Biomaterials (v.33, #5)

Protein adsorption in three dimensions by Erwin A. Vogler (1201-1237).
Recent experimental and theoretical work clarifying the physical chemistry of blood-protein adsorption from aqueous-buffer solution to various kinds of surfaces is reviewed and interpreted within the context of biomaterial applications, especially toward development of cardiovascular biomaterials. The importance of this subject in biomaterials surface science is emphasized by reducing the “protein-adsorption problem” to three core questions that require quantitative answer. An overview of the protein-adsorption literature identifies some of the sources of inconsistency among many investigators participating in more than five decades of focused research. A tutorial on the fundamental biophysical chemistry of protein adsorption sets the stage for a detailed discussion of the kinetics and thermodynamics of protein adsorption, including adsorption competition between two proteins for the same adsorbent immersed in a binary-protein mixture. Both kinetics and steady-state adsorption can be rationalized using a single interpretive paradigm asserting that protein molecules partition from solution into a three-dimensional (3D) interphase separating bulk solution from the physical-adsorbent surface. Adsorbed protein collects in one-or-more adsorbed layers, depending on protein size, solution concentration, and adsorbent surface energy (water wettability). The adsorption process begins with the hydration of an adsorbent surface brought into contact with an aqueous-protein solution. Surface hydration reactions instantaneously form a thin, pseudo-2D interface between the adsorbent and protein solution. Protein molecules rapidly diffuse into this newly formed interface, creating a truly 3D interphase that inflates with arriving proteins and fills to capacity within milliseconds at mg/mL bulk-solution concentrations C B. This inflated interphase subsequently undergoes time-dependent (minutes-to-hours) decrease in volume V I by expulsion of either-or-both interphase water and initially adsorbed protein. Interphase protein concentration C I increases as V I decreases, resulting in slow reduction in interfacial energetics. Steady state is governed by a net partition coefficient P = ( C I / C B ) . In the process of occupying space within the interphase, adsorbing protein molecules must displace an equivalent volume of interphase water. Interphase water is itself associated with surface-bound water through a network of transient hydrogen bonds. Displacement of interphase water thus requires an amount of energy that depends on the adsorbent surface chemistry/energy. This “adsorption–dehydration” step is the significant free energy cost of adsorption that controls the maximum amount of protein that can be adsorbed at steady state to a unit adsorbent surface area (the adsorbent capacity). As adsorbent hydrophilicity increases, adsorbent capacity monotonically decreases because the energetic cost of surface dehydration increases, ultimately leading to no protein adsorption near an adsorbent water wettability (surface energy) characterized by a water contact angle θ → 65 ° . Consequently, protein does not adsorb (accumulate at interphase concentrations greater than bulk solution) to more hydrophilic adsorbents exhibiting θ < 65 ° . For adsorbents bearing strong Lewis acid/base chemistry such as ion-exchange resins, protein/surface interactions can be highly favorable, causing protein to adsorb in multilayers in a relatively thick interphase. A straightforward, three-component free energy relationship captures salient features of protein adsorption to all surfaces predicting that the overall free energy of protein adsorption Δ G ads o is a relatively small multiple of thermal energy for any surface chemistry (except perhaps for bioengineered surfaces bearing specific ligands for adsorbing protein) because a surface chemistry that interacts chemically with proteins must also interact with water through hydrogen bonding. In this way, water moderates protein adsorption to any surface by competing with adsorbing protein molecules. This Leading Opinion ends by proposing several changes to the protein-adsorption paradigm that might advance answers to the three core questions that frame the “protein-adsorption problem” that is so fundamental to biomaterials surface science.
Keywords: Review; Protein adsorption; Blood; Solution depletion; QCM; Tensiometry;

The cytotoxicity of cadmium-based quantum dots by Nan Chen; Yao He; Yuanyuan Su; Xiaoming Li; Qing Huang; Haifeng Wang; Xiangzhi Zhang; Renzhong Tai; Chunhai Fan (1238-1244).
Semiconductor Quantum dots (QDs) have raised great attention because of their superior optical properties and wide utilization in biological and biomedical studies. More recently, there have been intense concerns on cytotoxicity assessment of QDs. Most QDs are made of heavy metal ions (e.g., Cd2+), which may result in potential in vitro toxicity that hampers their practical applications. In this article, we aim to summarize recent progress on mechanistic studies of cytotoxicity of II–IV QDs. We have studied the cytotoxicity of a series of aqueous synthesized QDs (aqQDs), i.e. CdTe, CdTe/CdS core-shell structured and CdTe/CdS/ZnS core-shell-shell structured aqQDs. Our results suggested that released cadmium ions are responsible for the observed cytotoxicity of cadmium-based QDs. The fact that CdTe/CdS/ZnS core-shell-shell structured QDs are nearly nontoxic to cells further confirmed the role of released cadmium ions on cytotoxicity, and the effective protection of the ZnS shell. However, intracellular level of Cd2+ ions cannot be the only reason since the comparison with CdCl2-treated cells suggests there are other factors contributed to the cytotoxicity of aqQDs. Our studies on genome-wide gene expression profiling and subcellular localization of aqQDs with synchrotron-based scanning transmission X-ray microscopy (STXM) further suggest that the cytotoxicity of CdTe QDs not only comes from the release of Cd2+ ions but also intracellular distribution of QD nanoparticles in cells and the associated nanoscale effects.
Keywords: Cadmium based quantum dots; Cytotoxicity; Metallothioneins; Nanomaterials;

Hydrophobic polycationic coatings that inhibit biofilms and support bone healing during infection by Thomas P. Schaer; Suzanne Stewart; Bryan B. Hsu; Alexander M. Klibanov (1245-1254).
Adhesion of microorganisms to biomaterials with subsequent formation of biofilms on such foreign bodies as orthopedic trauma hardware is a critical factor in implant-associated infections; once a biofilm has been established, its microorganisms become recalcitrant to the host’s immune surveillance and markedly resistant to drugs. We have previously reported that painting with the hydrophobic polycation N,N-dodecyl,methyl-PEI (PEI = polyethylenimine) renders solid surfaces bactericidal in vitro. Herein we observe that N,N-dodecyl,methyl-PEI-derivatized titanium and stainless steel surfaces resist biofilm formation by Staphylococcus aureus compared to the untreated ones. Using imaging, microbiology-, histopathology-, and scanning electron microscopy (SEM) experiments in a clinically relevant large-animal (sheep) trauma model, we subsequently demonstrate in vivo that orthopedic fracture hardware painted with N,N-dodecyl,methyl-PEI not only prevents implant colonization with biofilm but also promotes bone healing. Functionalizing orthopedic hardware with hydrophobic polycations thus holds promise in supporting bone healing in the presence of infection in veterinary and human orthopedic patients.
Keywords: Orthopedics; Infection; Hardware; Bactericidal; Animal model;

Circulating activated platelets roll and make transient contacts before ultimately adhering to a substrate. However, despite the dynamic nature of platelet adhesion, most in vitro adhesion and activation studies have focused on establishing local cause and effect relationships. Here, we determined the effect of exposing platelets to immobilized upstream human fibrinogen on downstream adhesion and activation. Microcontact printing was used to prepare substrates that contained well defined fibrinogen priming regions. Washed platelets were perfused over the substrates and adhesion and activation in a downstream capture region were compared with samples that did not contain a fibrinogen priming region. It was found that samples containing an upstream priming region resulted in higher adhesion, platelet spreading areas and aggregation than samples that lacked the priming region. Also, when the priming region was selectively blocked with a polyclonal anti-fibrinogen antibody, the platelet response was attenuated. To characterize this phenomenon further, flow cytometry was used to assess bulk platelet activation following fibrinogen priming. The expression of two activation markers, PAC-1 and P-selectin were quantified. Expression of both activation markers was found to be higher after perfusion over fibrinogen versus albumin-coated substrates.
Keywords: Upstream platelet activation; Platelet adhesion; Surface fibrinogen; Microcontact printing;

Three critical aspects of searching for and understanding how to find highly resistant surfaces to protein adhesion are addressed here with specific application to synthetic membrane filtration. They include the (i) discovery of a series of previously unreported monomers from a large library of monomers with high protein resistance and subsequent low fouling characteristics for membrane ultrafiltration of protein-containing fluids, (ii) development of a new approach to investigate protein-resistant mechanisms from structure-property relationships, and (iii) adaptation of a new surface modification method, called atmospheric pressure plasma-induced graft polymerization (APP), together with a high throughput platform (HTP), for low cost vacuum-free synthesis of anti-fouling membranes. Several new high-performing chemistries comprising two polyethylene glycol (PEG), two amines and one zwitterionic monomers were identified from a library (44 commercial monomers) of five different classes of monomers as strong protein-resistant monomers. Combining our analysis here, using the Hansen solubility parameters (HSP) approach, and data from the literature, we conclude that strong interactions with water (hydrogen bonding) and surface flexibility are necessary for producing the highest protein resistance. Superior protein-resistant surfaces and subsequent anti-fouling performance was obtained with the HTP-APP as compared with our earlier HTP-photo graft-induced polymerization (PGP).
Keywords: Protein-resistant surfaces; Hansen solubility parameters; Atmospheric pressure plasma-induced graft polymerization; High throughput platform; Fouling;

Systematic engineering of 3D pluripotent stem cell niches to guide blood development by Kelly A. Purpura; Andrés M. Bratt-Leal; Katy A. Hammersmith; Todd C. McDevitt; Peter W. Zandstra (1271-1280).
Pluripotent stem cells (PSC) provide insight into development and may underpin new cell therapies, yet controlling PSC differentiation to generate functional cells remains a significant challenge. In this study we explored the concept that mimicking the local in vivo microenvironment during mesoderm specification could promote the emergence of hematopoietic progenitor cells from embryonic stem cells (ESCs). First, we assessed the expression of early phenotypic markers of mesoderm differentiation (E-cadherin, brachyury (T-GFP), PDGFRα, and Flk1: +/−ETPF) to reveal that E−T+P+F+ cells have the highest capacity for hematopoiesis. Second, we determined how initial aggregate size influences the emergence of mesodermal phenotypes (E−T+P+F+, E−T−P+/−F+, and E−T−P+F−) and discovered that colony forming cell (CFC) output was maximal with ∼100 cells per PSC aggregate. Finally, we introduced these 100-cell PSC aggregates into a low oxygen environment (5%; to upregulate endogenous VEGF secretion) and delivered two potent blood-inductive molecules, BMP4 and TPO (bone morphogenetic protein-4 and thrombopoietin), locally from microparticles to obtain a more robust differentiation response than soluble delivery methods alone. Approximately 1.7-fold more CFCs were generated with localized delivery in comparison to exogenous delivery, while combined growth factor use was reduced ∼14.2-fold. By systematically engineering the complex and dynamic environmental signals associated with the in vivo blood developmental niche we demonstrate a significant role for inductive endogenous signaling and introduce a tunable platform for enhancing PSC differentiation efficiency to specific lineages.
Keywords: Stem cells; Blood progenitor cell; Gelatin; Heparin; Microparticles;

Enzyme-catalyzed crosslinkable hydrogels: Emerging strategies for tissue engineering by Liliana S. Moreira Teixeira; Jan Feijen; Clemens A. van Blitterswijk; Pieter J. Dijkstra; Marcel Karperien (1281-1290).
State-of-the-art bioactive hydrogels can easily and efficiently be formed by enzyme-catalyzed mild-crosslinking reactions in situ. Yet this cell-friendly and substrate-specific method remains under explored. Hydrogels prepared by using enzyme systems like tyrosinases, transferases and lysyl oxidases show interesting characteristics as dynamic scaffolds and as systems for controlled release. Increased attention is currently paid to hydrogels obtained via crosslinking of precursors by transferases or peroxidases as catalysts. Enzyme-mediated crosslinking has proven its efficiency and attention has now shifted to the development of enzymatically crosslinked hydrogels with higher degrees of complexity, mimicking extracellular matrices. Moreover, bottom-up approaches combining biocatalysts and self-assembly are being explored for the development of complex nano-scale architectures. In this review, the use of enzymatic crosslinking for the preparation of hydrogels as an innovative alternative to other crosslinking methods, such as the commonly used UV-mediated photo-crosslinking or physical crosslinking, will be discussed. Photo-initiator-based crosslinking may induce cytotoxicity in the formed gels, whereas physical crosslinking may lead to gels which do not have sufficient mechanical strength and stability. These limitations can be overcome using enzymes to form covalently crosslinked hydrogels. Herewith, we report the mechanisms involved and current applications, focusing on emerging strategies for tissue engineering and regenerative medicine.
Keywords: Enzymes; Crosslinking; Injectable hydrogels; Tissue engineering;

Dental follicle cells and treated dentin matrix scaffold for tissue engineering the tooth root by Weihua Guo; Kun Gong; Haigang Shi; Guoxiong Zhu; Yong He; Bofu Ding; Lingying Wen; Yan Jin (1291-1302).
Tissue engineering strategies to reconstruct tooth roots are an effective therapy for the treatment of tooth loss. However, strategies to successfully regenerate tooth roots have not been developed and optimized. In the present study, rat dental follicle stem cells (DFCs) were characterized, followed by a thorough investigation of tooth roots regeneration for a combination of DFCs seeding cells, treated dentin matrix (TDM) scaffolds, and an inductive alveolar fossa microenvironment. Eighteen clones derived from single DFCs were harvested; however, only three clones were amplified successfully more than five passages and 90–95 days in culture. Following 270 days or 30 passages, the heterogeneous DFCs showed suitable characteristics for seeding cells to regenerate tooth roots. However, various features, such as variable proliferation rates, differentiation characteristics, apoptosis rates, and total lifespan were observed in DFCs and the three clones. Importantly, upon transplantation of DFCs combined with TDM for four weeks, root-like tissues stained positive for markers of dental pulp and periodontal tissues were regenerated in the alveolar fossa, but not in the skull and omental pockets. These results indicate that tooth roots were successfully regenerated and suggest that the combination of DFCs with TDM in the alveolar fossa is a feasible strategy for tooth roots regeneration. This strategy could be a promising approach for the treatment of clinical tooth loss and provides a perspective with potential applications to regeneration of other tissues and organs.
Keywords: Dental follicle cells; Tooth root; Regeneration; Scaffold; Tooth;

Recovery of cardiac function mediated by MSC and interleukin-10 plasmid functionalised scaffold by Carolyn A. Holladay; Aoife M. Duffy; Xizhe Chen; Michael V. Sefton; Timothy D. O’Brien; Abhay S. Pandit (1303-1314).
Stem cell transplantation has been suggested as a treatment for myocardial infarction, but clinical studies have yet to demonstrate conclusive, positive effects. This may be related to poor survival of the transplanted stem cells due to the inflammatory response following myocardial infarction. To address this, a scaffold-based stem cell delivery system was functionalised with anti-inflammatory plasmids (interleukin-10) to improve stem cell retention and recovery of cardiac function. Myocardial infarction was induced and these functionalised scaffolds were applied over the infarcted myocardium. Four weeks later, stem cell retention, cardiac function, remodelling and inflammation were quantified. Interleukin-10 gene transfer improved stem cell retention by more than five-fold and the hearts treated with scaffold, stem cells and interleukin-10 had significant functional recovery compared to the scaffold control (scaffold: −10 ± 7%, scaffold, interleukin-10 and stem cells: +7 ± 6%). This improved function was associated with increased infarcted wall thickness and increased ratios of collagen type III/type I, decreased cell death, and a change in macrophage markers from mainly cytotoxic in the scaffold group to mainly regulatory in scaffold, stem cells and interleukin-10 group. Thus, treatment of myocardial infarction with stem cells and interleukin-10 gene transfer significantly improved stem cell retention and ultimately improved overall cardiac function.
Keywords: Anti-inflammatory gene transfer; Mesenchymal stem cells; Cardiac tissue engineering; Scaffold; Interleukin-10;

Effect of oligonucleotide mediated immobilization of bone morphogenic proteins on titanium surfaces by Henning Schliephake; Christian Bötel; Anne Förster; Bernd Schwenzer; Judith Reichert; Dieter Scharnweber (1315-1322).
The aim of the present study was to test the hypothesis that oligonucleotides can be used for anchorage and slow release of osteogenic growth factors such as BMP to enhance the osteogenic activity of a titanium implant surface. Strands of 60-mer non-coding DNA oligonucleotides (ODN) were bound to an acid-etched sandblasted cp Ti-surface by nanomechanical fixation using anodic polarization. RhBMP2 that had been conjugated to complementary strands of DNA oligonucleotides was then bound to the anchored ODN strands by hybridization. Binding studies showed a higher binding capacity compared to non-conjugated BMP2. Long term release experiments demonstrated a continuous release from all surfaces that was lowest for the conjugated BMP2 bound to the ODN anchor strands. Proliferation of human bone marrow stroma cells (hBMSC) was significantly increased on these surfaces. Immunofluorescence showed that hBMSC grown on surfaces coated with specifically bound conjugated BMP2 developed significantly higher numbers of focal adhesion points and exhibited significantly higher levels of transcription of osteogenic markers alkaline phosphatase and osteopontin at early intervals. Biological activity (induction of alkaline phosphatase) of conjugated BMP2 released from the surface was comparable to released non-conjugated BMP2, indicating that conjugation did not negatively affect the activity of the released molecules. In conclusion the present study has shown that BMP2 conjugated to ODN strands and hybridized to complementary ODN strands anchored to a titanium surface has led to slow growth factor release and can enhance the osteogenic activity of the titanium surface.
Keywords: Bone morphogenic proteins; Oligonucleotides; Titanium; Controlled release; Recombinant proteins; Biofunctionalization;

Inducing alignment in astrocyte tissue constructs by surface ligands patterned on biomaterials by Fanwei Meng; Vladimir Hlady; Patrick A. Tresco (1323-1335).
Planar substrates with patterned ligands were used to induce astrocyte alignment whereas substrates with uniform fields of ligand were used to produce random cell orientation. DRG neurons plated on top of oriented astrocyte monolayers exhibited directional outgrowth along aligned astrocytes, demonstrating that purely biological cues provided by the oriented astrocytes were sufficient to provide guidance cues. Antibody blocking studies demonstrated that astrocyte associated FN played a major mechanistic role in directing engineered neurite extension. Our results show that nanometer level surface cues are sufficient to direct nerve outgrowth through an intervening organized astrocyte cell layer. In other studies, we showed that patterned ligands were able to transmit organization cues through multiple cell layers to control the overall alignment of an astrocyte tissue construct, demonstrating how natural scar tissue may develop in situ into potent barriers. In such constructs the spatial organization of astrocyte derived FN maintained its organizational anisotropy throughout the thickness of multilayered astrocyte constructs. These in vitro studies suggest possible roles for such constructs as bridging substrates for neuroregenerative applications.
Keywords: Astrocyte; Extracellular matrix; Axonal regeneration; Anisotropy; 3-D culture; Glial scar;

Control of cell attachment on pH-responsive chitosan surface by precise adjustment of medium pH by Yi-Hsin Chen; Yi-Chen Chung; I-Jong Wang; Tai-Horng Young (1336-1342).
The purpose of this study is to demonstrate pH-responsive chitosan is able to control cell behavior in response to small changes in environmental pH, which is at useful pH suitable for recovering cultured cells without additional enzymatic treatment and extensive washing steps. HeLa cells attached and spread well on chitosan at pH 6.99 and 7.20. When the pH was increased to 7.65, over 90% of cells would rapidly detached from chitosan surface within 1 h. Similarly, fibronectin adsorbed on chitosan at pH 7.20 also rapidly desorbed after increasing the medium pH. Most importantly and interestingly, medium pH adjustment could be facilitated by altering environment pCO2. It was found over 80% of HeLa cells could be recovered from chitosan surface within 1 h and the viability of detached cells was more than 95% by transferring the culture plate from incubator to atmospheric condition. Additionally, chitosan substrate could effectively control attachment/detachment of various types of cells including cell lines HaCaT, H1299, NIH-3T3, and primary corneal fibroblasts, indicating the technology described here is easily reproducible and should be promising for controlling rapid fibronectin adsorption/desorption and cell attachment/detachment for tissue engineering applications.
Keywords: Chitosan; Fibronectin; Cell adhesion; pH-responsive materials; Cell culture system;

The engineering of organized human corneal tissue through the spatial guidance of corneal stromal stem cells by Jian Wu; Yiqin Du; Simon C. Watkins; James L. Funderburgh; William R. Wagner (1343-1352).
Corneal stroma is an avascular connective tissue characterized by layers of highly organized parallel collagen fibrils, mono-disperse in diameter with uniform local interfibrillar spacing. Reproducing this level of structure on a nano- and micro-scale may be essential to engineer corneal tissue with strength and transparency similar to that of native cornea. A substrate of aligned poly(ester urethane) urea (PEUU) fibers, 165 ± 55 nm in diameter, induced alignment of cultured human corneal stromal stem cells (hCSSCs) which elaborated a dense collagenous matrix, 8–10 μm in thickness, deposited on the PEUU substratum. This matrix contained collagen fibrils with uniform diameter and regular interfibrillar spacing, exhibiting global parallel alignment similar to that of native stroma. The cells expressed high levels of gene products unique to keratocytes. hCSSCs cultured on PEUU fibers of random orientation or on a cast film of PEUU also differentiated to keratocytes and produced abundant matrix, but lacked matrix organization. These results demonstrate the importance of topographic cues in instructing organization of the transparent connective tissue of the corneal stroma by differentiated keratocytes. This important information will help with design of biomaterials for a bottom-up strategy to bioengineer spatially complex, collagen-based nano-structured constructs for corneal repair and regeneration.
Keywords: Stem cell; Collagen; Nano-structured; Topographic cues; Electrospinning;

Cell adhesion on an artificial extracellular matrix using aptamer-functionalized PEG hydrogels by Niancao Chen; Zhaoyang Zhang; Boonchoy Soontornworajit; Jing Zhou; Yong Wang (1353-1362).
The development of an artificial extracellular matrix (ECM) is important to regenerative medicine because the ECM plays complex and dynamic roles in the regulation of cell behavior. In this study, nucleic acid aptamers were applied to functionalize hydrogels for mimicking the adhesion sites of the ECM. The results showed that nucleic acid aptamers could be incorporated into polyethylene glycol (PEG) hydrogels via free radical polymerization. The incorporation of the aptamers produced only a moderate effect on the mechanical properties of the PEG hydrogels. Importantly, the results also showed that the aptamers effectively induced cell type-specific adhesion to the PEG hydrogels without affecting cell viability. The cell adhesion was a function of the aptamer concentration, the spacer length and the cell seeding time. In addition, cell adhesion to the aptamer-functionalized hydrogel could be attenuated by means of aptamer inactivation in a physiological condition. Thus, aptamer-functionalized hydrogels are promising biomaterials for the development of artificial ECMs.
Keywords: Hydrogel; Biomimetic material; Extracellular matrix; Affinity; Adhesion molecules;

The role of microstructured and interconnected pore channels in a collagen-based nerve guide on axonal regeneration in peripheral nerves by Ahmet Bozkurt; Franz Lassner; Dan O’Dey; Ronald Deumens; Arne Böcker; Tilman Schwendt; Christoph Janzen; Christoph V. Suschek; Rene Tolba; Eiji Kobayashi; Bernd Sellhaus; S. Tholl; Lizette Eummelen; Frank Schügner; Leon Olde Damink; Joachim Weis; Gary A. Brook; Norbert Pallua (1363-1375).
The use of bioengineered nerve guides as alternatives for autologous nerve transplantation (ANT) is a promising strategy for the repair of peripheral nerve defects. In the present investigation, we present a collagen-based micro-structured nerve guide (Perimaix) for the repair of 2 cm rat sciatic nerve defects. Perimaix is an open-porous biodegradable nerve guide containing continuous, longitudinally orientated channels for orientated nerve growth. The effects of these nerve guides on axon regeneration by six weeks after implantation have been compared with those of ANT. Investigation of the regenerated sciatic nerve indicated that Perimaix strongly supported directed axon regeneration. When seeded with cultivated rat Schwann cells (SC), the Perimaix nerve guide was found to be almost as supportive of axon regeneration as ANT. The use of SC from transgenic green-fluorescent-protein (GFP) rats allowed us to detect the viability of donor SC at 1 week and 6 weeks after transplantation. The GFP-positive SC were aligned in a columnar fashion within the longitudinally orientated micro-channels. This cellular arrangement was not only observed prior to implantation, but also at one week and 6 weeks after implantation. It may be concluded that Perimaix nerve guides hold great promise for the repair of peripheral nerve defects.
Keywords: Nerve regeneration; Schwann cells; Scaffold; Neurotmesis; Sciatic nerve; Bands of Büngner;

Enhancement of MSC adhesion and therapeutic efficiency in ischemic heart using lentivirus delivery with periostin by Yun-Hyeong Cho; Min-Ji Cha; Byeong-Wook Song; Il-Kwon Kim; Heesang Song; Woochul Chang; Soyeon Lim; Onju Ham; Se-Yeon Lee; Eunmi Choi; Hyuck Moon Kwon; Ki-Chul Hwang (1376-1385).
Many approaches have shown beneficial effects of modified mesenchymal stem cells (MSCs) for treatment of infarcted myocardium, but have primarily focused on enhancing the survival of transplanted MSCs. Here, we show the dual benefits of periostin-overexpressing MSCs (p-MSCs) for infarcted myocardium. P-MSCs led to the marked histological and functional recovery of infarcted myocardium by enhancing survival of MSCs and directly preventing apoptosis of cardiomyocytes. Survival of p-MSCs themselves and cardiomyocytes co-cultured with p-MSCs or treated with the conditioned media from p-MSCs was significantly increased under hypoxic conditions. Decreases in adhesion-related integrins were reversed in cardiomyocytes co-cultured with p-MSCs, followed by increases in p-PI3K and Akt, indicating that periostin activates the PI3K pathway through adhesion-related integrins. When p-MSCs were injected into myocardial infarcted rats, histological pathology and cardiac function were significantly improved compared to MSC-injected controls. Thus, periostin might be a new target of therapeutic treatments using MSCs as carriers for infarcted myocardium.
Keywords: Adhesion; Infarcted myocardium; Lentivirus; Mesenchymal stem cells; Periostin;

Neuronal regeneration and protection by collagen-binding BDNF in the rat middle cerebral artery occlusion model by Jian Guan; Weimin Tong; Wenyong Ding; Shiwei Du; Zhifeng Xiao; Qianqian Han; Zhaohui Zhu; Xinjie Bao; Ximin Shi; Chenxi Wu; Jiani Cao; Yi Yang; Wenbin Ma; Guilin Li; Yong Yao; Jun Gao; Junji Wei; Jianwu Dai; Renzhi Wang (1386-1395).
It has been well confirmed that brain-derived neurotrophic factor (BDNF) has therapeutic effects following stroke. However, it is difficult to be maintained at a sufficient concentration of BDNF in the infarcted hemisphere. We have shown in our previous work that BDNF fused with a collagen-binding domain (CBD-BDNF) could specifically bind to collagen. The ventricular ependyma of the brain is rich in collagen. Therefore, we have speculated that in the infarcted hemisphere, CBD-BDNF will bind to the collagen of the ventricular ependyma and stimulate the cell proliferation in the subventricular zone (SVZ). Using a rat middle cerebral artery occlusion model (MCAO), we injected CBD-BDNF into the lateral ventricle of MCAO rats. The results demonstrated that CBD-BDNF was retained at high levels in the infarcted hemisphere, promoted neural regeneration and angiogenesis, reduced cell loss, decreased apoptosis, and improved functional recovery. In addition, brain perfusion and metabolism, as evaluated by SPECT and PET, were improved in the CBD-BDNF treated group.
Keywords: Collagen; Brain-derived neurotrophic factor; Drug delivery; Nerve regeneration; Wound healing;

Embryonic stem (ES) cell-derived photoreceptors are a promising cell source for enhanced in vitro models of retinal degenerative diseases, but the more differentiated characteristics of retinal cells do not typically develop in dissociated cell cultures. Therefore, we have reconstructed organized retinal tissue by seeding dissociated cells into an array of aligned units that more faithfully mimics the retina. We solvent-processed poly(lactic-co-glycolic acid) (PLGA) into a microchannel scaffold format to achieve this geometric constraint. We compared the effect of PLGA concentration on channel morphology and, along with other culture conditions, on the infiltration of dissociated newborn mouse retinal cells into the channels. Culturing scaffolds at the gas–liquid interface with low serum media increased infiltrated rod photoreceptor viability 18-fold over submerged, high serum cultures when evaluated after seven days. Rod photoreceptors and Müller glia aligned processes parallel to the microchannel walls. Otx2+ and Pax6+ subpopulations recapitulated lamination behavior. Further, we constructed scaffold/retinal pigment epithelium (RPE) co-cultures and observed rods extending rhodopsin-positive processes toward RPE cells, mimicking normal rod polarization and morphology. Finally, human embryonic stem cell-derived photoreceptors exhibited infiltration and morphological characteristics similar to mouse retinal cells inside the scaffolds. These findings constitute an important advance in generating tissue-level retinal models from dissociated cells for use as drug screening platforms and in regenerative medicine.
Keywords: Retina; Scaffold; Micropatterning; Stem cell; Cell morphology; Biomimetic material;

Preserved liver-specific functions of hepatocytes in 3D co-culture with endothelial cell sheets by Kyungsook Kim; Kazuo Ohashi; Rie Utoh; Kyoko Kano; Teruo Okano (1406-1413).
Hepatocyte-based tissue engineering is an attractive method that is being developed to treat liver diseases. However, this method is limited by the relatively short lifespan of cultured hepatocytes to maintain their normal function. For this reason, the present study was designed to develop a cell sheet-based hepatocyte co-culture system that enables cultured hepatocytes to preserve their functions for a longer period of time. To achieve this goal, a monolayer cell sheet composed of endothelial cells (EC) was placed on top of a monolayer of hepatocytes (Hep). In this hybrid cell sheet format, histological examination revealed that bile canaliculi networks were formed and well developed among the hepatocytes in the layered Hep-EC sheet group. The albumin secretion level was highly preserved at least for 28 days in the hybrid Hep-EC sheet, whereas the monolayer of hepatocytes exhibited a markedly reduced time course of secretion. The expression levels of hepatocyte-specific genes including albumin, hepatocyte nucleus factor 4 (HNF 4), multidrug resistance-associated protein 2 (MRP 2), and claudin-3 were significantly higher in the Hep-EC sheet compared to the Hep sheet alone after 14-days in culture. In all, this culture system provides a valuable technology to prolong hepatocyte functionality and enable more efficient development of liver tissue engineering approaches to create liver-targeted regenerative therapies.
Keywords: Liver; Hepatocyte; Cell culture; Cell morphology; Endothelial cell; Co-culture;

The differentiation and isolation of mouse embryonic stem cells toward hepatocytes using galactose-carrying substrata by Qingyuan Meng; Amranul Haque; Bayar Hexig; Toshihiro Akaike (1414-1427).
A simple culture system to achieve the differentiation of embryonic stem (ES) cells toward hepatocytes with high efficiency is crucial in providing a cell source for the medical application. In this study, we report the effect of a matrix-dependent enrichment of ES cell-derived hepatocytes using immobilized poly(N-p-vinylbenzyl-4-O-β-d-galactopyranosyl-d-gluconamide) (PVLA) with E-cadherin-IgG Fc (E-cad-Fc) as a galactose-carrying substratum. PVLA and E-cad-Fc were confirmed to be stably co-adsorbed onto polystyrene surface by quartz crystal microbalance (QCM). We showed that the E-cad-Fc/PVLA hybrid substratum was efficient in culturing primary hepatocytes and maintaining liver functions, on which the undifferentiated ES cells also maintained high proliferative capability. Furthermore, ES cell-derived hepatocytes on this hybrid matrix expressed elevated level of liver specific genes and functions together with early expression of definitive hepatocyte marker, asialoglycoprotein receptor (ASGPR). Finally, we isolated a high percentage of cells (about 60%) with ASGPR expression after re-seeding onto PVLA-coated surface, and observed the elimination of the poorly differentiated cells (Gata6+ and Sox17+) and the ones toward another cell lineage (brachyury+ and Pdx1+). The system uses a glycopolymer as an extracellular substratum for isolation and enrichment of ES cell-derived hepatocytes with adequate homogeneity and functionality.
Keywords: Embryonic stem cells; Hepatocyte differentiation; Galactose-carrying polymer; E-cadherin; Extracellular matrix;

In vivo treatment of tumors using host-guest conjugated nanoparticles functionalized with doxorubicin and therapeutic gene pTRAIL by Hui Fan; Qi-Da Hu; Fu-Jian Xu; Wen-Quan Liang; Gu-Ping Tang; Wan-Tai Yang (1428-1436).
The combination of gene therapy and chemotherapy may increase the therapeutic efficacy in the treatment of patients. In this work, the anti-cancer drug Dox and therapeutic gene pTRAIL-loaded host−guest co-delivery system was assayed for the possibility of in vivo synergistically treating tumors. The introduced Dox could act as an auxiliary component to human tumor necrosis factor-related apoptosis-inducing ligand-encoding plasmid gene pTRAIL. Such delivery system possessed the good ability of in vivo retention of chemotherapeutic drugs, achieved good therapeutic effects in the inhibition of tumor growth and significantly prolonged the survival time of tumor-bearing mice. With the efficient ability to co-deliver drug and gene, such host−guest assembly should have great potential applications in cancer therapy.
Keywords: DNA; Gene therapy; Vectors; Doxorubicin; pTRAIL;

The enhancement of cancer stem cell properties of MCF-7 cells in 3D collagen scaffolds for modeling of cancer and anti-cancer drugs by Lei Chen; Zhifeng Xiao; Yue Meng; Yannan Zhao; Jin Han; Guannan Su; Bing Chen; Jianwu Dai (1437-1444).
Three-dimensional (3D) culture could partially simulate in vivo conditions. In this work, we developed a 3D collagen scaffold to investigate cellular properties of MCF-7 cells. The porous scaffolds not only induced the diversification of cell morphologies but also extended cell proliferation. The expression of pro-angiogenic growth factors and the transcriptions of matrix metalloproteinases (MMPs) were significantly increased in cells cultured in 3D collagen scaffolds. In addition, 3D collagen scaffolds could generate a cell population with the properties of cancer stem cells (CSCs). The upregulation of EMT markers and the downregulation of the epithelial cell marker were observed in cells cultured in collagen scaffolds. The expression of stem cell markers, including OCT4A and SOX2, and breast cancer stem cell signatures, including SOX4, JAG1 and CD49F, was significantly unregulated in 3D collagen scaffolds. The proportion of cells with CSC-like CD44+/CD24−/low phenotype was notably increased. High-level expression of CSC-associated properties of MCF-7 cells cultured in 3D was further confirmed by high tumorigenicity in vivo. Moreover, xenografts with 3D cells formed larger tumors. The properties of MCF-7 cells in 3D may have partially simulated their in vivo behaviors. Thus, 3D collagen scaffolds might provide a useful platform for anti-cancer therapeutics and CSC research.
Keywords: Three-dimensional culture; Breast cancer stem cells; EMT; Collagen scaffolds; MCF-7;

We have developed a polymer conjugate (Cellax) composed of acetylated carboxymethylcellulose (CMC), docetaxel (DTX), and PEG, designed to enhance the pharmacokinetics (PK) and antitumor efficacy of DTX. Our design placed an emphasis on nanoparticle self-assembly to protect DTX during blood transport, stability of the nanoparticle, and PEGylation to enhance PK. Compared to Taxotere, Cellax exhibited a 38.6 times greater area under the curve (AUC), and significantly lower clearance (2.5%) in PK. Less than 10% of DTX was released from Cellax in the blood circulation, indicating that Cellax were stable during blood transport. Cellax reduced non-specific distribution of DTX to the heart, lung and kidney by 48, 90, and 90%, respectively, at 3 h, compared to Taxotere. The uptake of Cellax at 3 h in the liver and spleen was high (15–45 μg DTX/g) but declined rapidly to <10 μg DTX/g in 24 h, and induced no measurable toxicity at 170 mg DTX/kg. Taxotere, on the other hand, displayed non-specific uptake in all the examined normal tissues and induced significant apoptosis in the lung and kidney at 40 mg DTX/kg. The tumor uptake of Cellax was 5.5-fold more than that by Taxotere and the uptake occurred within 3 h after injection and persisted for 10 days. The conjugate exhibited enhanced efficacy in a panel of primary and metastatic mouse tumor models. These results clearly demonstrated that Cellax improved the pharmacokinetics, biodistribution and efficacy of DTX compared to Taxotere with reduced toxicity.
Keywords: Docetaxel; Carboxymethylcellulose; Conjugated polymers; Pharmacokinetics; Biodistribution;

Cisplatin@US-tube carbon nanocapsules for enhanced chemotherapeutic delivery by Adem Guven; Irene A. Rusakova; Michael T. Lewis; Lon J. Wilson (1455-1461).
The use of chemotherapeutic drugs in cancer therapy is often limited by problems with administration such as insolubility, inefficient biodistribution, lack of selectivity, and inability of the drug to cross cellular barriers. To overcome these limitations, various types of drug delivery systems have been explored, and recently, carbon nanotube (CNT) materials have also garnered attention in the area of drug delivery. In this study, we describe the preparation, characterization, and in vitro testing of a new ultra-short single-walled carbon nanotube (US-tube)-based drug delivery system for the treatment of cancer. In particular, the encapsulation of cisplatin (CDDP), a widely-used anticancer drug, within US-tubes has been achieved, and the resulting CDDP@US-tube material characterized by high-resolution transmission electron microscopy (HR-TEM), energy-dispersive spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), and inductively-coupled optical emission spectrometry (ICP-OES). Dialysis studies performed in phosphate-buffered saline (PBS) at 37 °C have demonstrated that CDDP release from CDDP@US-tubes can be controlled (retarded) by wrapping the CDDP@US-tubes with Pluronic-F108 surfactant. Finally, the anticancer activity of pluronic-wrapped CDDP@US-tubes has been evaluated against two different breast cancer cell lines, MCF-7 and MDA-MB-231, and found to exhibit enhanced cytotoxicity over free CDDP after 24 h. These studies have laid the foundation for developing US-tube-based delivery of chemotherapeutics, with drug release mainly limited to within cancer cells only.
Keywords: Cisplatin; Cancer chemotherapy; Nanotechnology; Single-walled carbon nanotubes; Drug delivery;

Inhibition of cancer stem cell-like properties and reduced chemoradioresistance of glioblastoma using microRNA145 with cationic polyurethane-short branch PEI by Yi-Ping Yang; Yueh Chien; Guang-Yuh Chiou; Jong-Yuh Cherng; Mong-Lien Wang; Wen-Liang Lo; Yuh-Lih Chang; Pin-I Huang; Yi-Wei Chen; Yang-Hsin Shih; Ming-Teh Chen; Shih-Hwa Chiou (1462-1476).
Glioblastomas (GBMs) are the most common primary brain tumors with poor prognosis. CD133 has been considered a putative marker of cancer stem cells (CSCs) in malignant cancers, including GBMs. MicroRNAs (miRNAs), highly conserved small RNA molecules, may target oncogenes and have potential as a therapeutic strategy against cancer. However, the role of miRNAs in GBM-associated CSCs remains mostly unclear. In this study, our miRNA/mRNA-microarray and RT-PCR analysis showed that the expression of miR145 (a tumor-suppressive miRNA) is inversely correlated with the levels of Oct4 and Sox2 in GBM-CD133+ cells and malignant glioma specimens. We demonstrated that miR145 negatively regulates GBM tumorigenesis by targeting Oct4 and Sox2 in GBM-CD133+. Using polyurethane-short branch polyethylenimine (PU-PEI) as a therapeutic-delivery vehicle, PU-PEI-mediated miR145 delivery to GBM-CD133+ significantly inhibited their tumorigenic and CSC-like abilities and facilitated their differentiation into CD133-non-CSCs. Furthermore, PU-PEI-miR145-treated GBM-CD133+ effectively suppressed the expression of drug-resistance and anti-apoptotic genes and increased the sensitivity of the cells to radiation and temozolomide. Finally, the in vivo delivery of PU-PEI-miR145 alone significantly suppressed tumorigenesis with stemness, and synergistically improved the survival rate when used in combination with radiotherapy and temozolomide in orthotopic GBM-CD133+-transplanted immunocompromised mice. Therefore, PU-PEI-miR145 is a novel therapeutic approach for malignant brain tumors.
Keywords: Glioblastoma; Drug-resistance; miR145; Polyurethane-short branch polyethylenimine;

Induction of ROS, mitochondrial damage and autophagy in lung epithelial cancer cells by iron oxide nanoparticles by Mohd Imran Khan; Akbar Mohammad; Govil Patil; S.A.H. Naqvi; L.K.S. Chauhan; Iqbal Ahmad (1477-1488).
Autophagy has attracted a great deal of research interest in tumor therapy in recent years. An attempt was made in this direction and now we report that iron oxide NPs synthesized by us selectively induce autophagy in cancer cells (A549) and not in normal cells (IMR-90). It was also noteworthy that autophagy correlated with ROS production as well as mitochondrial damage. Protection of NAC against ROS clearly suggested the implication of ROS in hyper-activation of autophagy and cell death. Pre-treatment of cancer cells with 3-MA also exhibited protection against autophagy and promote cellular viability. Results also showed involvement of classical mTOR pathway in autophagy induction by iron oxide NPs in A549 cells. Our results had shown that bare iron oxide NPs are significantly cytotoxic to human cancer cells (A549) but not to the normal human lung fibroblast cells (IMR-90).In other words our nanoparticles selectively kill cancerous cells. It is encouraging to conclude that iron oxide NPs bear the potential of its applications in biomedicine, such as tumor therapy specifically by inducing autophagy mediated cell death of cancer cells.
Keywords: Iron oxide NPs; Autophagy; Necrosis; ROS; Mitochondrial damage;

Tumor accumulation and antitumor efficacy of docetaxel-loaded core-shell-corona micelles with shell-specific redox-responsive cross-links by Ahn Na Koo; Kyung Hyun Min; Hong Jae Lee; Sang-Uk Lee; Kwangmeyung Kim; Ick Chan Kwon; Sun Hang Cho; Seo Young Jeong; Sang Cheon Lee (1489-1499).
A robust core-shell-corona micelle bearing redox-responsive shell-specific cross-links was evaluated as a carrier of docetaxel (DTX) for cancer therapy. The polymer micelles of poly(ethylene glycol)-b-poly(l-lysine)-b-poly(l-phenylalanine) (PEG-PLys-PPhe) in the aqueous phase provided the three distinct functional domains: the PEG outer corona for prolonged circulation, the PLys middle shell for disulfide cross-linking, and the PPhe inner core for DTX loading. The shell cross-linking was performed by the reaction of disulfide-containing cross-linkers with Lys moieties in the middle shells. The shell cross-linking did not change the micelle size or the spherical morphology. The shell cross-linked micelles exhibited enhanced serum stability. The DTX release from the DTX-loaded disulfide cross-linked micelles (DTX-SSCLM) was facilitated by increasing the concentration of glutathione (GSH). At an intracellular GSH level, DTX release was facilitated due to the reductive cleavage of the disulfide cross-links in the shell domains. The in vivo tissue distribution and tumor accumulation of the DTX-SSCLM that were labeled with a near-infrared fluorescence (NIRF) dye, Cy5.5, were monitored in MDA-MB231 tumor-bearing mice. Non-invasive real-time optical imaging results indicated that the DTX-SSCLM exhibited enhanced tumor specificity due to the prolonged stable circulation in blood and the enhanced permeation and retention (EPR) effect compared with the DTX-loaded non-cross-linked micelles (DTX-NCLM). The DTX-SSCLM exhibited enhanced therapeutic efficacy in tumor-bearing mice compared with free DTX and DTX-NCLM. The domain-specific shell cross-linking that is described in this work may serve as a useful guidance for enhancing the antitumor therapeutic efficacy of various polymer micelles and nano-aggregates.
Keywords: Shell cross-linking; Disulfide; Redox-responsive; Polymer micelle; Docetaxel;

An activatable multimodal/multifunctional nanoprobe for direct imaging of intracellular drug delivery by Rajendra N. Mitra; Mona Doshi; Xiaolei Zhang; Jessica C. Tyus; Niclas Bengtsson; Steven Fletcher; Brent D.G. Page; James Turkson; Andre J. Gesquiere; Patrick T. Gunning; Glenn A. Walter; Swadeshmukul Santra (1500-1508).
Multifunctional nanoparticles integrated with imaging modalities (such as magnetic resonance and optical) and therapeutic drugs are promising candidates for future cancer diagnostics and therapy. While targeted drug delivery and imaging of tumor cells have been the major focus in engineering nanoparticle probes, no extensive efforts have been made towards developing sensing probes that can confirm and monitor intracellular drug release events. Here, we present quantum dot (Qdot)-iron oxide (IO) based multimodal/multifunctional nanocomposite probe that is optically and magnetically imageable, targetable and capable of reporting on intracellular drug release events. Specifically, the probe consists of a superparamagnetic iron oxide nanoparticle core (IONP) decorated with satellite CdS:Mn/ZnS Qdots where the Qdots themselves are further functionalized with STAT3 inhibitor (an anti-cancer agent), vitamin folate (as targeting motif) and m-polyethylene glycol (mPEG, a hydrophilic dispersing agent). The Qdot luminescence is quenched in this nanocomposite probe (“OFF” state) due to combined electron/energy transfer mediated quenching processes involving IONP, folate and STAT3 agents. Upon intracellular uptake, the probe is exposed to the cytosolic glutathione (GSH) containing environment resulting in restoration of the Qdot luminescence (“ON” state), which reports on uptake and drug release. Probe functionality was validated using fluorescence and MR measurements as well as in vitro studies using cancer cells that overexpress folate receptors.
Keywords: Magnetic nanoparticles; Quantum dots; Targeted drug delivery; Bioimaging; Biosensing;

ICP-MS analysis of lanthanide-doped nanoparticles as a non-radiative, multiplex approach to quantify biodistribution and blood clearance by Samuel H. Crayton; Drew R. Elias; Ajlan Al Zaki; Zhiliang Cheng; Andrew Tsourkas (1509-1519).
Recent advances in material science and chemistry have led to the development of nanoparticles with diverse physicochemical properties, e.g. size, charge, shape, and surface chemistry. Evaluating which physicochemical properties are best for imaging and therapeutic studies is challenging not only because of the multitude of samples to evaluate, but also because of the large experimental variability associated with in vivo studies (e.g. differences in tumor size, injected dose, subject weight, etc.). To address this issue, we have developed a lanthanide-doped nanoparticle system and analytical method that allows for the quantitative comparison of multiple nanoparticle compositions simultaneously. Specifically, superparamagnetic iron oxide (SPIO) with a range of different sizes and charges were synthesized, each with a unique lanthanide dopant. Following the simultaneous injection of the various SPIO compositions into tumor-bearing mice, inductively coupled plasma mass spectroscopy (ICP-MS) was used to quantitatively and orthogonally assess the concentration of each SPIO composition in serial blood samples and the resected tumor and organs. The method proved generalizable to other nanoparticle platforms, including dendrimers, liposomes, and polymersomes. This approach provides a simple, cost-effective, and non-radiative method to quantitatively compare tumor localization, biodistribution, and blood clearance of more than 10 nanoparticle compositions simultaneously, removing subject-to-subject variability.
Keywords: Superparamagnetic iron oxide; Nanoparticle; ICP-MS; XPS; Biodistribution; Clearance; Multiplex;

Structural and mechanical profiles of native collagen fibers in vaginal wall connective tissues by Indumathi Sridharan; Yin Ma; Taeyoung Kim; William Kobak; Jacob Rotmensch; Rong Wang (1520-1527).
Collagen, an ubiquitous biomaterial, confers robustness and resilience to connective tissues. In this study, we analyzed the structure and elasticity profile of collagen from the vaginal wall connective tissue of healthy pre-menopausal (pre-M) and postmenopausal (post-M) women. The histological staining assisted study with an atomic force microscope renders the examination of native collagen fibers on site of the connective tissue from nanoscopic scale to microscopic scale with high spatial resolution. Our results suggest that during menopause, collagen’s structure and elasticity are subject to changes at all levels of organization- between individual collagen fibers, between collagen and muscle, and between collagen and other matrix elements. The systematic analysis of the native structure and mechanical properties of collagen within a tissue provides a potential way to study non-fatal conditions such as pelvic organ prolapse and other genito-urinary disorders, where the initial symptoms are subtle and multivariate, and where early detection of patient’s condition may allow better non-invasive interventions and reduce the number of women undergoing surgical correction of these common disorders.
Keywords: Collagen; Fibro-muscle; Tissue elasticity; Atomic force microscopy;

Preactivated thiomers as mucoadhesive polymers for drug delivery by Javed Iqbal; Gul Shahnaz; Sarah Dünnhaupt; Christiane Müller; Fabian Hintzen; Andreas Bernkop-Schnürch (1528-1535).
This study was aimed to synthesize polymeric excipients with improved mucoadhesive, cohesive and in situ-gelling properties to assure a prolonged retention time of dosage forms at a given target site, thereby achieving an increased uptake and improved oral bioavailability of certain challenging therapeutic agents such as peptides and proteins. Accordingly, poly(acrylic acid)-cysteine-2-mercaptonicotinic acid (PAA-cys-2MNA) conjugates were synthesized by the oxidative S–S coupling of PAA-cys (100-, 250- and 450 kDa) with 2-mercaptonicotinic acid (2MNA). Unmodified PAAs, PAAs-cys (thiomers) and PAA-cys-2MNA (100-, 250- and 450 kDa) conjugates were compressed into tablets to perform disintegration tests, mucoadhesion studies and rheological measurements. Moreover, cytotoxicty of the polymers was determined using Caco-2 cells. The resulting PAA-cys-2MNA (100-, 250- and 450 kDa) conjugates displayed 113.5 ± 12.7, 122.7 ± 12.2 and 117.3 ± 4.6 μmol/g of 2-mercaptonicotinic acid, respectively. Due to the immobilization of 2MNA, the PAA-cys-2MNA (pre-activated thiomers) conjugates exhibit comparatively higher swelling properties and disintegration time to the corresponding unmodified and thiolated polymers. On the rotating cylinder, tablets based on PAA-cys-2MNA (100-, 250- and 450 kDa) conjugates displayed 5.0-, 5.4- and 960-fold improved mucoadhesion time in comparison to the corresponding unmodified PAAs. Results achieved from tensile studies were found in good agreement with the results obtained by rotating cylinder method. The apparent viscosity of PAA-cys-2MNA (100-, 250- and 450 kDa) conjugates was improved 1.6-, 2.5- and 206.2-fold, respectively, in comparison to the corresponding unmodified PAAs. Moreover, pre-activated thiomers/mucin mixtures showed a time dependent increase in viscosity up to 24 h, leading to 7.0-, 18.9- and 2678-fold increased viscosity in comparison to unmodified PAAs (100-, 250- and 450 kDa), respectively. All polymers were found non-toxic over Caco-2 cells. Thus, on the basis of achieved results the pre-activated thiomers seem to represent a promising generation of mucoadhesive polymers which are safe to use for prolonged residence time of drug delivery systems to target various mucosa.
Keywords: Thiomers; Poly(acrylic acid)-cysteine; 2-Mercaptonictic acid; Pre-activated thiomers; Mucoadhesion; Oral drug delivery;

Doxorubicin-loaded human serum albumin nanoparticles surface-modified with TNF-related apoptosis-inducing ligand and transferrin for targeting multiple tumor types by Sungho Bae; Kyungwan Ma; Tae Hyung Kim; Eun Seong Lee; Kyung Taek Oh; Eun-Seok Park; Kang Choon Lee; Yu Seok Youn (1536-1546).
Human serum albumin (HSA) nanoparticles (NPs) surface modified with tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and transferrin, and containing doxorubicin were designed and prepared. Surface amines of HSA were reversibly protected with dimethylmaleic anhydride (DMMA), and HSA-NPs were prepared using a desolvation technique. Furthermore, the surfaces of HSA-NPs were modified with thiolated TRAIL or transferrin using sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (Sulfo-SMCC). The prepared TRAIL/transferrin plus doxorubicin HSA-NPs were characterized by TEM, FE-SEM, and particle size analysis, and their cytotoxic and apoptotic activities were evaluated in several cancer cell lines, namely, HCT 116, doxorubicin-resistant MCF-7, and CAPAN-1. In addition, the tumor-targeting abilities of NPs were assessed using an infrared imaging system in HCT 116-xenografted nu/nu mice. Results showed that the TRAIL/transferrin/doxorubicin HSA-NPs had remarkable cytotoxic and apoptotic activities in all cancer cells examined with a general or a drug-resistant character, and that these NPs had obvious synergistic cytotoxic effects particularly on CAPAN-1 cells. Moreover, these HSA-NPs were effectively localized to tumors in a HCT 116-xenografted nu/nu mouse over 32 h. The findings of this study suggest that the described TRAIL/transferrin/doxorubicin HSA-NPs are a useful targeting agent capable of killing different types of tumor cells in various tissue organs.
Keywords: TRAIL; Albumin nanoparticles; Apoptosis; Doxorubicin; Tumor targeting;

Anti-inflammatory effect with high intensity focused ultrasound-mediated pulsatile delivery of diclofenac by Chih-Yu Wang; Chih-Hui Yang; Yung-Sheng Lin; Chih-Hsin Chen; Keng-Shiang Huang (1547-1553).
A pulsatile ultrasound controlled drug release platform with diclofenac-loaded alginate microcapsules (fabricated with a home-made electrostatic device, 75% embedded rate) was established to evaluate anti-inflammation efficiency. Better anti-inflammation efficiency was found using the ultrasound system and the drug delivery can be adjusted based on the programmed ultrasound cycle. The results of the in vitro study show that an approx. 30% higher drug release rate was obtained by using continuous ultrasound irradiation (9-Watt, 180 min), and an approx. 16% higher drug release rate was obtained by using pulsatile ultrasound irradiation (9-Watt, 60 min) compared to without ultrasound activation. For the in vivo study, the anti-inflammatory test with carrageenan-induced rat’s paw edema shows that diclofenac-loaded microcapsules followed by ultrasound irradiation (9-Watt, 60 min) contributed to an 81% inhibition rate, which was significantly higher than diclofenac only (approx. 60% higher). In addition, because of their heat conducting properties, gold nanoparticles encapsulated in the diclofenac-loaded microcapsules resulted in better drug release efficiency, but tended to depress the anti-inflammation effect.
Keywords: Ultrasound enhanced delivery; Anti-inflammation effect; Alginate microcapsule; Diclofenac;

Recombinant spider silk particles for controlled delivery of protein drugs by Markus Hofer; Gerhard Winter; Julia Myschik (1554-1562).
The engineered and recombinant spider silk protein eADF4(C16) has been shown to be a promising biomaterial for the use as drug delivery system. In previous studies, eADF4(C16) particles were loaded with low molecular weight drugs exhibiting a positive net-charge and sufficient hydrophobicity. Here, we demonstrate that also macromolecular drugs like proteins can be loaded on eADF4(C16) particles. Using lysozyme as a model protein, remarkably high loading of up to 30% [w/w] was feasible and high loading efficiencies of almost 100% were obtained. Furthermore, using confocal laser scanning microscopy, it is demonstrated that fluorescently labeled lysozyme is not only adsorbed to the negatively charged particles’ surface, but also diffusing into the matrix of eADF4(C16) particles. The release of lysozyme is shown to be dependent on the ionic strength and pH of the release medium. To improve the long-term stability of eADF4(C16) containing dispersions, lyophilization is shown as a suitable tool. Disaccharides (sucrose, trehalose) and mannitol served as stabilizers to prevent aggregation and/or particle degradation during freeze-drying. The slowly biodegradable eADF4(C16) particles are a promising new particulate drug carrier system for the delivery of susceptible drugs like therapeutic proteins.
Keywords: Biomimetic material; Spider silk; Nanoparticle; Lysozyme; Drug delivery;

Alleviation of rheumatoid arthritis by cell-transducible methotrexate upon transcutaneous delivery by Sang-Won Lee; Ji-Hye Kim; Min-Chan Park; Yong-Beom Park; Wook Jin Chae; Tomohiro Morio; Dong-Ho Lee; Sang-Hwa Yang; Seung-Kyou Lee; Soo-Kon Lee; Sang-Kyou Lee (1563-1572).
Rheumatoid arthritis (RA) is a systemic autoimmune disease that is initiated and maintained by various inflammatory/immune cells and their cytokines, leading to cartilage degradation and bone erosion. Despite its potent therapeutic efficacy on RA, the oral administration of methotrexate (MTX) provokes serious adverse systemic complications, thus necessitating the local application of MTX. Here, we show that transcutaneous MTX (TC-MTX) can efficiently penetrate joint skin ex vivo and in vivo, and that TC-MTX can significantly improve the various inflammatory symptoms associated with RA. Further, TC-MTX preserved the joint-structures in mice with collagen-induced arthritis (CIA), which was also confirmed by three-dimensional micro-computed tomography scan. TC-MTX markedly decreased the secretion of inflammatory cytokines both in the serum and in inflamed joints of CIA mice. Further, its therapeutic potential is comparable to that of etanercept, a biological agent that block tumor necrosis factor (TNF)-α. Importantly, the systemic cytotoxicity of TC-MTX was not detected. Thus, TC-MTX can be a new therapeutic modality for RA patients without systemic complications.
Keywords: Arthritis; Drug delivery; Inflammation; Immunomodulation; Cytotoxicity;

Goblet cell-targeting nanoparticles for oral insulin delivery and the influence of mucus on insulin transport by Yun Jin; Yupin Song; Xi Zhu; Dan Zhou; Chunhui Chen; Zhirong Zhang; Yuan Huang (1573-1582).
The present study was to demonstrate the effects of goblet cell-targeting nanoparticles on the oral absorption of insulin in vitro, ex vivo and in vivo, and identify the targeting mechanism as well as the influence of mucus. The insulin loaded nanoparticles were prepared using trimethyl chitosan chloride (TMC) modified with a CSKSSDYQC (CSK) targeting peptide. Compared with unmodified nanoparticles, the CSK peptide modification could facilitate the uptake of nanoparticles in villi, enhance the permeation of drugs across the epithelium, meanwhile, induce a significantly higher internalization of drugs via clathrin and caveolae mediated endocytosis on goblet cell-like HT29-MTX cells. In transport studies across Caco-2/HT29-MTX co-cultured cell monolayer (simulating intestinal epithelium), the CSK peptide modification also showed enhanced transport ability, even if the targeting recognition was partially affected by mucus. Moreover, it was found the existence of mucus was propitious to the transport of insulin from both modified and unmodified nanoparticles. In the pharmacological and pharmacokinetic studies in diabetic rats, the orally administrated CSK peptide modified nanoparticles produced a better hypoglycemic effect with a 1.5-fold higher relative bioavailability compared with unmodified ones. In conclusion, CSK peptide modified TMC nanoparticles showed sufficient effectiveness as goblet cell-targeting nanocarriers for oral delivery of insulin.
Keywords: CSKSSDYQC peptide; Goblet cell-targeting; Mucus; Caco-2/HT29-MTX co-cultured cells; N-trimethyl chitosan chloride nanoparticles;

A rapid-acting, long-acting insulin formulation based on a phospholipid complex loaded PHBHHx nanoparticles by Qiang Peng; Zhi-Rong Zhang; Tao Gong; Guo-Qiang Chen; Xun Sun (1583-1588).
The application of poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) for sustained and controlled delivery of hydrophilic insulin was made possible by preparing insulin phospholipid complex loaded biodegradable PHBHHx nanoparticles (INS-PLC-NPs). The INS-PLC-NPs produced by a solvent evaporation method showed a spherical shape with a mean particle size, zeta potential and entrapment efficiency of 186.2 nm, −38.4 mv and 89.73%, respectively. In vitro studies demonstrated that only 20% of insulin was released within 31 days with a burst release of 5.42% in the first 8 h. The hypoglycaemic effect in STZ induced diabetic rats lasted for more than 3 days after the subcutaneous injection of INS-PLC-NPs, which significantly prolonged the therapeutic effect compared with the administration of insulin solution. The pharmacological bioavailability (PA) of INS-PLC-NPs relative to insulin solution was over 350%, indicating that the bioavailability of insulin was significantly enhanced by INS-PLC-NPs. Therefore, the INS-PLC-NPs system is promising to serve as a long lasting insulin release formulation, by which the patient compliance can be enhanced significantly. This study also showed that phospholipid complex loaded biodegradable nanoparticles (PLC-NPs) have a great potential to be used as a sustained delivery system for hydrophilic proteins to be encapsulated in hydrophobic polymers.
Keywords: PHBHHx; Insulin; Diabetes; Phospholipid complex; Biodegradable nanoparticles; Sustained and controlled release;

Mitochondrial dysfunction has been implicated in a variety of human diseases. It is now well accepted that mutations and defects in the mitochondrial genome form the basis of these diseases. Therefore, mitochondrial gene therapy and diagnosis would be expected to have great medical benefits. To achieve such a strategy, it will be necessary to deliver therapeutic agents into mitochondria in living cells. We report here on an approach to accomplish this via the use of a Dual Function (DF)-MITO-Porter, aimed at the mitochondrial genome, so-called mitochondrial DNA (mtDNA). The DF-MITO-Porter, a nano carrier for mitochondrial delivery, has the ability to penetrate the endosomal and mitochondrial membranes via step-wise membrane fusion. We first constructed a DF-MITO-Porter encapsulating DNase I protein as a bioactive cargo. It was expected that mtDNA would be digested, when the DNase I was delivered to the mitochondria. We observed the intracellular trafficking of the carriers, and then measured mitochondrial activity and mtDNA-levels after the delivery of DNase I by the DF-MITO-Porter. The findings confirm that the DF-MITO-Porter effectively delivered the DNase I into the mitochondria, and provides a demonstration of its potential use in therapies that are selective for the mitochondrial genome.
Keywords: Mitochondria; Mitochondrial drug delivery; Mitochondrial gene therapy; MITO-Porter; Membrane fusion; Mitochondrial DNA (mtDNA);

Cholesterol-based anionic long-circulating cisplatin liposomes with reduced renal toxicity by Ying Kuang; Jia Liu; Zhilan Liu; Renxi Zhuo (1596-1606).
Cholesterol anchored derivatives of 5-Cholestene-3-beta-ol 3-hemisuccinate (CHO-HS) and 1-cholesteryl-4-ω-methoxy-polyethylene glycol succinate (CHO-PEG) have been synthesized via esterification and employed at various ratios with di-stearoylphosphatidylcholine (DSPC) in the preparation of anionic long-circulating nanoliposmes for cisplatin (CDDP) delivery. In the present study, CHO-HS and CHO-PEG were characterized by FTIR and 1H NMR. The particle size and zeta potential of liposomes were determined by Dynamic lights scattering (DLS). The obtained liposomes have concentratedly distributed nanosizes around 100 nm and proper zeta potentials between −39.7 mV and −3.18 mV and good physical stability in test period of 28 days. Fine morphology of the liposomal vesicles can be observed via transmission electron microscopy (TEM). The CDDP encapsulating percentage of liposomes was 43–94% and loading efficiency was 7.5–29.3%, depending on the presence or absence of CHO-HS and CHO-PEG. In addition, the in vitro drug release behaviors, in vitro cytotoxicity against HeLa cells and 293T cells and in vivo CDDP distribution of CDDP loaded CHO-HS/CHO-PEG liposomes were evaluated. The results suggest that CHO-HS/CHO-PEG nanoliposomes represent a promising strategy for the CDDP delivery as an effective long-circulating drug carrier system which may reduce the acute renal toxicity.
Keywords: Cisplatin; Liposome; Long-circulating; Renal toxicity; Biodistribution;

Skin permeating nanogel for the cutaneous co-delivery of two anti-inflammatory drugs by Punit P. Shah; Pinaki R. Desai; Apurva R. Patel; Mandip S. Singh (1607-1617).
The aim of this study was to develop an effective drug delivery system for the simultaneous topical delivery of two anti-inflammatory drugs, spantide II (SP) and ketoprofen (KP). To achieve this primary goal, we have developed a skin permeating nanogel system (SPN) containing surface modified polymeric bilayered nanoparticles along with a gelling agent. Poly-(lactide-co-glycolic acid) and chitosan were used to prepare bilayered nanoparticles (NPS) and the surface was modified with oleic acid (NPSO). Hydroxypropyl methyl cellulose (HPMC) and Carbopol with the desired viscosity were utilized to prepare the nanogels. The nanogel system was further investigated for in vitro skin permeation, drug release and stability studies. Allergic contact dermatitis (ACD) and psoriatic plaque like model were used to assess the effectiveness of SPN. Dispersion of NPSO in HPMC (SPN) produced a stable and uniform dispersion. In vitro permeation studies revealed increase in deposition of SP for the SP-SPN or SP+KP-SPN in the epidermis and dermis by 8.5 and 9.5 folds, respectively than SP-gel. Further, the deposition of KP for KP-SPN or SP+KP-SPN in epidermis and dermis was 9.75 and 11.55 folds higher, respectively than KP-gel. Similarly the amount of KP permeated for KP-SPN or SP+KP-SPN was increased by 9.92 folds than KP-gel. The ear thickness in ACD model and the expression of IL-17 and IL-23; PASI score and TEWL values in psoriatic plaque like model were significantly less (p < 0.001) for SPN compared to control gel. Our results suggest that SP+KP-SPN have significant potential for the percutaneous delivery of SP and KP to the deeper skin layers for treatment of various skin inflammatory disorders.
Keywords: Skin permeating nanogel; Surface modified PLGA-chitosan nanoparticles; Skin delivery; Psoriasis; Imiquimod; Allergic contact dermatitis;

Multifunctional, multichannel bridges that deliver neurotrophin encoding lentivirus for regeneration following spinal cord injury by Hannah M. Tuinstra; Misael O. Aviles; Seungjin Shin; Samantha J. Holland; Marina L. Zelivyanskaya; Alan G. Fast; Sarah Y. Ko; Daniel J. Margul; Anne K. Bartels; Ryan M. Boehler; Brian J. Cummings; Aileen J. Anderson; Lonnie D. Shea (1618-1626).
Therapeutic strategies following spinal cord injury must address the multiple barriers that limit regeneration. Multiple channel bridges have been developed that stabilize the injury following implantation and provide physical guidance for regenerating axons. These bridges have now been employed as a vehicle for localized delivery of lentivirus. Implantation of lentivirus loaded multiple channel bridges produced transgene expression that persisted for at least 4 weeks. Expression was maximal at the implant at the earliest time point, and decreased with increasing time of implantation, as well as rostral and caudal to the bridge. Immunohistochemical staining indicated transduction of macrophages, Schwann cells, fibroblasts, and astrocytes within the bridge and adjacent tissue. Subsequently, the delivery of lentivirus encoding the neurotrophic factors NT-3 or BDNF significantly increased the extent of axonal growth into the bridge relative to empty scaffolds. In addition to promoting axon growth, the induced expression of neurotrophic factors led to myelination of axons within the channels of the bridge, where the number of myelinated axons was significantly enhanced relative to control. Combining gene delivery with biomaterials to provide physical guidance and create a permissive environment can provide a platform to enhance axonal growth and promote regeneration.
Keywords: Spinal cord injury; Nerve regeneration; Gene therapy; Neurotrophic factors; Lentivirus;

Targeted delivery strategies are becoming increasingly important. Herein, a novel hyperbranched amphiphilic poly[(amine-ester)-co-(d,l-lactide)]/1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine copolymer (HPAE-co-PLA/DPPE) with RGD peptide (cRGDfK) and transferrin (Tf) on the periphery was synthesized and used to prepare paclitaxel-loaded nanoparticles (NPs) for dual-targeting chemotherapy. These NPs show satisfactory size distribution, high encapsulated efficiency and a pH-dependent release profile. The intrinsic fluorescence of the hyperbranched copolymer renders the detection and tracking of NPs in vitro and in vivo conveniently. In vitro cytotoxicity studies proved that the presence of cRGDfK enhanced the cytotoxic efficiency by 10 folds in ανβ3 integrin over-expressed human umbilical vein endothelial cells, while Tf improved cytotoxicity by 2 folds in Tf receptor over-expressed human cervical carcinoma cells. The drug-loaded NPs can be efficiently transported into the vascular endothelial cells and the target tumor cells. These results indicate that the cRGDfK and Tf decorated HPAE-co-PLA/DPPE could deliver chemotherapies specifically inside the cell via receptor-mediated endocytosis with greater efficacy. Therefore, such a fluorescent nanocarrier prepared from non-cytotoxic and biodegradable polymers is promising for drug delivery in tumor therapy.
Keywords: Hyperbranched copolymer nanoparticle; Tumor-targeting; cRGDfK; Transferrin; Intrinsic fluorescence; Controlled drug release;

Arginine-engrafted biodegradable polymer for the systemic delivery of therapeutic siRNA by Jagadish Beloor; Chang Seon Choi; Hye Yeong Nam; Minsun Park; Sung Hwa Kim; Andrew Jackson; Kuen Yong Lee; Sung Wan Kim; Priti Kumar; Sang-Kyung Lee (1640-1650).
Small interfering RNA (siRNA) represent an interesting class of developmental nucleic acid-based therapeutics. Cationic carriers for deoxyribonucleic acids (DNA) are potential vehicles for siRNA delivery. However, in contrast to supercoiled plasmid DNA, the physical properties of siRNA molecules induces the formation of larger, loosely–packed complexes with most polycationic carriers, and consequently, poor target silencing. Here, we investigate a gene delivery agent, arginine-grafted bioreducible poly (disulfide amine) polymer (ABP) for siRNA delivery as it contains arginine residues with siRNA binding properties. ABP combines the attributes of polycations and poly disulfide-amines namely- excellent cell-penetrability and rapid release after disulphide bond reduction in the intracellular compartment. ABP bound siRNA, assembled into stable 150 nm sized nanoparticles and efficiently released complexed siRNA upon cellular entry. We investigated the utility of ABP in a combinatorial RNAi strategy for solid cancer therapy. Systemic administration of ABP-siRNA resulted in a preferential and enhanced accumulation of carrier-siRNA complexes in the tumor tissue. Two administrations of the formulation with a siRNA cocktail targeting Bcl-2, VEGF and Myc at 0.3 mg total siRNA/kg body weight could effectively regress advanced stage tumors. Our results establish the promise of ABP as a common systemic delivery platform for both siRNA and DNA therapeutics.
Keywords: Cationic carriers; siRNA; Combinatorial RNAi; Arginine-engrafted polymer; Cancer therapeutics;

The effect of environmental pH on polymeric transfection efficiency by Han Chang Kang; Olga Samsonova; Sun-Woong Kang; You Han Bae (1651-1662).
Although polymers, polyplexes, and cells are exposed to various extracellular and intracellular pH environments during polyplex preparation and polymeric transfection, the impact of environmental pH on polymeric transfection has not yet been investigated. This study aims to understand the influence of environmental pH on polymeric transfection by modulating the pH of the transfection medium or the culture medium. Changes in the extracellular pH affected polymeric transfection by way of complex factors such as pH-induced changes in polymer characteristics (e.g., proton buffering capacity and ionization), polyplex characteristics (e.g., size, surface charge, and decomplexation), and cellular characteristics (e.g., cellular uptake, cell cycle phases, and intracellular pH environment). Notably, acidic medium delayed endocytosis, endosomal acidification, cytosolic release, and decomplexation of polyplexes, thereby negatively affecting gene expression. However, acidic medium inhibited mitosis and reduced dilution of gene expression, resulting in increased transfection efficiency. Compared to pH 7.4 medium, acidic transfection medium reduced gene expression 1.6–7.7-fold whereas acidic culture medium enhanced transfection efficiency 2.1–2.6-fold. Polymeric transfection was affected more by the culture medium than by the transfection medium. Understanding the effects of extracellular pH during polymeric transfection may stimulate new strategies for determining effective and safe polymeric gene carriers.
Keywords: Extracellular pH; Polymeric gene delivery; Polyethyleneimine; Poly(l-lysine); Solution pH;

PEGylated silicon nanowire coated silica microparticles for drug delivery across intestinal epithelium by Vuk Uskoković; Phin Peng Lee; Laura A. Walsh; Kathleen E. Fischer; Tejal A. Desai (1663-1672).
Composite particles made by growing nanoscopic silicon wires from the surface of monodispersed, microsized silica beads were tested in this study for their ability to affect the integrity and permeability of an epithelial cell layer. Polyethylene glycol (PEG) is known to sterically stabilize particles and prevent protein binding; as such, it is a routine way to impart in vivo longevity to drug carriers. The effect of the silica beads, both with and without silicon nanowires and PEG, on the disruption of the tight junctions in Caco-2 cells was evaluated by means of: (a) analysis of the localization of zonula occludens-1 (ZO-1), claudin-1 and f-actin; (b) measurements of trans-epithelial electrical resistance (TEER); (c) real-time quantitative RT-PCR analysis of the expression of PKC-α and PKC-z, which regulate the fluidity of cell membranes, and RhoA and Rac1, which are mainly involved in mechanotransduction processes; and (d) drug permeability experiments with fluorescein-sodium. The results have shown that Si-nanowire-coated silica microparticles added to Caco-2 cells in culture lead to alterations in tight junction permeability and the localization of ZO-1 and f-actin, as well as to decreased width of ZO-1 and claudin-1 at the tight junction and increased expression of PKC transcripts. Si-nanowire-coated silica microparticles increased the permeability of Caco-2 cell monolayers to fluorescein-sodium in proportion to their amount. Effects indicative of loosening the Caco-2 cell monolayers and increasing their permeability were less pronounced for PEGylated particles, owing to their greater supposed inertness in comparison with the non-functionalized beads and nanowires. The analyzed Si-nanowire-coated silica microparticles have thus been shown to affect membrane barrier integrity in vitro, suggesting the possibility of using nanostructured microparticles to enhance drug permeability through the intestinal epithelium in vivo.
Keywords: Drug delivery; Caco-2; Epithelium; Nanowire;

The brain targeting mechanism of Angiopep-conjugated poly(ethylene glycol)-co-poly(ɛ-caprolactone) nanoparticles by Hongliang Xin; Xianyi Sha; Xinyi Jiang; Liangcen Chen; Kitki Law; Jijin Gu; Yanzuo Chen; Xiao Wang; Xiaoling Fang (1673-1681).
In order to evaluate the potential and mechanism of Angiopep-conjugated poly(ethylene glycol)-co-poly(ɛ-caprolactone)nanoparticles (ANG-PEG-NP) as brain targeting drug delivery system, Rhodamine B isothiocyanate (RBITC) was used as a fluorescent probe molecule to label ANG-PEG-NP through covalent bonding. The brain transcytosis across the blood–brain barrier (BBB) and brain delivery in mice of RBITC labeled ANG-PEG-NP were investigated in this paper. Results showed that ANG-PEG-NP enhanced significantly the uptake by BCECs compared with that of PEG-NP through caveolae- and clathrin-mediated endocytosis, involving a time-dependent, concentration-dependent and energy-dependent mode. The transport of ANG-PEG-NP across the in vitro BBB model was significantly increased than that of PEG-NP. After injection a dose of 100 mg/kg RBITC labeled ANG-PEG-NP or PEG-NP in mouse caudal vein, the brain coronal section showed a higher accumulation of ANG-PEG-NP in the cortical layer, lateral ventricle, third ventricles and hippocampus than that of PEG-NP. By using an excess of free LRP ligand (Angiopep-2 and/or Aprotinin) as a specific receptor inhibitor, it was evidenced that the uptake by BCECs in vitro, transport across in vitro BBB model and penetration into brain tissue in vivo of RBITC labeled ANG-PEG-NP could be inhibited significantly, which demonstrated the brain targeting mechanism of Angiopep-conjugated poly(ethylene glycol)-co-poly(ɛ-caprolactone)nanoparticles might be a LRP receptor mediated transcytosis process. Understanding these issues is important for the future development of ANG-PEG-NP as a brain targeting drug delivery system for neurodegenerative disorders including glioma and Alzheimer’s disease.
Keywords: Mechanism; Angiopep; Nanoparticles; Blood–brain barrier; Target; Biodistribution;

A magnetically guided anti-cancer drug delivery system using porous FePt capsules by Teruaki Fuchigami; Ryo Kawamura; Yoshitaka Kitamoto; Masaru Nakagawa; Yoshihisa Namiki (1682-1687).
Magnetic carriers with efficient loading, delivery, and release of drugs are required for magnetically guided drug delivery system (DDS) as the potential cancer therapy. The present article describes the fabrication of porous FePt capsules approximately 340 nm in diameter with large pores of 20 nm in an ultrathin shell of 10 nm and demonstrates their application to a magnetically guided DDS in vitro. An aqueous anti-cancer drug is easily introduced in the hollow space of the capsules without external stimuli and released to cancer cells on cue through the magnetic shell composed of an ordered-alloy FePt network structure, which exhibits superparamagnetic features at approximately body temperature. The drug-loaded magnetic capsules coated with a lipid membrane are efficiently guided to the cancer cells within 15 min using a NdFeB magnet (0.2 T), and more than 70% of the cancer cells are destroyed.
Keywords: Drug delivery; Magnetic targeting therapy; Magnetic nanoparticles; Medical applications; Porous capsule;