Biomaterials (v.33, #6)

Single walled carbon nanotubes as drug delivery vehicles: Targeting doxorubicin to tumors by Lingjie Meng; Xiaoke Zhang; Qinghua Lu; Zhaofu Fei; Paul J. Dyson (1689-1698).
Single walled carbon nanotubes (SWNTs) are emerging as promising delivery vehicles for cancer diagnostics and chemotherapies due to their unique properties, including, remarkable cell membrane penetrability, high drug-carrying capacities, pH-dependent therapeutic unloading, prolonged circulating times and intrinsic fluorescent, photothermal, photoacoustic and Raman properties. In this leading opinion paper, we systemically discuss and evaluate the relationship of the biological safety of SWNTs with their physicochemical properties such as their length, purity, agglomeration state, concentration and surface functionalization. Other relevant issues, including the cellular uptake mechanism, biodistribution and metabolism of SWNTs are also reviewed. The design and preparation of SWNT-based drug delivery systems (DDSs) and their pharmacokinetic, cancer targeting and therapeutic properties both in vitro and in vivo are highlighted. Future opportunities and challenges of SWNT-based DDSs are also discussed.
Keywords: Single walled carbon nanotubes (SWNTs); Doxorubicin (DOX); Polysaccharides; Drug delivery;

Predicting biomaterial property-dendritic cell phenotype relationships from the multivariate analysis of responses to polymethacrylates by Peng Meng Kou; Narayanan Pallassana; Rebeca Bowden; Barry Cunningham; Abraham Joy; Joachim Kohn; Julia E. Babensee (1699-1713).
Dendritic cells (DCs) play a critical role in orchestrating the host responses to a wide variety of foreign antigens and are essential in maintaining immune tolerance. Distinct biomaterials have been shown to differentially affect the phenotype of DCs, which suggested that biomaterials may be used to modulate immune response toward the biologic component in combination products. The elucidation of biomaterial property-DC phenotype relationships is expected to inform rational design of immuno-modulatory biomaterials. In this study, DC response to a set of 12 polymethacrylates (pMAs) was assessed in terms of surface marker expression and cytokine profile. Principal component analysis (PCA) determined that surface carbon correlated with enhanced DC maturation, while surface oxygen was associated with an immature DC phenotype. Partial square linear regression, a multivariate modeling approach, was implemented and successfully predicted biomaterial-induced DC phenotype in terms of surface marker expression from biomaterial properties with R prediction 2  = 0.76. Furthermore, prediction of DC phenotype was effective based on only theoretical chemical composition of the bulk polymers with R prediction 2  = 0.80. These results demonstrated that immune cell response can be predicted from biomaterial properties, and computational models will expedite future biomaterial design and selection.
Keywords: Dendritic cells; Polymethacrylate; Combinatorial library; Material properties; Principal component analysis; Partial linear squares regression;

Controllable synthesis of monodispersed silver nanoparticles as standards for quantitative assessment of their cytotoxicity by Lun Li; Jie Sun; Xiaoran Li; Yan Zhang; Zhaoxu Wang; Chunren Wang; Jianwu Dai; Qiangbin Wang (1714-1721).
Silver nanoparticles (Ag NPs) are appealing due to their excellent antibacterial/antivirus properties. At the meantime, the wide applications of Ag NPs as antibacterial/antivirus agents arise the concern of Ag NPs’ toxicity. However, quantitative understanding of the cytotoxicity of Ag NPs is minimum since that the Ag NPs in current studies have wide size distributions, in which the size effect of Ag NPs on cytotoxicity was unable to be accurately evaluated. In this work, unprecedentedly monodispersed Ag NPs with sizes of 25, 35, 45, 60 and 70 nm were obtained, respectively, by using an optimized polyol method with poly(vinyl pyrrolidone) (PVP) as surfactant. It was found that the reaction temperature, reaction time, concentration of the surfactant and reactants are playing important roles in determining the size and size distribution of Ag NPs. With the monodispersed Ag NPs as standard samples, the size- and dose- dependent cytotoxicity of Ag NPs against Human lung fibroblast (HLF) cells was accurately accomplished in terms of cell viability, apoptosis and necrosis, reactive oxygen species, etc. We expect that the monodispersed Ag NPs will act as the standard samples for quantitatively characterizing the toxicity of Ag NPs in vitro and in vivo.
Keywords: Silver nanoparticle; Particle size; Monodisperse; Cytotoxicity; Standard sample;

Mimicking the human smell sensing mechanism with an artificial nose platform by Sang Hun Lee; Oh Seok Kwon; Hyun Seok Song; Seon Joo Park; Jong Hwan Sung; Jyongsik Jang; Tai Hyun Park (1722-1729).
Sensing smell is a highly complex biological process, and characterizing and mimicking the interaction between the olfactory receptor (OR) protein and its ligands is extremely challenging. Herein, we report a highly sensitive and selective human nose-like nanobioelectronic nose (nbe-nose), which responds to gaseous odorants sensitively and selectively, has a signal specificity pattern similar to that in the cellular signal transduction pathway, and maintains an antagonistic behavior similar to the human nose. The human olfaction mechanism was mimicked by using carboxylated polypyrrole nanotubes (CPNTs) functionalized with human OR protein. The nbe-nose was able to detect gaseous odorants at a concentration as low as 0.02 parts-per-trillion (ppt), which was comparable to a highly trained, human expert’s nose. The nbe-nose can be used scientifically for smell mechanism studies. It can be also applied to various fields that rely on smell monitoring for industrial and public purposes.
Keywords: Nanobioelectronic nose; Human olfactory receptor; Conducting polymer nanotube; Antagonism;

Control of cell nucleus shapes via micropillar patterns by Zhen Pan; Ce Yan; Rong Peng; Yingchun Zhao; Yao He; Jiandong Ding (1730-1735).
We herein report a material technique to control the shapes of cell nuclei by the design of the microtopography of substrates to which the cells adhere. Poly(d,l-lactide-co-glycolide) (PLGA) micropillars or micropits of a series of height or depth were fabricated, and some surprising self deformation of the nuclei of bone marrow stromal cells (BMSCs) was found in the case of micropillars with a sufficient height. Despite severe nucleus deformation, BMSCs kept the ability of proliferation and differentiation. We further demonstrated that the shapes of cell nuclei could be regulated by the appropriate micropillar patterns. Besides circular and elliptoid shapes, some unusual nucleus shapes of BMSCs have been achieved, such as square, cross, dumbbell, and asymmetric sphere-protrusion.
Keywords: Polymeric materials; Surface Micropatterning; Cell nucleus; Stem cells;

3D co-culture of hematopoietic stem and progenitor cells and mesenchymal stem cells in collagen scaffolds as a model of the hematopoietic niche by Isabelle Leisten; Rafael Kramann; Mónica S. Ventura Ferreira; Manfred Bovi; Sabine Neuss; Patrick Ziegler; Wolfgang Wagner; Ruth Knüchel; Rebekka K. Schneider (1736-1747).
Here, we propose a collagen-based three-dimensional (3D) environment for hematopoietic stem and progenitor cells (HPC) with mesenchymal stem cells (MSC) derived either from bone marrow (BM) or umbilical cord (UC), to recapitulate the main components of the BM niche. Mechanisms described for HPC homeostasis were systematically analyzed in comparison to the conventional liquid HPC culture. The 3D-cultivation allows dissecting two sub-populations of HPC: (I) HPC in suspension above the collagen gel and (II) migratory HPC in the collagen fibres of the collagen gel. The different sites represent distinct microenvironments with significant impact on HPC fate. HPC in niche I (suspension) are proliferative and a dynamic culture containing HPC (CD34+/CD38-), maturing myeloid cells (CD38+, CD13+, CAE+) and natural killer (NK) cells (CD56+). In contrast, HPC in niche II showed clonal growth with significant high levels of the primitive CD34+/CD38- phenotype with starting myeloid (CD13+, CAE+) differentiation, resembling the endosteal part of the BM niche. In contrast, UC-MSC are not adequate for HSC expansion as they significantly enhance HPC proliferation and lineage commitment. In conclusion, the 3D-culture system using collagen and BM-MSC enables HPC expansion and provides a potential platform to dissect regulatory mechanisms in hematopoiesis.
Keywords: Hematopoietic niche; Collagen gel; Hematopoietic progenitor cells; Mesenchymal stem cells; Endosteal part bone marrow niche;

Adipose-derived stem cells (ASCs) have valuable applications in regenerative medicine, but maintaining the stemness of ASCs during in vitro culture is still a challenging issue. In this study, human ASCs spontaneously formed three-dimensional spheroids on chitosan films. Most ASCs within the spheroid were viable, and the cells produced more extracellular molecules, like laminin and fibronectin. Comparing to monolayer culture, ASC spheroids also exhibited enhanced cell survival in serum deprivation condition. Although cell proliferation was inhibited in spheroids, ASCs readily migrated out and proliferated upon transferring spheroids to another adherent growth surface. Moreover, spheroid-derived ASCs exhibited higher expansion efficiency and colony-forming activity. Importantly, we demonstrated that spheroid formation of human ASCs on chitosan films induced significant upregulation of pluripotency marker genes (Sox-2, Oct-4 and Nanog). By culturing the ASC spheroids in proper induction media, we found that ASC differentiation capabilities were significantly enhanced after spheroid formation, including increased transdifferentiation efficiency into neuron and hepatocyte-like cells. In a nude mice model, we further showed a significantly higher cellular retention ratio of ASC spheroids after intramuscular injection of spheroids and dissociated ASCs. These results suggested that ASCs cultured as spheroids on chitosan films can increase their therapeutic potentials.
Keywords: Spheroid; Adipose-derived stem cell; Chitosan; Stemness; Differentiation;

