Biomaterials (v.33, #10)

Balancing osteoblast functions and bacterial adhesion on functionalized titanium surfaces by Koon Gee Neoh; Xuefeng Hu; Dong Zheng; En Tang Kang (2813-2822).
The demand for orthopedic and dental implants will continue to grow, and for these applications, titanium and its alloys have been used extensively. While these implants have achieved high success rates, two major complications may be encountered: the lack of bone tissue integration and implant-centered infection. The surface of the implant, through its interactions with proteins, bacteria and tissue cells, plays a determining role in the success or failure of the implant. Ideally, to enhance the success of implants, their surfaces should inhibit bacterial colonization and concomitantly promote osteoblast functions. In this article, we discuss strategies for tailoring implant surfaces by exploiting the differences in the response of bacteria and osteoblasts to proteins and surface structures. Nevertheless, limitations still exist in the quest for an ideal implant surface. Further advances in this field will require concurrent development in surface modification techniques and a better understanding of the complex and highly inter-related events occurring at the implant surface after implantation.
Keywords: Titanium implant; Infection; Osteoblast; Bacterial adhesion; Surface modification;

An injectable biodegradable temperature-responsive gel with an adjustable persistence window by Jae Il Kim; Da Yeon Kim; Doo Yeon Kwon; Hwi Ju Kang; Jae Ho Kim; Byoung Hyun Min; Moon Suk Kim (2823-2834).
ɛ-Caprolactone (CL) and 3-benzyloxymethyl-6-methyl-1,4-dioxane-2,5-dion (fLA), with a benzyloxymethyl group at the 3-position of the lactide, were randomly copolymerized. The methoxy polyethylene glycol (MPEG)-b-[poly(ɛ-caprolactone)-ran-poly(3-benzyloxymethyl lactide) (PCL-ran-PfLA)] diblock copolymers were designed such that the PfLA content (0–15 mol%) in the PCL segment was varied. The MPEG-b-(PCL-ran-PfLA) diblock copolymers were derivatized by introducing a pendant benzyl group (MCxLy-OBn), hydroxyl group (MCxLy-OH), or carboxylic acid group (MCxLy-COOH) at the PfLA segment. The derivatized MPEG-b-(PCL-ran-PfLA) diblock copolymer solutions exhibited sol-to-gel phase transitions upon a temperature increase. The sol-to-gel phase transition depended on both the type of functional pendant group on the PfLA and the PfLA content in the PCL segment. MCxLy-COOH diblock copolymer solutions formed gels immediately after injection into Fischer rats. The gels gradually degraded over a period of 0–6 weeks after the initial injection, and the rate of degradation increased for higher concentrations of PfLA. Immunohistochemical characterization showed that the in vivo MPEG-b-(PCL-ran-PfLA) diblock copolymer gels provoked only a modest inflammatory response. These results show that the MPEG-b-(PCL-ran-PfLA) diblock copolymer gel described here may serve as a minimally invasive therapeutic, in situ-forming gel system with an adjustable temperature-responsive and in vivo biodegradable window.
Keywords: In situ forming gel; Phase transition; MPEG-b-(PCL-ran-PfLA) diblock copolymer; Crystallinity; Hydrophobicity;

The role of microtopography in cellular mechanotransduction by Laura E. McNamara; Richard Burchmore; Mathis O. Riehle; Pawel Herzyk; Manus J.P. Biggs; Chris D.W. Wilkinson; Adam S.G. Curtis; Matthew J. Dalby (2835-2847).
Mechanotransduction is crucial for cellular processes including cell survival, growth and differentiation. Topographically patterned surfaces offer an invaluable non-invasive means of investigating the cell response to such cues, and greater understanding of mechanotransduction at the cell–material interface has the potential to advance development of tailored topographical substrates and new generation implantable devices. This study focuses on the effects of topographical modulation of cell morphology on chromosomal positioning and gene regulation, using a microgrooved substrate as a non-invasive mechanostimulus. Intra-nuclear reorganisation of the nuclear lamina was noted, and the lamina was required for chromosomal repositioning. It appears that larger chromosomes could be predisposed to such repositioning. Microarrays and a high sensitivity proteomic approach (saturation DiGE) were utilised to identify transcripts and proteins that were subject to mechanoregulated changes in abundance, including mediators of chromatin remodelling and DNA synthesis linked to the changes in nucleolar morphology and the nucleoskeleton.
Keywords: Surface topography; Gene expression; Cell signalling; Molecular biology; Fibroblast;

Adult bone marrow derived mesenchymal stem cells are undifferentiated, multipotential cells and have the potential to differentiate into multiple lineages like bone, cartilage or fat. In this study, polyelectrolyte complex silk fibroin/chitosan blended porous scaffolds were fabricated and examined for its ability to support in vitro chondrogenesis of mesenchymal stem cells. Silk fibroin matrices provide suitable substrate for cell attachment and proliferation while chitosan are promising biomaterial for cartilage repair due to it’s structurally resemblance with glycosaminoglycans. We compared the formation of cartilaginous tissue in the silk fibroin/chitosan blended scaffolds with rat mesenchymal stem cells and cultured in vitro for 3 weeks. Additionally, pure silk fibroin scaffolds of non-mulberry silkworm, Antheraea mylitta and mulberry silkworm, Bombyx mori were also utilized for comparative studies. The constructs were analyzed for cell attachment, proliferation, differentiation, histological and immunohistochemical evaluations. Silk fibroin/chitosan blended scaffolds supported the cell attachment and proliferation as indicated by SEM observation, Confocal microscopy and metabolic activities. Alcian Blue and Safranin O histochemistry and expression of collagen II indicated the maintenance of chondrogenic phenotype in the constructs after 3 weeks of culture. Glycosaminoglycans and collagen accumulated in all the scaffolds and was highest in silk fibroin/chitosan blended scaffolds and pure silk fibroin scaffolds of A. mylitta. Chondrogenic differentiation of MSCs in the silk fibroin/chitosan and pure silk fibroin scaffolds was evident by real-time PCR analysis for cartilage-specific ECM gene markers. The results represent silk fibroin/chitosan blended 3D scaffolds as suitable scaffold for mesenchymal stem cells-based cartilage repair.
Keywords: Silk fibroin; Chitosan; Mesenchymal stem cells; Cartilage; Tissue engineering;

Non-invasive imaging of transplanted human neural stem cells and ECM scaffold remodeling in the stroke-damaged rat brain by 19F- and diffusion-MRI by Ellen Bible; Flavio Dell’Acqua; Bhavana Solanky; Anthony Balducci; Peter M. Crapo; Stephen F. Badylak; Eric T. Ahrens; Michel Modo (2858-2871).
Transplantation of human neural stem cells (hNSCs) is emerging as a viable treatment for stroke related brain injury. However, intraparenchymal grafts do not regenerate lost tissue, but rather integrate into the host parenchyma without significantly affecting the lesion cavity. Providing a structural support for the delivered cells appears important for cell based therapeutic approaches. The non-invasive monitoring of therapeutic methods would provide valuable information regarding therapeutic strategies but remains a challenge. Labeling transplanted cells with metal-based 1H-magnetic resonance imaging (MRI) contrast agents affects the visualization of the lesion cavity. Herein, we demonstrate that a 19F-MRI contrast agent can adequately monitor the distribution of transplanted cells, whilst allowing an evaluation of the lesion cavity and the formation of new tissue on 1H-MRI scans. Twenty percent of cells labeled with the 19F agent were of host origin, potentially reflecting the re-uptake of label from dead transplanted cells. Both T2- and diffusion-weighted MRI scans indicated that transplantation of hNSCs suspended in a gel form of a xenogeneic extracellular matrix (ECM) bioscaffold resulted in uniformly distributed cells throughout the lesion cavity. However, diffusion MRI indicated that the injected materials did not yet establish diffusion barriers (i.e. cellular network, fiber tracts) normally found within striatal tissue. The ECM bioscaffold therefore provides an important support to hNSCs for the creation of de novo tissue and multi-nuclei MRI represents an adept method for the visualization of some aspects of this process. However, significant developments of both the transplantation paradigm, as well as regenerative imaging, are required to successfully create new tissue in the lesion cavity and to monitor this process non-invasively.
Keywords: Tissue engineering; Stem cell; Stroke; MRI; 19F; Extracellular matrix;

The emerging field of stem cell therapy and biomaterials has begun to provide promising strategies for the treatment of ischemic cardiomyopathy. Platelet gel and cardiosphere-derived cells (CDCs) are known to be beneficial when transplanted separately post-myocardial infarction (MI). We hypothesize that pre-seeding platelet gel with CDCs can enhance therapeutic efficacy. Platelet gel and CDCs were derived from venous blood and heart biopsies of syngeneic rats, respectively. In vitro, the viability, growth, and morphology of CDCs cultured in platelet gel were characterized. When delivered into infarcted rat hearts, platelet gel pre-seeded with CDCs was more efficiently populated with endogenous cardiomyocytes and endothelial cells than platelet gel alone. Recruitment of endogenous c-kit positive cells was enhanced in the hearts treated with gel with CDC. At 3 weeks, the hearts treated with CDC-seeded platelet gel exhibited the greatest attenuation of adverse left ventricular (LV) remodeling and the highest cardiac function (i.e., LV ejection fraction) as compared to hearts transplanted with Gel only or vehicle controls. Histological analysis revealed that, though some transplanted CDCs differentiated into cardiomyocytes and endothelial cells in the recipients’ hearts, most of the incremental benefit arose from CDC-mediated endogenous repair. Pre-seeding platelet gel with CDCs enhanced the functional benefit of biomaterial therapy for treating myocardial infarction.
Keywords: Cardiac stem cells; Platelet gel; Myocardial infarction; Cardiac regeneration;

Endothelialization and patency of RGD-functionalized vascular grafts in a rabbit carotid artery model by Wenting Zheng; Zhihong Wang; Lijie Song; Qiang Zhao; Jun Zhang; Dong Li; Shufang Wang; Jihong Han; Xi-Long Zheng; Zhimou Yang; Deling Kong (2880-2891).
To address the growing demand of small-diameter vascular grafts for cardiovascular disease, it is necessary to develop substitutes with bio-functionalities, such as anticoagulation, rapid endothelialization, and smooth muscle regeneration. In this study, the small-diameter tubular grafts (2.2 mm) were fabricated by electrospinning of biodegradable polymer polycaprolactone (PCL) followed by functional surface coating with an arginine-glycine-aspartic acid (RGD)-containing molecule. The healing characteristics of the grafts were evaluated by implanting them in rabbit carotid arteries for 2 and 4 weeks. Results showed that at both time points, all 10 of the RGD-modified PCL grafts (PCL-RGD) were patent, whereas 4 of the 10 non-modified PCL grafts were occluded due to thrombus formation. Scanning electron microscopy (SEM) data showed abundant platelets adhering on the surface of the midportion of the PCL grafts. In contrast, only few platelets were observed on the PCL-RGD surface, suggesting that RGD modification significantly improved the hemocompatibility of the PCL grafts. Histological analysis demonstrated enhanced cell infiltration and homogeneous distribution within the PCL-RGD grafts in comparison with the PCL grafts. Furthermore, immunofluorescence staining also showed a 3-fold increase of endothelial coverage of the PCL-RGD grafts than that of PCL grafts at those two time points. After 4-week implantation, 65.3 ± 7.6% of the surface area of the PCL-RGD grafts was covered by smooth muscle cell layer, which is almost 23% more than that on the PCL grafts. The present study indicates that RGD-modified PCL grafts exhibit an improved remodeling and integration capability in revascularization.
Keywords: Vascular graft; RGD peptide; Endothelialization; Smooth muscle regeneration; Surface modification;

The effect of long-term release of Shh from implanted biodegradable microspheres on recovery from spinal cord injury in mice by Natalia Lowry; Susan K. Goderie; Patricia Lederman; Carol Charniga; Michael R. Gooch; Kristina D. Gracey; Akhilesh Banerjee; Supriya Punyani; Jerry Silver; Ravi S. Kane; Jeffrey H. Stern; Sally Temple (2892-2901).
After spinal cord injury (SCI), loss of cells and damage to ascending and descending tracts can result in paralysis. Current treatments for SCI are based on patient stabilization, and much-needed regenerative therapies are still under development. To activate and instruct stem and progenitor cells or injured tissue to aid SCI repair, it is important to modify the injury environment for a protracted period, to allow time for cell activation, proliferation and appropriate fate differentiation. Shh plays a critical role in spinal cord formation, being involved in multiple processes: it promotes production of motor neurons and oligodendrocytes from ventral cord progenitor cells and serves as an axon guidance molecule. Hence Shh is a candidate pleiotropic beneficial environmental factor for spinal cord regeneration. Here we show that administration of biodegradable microspheres that provide sustained, controlled delivery of Shh resulted in significant functional improvement in two different mouse models of SCI: contusion and dorsal hemioversection. The mechanism is multifactorial, involving increased proliferation of endogenous NG2+ oligodendrocyte lineage cells, decreased astrocytic scar formation and increased sprouting and growth of corticospinal (CST) and raphespinal tract (RST) fibers. Thus, long-term administration of Shh is a potential valuable therapeutic intervention for SCI.
Keywords: Mouse; Spinal cord injury; Shh; Biodegradable microspheres;

The proliferation of anchorage-dependent cells of mesenchymal origin requires the attachment of the cells to substrates. Thus, cells that are poorly attached to substrates exhibit retarded cell cycle progression or apoptotic death. A major disadvantage of most polymers used in tissue engineering is their hydrophobicity; hydrophobic surfaces do not allow cells to attach firmly and, therefore, do not allow normal proliferation rates. In this study, we investigated the molecular mechanism underlying the reduced proliferation rate of cells that are poorly attached to substrates. There was an inverse relationship between the activity of the small GTPase RhoA (RhoA) and the cell proliferation rate. RhoA activity correlated inversely with the strength of cell adhesion to the substrates. The high RhoA activity in the cells poorly attached to substrates caused an increase in the activity of Rho-associated kinase (ROCK), a well-known effector of RhoA that upregulated the activity of phosphatase and tensin homolog (PTEN). The resulting activated PTEN downregulated Akt activity, which is essential for cell proliferation. Thus, the cells that were poorly attached to substrates showed low levels of cell proliferation because the RhoA-ROCK-PTEN pathway was hyperactive. In addition, RhoA activity seemed to be related to focal adhesion kinase (FAK) activity. Weak FAK activity in these poorly attached cells failed to downregulate the high RhoA activity that restrained cell proliferation. Interestingly, reducing the expression of any component of the RhoA-ROCK-PTEN pathway rescued the proliferation rate without physico-chemical surface modifications. Based on these results, we suggest that the RhoA-ROCK-PTEN pathway acts as a molecular switch to control cell proliferation and determine anchorage dependence. In cells that are poorly attached to substrates, its inhibition is sufficient to restore cell proliferation without the need for physico-chemical modification of the material surface.
Keywords: Cell adhesion; Cell proliferation; Cell signaling; Wettability;

Biologic scaffold composed of skeletal muscle extracellular matrix by Matthew T. Wolf; Kerry A. Daly; Janet E. Reing; Stephen F. Badylak (2916-2925).
Biologic scaffolds prepared from the extracellular matrix (ECM) of decellularized mammalian tissues have been shown to facilitate constructive remodeling in injured tissues such as skeletal muscle, the esophagus, and lower urinary tract, among others. The ECM of every tissue has a unique composition and structure that likely has direct effects on the host response and it is plausible that ECM harvested from a given tissue would provide distinct advantages over ECM harvested from nonhomologous tissues. For example, a tissue specific muscle ECM scaffold may be more suitable for constructive remodeling of skeletal muscle than non-homologous ECM tissue sources. The present study describes an enzymatic and chemical decellularization process for isolating skeletal muscle ECM scaffolds using established decellularization criteria and characterized the structure and chemical composition of the resulting ECM. The results were compared to those from a non-muscle ECM derived from small intestine (SIS). Muscle ECM was shown to contain growth factors, glycosaminoglycans, and basement membrane structural proteins which differed from those present in SIS. Myogenic cells survived and proliferated on muscle ECM scaffolds in vitro, and when implanted in a rat abdominal wall injury model in vivo was shown to induce a constructive remodeling response associated with scaffold degradation and myogenesis in the implant area; however, the remodeling outcome did not differ from that induced by SIS by 35 days post surgery. These results suggest that superior tissue remodeling outcomes are not universally dependent upon homologous tissue derived ECM scaffold materials.
Keywords: ECM (extracellular matrix); Muscle; Scaffold; Regenerative medicine; Animal model;

The impact of PLGA scaffold orientation on in vitro cartilage regeneration by Yingying Zhang; Fei Yang; Kai Liu; Hong Shen; Yueqian Zhu; Wenjie Zhang; Wei Liu; Shenguo Wang; Yilin Cao; Guangdong Zhou (2926-2935).
The success of in vitro cartilage regeneration provides a promising approach for cartilage repair. However, the currently engineered cartilage in vitro is unsatisfactory for clinical application due to non-homogeneous structure, inadequate thickness, and poor mechanical property. It has been widely reported that orientation of scaffolds can promote cell migration and thus probably contributes to improving tissue regeneration. This study explored the impact of microtubular oriented scaffold on in vitro cartilage regeneration. Porcine articular chondrocytes were seeded into microtubule-oriented PLGA scaffolds and non-oriented scaffolds respectively. A long-term in vitro culture followed by a long-term in vivo implantation was performed to evaluate the influence of scaffold orientation on cartilage regeneration. The current results showed that the oriented scaffolds could efficiently promote cell migration towards the inner region of the constructs. After 12 weeks of in vitro culture, the chondrocyte-scaffold constructs in the oriented group formed thicker cartilage with more homogeneous structure, stronger mechanical property, and higher cartilage matrix content compared to the non-oriented group. Furthermore, the in vitro engineered cartilage based on oriented scaffolds showed better cartilage formation in terms of size, wet weight, and homogeneity after 12-week in vivo implantation in nude mice. These results indicated that the longitudinal microtubular orientation of scaffolds can efficiently improve the structure and function of in vitro engineered cartilage.
Keywords: Scaffold orientation; PLGA; Cartilage; Tissue engineering;

The transport of non-surfactant based paclitaxel loaded magnetic nanoparticles across the blood brain barrier in a rat model by Fahima Dilnawaz; Abhalaxmi Singh; Sujeet Mewar; Uma Sharma; N.R. Jagannathan; Sanjeeb Kumar Sahoo (2936-2951).
There is much interest in utilizing the intrinsic properties of magnetic nanoparticles (MNPs) for the theranostic approaches in medicine. With an aim to develop a potential therapeutics for glioma treatment, efficacy of aqueous dispersible paclitaxel loaded MNPs (Pac-MNPs) were studied in glioblastoma cell line (U-87). The identified potential receptor, glycoprotein non-metastatic melanoma protein B (GPNMB) overexpressed by glioblastoma cells, was actively targeted using GPNMB conjugated Pac-MNPs in U-87 cells. As blood brain barrier (BBB) is the primary impediment in the treatment of glioblastoma, therefore, an attempt was taken to evaluate the biodistribution and brain uptake of Pac-MNPs in rats. The bioavailability of Pac-MNPs illustrated a prolonged blood circulation in vivo, which demonstrated the presence of significant amounts of drug in rat brain tissues as compared to native paclitaxel. Further, the transmission electron microscopy (TEM) study revealed significant accumulation of the Pac-MNPs in the brain tissues. Being an effective contrast enhancement agent for magnetic resonance imaging (MRI) at tissue levels, the MNPs devoid of any surfactant demonstrated enhanced contrast effect in liver and brain imaging. Hence, the significant prevalence of drugs in the rat brain tissues, in vitro targeting potentiality as well as the augmented contrast effect elicit the non-invasive assessment and theranostic applications of MNPs for brain tumor therapy.
Keywords: Magnetic nanoparticles; Glioblastoma; Blood brain barrier; Biodistribution;

Internalization of C60 fullerenes into cancer cells with accumulation in the nucleus via the nuclear pore complex by Mustafa Raoof; Yuri Mackeyev; Matthew A. Cheney; Lon J. Wilson; Steven A. Curley (2952-2960).
A highly water-soluble, non-ionic, and non-cytotoxic fullerene malonodiserinolamide-derivatized fullerene C60 (C60-ser) is under investigation as a potential nanovector to deliver biologic and cancer drugs across biological barriers. Using laser-scanning confocal microscopy and flow cytometry, we find that PF-633 fluorophore conjugated C60-ser nanoparticles (C60-serPF) are internalized within living cancer cells in association with serum proteins through multiple energy-dependent pathways, and escape endocytotic vesicles to eventually localize and accumulate in the nucleus of the cells through the nuclear pore complex. Furthermore, in a mouse model of liver cancer, the C60-serPF conjugate is detected in most tissues, permeating through the altered vasculature of the tumor and the tightly-regulated blood brain barrier while evading the reticulo-endothelial system.
Keywords: Fullerenes; Nucleus; Cancer; Liver; Nuclear pore complex; Biodistribution;

The resistance of breast cancer stem cells to conventional hyperthermia and their sensitivity to nanoparticle-mediated photothermal therapy by Andrew R. Burke; Ravi N. Singh; David L. Carroll; James C.S. Wood; Ralph B. D’Agostino; Pulickel M. Ajayan; Frank M. Torti; Suzy V. Torti (2961-2970).
Breast tumors contain a small population of tumor initiating stem-like cells, termed breast cancer stem cells (BCSCs). These cells, which are refractory to chemotherapy and radiotherapy, are thought to persist following treatment and drive tumor recurrence. We examined whether BCSCs are similarly resistant to hyperthermic therapy, and whether nanoparticles could be used to overcome this resistance. Using a model of triple-negative breast cancer stem cells, we show that BCSCs are markedly resistant to traditional hyperthermia and become enriched in the surviving cell population following treatment. In contrast, BCSCs are sensitive to nanotube-mediated thermal treatment and lose their long-term proliferative capacity after nanotube-mediated thermal therapy. Moreover, use of this therapy in vivo promotes complete tumor regression and long-term survival of mice bearing cancer stem cell-driven breast tumors. Mechanistically, nanotube thermal therapy promotes rapid membrane permeabilization and necrosis of BCSCs. These data suggest that nanotube-mediated thermal treatment can simultaneously eliminate both the differentiated cells that constitute the bulk of a tumor and the BCSCs that drive tumor growth and recurrence.
Keywords: Breast cancer; Cancer stem cells; Nanotube; Hyperthermia; Near-infrared radiation; Photothermal therapy;

The role of non-covalent interactions in anticancer drug loading and kinetic stability of polymeric micelles by Chuan Yang; Amalina B. Ebrahim Attia; Jeremy P.K. Tan; Xiyu Ke; Shujun Gao; James L. Hedrick; Yi-Yan Yang (2971-2979).
A new series of acid- and urea-functionalized polycarbonate block copolymers were synthesized via organocatalytic living ring-opening polymerization using methoxy poly(ethylene glycol) (PEG) as a macroinitiator to form micelles as drug delivery carriers. The micelles were characterized for critical micelle concentration, particle size and size distribution, kinetic stability and loading capacity for a model anticancer drug, doxorubicin (DOX) having an amine group. The acid/urea groups were placed in block forms (i.e. acid as the middle block or the end block) or randomly distributed in the polycarbonate block to investigate molecular structure effect. The micelles formed from the polymers in both random and block forms provided high drug loading capacity due to strong ionic interaction between the acid in the polymer and the amine in DOX. However, the polymers with acid and urea groups placed in the block forms formed micelles with wider size distribution (two size populations), and their DOX-loaded micelles were less stable. The number of acid/urea groups in the random form was further varied from 5 to 8, 13 and 19 to study its effects on self-assembly behaviors and DOX loading. An increased number of acid/urea groups yielded DOX-loaded micelles with smaller size and enhanced kinetic stability because of improved inter-molecular polycarbonate–polycarbonate (urea–urea and urea–acid) hydrogen-bonding and polycarbonate–DOX (acid–amine) ionic interactions. However, when the number of acid/urea groups was 13 or higher, micelles aggregated in a serum-containing medium, and freeze-dried DOX-loaded micelles were unable to re-disperse in an aqueous solution. Among all the polymers synthesized in this study, 1b with 8 acid/urea groups in the random form had the optimum properties. In vitro release studies showed that DOX release from 1b micelles was sustained over 7 h without significant initial burst release. MTT assays demonstrated that the polymer was not toxic towards HepG2 and HEK293 cells. Importantly, DOX-loaded micelles were potent against HepG2 cells with IC50 of 0.26 mg/L, comparable to that of free DOX (IC50: 0.20 mg/L). In addition, DOX-loaded 1b micelles yielded lower DOX content in the heart tissue of the tested mice as compared to free DOX formulation after i.v. injection. These findings signify that 1b micelles may be a promising carrier for delivery of anticancer drugs that contain amine groups.
Keywords: Micelles; Carboxylic acid group; Urea group; Non-covalent interaction; Kinetic stability; Drug loading;

Gene transfer by chemical vectors, and endocytosis routes of polyplexes, lipoplexes and lipopolyplexes in a myoblast cell line by Ludivine Billiet; Jean-Pierre Gomez; Mathieu Berchel; Paul-Alain Jaffrès; Tony Le Gall; Tristan Montier; Emilie Bertrand; Hervé Cheradame; Philippe Guégan; Mathieu Mével; Bruno Pitard; Thierry Benvegnu; Pierre Lehn; Chantal Pichon; Patrick Midoux (2980-2990).
Chemical vectors are widely developed for providing safe DNA delivery systems. It is well admitted that their endocytosis and intracellular trafficking are critical for the transfection efficiency. Here, we have compared the endocytic pathways of lipoplexes, polyplexes and lipopolyplexes formed with carriers of various chemical compositions. Engineered C2C12 mouse myoblast cells expressing Rab5-EGFP, Rab7-EGFP or Cav1-GFP were used to monitor the location of the plasmid DNA into the endocytic compartments by real time fluorescence confocal microscopy. We observed that (i) DNA complexes made with dioleyl succinyl paromomycin:O,O-dioleyl-N-histamine phosphoramidate (DOSP/MM27) liposomes or histidinylated lPEI (His-lPEI) allowing the highest transfection efficiency displayed a positive ζ potential and were internalized by clathrin-mediated endocytosis, (ii) DOSP/MM27 lipoplexes were 6-times more internalized than His-lPEI polyplexes, (iii) all negatively charged DNA complexes lead to less efficient transfection and entered the cells via caveolae and (iv) lipopolyplexes allowing high transfection efficiency were weakly internalized via caveolae. Our results indicate that the transfection efficiency is better correlated with the nature of the endocytic pathway than with the uptake efficacy. This study shows also that engineered cells expressing specific fluorescent compartments are convenient tools to monitor endocytosis of a fluorescent plasmid DNA by real time fluorescence confocal microscopy.
Keywords: Gene transfer; DNA, liposomes; Polymers; Confocal microscopy;

The use of nano-quercetin to arrest mitochondrial damage and MMP-9 upregulation during prevention of gastric inflammation induced by ethanol in rat by Somsuta Chakraborty; Sami Stalin; Nirmalendu Das; Somsubhra Thakur Choudhury; Swarupa Ghosh; Snehasikta Swarnakar (2991-3001).
Gastric ulcer is a multifaceted process that involves reactive oxygen species (ROS) generation, extracellular matrix degradation and mitochondrial damage. Mitochondria play a crucial role for homeostasis of ROS and cell survival. In our study, we investigated the efficacy and mechanism of polymeric nanocapsuled quercetin (NQC) over the free quercetin (QC) molecule in prevention of ethanol-induced gastric ulcer in rat. NQC possessed significantly higher efficacy (∼20 fold) than free QC while preventing gastric ulcers. