Biomaterials (v.33, #18)

Effect of surface-functionalized nanoparticles on the elongation phase of beta-amyloid (1–40) fibrillogenesis by Ho-Man Chan; Lehui Xiao; Kai-Ming Yeung; See-Lok Ho; Dan Zhao; Wing-Hong Chan; Hung-Wing Li (4443-4450).
The influence of nanoparticles of various sizes and surface functionalities on the self-assembling fibrillogenesis of beta-amyloid (1–40) peptide was investigated. Functionalized nanoparticles including quantum dots and gold nanoparticles were co-incubated with monomeric Aβ1–40 peptides under seed-mediated growth method to study their influences on the elongation phase of the fibrillogenesis. It is observed that charge-to-surface area ratio of the nanoparticles and the functional moiety and electrostatic charges of the conjugated ligands on the particle surfaces took crucial regulatory role in the Aβ1–40 fibrillogenesis.
Keywords: Nanoparticle; Self-assembly; Peptide; Surface modification;

Biomimetic hydration lubrication with various polyelectrolyte layers on cross-linked polyethylene orthopedic bearing materials by Masayuki Kyomoto; Toru Moro; Kenichi Saiga; Masami Hashimoto; Hideya Ito; Hiroshi Kawaguchi; Yoshio Takatori; Kazuhiko Ishihara (4451-4459).
Natural joints rely on fluid thin-film lubrication by the hydrated polyelectrolyte layer of cartilage. However, current artificial joints with polyethylene (PE) surfaces have considerably less efficient lubrication and thus much greater wear, leading to osteolysis and aseptic loosening. This is considered a common factor limiting prosthetic longevity in total hip arthroplasty (THA). However, such wear could be mitigated by surface modification to mimic the role of cartilage. Here we report the development of nanometer-scale hydrophilic layers with varying charge (nonionic, cationic, anionic, or zwitterionic) on cross-linked PE (CLPE) surfaces, which could fully mimic the hydrophilicity and lubricity of the natural joint surface. We present evidence to support two lubrication mechanisms: the primary mechanism is due to the high level of hydration in the grafted layer, where water molecules act as very efficient lubricants; and the secondary mechanism is repulsion of protein molecules and positively charged inorganic ions by the grafted polyelectrolyte layer. Thus, such nanometer-scaled hydrophilic polymers or polyelectrolyte layers on the CLPE surface of acetabular cup bearings could confer high durability to THA prosthetics.
Keywords: Joint replacement; Polyethylene; Surface modification; Biomimetic material; Wear mechanism;

Hematopoietic stem cells (HSCs) are adult stem cells with the capacity to give rise to all blood and immune cells in the body. HSCs are housed in a specialized microenvironment known as the stem cell niche, which provides intrinsic and extrinsic signals to regulate HSC fate: quiescence, self-renewal, differentiation, mobilization, homing, and apoptosis. These niches provide a complex, three dimensional (3D) microenvironment consisting of cells, the extracellular matrix (ECM), and ECM-bound or soluble biomolecules that provides cellular, structural, and molecular signals that regulate HSC fate decisions. In this study, we examined the decoupled effects of substrate elasticity, construct dimensionality, and ligand concentration on the biophysical properties of primary hematopoietic stem and progenitor cells (HSPCs) using homologous series of two and three dimensional microenvironments. Microenvironments were chosen to span the range of biophysical environments presented physiologically within the bone marrow, ranging from soft marrow and adipose tissue (<1 kPa), to surrounding cell membranes (1–3 kPa), to developing osteoid (>30 kPa). We additionally investigated the influence of collagen ligand density on HSPC biophysical parameters and compared these behaviors to those observed in HSPCs grown in culture on stiff glass substrates. This work suggests the potential for substrate stiffness and ligand density to directly affect the biophysical properties of primary hematopoietic stem and progenitor cells at the single cell level and that these parameters may be critical design criteria for the development of artificial HSC niches.
Keywords: Hematopoietic stem cell; Substrate stiffness; Ligand density; Cytoskeleton; Fate;

The isolation and in situ identification of MSCs residing in loose connective tissues using a niche-preserving organ culture system by Min-Young Choi; Hyeong-In Kim; Young-Il Yang; Jong Tae Kim; Soo Hwa Jang; Chung Mu Park; Won Hee Jang; Young-Chul Youn; Soon-Ho Cheong; Chang Soo Choi; Dae Kyeong Kim; Seung Jin Lee (4469-4479).
Mesenchymal stem cells (MSCs) have been discovered in a multitude of organs, but their distribution and identity are still uncertain. Furthermore, loose connective tissue (LCT) is dispersed throughout virtually all organs, but its biological role in tissue homeostasis is unclear. Here, we describe a unique organ culture system to explore the omnipresence and in situ identity of MSCs among the LCTs. This culture system included the use of the fibrin hydrogel coupled with dynamic culture conditions, using native LCTs obtained from various organs as starting materials. This culture allowed MSC outgrowth into the hydrogel to be robustly supported, while maintaining the structural integrity of LCTs during in vitro culture. Subcultured outgrown cells fulfilled the minimal requirements for defining MSCs on the basis of clonogenicity, multipotency, and immunophenotypic characteristics. In vitro label-retaining assay demonstrated that the numbers of mobilized and proliferated cells in situ increased in the pericapillary region and expressed both MSCs and pericytes markers, indicating that the in situ identity of MSCs represents a certain population of pericapillary pericytes. Our results indicate that this culture system affords a unique strategy for both isolating MSCs and recapitulating their niche in LCTs.
Keywords: Loose connective tissues; Mesenchymal stem cells; Stem cell niche; Fibrin; Pericytes; Organ culture;

Developing biomaterials to promote stem cell proliferation and differentiation is a critical requirement in tissue engineering and regeneration. Extracellular matrix (ECM) derived from mesenchymal stem cells (MSCs) has recently been shown to be able to maintain the differentiation potential of MSCs during culture expansion and to restore the activities of aging MSCs, suggesting that MSC ECM (MECM) may be a suitable culture substrate to enhance the bioactivity of biomaterial scaffolds for MSCs. This investigation aims to characterize the biological nature and specificity of the influence of the MECM on MSCs. Native ECM produced by human MSC in vitro was extracted in urea, and the residual pellet was further processed with pepsin digestion (denoted as U-MECM and HP-MECM, respectively). The MECM products were then coated as a substrate on standard tissue culture plastic, and the behavior of MSCs seeded on the coated surfaces was studied. Our results showed that U-MECM coating dramatically accelerated MSC proliferation, attachment, spread, migration and multi-lineage differentiation (i.e., osteogenesis and adipogenesis), compared to collagen type I and HP-MECM coating. Non-collagenous proteins are likely the bioactive components in U-MECM, as MSCs cultured on collagen type I and HP-MECM showed similar biological activities, and collagen type I appeared to be the major protein components remaining in HP-MECM based on SDS-PAGE. These findings support the biological utility of MECM in the formulation of biomaterial scaffolds to enhance MSC bioactivities, including proliferation, migration and multi-lineage differentiation, for tissue regeneration applications.
Keywords: Decellularized matrix; Adult stem cells; Cell migration; Differentiation; Cell–matrix interaction;