Inducing functional radial glia-like progenitors from cortical astrocyte cultures using micropatterned PMMA by Marta Mattotti; Zaida Alvarez; Juan A. Ortega; Josep A. Planell; Elisabeth Engel; Soledad Alcántara (1759-1770).
Radial glia cells (RGC) are multipotent progenitors that generate neurons and glia during CNS development, and which also served as substrate for neuronal migration. After a lesion, reactive glia are the main contributor to CNS regenerative blockage, although some reactive astrocytes are also able to de-differentiate in situ into radial glia-like cells (RGLC), providing beneficial effects in terms of CNS recovery. Thus, the identification of substrate properties that potentiate the ability of astrocytes to transform into RGLC in response to a lesion might help in the development of implantable devices that improve endogenous CNS regeneration. Here we demonstrate that functional RGLC can be induced from in vitro matured astrocytes by using a precisely-sized micropatterned PMMA grooved scaffold, without added soluble or substrate adsorbed biochemical factors. RGLC were extremely organized and aligned on 2 μm line patterned PMMA and, like their embryonic counterparts, express nestin, the neuron-glial progenitor marker Pax6, and also proliferate, generate different intermediate progenitors and support and direct axonal growth and neuronal migration. Our results suggest that the introduction of line patterns in the size range of the RGC processes in implantable scaffolds might mimic the topography of the embryonic neural stem cell niche, driving endogenous astrocytes into an RGLC phenotype, and thus favoring the regenerative response in situ.
Keywords: Polymethylmethacrylate; Micropatterning; Surface topography; Astrocyte; Nerve guide; Co-culture;

Consequences of ineffective decellularization of biologic scaffolds on the host response by Timothy J. Keane; Ricardo Londono; Neill J. Turner; Stephen F. Badylak (1771-1781).
Biologic scaffold materials composed of extracellular matrix (ECM) are routinely used for a variety of clinical applications. Despite known variations in tissue remodeling outcomes, quantitative criteria by which decellularization can be assessed were only recently described and as a result, the amount of retained cellular material varies widely among commercial products. The objective of this study was to evaluate the consequences of ineffective decellularization on the host response. Three different methods of decellularization were used to decellularize porcine small intestinal ECM (SIS-ECM). The amount of cell remnants was quantified by the amount and fragmentation of DNA within the scaffold materials. The M1/M2 phenotypic polarization profile of macrophages, activated in response to these ECM scaffolds, was assessed in vitro and in vivo using a rodent model of body wall repair. The results show that, in vitro, more aggressive decellularization is associated with a shift in macrophage phenotype predominance from M1 to M2. While this shift was not quantitatively apparent in vivo, notable differences were found in the distribution of M1 vs. M2 macrophages within the various scaffolds. A clear association between macrophage phenotype and remodeling outcome exists and effective decellularization remains an important component in the processing of ECM-based scaffolds.
Keywords: Extracellular matrix; Macrophage; Decellularization; Immune response; Scaffold;

Cardiac tissue engineering using tissue printing technology and human cardiac progenitor cells by Roberto Gaetani; Peter A. Doevendans; Corina H.G. Metz; Jacqueline Alblas; Elisa Messina; Alessandro Giacomello; Joost P.G. Sluijter (1782-1790).
Tissue engineering is emerging as a potential therapeutic approach to overcome limitations of cell therapy, like cell retention and survival, as well as to mechanically support the ventricular wall and thereby prevent dilation. Tissue printing technology (TP) offers the possibility to deliver, in a defined and organized manner, scaffolding materials and living cells. The aim of our study was to evaluate the combination of TP, human cardiac-derived cardiomyocyte progenitor cells (hCMPCs) and biomaterials to obtain a construct with cardiogenic potential for in vitro use or in vivo application. With this approach, we were able to generate an in vitro tissue with homogenous distribution of cells in the scaffold. Cell viability was determined after printing and showed that 92% and 89% of cells were viable at 1 and 7 days of culturing, respectively. Moreover, we demonstrated that printed hCMPCs retained their commitment for the cardiac lineage. In particular, we showed that 3D culture enhanced gene expression of the early cardiac transcription factors Nkx2.5, Gata-4 and Mef-2c as well as the sarcomeric protein TroponinT. Printed cells were also able to migrate from the alginate matrix and colonize a matrigel layer, thereby forming tubular-like structures. This indicated that printing can be used for defined cell delivery, while retaining functional properties.
Keywords: Cardiac progenitor cells; Tissue printing; Alginate; Cardiac tissue engineering;

Utilization of nanofibrous matrices for skin wound repair holds great promise due to their morphological and dimensional similarity to native extracellular matrix (ECM). It becomes highly desired to understand how various nanofibrous matrices regulate skin cell behaviors and intracellular signaling pathways, important to tuning the functionality of tissue-engineered skin grafts and affecting the wound healing process. In this study, the phenotypic expressions of normal human dermal fibroblasts (NHDFs) on collagen-containing nanofibrous matrices with either isotropic (i.e., fibers collected randomly with no alignment) or anisotropic (i.e., fibers collected with alignment) fiber organizations were studied by immunostaining, migration assay and molecular analyses. Results showed that both nanofibrous matrices supported the attachment and growth of NHDFs similarly, while showing different cell morphology with distinct variation in focal adhesion formation and distribution. Anisotropic nanofibers significantly triggered the integrin β1 signaling pathway in NHDFs as evidenced by an increase of active integrin β1 (130 kD mature form) and phosphorylation of focal adhesion kinase (FAK) at Tyr-397. Anisotropic matrices also promoted the migration of NHDFs along the fibers, while neutralization of the integrin β1 activity abolished this promotion. Moreover, the fibroblast-to-myofibroblast differentiation was greatly enhanced for the NHDFs cultured on anisotropic nanofibrous matrices over a period of 48 h. Inhibition of cellular integrin β1 activity by neutralizing antibody eliminated this enhancement. These findings suggest the important role of integrin β1 signaling pathway in regulating the nanofiber-induced fibroblast phenotypic alteration and providing insightful understanding of the possible application of collagen-containing nanofibrous matrices for skin regeneration.
Keywords: Anisotropic nanofibers; Normal human dermal fibroblasts; Fibroblast-to-myofibroblast differentiation; Integrin β1 signaling;

