up to 6 months as interposed carotid artery grafts in the same animals from which the cells were harvested. Explanted grafts ( n = 6) were patent in each of the study groups (1 month, 3 months, 6 months), with a significant stenosis in one explant (3 months). There was a complete absence of thrombus formation on the luminal surface of grafts, with no evidence for aneurysm formation or calcification after 6 months in vivo. Histological analyses revealed remodeling of the fibrin scaffold with mature autologous proteins, and excellent cell distribution within the graft wall. Positive vWf and eNOS staining, in addition to scanning electron microscopy, revealed a confluent monolayer of endothelial cells lining the luminal surface of the grafts. The present study demonstrates the successful production and mid-term application of an autologous, fibrin-based small-calibre vascular graft in the arterial circulation, and highlights the potential for the creation of autologous implantable arterial grafts in a number of settings.
Keywords: Tissue engineering; Grafting; Fibrin; Artery; Animal experiment
A high-performance VEGF aptamer functionalized polypyrrole nanotube biosensor
by Oh Seok Kwon; Seon Joo Park; Jyongsik Jang (pp. 4740-4747).
In this study, we examined the in vitro electrochemical detection of Vascular Endothelial Growth Factor (VEGF) as cancer biomarker using p-type field-effect transistor (FET) biosensor. We demonstrated the high-performance FET sensor, which could detect ca. 400 fM of VEGF concentration, based on anti-VEGF RNA aptamer conjugated carboxylated polypyrrole nanotubes (CPNTs). The CPNTs used as high-performance transducers of this FET system were successfully fabricated by cylindrical micelle templates in a water-in-oil emulsion system. The functional carboxyl group (–COOH) was effectively incorporated into the polymer backbone during the polymerization by using pyrrole-3-carboxylic acid (P3CA) as a co-monomer. Two types of CPNTs (CPNT1: ca. 200 nm in diameter, CPNT2: ca. 120 nm in diameter) demonstrated the excellent conductivity performance in this FET system. Based on CPNTs conjugated with anti-VEGF RNA aptamer (CPNTs-aptamer), VEGF (target molecule) acts as the gate dielectrics of p-type FET sensor and specifically interacts with anti-VEGF aptamer attached to CPNT surfaces. Importantly, the VEGF detection limit of the FET sensor based on CPNT2-aptamer was found to be near 400 fM in real-time. Moreover, the CPNTs-aptamer FET sensors can be repeatedly used for various concentrations of the target molecule (VEGFs) through the washing and rinsing processes.
Keywords: Vascular endothelial cell growth factors (VEGFs); Aptamer; Biosensor; Field-effect transistors; Conducting polymer
Glycyrrhetinic acid-modified chitosan/poly(ethylene glycol) nanoparticles for liver-targeted delivery
by Qin Tian; Chuang-Nian Zhang; Xiu-Hua Wang; Wei Wang; Wei Huang; Rui-Tao Cha; Chun-Hong Wang; Zhi Yuan; Min Liu; Hai-Ying Wan; Hua Tang (pp. 4748-4756).
A liver-targeted drug delivery carrier, composed of chitosan/poly(ethylene glycol)–glycyrrhetinic acid (CTS/PEG–GA) nanoparticles, was prepared by an ionic gelation process, in which glycyrrhetinic acid (GA) acted as the targeting ligand. The formation and characterization of these nanoparticles were confirmed by FT-IR, dynamic light scattering (DLS) and zeta potential measurements. The biodistribution of the nanoparticles was assessed by single-photon emission computed tomography (SPECT), and the cellular uptake was evaluated using human hepatic carcinoma cells (QGY-7703 cells). The anti-neoplastic effect of the doxorubicin·HCl-loaded nanoparticles (DOX-loaded nanoparticles) was also investigated in vitro and in vivo. The results showed that the CTS/PEG–GA nanoparticles were remarkably targeted to the liver, and keep at a high level during the experiment. The accumulation in the liver was 51.3% at 3 h after injection; this was nearly 2.6 times that obtained with the CTS/PEG nanoparticles. The DOX-loaded nanoparticles were greatly cytotoxic to QGY-7703 cells, and the IC50 (50% inhibitory concentration) for the free doxorubicin·HCl (DOX·HCl) and the DOX-loaded CTS/PEG–GA nanoparticles were 47 and 79 ng/mL, respectively. Moreover, the DOX-loaded CTS/PEG–GA nanoparticles could effectively inhibit tumor growth in H22 cell-bearing mice.
Keywords: Glycyrrhetinic acid; Chitosan; Poly(ethylene glycol); Liver-targeting
Effects of cleavable PEG-cholesterol derivatives on the accelerated blood clearance of PEGylated liposomes
by Huan Xu; Kai Q. Wang; Yi H. Deng; Da W. Chen (pp. 4757-4763).
The “accelerated blood clearance” (ABC) phenomenon describes a syndrome whereby the circulation time of a second dose of injected liposomes is substantially reduced, while the hepatic and splenic accumulations increase. To avoid this unexpected immune response, we have modified liposomes with cleavable PEG-lipid derivatives (PEG-CHEMS and PEG-CHMC). The ABC phenomenon was induced by repeated injection of conventional PEG-DSPE-liposomes in rat body and was accompanied by a greatly increased uptake in the liver. In contrast, only a slight ABC phenomenon was induced by repeated injection of PEG-CHMC-liposomes and was accompanied by increased uptake in the liver. Repeated injection of PEG-CHEMS-liposomes did not induce either an ABC phenomenon or an increase in liver accumulation. Repeated injection of the cleavable PEG-lipid modified vesicles (tris (hydroxymethyl) aminomethane salt of cholesteryl hemisuccinate(CHST)) showed similar results to those found with the cleavable PEG-lipid modified liposomes. These cleavable PEG-lipid derivatives could lessen or eliminate the ABC phenomenon produced by repeated injection of PEGylated liposomes or vesicles. Consequently, these two cleavable PEG-lipid derivatives may not only prolong the circulation time of nanoparticles, but may also represent a solution to the ABC phenomenon.
Keywords: Accelerated blood clearance (ABC); Cleavable; Vesicles; Liposomes
Enhanced transfection with silica-coated polyplexes loading plasmid DNA
by Kanjiro Miyata; Noha Gouda; Hiroyasu Takemoto; Makoto Oba; Yan Lee; Hiroyuki Koyama; Yuichi Yamasaki; Keiji Itaka; Nobuhiro Nishiyama; Kazunori Kataoka (pp. 4764-4770).
Silica-coating of positively charged polyplexes was demonstrated through silicic acid condensation to improve the polyplexes for enhanced complex stability and transfection efficiency. Silicic acid was efficiently condensed by polycations to form a silica network in the polyplex through electrostatic interaction and hydrogen bonding. The silica-coated (SC) polyplexes had an anionic surface charge of −20 mV and were 10–20 nm larger in size compared to the non-silica-coated control (+33.4 mV, 106 nm). Silica-coating significantly improved the polyplex stability against both dissociations by counter polyanion exchange and aggregation by salt. The silica network was dissolved to form silicic acid by removing free silicic acid based on the equilibrium, SiO2 + 2H2O ⇄ Si(OH)4. Indeed, dialysis of the SC polyplex solution against excess silica-free buffer permitted plasmid DNA release from the silica-coated polyplex, indicating the reversible nature of the silica-layer. The SC polyplex achieved significantly higher transfection efficiency without serious cytotoxicity compared to the polyplex without silica-coating. Detailed examinations of transfection using SC polyplexes revealed that the enhanced transfection efficiency was because of facilitated endosomal escape, possibly due to the protonation of the silica in acidic endosomal compartments. These findings demonstrate the utility of the silica-coating technique for polyplex-mediated gene delivery.
Keywords: Gene transfer; Polyamino acid; Plasmid DNA; Polyplex; Silica
Biotinylated transferrin/avidin/biotinylated disulfide containing PEI bioconjugates mediated p53 gene delivery system for tumor targeted transfection
by Xuan Zeng; Yun-Xia Sun; Wei Qu; Xian-Zheng Zhang; Ren-Xi Zhuo (pp. 4771-4780).
As mutation and dysfunction of p53 gene could induce most of human cancers, the p53 tumor suppressor gene was used to replace them and recover their normal functions in cancer cells. In this paper, biotinylated transferrin/avidin/biotinylated disulfide containing PEI bioconjugates (TABP-SS) mediated p53 gene delivery system was formed attributed to the ‘avidin–biotin bridge’. Characteristics of the obtained TABP-SS and its p53 complexes were evaluated in terms of acid–base titration, agarose gel electrophoresis, SEM, particle size and ζ-potential measurements. The acid–base titration results showed that TABP-SS had good buffer capability. The results of gel electrophoresis indicated that TABP-SS could fully condensed DNA and would be degraded by reducing agent inside cells. In vitro cell viability and transfection of TABP-SS were investigated in COS7, HepG2, and HeLa cells. Among the three different cell lines, TABP-SS exhibited much lower cytotoxicity and higher transfection efficacy in HepG2 and HeLa cells due to the specific interactions between transferrin ligands and their receptors on tumor cells. Apoptotic morphology was observed using confocal microscopy, and the expression of p53 protein in transfected cells was evaluated by western blotting. All the results indicated that TABP-SS/p53 complex could be considered as a low toxic and high efficient tumor targeted gene delivery system, which has great potential for further clinical application.
Keywords: Gene vector; Tumor target; Biotin–avidin system; Transferrin
Nucleic acid transfer with hemifluorinated polycationic lipids
by Emmanuel Klein; Christian Leborgne; Miahala Ciobanu; Jérôme Klein; Benoît Frisch; Françoise Pons; Guy Zuber; Daniel Scherman; Antoine Kichler; Luc Lebeau (pp. 4781-4788).
In this study, the ability of synthetic fluorinated lipospermines to bind DNA and siRNA was investigated and the transfection efficiency and toxicity of the resulting lipoplexes in cell lines were evaluated. Three lipopolyamines displaying fluorous tags close to their cationic polar head (“HFP” polyamines) were synthesized. Their ability to condense pDNA and siRNA, and to form nanoparticles were characterized. Lipoplex stability was investigated in the presence of different surface active compounds and was shown to be significantly improved due to the presence of the fluorous tags. Transfection efficiencies were studied in HepG2 and 911 cell lines, and compared to that of DOGS, DOTAP, and Lipofectamine™ 2000. Also, the ability of these compounds to deliver nucleic acids into cells in the presence of high concentration of serum was quantified. By incorporating two fluorous tags in the direct vicinity of the polycationic head group of the lipospermines, we show efficient pDNA and siRNA formulation, and delivery to cultured cells. Fluorinated lipoplexes exhibit improved stability in the presence of amphiphilic compounds and retain high transfection efficiency in the presence of 50–75% serum. These results demonstrate that lipospermines displaying fluorous tags close to their cationic polar head bind to and deliver pDNA and siRNA with high cell viability in different cell lines. They are efficient non-viral vectors that exhibit remarkable serum compatibility.
Keywords: Fluorocarbon; Gene transfer; Lipid; Self-assembly; DNA; siRNA