The performance of decellularized adipose tissue microcarriers as an inductive substrate for human adipose-derived stem cells by Allison E.B. Turner; Claire Yu; Juares Bianco; John F. Watkins; Lauren E. Flynn (4490-4499).
With the aim of developing a clinically-translatable cell expansion and delivery vehicle for adipose tissue engineering, the adipogenic differentiation of human adipose-derived stem cells (ASCs) was investigated on microcarriers fabricated from human decellularized adipose tissue (DAT). ASCs seeded on the DAT microcarriers and cultured in adipogenic differentiation medium within a low-shear spinner culture system demonstrated high levels of adipogenic differentiation, as measured by the expression of adipogenic genes, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, and intracellular lipid accumulation. In contrast, gelatin microcarrier controls did not demonstrate significant adipogenesis, emphasizing the role of the native matrix in mediating ASC differentiation. Interestingly, ASCs cultured on the DAT microcarriers in proliferation medium expressed elevated levels of the adipogenic markers, suggesting that the DAT provided an adipo-inductive substrate for the human ASCs. In vivo testing of the DAT and gelatin microcarriers in a subcutaneous Wistar rat model confirmed injectability and demonstrated stable volume retention over 28 days. Under histological analysis, the DAT microcarriers demonstrated no evidence of immunogenicity or cytotoxicity, with the DAT supporting cellular infiltration and tissue remodeling. Pre-seeding the DAT microcarriers with allogenic rat ASCs enhanced cellularity and angiogenesis within the implant region.
Keywords: Adipose tissue engineering; Cell culture; Differentiation; ECM (extracellular matrix); Microcarrier; Stem cell;

The role of microRNA-23b in the differentiation of MSC into chondrocyte by targeting protein kinase A signaling by Onju Ham; Byeong-Wook Song; Se-Yeon Lee; Eunmi Choi; Min-Ji Cha; Chang Youn Lee; Jun-Hee Park; Il-Kwon Kim; Woochul Chang; Soyeon Lim; Chang Hyun Lee; Soonhag Kim; Yangsoo Jang; Ki-Chul Hwang (4500-4507).
Chondrogenic differentiation of mesenchymal stem cells (MSCs) is critical for successful cartilage regeneration. Several methods have been developed to attempt to chondrogenic differentiation, because chondrogenic differentiated cells can form stable cartilage and induce expression of a cartilage-specific phenotype. In this study, we found that both H-89 and microRNA-23b induced differentiation into chondrocyte of hMSCs through down-regulation of protein kinase A (PKA) signaling. The small molecule, H-89, was identified by PCA analysis as a potential mediator of chondrogenic differentiation. H-89 induced the expression of the chondrocyte marker, aggrecan, as well as miR-23b. We searched that miR-23b regulates protein level of PKA. When miR-23b was transfected into hMSCs, chondrogenic differentiation was induced. We confirmed the target of miR-23b using a reporter gene assay. Furthermore, not only H-89 or miR-23b-treated cells, but also cell co-treated with H-89 and miR-23b differentiated into chondrocytes. Our results indicate that H-89 induces the expression of endogenous miR-23b, thereby inducing chondrogenic differentiation by negatively inhibition of PKA signaling.
Keywords: Chondrocyte; Differentiation; Mesenchymal stem cells; MicroRNA; PKA;

Rapid aggregation of heterogeneous cells and multiple-sized microspheres in methylcellulose medium by Nobuhiko Kojima; Shoji Takeuchi; Yasuyuki Sakai (4508-4514).
We report a method for the rapid production of cellular aggregates without electric power and cell modification. We focused on the swelling property of a solution containing a high molecular material, methylcellulose (MC), which immediately absorbs a small amount of solvent and fills the space occupied by the solvent. When 1 μl of a suspension of 1000 animal cells in normal culture medium was injected into the 3% MC medium, the normal medium was rapidly absorbed by the surrounding MC medium. Suspended cells were simultaneously trapped on the interfaces between the normal and MC media; they were finally pulled together and held in the MC medium. This event was nearly complete within the first 10 min. Moreover, MC medium-dependent aggregation was observed when polystyrene microspheres of different sizes (diameter, 100 nm–100 μm) were added. Furthermore, we demonstrated the stepwise fabrication of multi-layered aggregates with embedded structures. These methods for creating engineered aggregates should enhance the study of three-dimensional cultures comprising two or more cell types with well-designed structures.
Keywords: Swelling; Absorption; Microsphere; Nanoparticle;

Highly efficient magnetic stem cell labeling with citrate-coated superparamagnetic iron oxide nanoparticles for MRI tracking by Kristin Andreas; Radostina Georgieva; Mechthild Ladwig; Susanne Mueller; Michael Notter; Michael Sittinger; Jochen Ringe (4515-4525).
Tracking of transplanted stem cells is essential to monitor safety and efficiency of cell-based therapies. Magnetic resonance imaging (MRI) offers a very sensitive, repetitive and non-invasive in vivo detection of magnetically labeled cells but labeling with commercial superparamagnetic iron oxide nanoparticles (SPIONs) is still problematic because of low labeling efficiencies and the need of potentially toxic transfection agents. In this study, new experimental citrate-coated SPIONs and commercial Endorem and Resovist SPIONs were investigated comparatively in terms of in vitro labeling efficiency, effects on stem cell functionality and in vivo MRI visualization. Efficient labeling of human mesenchymal stem cells (MSCs) without transfection agents was only achieved with Citrate SPIONs. Magnetic labeling of human MSCs did not affect cell proliferation, presentation of typical cell surface marker antigens and differentiation into the adipogenic and osteogenic lineages. However, chondrogenic differentiation and chemotaxis were significantly impaired with increasing SPION incorporation. Transplanted SPION-labeled MSCs were visualized in vivo after intramuscular injection in rats by 7T-MRI and were retrieved ex vivo by Prussian Blue and immunohistochemical stainings. Though a careful titration of SPION incorporation, cellular function and MRI visualization is essential, Citrate SPIONs are very efficient intracellular magnetic labels for in vivo stem cell tracking by MRI.
Keywords: Mesenchymal stem cell; Magnetic cell labeling; Superparamagnetic iron oxide nanoparticles; Magnetic resonance imaging; In vivo cell tracking; Tissue engineering;

The promotion of hepatic maturation of human pluripotent stem cells in 3D co-culture using type I collagen and Swiss 3T3 cell sheets by Yasuhito Nagamoto; Katsuhisa Tashiro; Kazuo Takayama; Kazuo Ohashi; Kenji Kawabata; Fuminori Sakurai; Masashi Tachibana; Takao Hayakawa; Miho Kusuda Furue; Hiroyuki Mizuguchi (4526-4534).
Hepatocyte-like cells differentiated from human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs) are known to be a useful cell source for drug screening. We recently developed an efficient hepatic differentiation method from hESCs and hiPSCs by sequential transduction of FOXA2 and HNF1α. It is known that the combination of three-dimensional (3D) culture and co-culture, namely 3D co-culture, can maintain the functions of primary hepatocytes. However, hepatic maturation of hESC- or hiPSC-derived hepatocyte-like cells (hEHs or hiPHs, respectively) by 3D co-culture systems has not been examined. Therefore, we utilized a cell sheet engineering technology to promote hepatic maturation. The gene expression levels of hepatocyte-related markers (such as cytochrome P450 enzymes and conjugating enzymes) and the amount of albumin secretion in the hEHs or hiPHs, which were 3D co-cultured with the Swiss 3T3 cell sheet, were significantly up-regulated in comparison with those in the hEHs or hiPHs cultured in a monolayer. Furthermore, we found that type I collagen synthesized in Swiss 3T3 cells plays an important role in hepatic maturation. The hEHs or hiPHs that were 3D co-cultured with the Swiss 3T3 cell sheet would be powerful tools for medical applications, such as drug screening.
Keywords: Hepatocyte; Co-culture; Collagen; Fibroblast; Liver; ECM (extracellular matrix);

Effects of co-culturing BMSCs and auricular chondrocytes on the elastic modulus and hypertrophy of tissue engineered cartilage by Ning Kang; Xia Liu; Yue Guan; Jian Wang; Fuxing Gong; Xun Yang; Li Yan; Qian Wang; Xin Fu; Yilin Cao; Ran Xiao (4535-4544).
Co-culture of BMSCs and chondrocytes is considered as a promising strategy to generate tissue engineered cartilage as chondrocytes induce the chondrogenesis of BMSCs and inhibit the hypertrophy of engineered cartilage. Because the tissue specific stem/progenitor cells have been isolated from mature tissues including auricular cartilage, we hypothesized that adding stem cells to auricular chondrocytes in co-culture would also enhance the quality of engineered cartilage. In the present study, using the histological assay, biomechanical evaluation, and quantitative analysis of gene expression, we compared different strategies of auricular chondrocytes, BMSCs induction, and co-culture at different ratios on PGA/PLA scaffolds to construct tissue engineered elastic cartilage in vitro and in vivo. The up-regulation of RUNX2 and down-regulation of SOX9 were found in BMSCs chondrogenic induction group, which might imply a regulatory mechanism for the hypertrophy and potential osteogenic differentiation. Engineered cartilage in co-culture 5:5 group showed the densest elastic fibers and the highest Young's modulus, which were consistent with the expression profile of cartilage matrix-related genes including DCN and LOXL2 genes. Moreover, the better proliferative and chondrogenic potentials of engineered cartilage in co-culture 5:5 group were demonstrated by the stronger expression of Ki67 and Dlk1.
Keywords: Cartilage tissue engineering; Auricular chondrocytes; Bone marrow mesenchymal stem cells; Co-culture system;

Decellularized homologous tissue-engineered heart valves as off-the-shelf alternatives to xeno- and homografts by Petra E. Dijkman; Anita Driessen-Mol; Laura Frese; Simon P. Hoerstrup; Frank P.T. Baaijens (4545-4554).
Decellularized xenogenic or allogenic heart valves have been used as starter matrix for tissue-engineering of valve replacements with (pre-)clinical promising results. However, xenografts are associated with the risk of immunogenic reactions or disease transmission and availability of homografts is limited. Alternatively, biodegradable synthetic materials have been used to successfully create tissue-engineered heart valves (TEHV). However, such TEHV are associated with substantial technological and logistical complexity and have not yet entered clinical use. Here, decellularized TEHV, based on biodegradable synthetic materials and homologous cells, are introduced as an alternative starter matrix for guided tissue regeneration. Decellularization of TEHV did not alter the collagen structure or tissue strength and favored valve performance when compared to their cell-populated counterparts. Storage of the decellularized TEHV up to 18 months did not alter valve tissue properties. Reseeding the decellularized valves with mesenchymal stem cells was demonstrated feasible with minimal damage to the reseeded valve when trans-apical valve delivery was simulated. In conclusion, decellularization of in-vitro grown TEHV provides largely available off-the-shelf homologous scaffolds suitable for reseeding with autologous cells and trans-apical valve delivery.
Keywords: Heart valve tissue engineering; Tissue regeneration; Scaffold; Decellularization; Retraction; Mesenchymal stem cells;

The effects of intrathecal injection of a hyaluronan-based hydrogel on inflammation, scarring and neurobehavioural outcomes in a rat model of severe spinal cord injury associated with arachnoiditis by James W. Austin; Catherine E. Kang; M. Douglas Baumann; Lisa DiDiodato; Kajana Satkunendrarajah; Jefferson R. Wilson; Greg J. Stanisz; Molly S. Shoichet; Michael G. Fehlings (4555-4564).
Traumatic spinal cord injury (SCI) comprises a heterogeneous condition caused by a complex array of mechanical forces that damage the spinal cord – making each case somewhat unique. In addition to parenchymal injury, a subset of patients experience severe inflammation in the subarachnoid space or arachnoiditis, which can lead to the development of fluid-filled cavities/syringes, a condition called post-traumatic syringomyelia (PTS). Currently, there are no therapeutic means to address this devastating complication in patients and furthermore once PTS is diagnosed, treatment is often prone to failure. We hypothesized that reducing subarachnoid inflammation using a novel bioengineered strategy would improve outcome in a rodent model of PTS. A hydrogel of hyaluronan and methyl cellulose (HAMC) was injected into the subarachnoid space 24 h post PTS injury in rats. Intrathecal injection of HAMC reduced the extent of fibrosis and inflammation in the subarachnoid space. Furthermore, HAMC promoted improved neurobehavioural recovery, enhanced axonal conduction and reduced the extent of the lesion as assessed by MRI and histomorphometric assessment. These findings were additionally associated with a reduction in the post-traumatic parenchymal fibrous scar formation as evidenced by reduced CSPG deposition and reduced IL-1α cytokine levels. Our data suggest that HAMC is capable of modulating inflammation and scarring events, leading to improved functional recovery following severe SCI associated with arachnoiditis.
Keywords: Hyaluronan; Inflammation; Fibrosis; Spinal cord injury; Hydrogel;

Tissue specific synthetic ECM hydrogels for 3-D in vitro maintenance of hepatocyte function by Aleksander Skardal; Leona Smith; Shantaram Bharadwaj; Anthony Atala; Shay Soker; Yuanyuan Zhang (4565-4575).
Despite recent advances in biomaterial science, there is yet no culture system that supports long-term culture expansion of human adult hepatocytes, while preserving continued function. Previous studies suggested that acellular liver extracellular matrix (ECM), employed as a substrate, improved proliferation and function of liver cells. Here we investigated whether extracts prepared from acellular liver ECM (liver ECM extract, LEE), or from whole (fresh) liver tissue (liver tissue extract, LTE), could be combined with collagen Type I, hyaluronic acid (HA), or heparin-conjugated HA (HP) hydrogels to enhance survival and functional output of primary human hepatocytes. The liver-specific semi-synthetic ECMs (sECMs) were prepared by incorporating LEE or LTE into the gel matrices. Subsequently, primary human hepatocytes were maintained in sandwich-style hydrogel cultures for 4 weeks. Progressive increase in hepatocyte metabolism was observed in all HA and HP groups. Hepatocytes cultured in HA and HP hydrogels containing LEE or LTE synthesized and secreted steady levels of albumin and urea and sustained cytochrome p450-dependent drug metabolism of ethoxycoumarin. Collectively, these results indicate that customized HA hydrogels with liver-specific ECM components may be an efficient method for expansion human hepatocytes in vitro for cell therapy and drug and toxicology screening purposes.
Keywords: Hyaluronic acid; Heparin; Hepatocyte; Extracellular matrix; Growth factors;

The accumulation of dual pH and temperature responsive micelles in tumors by Yi-Chun Chen; Li-Chi Liao; Pei-Lin Lu; Chun-Liang Lo; Hsieh-Chih Tsai; Chiung-Yin Huang; Kuo-Chen Wei; Tzu-Chen Yen; Ging-Ho Hsiue (4576-4588).
An optimized, biodegradable, dual temperature- and pH-responsive micelle system conjugated with functional group Cy5.5 was prepared in order to enhance tumor accumulation. The Dynamic light scattering (DLS) measurements showed that these diblock copolymers form micelle in PBS buffer with a size of around 50 nm by heating of an aqueous polymer solution from below to above the cloud point (CP). Anticancer drug, doxorubicin was incorporated into the inner core of micelle by hot shock protocol. The size and stability of the micelle were controlled by the copolymer composition and is fine tuned to extracellular pH of tumor. The mechanism then caused pH change and at body temperature which induce doxorubicin release from micelles and have strong effects on the viability of HeLa, ZR-75-1, MCF-7 and H661 cancer cells. Our in vivo results revealed a clear distribution of Doxorubicin-loaded mixed micelle (Dox-micelle) and efficiency targeting tumor site with particles increasing size in the tumor interstitial space, and the particles could not diffuse throughout the tumor matrix. In vivo tumor growth inhibition showed that Dox-micelle exhibited excellent antitumor activity and a high rate of anticancer drug in cancer cells by this strategy.
Keywords: Polymeric micelles; Temperature-responsive; pH-responsive; EPR effect; Doxorubicin;

Hydrophobic poly (amino acid) modified PEI mediated delivery of rev-casp-3 for cancer therapy by Chunling Fu; Lin Lin; Hengliang Shi; Daxue Zheng; Wei Wang; Shiqian Gao; Yifei Zhao; Huayu Tian; Xiaojuan Zhu; Xuesi Chen (4589-4596).
Recent studies in amphiphilic cationic polymers have demonstrated their potential as gene carriers with high transfection efficiency and low cytotoxicity in the in vitro settings to deliver drug, siRNA and plasmid DNA. Yet their safety and efficacy in vivo remain to be a challenge, and require further investigation. In our previous work, PP80 was synthesized as a novel amphiphilic cationic polymer by grafting hydrophobic polyphenylalanine segment on PEI, which displayed higher transfection efficiency than PEI in a number of cell lines in vitro. Here, we reported the favorable biocompatibility displayed by PP80/pDNA complex both in vitro and in vivo. Furthermore, when therapeutic gene rev-casp-3 was conjugated to PP80 and administered intratumorally to a HeLa xenograft model, significant tumor apoptosis was induced with concurrent tumor growth inhibition, indicating that PP80 mediated expression of rev-casp-3 gene in solid tumors with not detectable side effects on the tumor-bearing mice. These data demonstrated that PP80 warrants further investigation as a promising cancer gene delivery vehicle.
Keywords: Amphiphilic cationic polymer; PP80; Rev-casp-3; Biocompatibility; Cancer therapy;

A gene nanocomplex conjugated with monoclonal antibodies for targeted therapy of hepatocellular carcinoma by J.L. Wang; G.P. Tang; J. Shen; Q.L. Hu; F.J. Xu; Q.Q. Wang; Z.H. Li; W.T. Yang (4597-4607).
To enhance tumor-targeting abilities and therapeutic efficiency, a monoclonal antibody-conjugated gene nanocomplex was herein designed. The biodegradable cationic polyethylenimine-grafted-α,β-poly(N-3-hydroxypropyl)-dl-aspartamide (PHPA-PEI) was used for complexing pDNA to form the PHPA-PEI/pDNA nanoparticle, and then 9B9 mAb, an anti-epidermal growth factor receptor (anti-EGFR) monoclonal antibody, was conjugated to produce the PHPA-PEI/pDNA/9B9 mAb (PP9mN) complex. The PP9mN complex with the diameter of around 300 nm at its optimal weight ratio could be uptaken effectively by SMMC-7721 cells. The cytotoxicity of the PP9mN complex was much lower than that of PEI 25 kD in SMMC-7721, HepG2, Bel-7404 and COS-7 cell lines. The PP9mN complex possessed the highly efficient in vitro gene delivery ability to the hepatocellular carcinoma cells. The in vivo gene expression indicated that PP9mN could target to the tumor tissues effectively. By using the therapeutic AChE gene, it was found that the PP9mN complexes significantly enhanced the anti-tumor effect on tumor-bearing nude mice. Such monoclonal antibody-conjugated gene complex should have great potential applications in liver cancer therapy.
Keywords: Liver cancer; Target gene therapy; Monoclonal antibody; AChE;

Drug-loaded temperature-sensitive liposomes (TSL) in combination with hyperthermia (HT) have attracted considerable attention for cancer treatment. Different TSL systems have been designed with wide variations in their temperature sensitivity and drug release profile. Low temperature-sensitive liposomes (LTSL) with the capacity for ultrafast drug release, traditional temperature-sensitive (TTSL) with intermediate drug release properties and non-temperature-sensitive liposomes (NTSL) (no drug release) were dual-labeled with 3H-cholesteryl hexadecyl ether (3H-CHE) lipid and loaded with 14C-doxorubicin (14C-Dox). Their blood profile, serum stability, tissue distribution and tumor localization (B16F10 melanoma) were studied after intravenous administration and mild HT treatment. LTSL showed higher affinity for the liver compared to TTSL and NTSL which were uptaken mainly by spleen. Under normal conditions (no HT) Dox leakage from liposomes was expected, higher for LTSL, less for TTSL and minimal for NTSL. Localized HT did not affect the overall blood circulation or organ accumulation for all TSL studied. Since LTSL showed ultrafast Dox release kinetics at 42 °C, the highest drug accumulation in tumors was observed using this system immediately after HT, however decreased significantly after 24 h. In contrast, TTSL and NTSL showed 2-3 fold increase in both liposome and Dox levels that indicated enhanced tumor extravasation of intact Dox-loaded liposomes during the 60 min HT applications. More interestingly, high levels of drug tumor accumulation were achieved 24 h post-HT. This study offers further understanding on how the mechanisms of drug release from temperature-sensitive liposomes affect their pharmacological profile under mild hyperthermia.
Keywords: Animal model; Drug delivery; Drug release; In vivo test; Liposomes; Phospholipid;

Core–shell Fe3O4@NaLuF4:Yb,Er/Tm nanostructure for MRI, CT and upconversion luminescence tri-modality imaging by Xingjun Zhu; Jing Zhou; Min Chen; Mei Shi; Wei Feng; Fuyou Li (4618-4627).
Core–shell Fe3O4@NaLuF4:Yb,Er/Tm nanostructure (MUCNP) with multifunctional properties has been developed using a step-wise synthetic method. The successful fabrication of MUCNP has been confirmed by transmission electron microscopy, powder X-ray diffraction, energy-dispersive X-ray analysis and X-ray photoelectron spectroscopy. The MUCNP exhibits superparamagnetic property with saturation magnetization of 15 emu g−1, and T 2-enhanced magnetic resonance (MR) effect with an r 2 value of 21.63 s−1 mM−1 at 0.5 T, resulting from the Fe3O4 cores. Moreover, the NaLuF4-based MUCNP provides excellent X-ray attenuation and upconversion luminescence (UCL) emission under excitation at 980 nm. In vivo MR, computed tomography (CT) and UCL images of tumor-bearing mice show that the MUCNP can be successfully used in multimodal imaging. In vitro tests reveal that the MUCNP is non-cytotoxic. These results suggest that the developed MUCNP could be served as an MR, CT and UCL probe for tri-modality imaging.
Keywords: Upconversion luminescence (UCL); Upconversion nanophosphors; Luminescence imaging; Core–shell; Tri-modality imaging;

In vivo renal clearance, biodistribution, toxicity of gold nanoclusters by Xiao-Dong Zhang; Di Wu; Xiu Shen; Pei-Xun Liu; Fei-Yue Fan; Sai-Jun Fan (4628-4638).
Gold nanoparticles have shown great prospective in cancer diagnosis and therapy, but they can not be metabolized and prefer to accumulate in liver and spleen due to their large size. The gold nanoclusters with small size can penetrate kidney tissue and have promise to decrease in vivo toxicity by renal clearance. In this work, we explore the in vivo renal clearance, biodistribution, and toxicity responses of the BSA- and GSH-protected gold nanoclusters for 24 h and 28 days. The BSA-protected gold nanoclusters have low-efficient renal clearance and only 1% of gold can be cleared, but the GSH-protected gold nanoclusters have high-efficient renal clearance and 36% of gold can be cleared after 24 h. The biodistribution further reveals that 94% of gold can be metabolized for the GSH-protected nanoclusters, but only less than 5% of gold can be metabolized for the BSA-protected nanoclusters after 28 days. Both of the GSH- and BSA-protected gold nanoclusters cause acute infection, inflammation, and kidney function damage after 24 h, but these toxicity responses for the GSH-protected gold nanoclusters can be eliminated after 28 days. Immune system can also be affected by the two kinds of gold nanoclusters, but the immune response for the GSH-protected gold nanoclusters can also be recovered after 28 days. These findings show that the GSH-protected gold nanoclusters have small size and can be metabolized by renal clearance and thus the toxicity can be significantly decreased. The BSA-protected gold nanoclusters, however, can form large compounds and further accumulate in liver and spleen which can cause irreparable toxicity response. Therefore, the GSH-protected gold nanoclusters have great potential for in vivo imaging and therapy, and the BSA-protected gold nanoclusters can be used as the agent of liver cancer therapy.
Keywords: Gold nanoclusters; Renal clearance; Biodistribution; In vivo toxicity;

The time courses of uptake and removal of silicon quantum dots (Si-QDs) by human umbilical endothelial cells (HUVECs) were observed via confocal laser scanning microscope. Si-QDs were internalized via endocytosis and transported to late endosomes/lysosomes. The number of internalized Si-QDs increased with time and gradually reached a plateau value. When Si-QD-internalized HUVECs were subsequently washed and exposed to fresh culture medium, HUVECs removed internalized Si-QDs via exocytosis. The number of internalized Si-QDs decreased with time and gradually reached a plateau value. Not all internalized Si-QDs were removed from the cell interior but large numbers of internalized Si-QDs remained accumulated inside cells. A kinetic model based on the mass balance of Si-QDs and receptors in a cell was proposed to describe the cellular uptake and removal of Si-QDs. Model calculation fitted well with experimental results. Using this model, the dissociation constant between receptors and Si-QDs in the endosome, K d,in, was found to be a determinant factor for Si-QD accumulation in cells after the removal process.
Keywords: Silicon; Nanoparticle; Fluorescence; Confocal microscopy; Modeling; Endothelial cell;

Sustained release of proteins from high water content supramolecular polymer hydrogels by Eric A. Appel; Xian Jun Loh; Samuel T. Jones; Cecile A. Dreiss; Oren A. Scherman (4646-4652).
Self-assembled hydrogels with extremely high water content (up to 99.5%) and highly tunable mechanical properties were prepared from renewable cellulose derivatives. These hydrogels are easily processed and the simplicity of their preparation, their availability from inexpensive renewable resources, and the tunability of their mechanical properties are distinguishing for important biomedical applications. The protein release characteristics were investigated to determine the effect of both the protein molecular weight and polymer loadings of the hydrogels on the protein release rate. Extremely sustained release of bovine serum albumin is observed over the course of 160 days from supramolecular hydrogels containing only 1.5 wt% polymeric constituents. This sustained release far surpasses the current state of the art for protein release from a hydrogel, highlighting these materials as important potential candidates for sustained therapeutic applications.
Keywords: Controlled drug release; Drug release; Growth factors; Hydrogel; Polyvinylalcohol; Cellulose;

Binary and ternary complexes based on polycaprolactone-graft-poly (N, N-dimethylaminoethyl methacrylate) for targeted siRNA delivery by Yuanyu Huang; Daoshu Lin; Qian Jiang; Wendi Zhang; Shutao Guo; Ping Xiao; Shuquan Zheng; Xiaoxia Wang; Hongbo Chen; Hong-Yan Zhang; Liandong Deng; Jinfeng Xing; Quan Du; Anjie Dong; Zicai Liang (4653-4664).
Small interfering RNA (siRNA) is a powerful gene silencing tool and has promising prospects in basic research and the development of therapeutic reagents. However, the lack of an effective and safe tool for siRNA delivery hampers its application. Here, we introduced binary and ternary complexes that effectively mediated siRNA-targeted gene silencing. Both complexes showed excellent siRNA loading even at the low N/P/C ratio of 3:1:0. FACS and confocal microscopy demonstrated that nearly all cells robustly internalized siRNAs into the cytoplasm, where RNA interference (RNAi) occurred. Luciferase assay and Western blot verified that silencing efficacy reached >80%, and introducing folate onto the ternary complexes further enhanced silencing efficacy by about 10% over those without folate at the same N/P/C ratio. In addition, the coating of PGA-g-mPEG decreased the zeta potential almost to electroneutrality, and the MTT assay showed decreased cytotoxicity. In vivo distribution measurement and histochemical analysis executed in C57BL/6 and Hela tumor-bearing BALB/c nude mice showed that complexes accumulated in the liver, lungs, pancreas and tumors and were released slowly for a long time after intravenous injection. Furthermore, ternary complexes showed higher siRNA fluorescence intensity than binary complexes at the same N/P ratio in tumor tissues, those with folate delivered more siRNAs to tumors than those without folate, and more folate induced more siRNA transport to tumors. In addition, in vivo functional study showed that both binary and ternary complexes mediated down-regulation of ApoB in liver efficiently and consequently blocked the secretion of fatty acids into the blood, resulted in lipid accumulation in liver, liver steatosis and hepatic dysfunction. In conclusion, these complexes provided a powerful means of administration for siRNA-mediated treatment of liver-related diseases and various cancers, especial for pancreatic and cervical cancer.
Keywords: siRNA delivery; Tumor targeting; Liver targeting; Ternary complex; PCL-g-PDMAEMA; Non-viral carriers;

A protein transduction method using oligo-arginine (3R) for the delivery of transcription factors into cell nuclei by Takashi Hitsuda; Hiroyuki Michiue; Mizuki Kitamatsu; Atsushi Fujimura; Feifei Wang; Takahiro Yamamoto; Xiao-Jian Han; Hiroshi Tazawa; Atsuhito Uneda; Iori Ohmori; Tei-ichi Nishiki; Kazuhito Tomizawa; Hideki Matsui (4665-4672).
Protein transduction with cell-penetrating peptides such as poly-arginine and HIV TAT peptides is widely used to deliver proteins, peptides, siRNA and biologically active compounds. It has been thought that poly-arginine peptides transduce proteins in a manner dependent on the number of arginine residues and oligo-peptides such as three arginines (3R) are ineffective. Here we showed that 3R-fused proteins were effectively delivered and functioned in cells co-treated with pyrenebutyrate, a counteranion bearing an aromatic hydrophobic moiety. Little 3R was transduced in glioma cells without pyrenebutyrate whereas the oligo-arginine was effectively delivered with pyrenebutyrate. Enhanced green fluorescence protein (eGFP) fused with 3R was effectively delivered into various kinds of cells including primary cultured cells and suspended cells in the presence of pyrenebutyrate. p53 fused with 3R (3R-p53) was delivered into glioma cells without pyrenebutyrate but could not be translocated into the nucleus. In contrast, 3R-p53 was observed in nuclei of glioma cells when co-applied with pyrenebutyrate. Although 3R-p53 was delivered less effectively than 11R-p53 with pyrenebutyrate, its transcriptional activity was higher than that of 11R-p53. Moreover, a single administration of 3R-p53 with pyrenebutyrate significantly inhibited the growth of cancer cells. These results suggest protein transduction using an oligo-arginine (3R) with pyrenebutyrate to be a good tool for the delivery of functional transcription factors and a promising method of treating cancer.
Keywords: Protein transduction; eGFP; p53; Arginine; Drug delivery; TAT;

Diaminododecane-based cationic bolaamphiphile as a non-viral gene delivery carrier by Majad Khan; Chung Yen Ang; Nikken Wiradharma; Lin-Kin Yong; Shaoqiong Liu; Lihong Liu; Shujun Gao; Yi-Yan Yang (4673-4680).
The advancement in gene therapy relies upon the discovery of safe and efficient delivery agents and methods. In this study, we report the design and synthesis of a cationic bolaamphiphile as a non-viral gene delivery agent. The bolaamphiphile is composed of 1,12-diaminododecane as the central hydrophobic unit linked to the hydrophilic pentaethylenehexamine via thioether-based glycidyl units. This bolaamphiphile condensed DNA efficiently into nanoparticles of sizes around 150–200 nm with positive zeta potential of 30–35 mV. In vitro luciferase expression levels and percentage of GFP expressing cells induced by the bolaamphiphile/DNA complexes were higher than those mediated by the often used “golden” standard of non-viral systems, polyethyleneimine (PEI, branched, 25 kDa) at its optimal N/P ratio in HEK293, HepG2, NIH3T3, HeLa and 4T1 cells. In vitro cytotoxicity testing revealed that the DNA complexes fabricated from this cationic bolaamphiphile displayed marginal toxicity towards all the cell lines tested. In addition, in vivo transfection studies carried out in a 4T1 mouse breast cancer model showed that the cationic bolaamphiphile delivered DNA more efficiently than PEI. This cationic bolaamphiphile may make a promising gene delivery vector for future gene therapy.
Keywords: Bolaamphiphile; Cationic; Gene delivery; Luciferase gene; GFP gene; In vivo gene transfection;

Microcapsules with intrinsic barium radiopacity for immunoprotection and X-ray/CT imaging of pancreatic islet cells by Dian R. Arifin; Sameer Manek; Emma Call; Aravind Arepally; Jeff W.M. Bulte (4681-4689).
Microencapsulation is a commonly used technique for immunoprotection of engrafted therapeutic cells. We investigated a library of capsule formulations to determine the most optimal formulation for pancreatic beta islet cell transplantation, using barium as the gelating ion and clinical-grade protamine sulfate (PS) as a new cationic capsule cross-linker. Barium-gelated alginate/PS/alginate microcapsules (APSA, diameter = 444 ± 21 μm) proved to be mechanically stronger and supported a higher cell viability as compared to conventional alginate/poly-l-lysine/alginate (APLLA) capsules. Human pancreatic islets encapsulated inside APSA capsules, gelated with 20 mm barium as optimal concentration, exhibited a sustained morphological integrity, viability, and functionality for at least 3–4 weeks in vitro, with secreted human C-peptide levels of 0.2–160 pg/ml/islet. Unlike APLLA capsules that are gelled with calcium, barium-APSA capsules are intrinsically radiopaque and, when engrafted into mice, could be readily imaged in vivo with micro-computed tomography (CT). Without the need of adding contrast agents, these capsules offer a clinically applicable alternative for simultaneous immunoprotection and real-time, non-invasive X-ray/CT monitoring of engrafted cells during and after in vivo administration.
Keywords: Microcapsule; Islet cell; Diabetes; CT imaging; Barium;

The effects of sub-lethal concentrations of silver nanoparticles on inflammatory and stress genes in human macrophages using cDNA microarray analysis by Dae-Hyoun Lim; Jiyoung Jang; Seungjae Kim; Taegyeong Kang; Kangtaek Lee; In-Hong Choi (4690-4699).
Because of the limited information on size-dependent particle-mediated effects, the present study was conducted to determine if the changes in induced protein expression between 5 nm silver nanoparticles and 100 nm particles after exposure to sub-lethal concentrations. A total of 28,000 cDNA profiles were screened using 5 nm silver nanoparticles and 100 nm silver nanoparticles in a macrophage cell line. Based on results obtained from cDNA microarray we also assessed protein levels of hemeoxygenase-1 (HO-1), heat shock protein-70 (HSP-70) and interleukin-8 (IL-8), which were shown to significantly increase. Together with results obtained using N-acetylcystein (NAC), we were able to clearly show that low level and early stage exposure to 5 nm silver nanoparticles, but not 100 nm, induces expression of IL-8 as well as stress genes against reactive oxygen species (ROS). Therefore, we provide important data to understand and identify the early effects of silver nanoparticles on the immune system.
Keywords: Nanoparticle; Silver; Macrophage; Interleukin; mRNA;

A 3-D organoid kidney culture model engineered for high-throughput nephrotoxicity assays by Anna I. Astashkina; Brenda K. Mann; Glenn D. Prestwich; David W. Grainger (4700-4711).
Cell–cell and cell-matrix interactions control cell phenotypes and functions in vivo. Maintaining these interactions in vitro is essential to both produce and retain cultured cell fidelity to normal phenotype and function in the context of drug efficacy and toxicity screening. Two-dimensional (2-D) cultures on culture plastics rarely recapitulate any of these desired conditions. Three dimensional (3-D) culture systems provide a critical junction between traditional, yet often irrelevant, in vitro cell cultures and more accurate, yet costly, in vivo models. This study describes development of an organoid-derived 3-D culture of kidney proximal tubules (PTs) that maintains native cellular interactions in tissue context, regulating phenotypic stability of primary cells in vitro for up to 6 weeks. Furthermore, unlike immortalized cells on plastic, these 3-D organoid kidney cultures provide a more physiologically-relevant response to nephrotoxic agent exposure, with production of toxicity biomarkers found in vivo. This biomimetic primary kidney model has broad applicability to high-throughput drug and biomarker nephrotoxicity screening, as well as more mechanistic drug toxicology, pharmacology, and metabolism studies.
Keywords: Nephrotoxicity; Primary proximal tubules; 3-D culture; Organoid culture; In vivo biomarkers; In vitro toxicity assessment;

Comparing predictive drug nephrotoxicity biomarkers in kidney 3-D primary organoid culture and immortalized cell lines by Anna I. Astashkina; Brenda K. Mann; Glenn D. Prestwich; David W. Grainger (4712-4721).
The cellular microenvironment is recognized to play a key role in stabilizing cell differentiation states and phenotypes in culture. This study addresses the hypothesis that preservation of in vivo-like tissue architecture in vitro produces a cell culture more capable of responding to environmental stimuli with clinically relevant toxicity biomarkers. This was achieved using kidney proximal tubules in three-dimensional organoid hydrogel culture, with comparisons to conventional monolayer kidney cell cultures on plastic. Kidney proximal tubule cultures and two immortalized kidney cell line monolayer cultures exposed to known nephrotoxic drugs were evaluated for inflammatory cytokines, nephrotoxicity-associated genes, Kim-1 protein, cytochrome enzymes, and characteristic cellular enzyme shedding. Significant similarities are shown for these traditional biomarkers of kidney toxicity between in vivo and 3-D organoid endpoints of drug toxicity, and significantly, a consistent lack of clinically relevant endpoints produced by traditional 2-D kidney cell cultures. These findings impact both in vitro bioreactor-based kidney functional and regenerative medicine models, as well as high-throughput cell-based drug screening validations.
Keywords: 3-D organoid culture; In vivo biomarkers; Primary proximal tubules; HEK 293; LLC-PK1; Drug screening;

Homo-catiomer integration into PEGylated polyplex micelle from block-catiomer for systemic anti-angiogenic gene therapy for fibrotic pancreatic tumors by Qixian Chen; Kensuke Osada; Takehiko Ishii; Makoto Oba; Satoshi Uchida; Theofilus A. Tockary; Taisuke Endo; Zhishen Ge; Hiroaki Kinoh; Mitsunobu R. Kano; Keiji Itaka; Kazunori Kataoka (4722-4730).
Homo-poly{N′-[N-(2-aminoethyl)-2-aminoehtyl]aspartamide} [PAsp(DET), H] was attempted to integrate into poly (ethylene glycol) (PEG)-b-PAsp(DET)] (B) formulated polyplex micelle with the aim of enhancing cell transfection efficiency for PEGylated polyplex micelle via H integration. In vitro evaluations verified H integration of potent stimulation in enhancing cell-transfecting activity of PEGylated polyplex micelles via promoted cellular uptake and facilitated endosome escape. In vivo anti-angiogenic tumor suppression evaluations validated the feasibility of H integration in promoting gene transfection to the affected cells via systemic administration, where loaded anti-angiogenic gene remarkably expressed in the tumor site, thereby imparting significant inhibitory effect on the growth of vascular endothelial cells, ultimately leading to potent tumor growth suppression. These results demonstrated potency of H integration for enhanced transfection activity and potential usage in systemic applications, which could have important implications on the strategic use of H integration in the non-viral gene carrier design.
Keywords: DNA; Micelle; Nanoparticle; Gene transfer; In vitro test; In vivo test;

Hyperbranched cationic amylopectin derivatives for gene delivery by Yanfang Zhou; Bin Yang; Xianyue Ren; Zhenzhen Liu; Zheng Deng; Luming Chen; Yubin Deng; Li-Ming Zhang; Liqun Yang (4731-4740).
A series of hyperbranched cationic amylopectin derivatives conjugated with 1,2-ethylenediamine, diethylenetriamine and 3-(dimethylamino)-1-propylamine residues, named as EDA-Amp, DETA-Amp and DMAPA-Amp, were synthesized by the N,N’-carbonyldiimidazole activation method at room temperature. Their structures were characterized by FTIR and 1H NMR analyses, and their buffering capability was assessed by acid-base titration. The amylopectin derivatives exhibited better blood compatibility and lower cytotoxicity when compared to branched polyethyleneimine (bPEI) in the hemolysis and MTT assays. Atomic force microscopy and optical microscopy confirmed that the amylopectin derivatives exhibited lower damage for erythrocytes than bPEI. The amylopectin derivatives could bind and condense plasmid DNA (pDNA) to form the complexes with the size ranging from 100 to 300 nm. The resultant complexes showed higher transfection efficiency in 293T cells than in A549 cells. The DMAPA-Amp derivative-mediated gene transfection for Forkhead box O1 exhibited higher protein expression than that of the EDA-Amp and DETA-Amp derivatives in 293T cells, which was analyzed by western blot, flow cytometry and Hoechst staining assay. On the basis of these data, amylopectin derivatives exhibit potential as nonviral gene vectors.
Keywords: Amylopectin; Oligoamine; Gene delivery; Transfection efficiency;

Poly(caprolactone)-modified Pluronic P105 micelles for reversal of paclitaxcel-resistance in SKOV-3 tumors by Yongzhong Wang; Junguo Hao; Yajuan Li; Zhiwen Zhang; Xianyi Sha; Limei Han; Xiaoling Fang (4741-4751).
Three poly(caprolactone)-modified Pluronic P105 polymers (P105/PCLs) were synthesized using commercially available ε-caprolactone monomers and Pluronic P105 copolymers. The chemical structures, compositions and molecular weights of the P105/PCLs were confirmed by FT-IR, 1H NMR and GPC measurements. Three paclitaxel (PTX)-loaded P105/PCL polymeric micelles were then prepared, and they showed average diameters in the range of 30–150 nm, drug-loading coefficients of 0.15%–5.43%, and encapsulation ratios of 2.1%–76.53%. The in vitro cytotoxicity assay demonstrated that three PTX-loaded P105/PCL micelles were able to sensitize the resistant SKOV-3/PTX tumor cells. The PTX-loaded P105/PCL50 micelle was then selected for an in vivo antitumor efficacy study. The tumor volumes in nude mice bearing s.c. resistant SKOV-3/PTX carcinoma treated with this micellar PTX were significantly less than the control group treated with Taxol. It was demonstrated that three PCL-modified P105 monomers and micelles inhibited P-gP efflux activity in the resistant SKOV-3/PTX cells via at least three intracellular events: 1) inhibition of ATPase of P-gP, 2) decrease of membrane microviscosity and 3) a loss of mitochondrial membrane potential and subsequent decrease of ATP levels at the concentration of monomers (0.001%) and/or micelles (0.01–1.0%). Considering other favorable characteristics, such as sustained PTX release in vitro, long-circulating time in vivo and increased PTX concentration in the tissues of ovaries and uterus in mice, the PCL-modified Pluronic P105 polymeric micelle system could have important clinical implications for delivery of paclitaxel and treatment of the resistant ovarian tumors.
Keywords: Pluronic; Ploy(caprolactone); Polymeric micelles; Multidrug resistance; Paclitaxel;

Therapeutic effect of orally administered microencapsulated oxaliplatin for colorectal cancer by Aleksandra M. Urbanska; Emmanouil D. Karagiannis; Gonzalo Guajardo; Robert S. Langer; Daniel G. Anderson (4752-4761).
Colorectal cancer is a significant source of morbidity and mortality in the United States and other Western countries. Oral delivery of therapeutics remains the most patient accepted form of medication. The development of an oral delivery formulation for local delivery of chemotherapeutics in the gastrointestinal tract can potentially alleviate the adverse side effects including systemic cytotoxicity, as well as focus therapy to the lesions. Here we develop an oral formulation of the chemotherapeutic drug oxaliplatin for the treatment of colorectal cancer. Oxaliplatin was encapsulated in pH sensitive, mucoadhesive chitosan-coated alginate microspheres. The microparticles were formulated to release the chemotherapeutics after passing through the acidic gastric environment thus targeting the intestinal tract. In vivo, these particles substantially reduced the tumor burden in an orthotopic mouse model of colorectal cancer, and reduced mortality.
Keywords: Oxaliplatin; Intestinal tumorigenesis; Colon cancer; Oral delivery; Alginate;

Tailoring the immune response by targeting C-type lectin receptors on alveolar macrophages using “pathogen-like” amphiphilic polyanhydride nanoparticles by Ana V. Chavez-Santoscoy; Rajarshi Roychoudhury; Nicola L.B. Pohl; Michael J. Wannemuehler; Balaji Narasimhan; Amanda E. Ramer-Tait (4762-4772).
C-type lectin receptors (CLRs) offer unique advantages for tailoring immune responses. Engagement of CLRs regulates antigen presenting cell (APC) activation and promotes delivery of antigens to specific intracellular compartments inside APCs for efficient processing and presentation. In these studies, we have designed an approach for targeted antigen delivery by decorating the surface of polyanhydride nanoparticles with specific carbohydrates to provide pathogen-like properties. Two conserved carbohydrate structures often found on the surface of respiratory pathogens, galactose and di-mannose, were used to functionalize the surface of polyanhydride nanoparticles and target CLRs on alveolar macrophages (AMϕ), a principle respiratory tract APC. Co-culture of functionalized nanoparticles with AMϕ significantly increased cell surface expression of MHC I and II, CD86, CD40 and the CLR CIRE over non-functionalized nanoparticles. Di-mannose and galactose functionalization also enhanced the expression of the macrophage mannose receptor (MMR) and the macrophage galactose lectin, respectively. This enhanced AMϕ activation phenotype was found to be dependent upon nanoparticle internalization. Functionalization also promoted increased AMϕ production of the pro-inflammatory cytokines IL-1β, IL-6 and TNF-α. Additional studies demonstrated the requirement of the MMR for the enhanced cellular uptake and activation provided by the di-mannose functionalized nanoparticles. Together, these data indicate that targeted engagement of MMR and other CLRs is a viable strategy for enhancing the intrinsic adjuvant properties of nanovaccine adjuvants and promoting robust pulmonary immunity.
Keywords: Polyanhydrides; Nanoparticles; Carbohydrates; Alveolar macrophages;

Surface conjugation of triphenylphosphonium to target poly(amidoamine) dendrimers to mitochondria by Swati Biswas; Namita S. Dodwadkar; Aleksandr Piroyan; Vladimir P. Torchilin (4773-4782).
Dendrimers have emerged as promising carriers for the delivery of a wide variety of pay-loads including therapeutic drugs, imaging agents and nucleic acid materials into biological systems. The current work aimed to develop a novel mitochondria-targeted generation 5 poly(amidoamine) (PAMAM) dendrimer (G(5)-D). To achieve this goal, a known mitochondriotropic ligand triphenylphosphonium (TPP) was conjugated on the surface of the dendrimer. A fraction of the cationic surface charge of G(5)-D was neutralized by partial acetylation of the primary amine groups. Next, the mitochondria-targeted dendrimer was synthesized via the acid-amine-coupling conjugation reaction between the acid group of (3-carboxypropyl)triphenyl-phosphonium bromide and the primary amines of the acetylated dendrimer (G(5)-D-Ac). These dendrimers were fluorescently labeled with fluorescein isothiocyanate (FITC) to quantify cell association by flow cytometry and for visualization under confocal laser scanning microscopy to assess the mitochondrial targeting in vitro. The newly developed TPP-anchored dendrimer (G(5)-D-Ac-TPP) was efficiently taken up by the cells and demonstrated good mitochondrial targeting. In vitro cytotoxicity experiments carried out on normal mouse fibroblast cells (NIH-3T3) had greater cell viability in the presence of the G(5)-D-Ac-TPP compared to the parent unmodified G(5)-D. This mitochondria-targeted dendrimer-based nanocarrier could be useful for imaging as well as for selective delivery of bio-actives to the mitochondria for the treatment of diseases associated with mitochondrial dysfunction.
Keywords: Dendrimer; TPP; Conjugation; Mitochondrial targeting; Cytotoxicity;