The effect of collagen-binding vascular endothelial growth factor on the remodeling of scarred rat uterus following full-thickness injury by Nacheng Lin; Xin’an Li; Tianran Song; Jingmei Wang; Kui Meng; Jun Yang; Xianglin Hou; Jianwu Dai; Yali Hu (1801-1807).
Serious injuries of uterine which lead to scar formation will finally result in infertility or pregnancy complications. There are few effective methods to treat such damages because of the shortage of native tissues. Vascular endothelial growth factor (VEGF) is important for the formation of new vessels and re-epithelialization of endometrium. Here we produced a collagen-binding VEGF by fusing a collagen-binding domain to the N-terminal of native VEGF. After injection into a rat scarred uterus model (partial of rat uterine horn was excised and left for scar formation) the collagen targeting VEGF promoted remodeling of the scarred uterus including the regeneration of endometrium, muscular cells, and vascularization and improved pregnancy outcomes. Thus, collagen-binding VEGF may be a pragmatic solution for the treatment of severe uterine damages.
Keywords: Vascular endothelial growth factor; Collagen-binding domain; Uterine horn scar regeneration; Tissue engineering;

Mitochondrial targeting topotecan-loaded liposomes for treating drug-resistant breast cancer and inhibiting invasive metastases of melanoma by Yang Yu; Zhao-Hui Wang; Liang Zhang; Hong-Juan Yao; Yan Zhang; Ruo-Jing Li; Rui-Jun Ju; Xiao-Xing Wang; Jia Zhou; Nan Li; Wan-Liang Lu (1808-1820).
Multidrug resistance and cancer metastases are two obstacles to a successful chemotherapy and metastases are closely associated with drug resistance. Mitochondrial targeting topotecan-loaded liposomes have been developed to overcome this resistance and resistance-related metastases. Investigations were performed on breast cancer MCF-7 and resistant MCF-7/adr cells, MCF-7 and resistant MCF-7/adr tumor spheroids, resistant MCF-7/adr cell xenografts in nude mice, and a naturally resistant B16 melanoma metastatic model in nude mice. The mitochondrial targeting topotecan-loaded liposomes were approximately 64 nm in size, and exhibited the strongest inhibitory effects on MCF-7 cells and resistant MCF-7/adr cells. Mitochondrial targeting effects were demonstrated by co-localization in mitochondria, enhanced drug content in mitochondria, dissipated mitochondrial membrane potential, opening of mitochondrial permeability transition pores, release of cytochrome C, and activation of caspase 9 and 3. The targeting liposomes had a stronger inhibitory effect on the resistant tumor spheroids in vitro, enhanced accumulation in resistant MCF-7/adr cell xenografts in mice, as well as being very effective on resistant MCF-7/adr cell xenografts in mice, and having a marked anti-metastastic effect on the naturally resistant B16 melanoma metastatic model in mice. In conclusion, mitochondrial targeting topotecan-loaded liposomes could be a promising strategy for treating resistant cancers and resistance-related metastases.
Keywords: Mitochondrial targeting topotecan-loaded liposomes; Multidrug resistance; Metastases; Breast cancer; Melanoma;

Plasmonic nanobubble-enhanced endosomal escape processes for selective and guided intracellular delivery of chemotherapy to drug-resistant cancer cells by Ekaterina Y. Lukianova-Hleb; Andrey Belyanin; Shruti Kashinath; Xiangwei Wu; Dmitri O. Lapotko (1821-1826).
Cancer chemotherapies suffer from multi drug resistance, high non-specific toxicity and heterogeneity of tumors. We report a method of plasmonic nanobubble-enhanced endosomal escape (PNBEE) for the selective, fast and guided intracellular delivery of drugs through a self-assembly by cancer cells of separately targeted gold nanoparticles and encapsulated drug (Doxil). The co-localized with Doxil plasmonic nanobubbles optically generated in cancer cells released the drug into the cytoplasm thus increasing the therapeutic efficacy against these drug-resistant cells by 31-fold, reducing drug dose by 20-fold, the treatment time by 3-fold and the non-specific toxicity by 10-fold compared to standard treatment. Thus the PNBEE mechanism provided selective, safe and efficient intracellular drug delivery in heterogeneous environment opening new opportunities for drug therapies.
Keywords: Liposome; Gold; Nanoparticle; Laser; Drug release; Drug delivery;

Stimulated release of photosensitizers from graft and diblock micelles for photodynamic therapy by Hsieh-Chih Tsai; Cheng-Hung Tsai; Shuian-Yin Lin; Chang-Rong Jhang; Yung-Sheng Chiang; Ging-Ho Hsiue (1827-1837).
To understand the effect of photosensitizer (PS) release from graft copolymer based micelles in photodynamic therapy (PDT), the two pH-sensitive and non-pH-sensitive graft copolymers, (poly(N-vinyly caprolactam)-g-poly(d,l-lactide) and poly(N-vinyly caprolactam-co-N-vinyl imidazole)-g-poly(d,l-lactide)), were synthesized and utilized for the encapsulation of protoporphyrin IX (PPIX) for in vitro and in vivo PDT studies. Photochemical internalization (PCI) was utilized to study the localization of pH- and non-pH-sensitive micelles uptake in the lysosome. After non-toxic light treatment, PPIX was found in the nucleus with pH-sensitive micelles, while PPIX was still localized in the lysosomal organism with the non-pH-sensitive micelles, as observed by confocal microscopy. Because the formation of singlet oxygen was observed for the block and graft micelles, dramatic differences in the cell viability could be ascribed to the damage occurring at the region where the PPIX was located. An in vivo study revealed that PPIX-loaded graft and diblock micelles presented prolonged blood circulation and enhanced tumor targeting ability. The PPIX released from g-CIM micelles on tumor site was further proved by ex vivo confocal image. In addition, non-pH-sensitive micelle-treated mice showed a better repression of tumor growth than PPIX-treated mice, which was likely due to the larger amount of PS localized in the tumor region still exhibiting therapeutic effects. Finally, effective PDT-induced inhibition of tumor growth was found in pH-sensitive micelle-treated mice. This work provides insight into PS-loaded graft and diblock micelles for the PDT of tumors.
Keywords: Graft copolymer; Photodynamic therapy; pH-sensitive; Polymeric micelles;

Enhancing the therapeutic efficacy of adenovirus in combination with biomaterials by Jaesung Kim; Pyung-Hwan Kim; Sung Wan Kim; Chae-Ok Yun (1838-1850).
With the reason that systemically administered adenovirus (Ad) is rapidly extinguished by innate/adaptive immune responses and accumulation in liver, in vivo application of the Ad vector is strictly restricted. For achieving to develop successful Ad vector systems for cancer therapy, the chemical or physical modification of Ad vectors with polymers has been generally used as a promising strategy to overcome the obstacles. With polyethylene glycol (PEG) first in order, a variety of polymers have been developed to shield the surface of therapeutic Ad vectors and well accomplished to extend circulation time in blood and reduce liver toxicity. However, although polymer-coated Ads can successfully evacuate from a series of guarding systems in vivo and locate within tumors by enhanced permeability and retention (EPR) effect, the possibility to entering into the target cell is few and far between. To endow targeting moiety to polymer-coated Ad vectors, a diversity of ligands such as tumor-homing peptides, growth factors or antibodies, have been introduced with avoiding unwanted transduction and enhancing therapeutic efficacy. Here, we will describe and classify the characteristics of the published polymers with respect to Ad vectors. Furthermore, we will also compare the properties of variable targeting ligands, which are being utilized for addressing polymer-coated Ad vectors actively.
Keywords: Adenovirus; Biomaterials; Passive targeting; Active targeting; Cancer gene therapy;

Cationic glycopolymers for the delivery of pDNA to human dermal fibroblasts and rat mesenchymal stem cells by Karina Kizjakina; Joshua M. Bryson; Giovanna Grandinetti; Theresa M. Reineke (1851-1862).
Progenitor and pluripotent cell types offer promise as regenerative therapies but transfecting these sensitive cells has proven difficult. Herein, a series of linear trehalose-oligoethyleneamine “click” copolymers were synthesized and examined for their ability to deliver plasmid DNA (pDNA) to two progenitor cell types, human dermal fibroblasts (HDFn) and rat mesenchymal stem cells (RMSC). Seven polymer vehicle analogs were synthesized in which three parameters were systematically varied: the number of secondary amines (4–6) within the polymer repeat unit (Tr433, Tr530, and Tr632 ), the end group functionalities [PEG (Tr4128PEG-a, Tr4118PEG-b), triphenyl (Tr4107-c), or azido (Tr499-d)], and the molecular weight (degree of polymerization of about 30 or about 100) and the biological efficacy of these vehicles was compared to three controls: Lipofectamine 2000, JetPEI, and Glycofect. The trehalose polymers were all able to bind and compact pDNA polyplexes, and promote pDNA uptake and gene expression [luciferase and enhanced green fluorescent protein (EGFP)] with these primary cell types and the results varied significantly depending on the polymer structure. Interestingly, in both cell types, Tr433 and Tr530 yielded the highest luciferase gene expression. However, when comparing the number of cells transfected with a reporter plasmid encoding enhanced green fluorescent protein, Tr433 and Tr4107-c yielded the highest number of HDFn cells positive for EGFP. Interestingly, with RMSCs, all of the higher molecular weight analogs (Tr4128PEG-a, Tr4118PEG-b, Tr4107-c, Tr499-d) yielded high percentages of cells positive for EGFP (30–40%).
Keywords: Biocompatibility; DNA; Drug Delivery; Nanoparticle; Polyethyleneoxide; Polymerization;

Improved treatments for prostate cancer are in great need to overcome lethal recurrence and metastasis. Targeting the tumorigenic cancer stem cells (CSCs) with self-renewal and differentiation capacity appears to be a promising strategy. Blockade of the hedgehog (Hh) signaling pathway, an important pathway involved in stem cell self-renewal, by cyclopamine leads to long-term prostate cancer regression without recurrence, strongly suggesting the connection between Hh pathway and prostate CSCs. Here we designed an HPMA (N-(2-hydroxypropyl)methacrylamide)-based cyclopamine delivery system as a CSC-selective macromolecular therapeutics with improved drug solubility and decreased systemic toxicity. To this end, HPMA and N-methacryloylglycylphenylalanylleucylglycyl thiazolidine-2-thione were copolymerized using the RAFT (reversible addition-fragmentation chain transfer) process, followed by polymer-analogous attachment of cyclopamine. The selectivity of the conjugate toward CSCs was evaluated on RC-92a/hTERT cells, the human prostate cancer epithelial cells with human telomerase reverse transcriptase transduction. The use of RC-92a/hTERT cells as an in vitro CSC model was validated by stem cell marker expression and prostasphere culture. The bioactivity of cyclopamine was retained after conjugation to the polymer. Furthermore, HPMA polymer-conjugated cyclopamine showed anti-CSC efficacy on RC-92a/hTERT cells as evaluated by decreased stem cell marker expression and CSC viability.
Keywords: Prostate cancer; Cancer stem cells; HPMA; Hedgehog; Cyclopamine;

Biocleavable comb-shaped gene carriers from dextran backbones with bioreducible ATRP initiation sites by Zeng-Hui Wang; Yun Zhu; Ming-Ying Chai; Wan-Tai Yang; Fu-Jian Xu (1873-1883).
It is of crucial importance to design reduction-sensitive polysaccharide-based copolymers for intracellular triggered gene and drug delivery. In this work, a simple two-step method involving the reaction of hydroxyl groups of dextran with cystamine was first developed to introduce reduction-sensitive disulfide linked initiation sites of atom transfer radical polymerization (ATRP) onto dextran. Well-defined biocleavable comb-shaped vectors consisting of nonionic dextran backbones and disulfide-linked cationic P(DMAEMA) side chains were subsequently prepared via ATRP for highly efficient gene delivery. The P(DMAEMA) side chains can be readily cleavable from the dextran backbones under reducible conditions. Moreover, the bioreducible P(DMAEMA) side chains can be functionalized by poly(poly(ethylene glycol)ethyl ether methacrylate) (P(PEGEEMA)) end blocks to reduce the cytotoxicity and further enhance the gene transfection efficiency. This present study demonstrated that properly grafting short bioreducible polycation side chains from a nonionic polysaccharide backbone with biocleavable ATRP initiation sites is an effective means to produce a class of polysaccharide-based gene delivery vectors.
Keywords: Gene delivery; Bioreducible vector; P(DMAEMA); Dextran; ATRP;

Electrostatic charge conversion processes in engineered tumor-identifying polypeptides for targeted chemotherapy by Nam Muk Oh; Dong Sup Kwag; Kyung Taek Oh; Yu Seok Youn; Eun Seong Lee (1884-1893).
One of the current challenges in cancer chemotherapy is the ultra-sensitive identification of in vivo tumors. Herein, we report a new class of tumor-identifying polypeptides that can home in on in vivo tumors via an electrostatic charge conversion process occurring in the acidic milieu of a verity of tumors, which can be distinguished from receptor-interacting conventional tumor-homing peptides. We exploit the chemical coupling between polypeptides and therapeutic objects (drugs or particles) to carry out an antitumor study in nude mice, and find a significant increase in the efficiency of polypeptide-tagged objects in tumor uptake and inhibition, which is more significant than any known tumor-homing peptide system thus far developed.
Keywords: Tumor-identifying polypeptide; Cancer chemotherapy; Tumor extracellular pH; Photodynamic therapy;

Suppression of tumor growth in H-ras12V liver cancer mice by delivery of programmed cell death protein 4 using galactosylated poly(ethylene glycol)-chitosan-graft-spermine by Ji-Hye Kim; Arash Minai-Tehrani; You-Kyoung Kim; Ji-Young Shin; Seong-Ho Hong; Hye-Joon Kim; Hee-Do Lee; Seung-Hee Chang; Kyeong-Nam Yu; Yong-Bin Bang; Chong-Su Cho; Tae-Jong Yoon; Dae-Yeul Yu; Hu-Lin Jiang; Myung-Haing Cho (1894-1902).
Non-viral gene delivery systems based on polyethyleneimine (PEI) are efficient due to their proton-sponge effect within endosomes, but they have poor physical characteristics such as slow dissociation, cytotoxicity, and non targeted gene delivery. To overcome many of the problems associated with PEI, we synthesized a galactosylated poly(ethylene glycol)-chitosan-graft-spermine (GPCS) copolymer with low cytotoxicity and optimal gene delivery to hepatocytes using an amide bond between galactosylated poly(ethylene glycol) and chitosan-graft-spermine. The GPCS copolymer formed complexes with plasmid DNA, and the GPCS/DNA complexes had well-formed spherical shapes. The GPCS/DNA complexes were nanoscale size with homogenous size distribution and a positive zeta potential by dynamic light scattering (DLS). The GPCS copolymer had lower cytotoxicity than that of PEI 25K in HepG2, HeLa, and A549 cell lines at various concentrations and showed good hepatocyte-targeting ability. Furthermore, GPCS/DNA complexes showed higher levels of GFP expression in the liver in model mice after intravenous injection than naked DNA and metoxy-poly(ethylene glycol)-chitosan-graft-spermine as controls without remarkable fibrosis, inflammation, lipidosis, or necrosis. In a tumor suppression study, an intravenous injection of the GPCS/Pdcd4 complexes significantly suppressed tumor growth, activated apoptosis, and suppressed proliferation and angiogenesis in liver tumor-bearing H-ras12V mice. Our results indicate that the GPCS copolymer has potential as a hepatocyte-targeting gene carrier.
Keywords: Gene therapy; Non-viral gene delivery; Hepatocyte targeting; Galactosylated poly(ethylene glycol)-chitosan-graft-spermine;

Molecular interactions of mussel protective coating protein, mcfp-1, from Mytilus californianus by Qingye Lu; Dong Soo Hwang; Yang Liu; Hongbo Zeng (1903-1911).
Protective coating of the byssus of mussels (Mytilus sp.) has been suggested as a new paradigm of medical coating due to its high extensibility and hardness co-existence without their mutual detriment. The only known biomacromolecule in the extensible and tough coating on the byssus is mussel foot protein-1 (mfp-1), which is made up with positively charged residues (∼20 mol%) and lack of negatively charged residues. Here, adhesion and molecular interaction mechanisms of Mytilus californianus foot protein-1 (mcfp-1) from California blue mussel were investigated using a surface forces apparatus (SFA) in buffer solutions of different ionic concentrations (0.2–0.7 M) and pHs (3.0–5.5). Strong and reversible cohesion between opposed positively charged mcfp-1 films was measured in 0.1 M sodium acetate buffer with 0.1 M KNO3. Cohesion of mcfp-1 was gradually reduced with increasing the ionic strength, but was not changed with pH variations. Oxidation of 3,4-dihydroxyphenylalanine (DOPA) residues of mcfp-1, a key residue for adhesive and coating proteins of mussel, didn’t change the cohesion strength of mcfp-1 films, but the addition of chemicals with aromatic groups (i.e., aspirin and 4-methylcatechol) increased the cohesion. These results suggest that the cohesion of mcfp-1 films is mainly mediated by cation-π interactions between the positively charged residues and benzene rings of DOPA and other aromatic amino acids (∼20 mol% of total amino acids of mcfp-1), and π-π interactions between the phenyl groups in mcfp-1. The adhesion mechanism obtained for the mcfp-1 proteins provides important insight into the design and development of functional biomaterials and coatings mimicking the extensible and robust mussel cuticle coating.
Keywords: Mcfp-1; Cation–π interaction; Bioadhesion; Protein interactions; Surface forces apparatus;

Photodynamic inactivation of viruses using upconversion nanoparticles by Meng Earn Lim; Yen-ling Lee; Yong Zhang; Justin Jang Hann Chu (1912-1920).
Photodynamic therapy (PDT) is a promising treatment modality that utilizes light of an appropriate wavelength to excite photosensitive materials called photosensitizers, which upon excitation, generate reactive oxygen species (ROS) that are cytocidal and virucidal. However, problems such as hydrophobicity of photosensitizers and limited tissue penetration ability of the current light sources impeded its promotion as a mainstay in medical technology. Here, by using near-infrared (NIR)-to-visible upconversion nanoparticles (UCNs), we demonstrate UCN-based photodynamic inactivation as a potential antiviral strategy. These UCNs are nanotransducers which not only act as carriers of photosensitizers but also active participants in PDT by transducing NIR radiation to visible emissions appropriate for excitation of the attached photosensitizers. The UCNs effectively reduced the infectious virus titers in vitro with no clear pathogenicity in murine model and increased target specificity to virus-infected cells. Hence, this is a promising antiviral approach with feasible applications in the treatments of virus-associated infections, lesions and cancers.
Keywords: Nanoparticle; Upconversion; Viral pathogen; Photodynamic therapy;

The use of cholesterol-containing biodegradable block copolymers to exploit hydrophobic interactions for the delivery of anticancer drugs by Ashlynn L.Z. Lee; Shrinivas Venkataraman; Syamilah B.M. Sirat; Shujun Gao; James L. Hedrick; Yi Yan Yang (1921-1928).
A series of biodegradable amphiphilic block copolymers with controlled composition and relatively low polydispersity index were synthesized from monomethoxy polyethylene glycol (mPEG-OH, 5 kDa) via organocatalytic ring opening polymerization of aliphatic cyclic carbonate monomers - trimethylene carbonate (TMC) or cholesteryl 2-(5-methyl-2-oxo-1,3-dioxane-5-carboxyloyloxy)ethyl carbamate (MTC-Chol) or a copolymer of both the monomers (TMC and MTC-Chol): mPEG113-b-PTMC67, mPEG113-b-P(MTC-Chol11) and mPEG113-b-P(MTC-Cholx-co-TMCy)x+y. These well-defined polymers were employed to study the role of molecular weight and composition of the hydrophobic block of the polymers in loading paclitaxel (PTX), an extremely hydrophobic anticancer drug with rigid structure and strong tendency of self-association to form long fibers. The PTX-loaded micelles were fabricated by simple self-assembly without sonication or homogenization procedures. The results demonstrated that the presence of both MTC-Chol and TMC in the hydrophobic block significantly increased PTX loading levels, and the micelles formed from the polymer with the optimized composition (i.e. mPEG113-b-P(MTC-Chol11-co-TMC30)) were in nanosize (36 nm) with narrow size distribution (PDI: 0.07) and high PTX loading capacity (15 wt.%). In vitro treatment of human liver hepatocellular carcinoma HepG2 cells with blank micelles showed that these polymeric carriers were non-cytotoxic with cell viability greater than 90% at ∼2400 mg/L. Importantly, PTX-loaded micelles were able to kill cancer cells much more effectively compared to free PTX. In addition, these nanocarriers also possessed exceptional kinetic stability. The results from non-invasive near-infrared fluorescence (NIRF) imaging studies showed that these micelles allowed effective passive targeting, and were preferably accumulated in tumor tissue with limited distribution to healthy organs.
Keywords: Cholesterol; Polycarbonate; Amphiphilic block copolymer; Micelles; Paclitaxel; Tumor targeting;

Predicting protein instability in sustained protein delivery systems using spectral-phase interference by Nina Seidel; Johannes Sitterberg; Wolfgang Vornholt; Udo Bakowsky; Michael Keusgen; Thomas Kissel (1929-1938).
Biodegradable and non-biodegradable polymers represent promising materials for sustained protein delivery systems. However, structural protein instabilities due to interactions with the polymer surface are often observed. Aim of the present study was to analyze and predict these instabilities by determination of adsorption pattern and extent via biomolecular interaction analysis. A new optical method based on spectral-phase interference successfully demonstrated its suitability for this new application scope. It was characterized in terms of sensitivity, reproducibility and dynamic range using bovine serum albumin (BSA) as model compound. For protein–polymer interaction studies, materials with different wettabilities and zeta potential were selected and successfully applied on the sensor chip: Glass, poly(styrene), poly(lactic acid), poly(lactic-co-glycolic acid), and poly(ethylene carbonate). Concentration dependent adsorption curves revealed two principal adsorption patterns based on the connection between BSA spreading and supply rate. This connection was stronger influenced by polymer hydrophobicity than surface charge. Association, dissociation and binding rate constants in the range from 0.15 to 34.19 × 10−6 M were obtained. Atomic force microscopy images of the films before and after adsorption confirmed the previous elaborated model. Poly(ethylene carbonate) emerged as highly promising biomaterial for protein delivery due to its favorable adsorption behavior based on low polymer–protein interactions.
Keywords: Biomolecular interaction analysis (BIA); Biosensor; Bovine serum albumin (BSA); Poly(ethylene carbonate) (PEC); Protein delivery; Protein adsorption;

Aptamer-conjugated and drug-loaded acoustic droplets for ultrasound theranosis by Chung-Hsin Wang; Shih-Tsung Kang; Ya-Hsuan Lee; Yun-Ling Luo; Yu-Fen Huang; Chih-Kuang Yeh (1939-1947).
Tumor therapy requires multi-functional treatment strategies with specific targeting of therapeutics to reduce general toxicity and increase efficacy. In this study we fabricated and functionally tested aptamer-conjugated and doxorubicin (DOX)-loaded acoustic droplets comprising cores of liquid perfluoropentane compound and lipid-based shell materials. Conjugation of sgc8c aptamers provided the ability to specifically target CCRF-CEM cells for both imaging and therapy. High-intensity focused ultrasound (HIFU) was introduced to trigger targeted acoustic droplet vaporization (ADV) which resulted in both mechanical cancer cell destruction by inertial cavitation and chemical treatment through localized drug release. HIFU insonation showed a 56.8% decrease in cell viability with aptamer-conjugated droplets, representing a 4.5-fold increase in comparison to non-conjugated droplets. In addition, the fully-vaporized droplets resulted in the highest DOX uptake by cancer cells, compared to non-vaporized or partially vaporized droplets. Optical studies clearly illustrated the transient changes that occurred upon ADV of droplet-targeted CEM cells, and B-mode ultrasound imaging revealed contrast enhancement by ADV in ultrasound images. In conclusion, our fabricated droplets functioned as a hybrid chemical and mechanical strategy for the specific destruction of cancer cells upon ultrasound-mediated ADV, while simultaneously providing ultrasound imaging capability.
Keywords: High-intensity focused ultrasound (HIFU); Aptamer-conjugated; Doxorubicin-loaded; Acoustic droplets; Ultrasound imaging; Acoustic droplet vaporization (ADV);

There have been two strategies developed in the recent literature for quantitative control of the targeting effects for drug delivery by ligand-conjugated nanoparticles of biodegradable copolymer blend such as PLGA/PLGA-PEG, i.e. the pre-conjugation strategy and the post-conjugation strategy, in which the ligand conjugation was made before and after the nanoparticle formulation respectively. This research developed another drug delivery system of the PLA-TPGS/TPGS-COOH copolymer blend and further improved the post-conjugation strategy to precisely control the targeting effects by two ways: one is to adjust the PLA-TPGS:TPGS-COOH copolymer blend ratio in the nanoparticle formulation process, which provides a way for coarse control, and another is to control the feeding concentration of the ligand in the herceptin conjugation process, which further provides a fine control. Herceptin conjugation was visualized by the FETEM with immumogold labeling and further quantified by the two techniques, i.e. the Bradford assay and the flow cytometry to confirm each other. The positive correlation between the surface density of the ligand and the cellular internalization as well as the cytotoxicity of the nanoparticle formulations was assessed, which demonstrated that the strategy developed in this research is simple and feasible, which can precisely control the targeting effects of the nanoparticles of biodegradable polymers as well as other nanocarriers such as micelles and liposomes.
Keywords: Biodegradable copolymers; Cancer nanotechnology; Chemotherapeutic engineering; Drug targeting; Nanomedicine; Herceptin®;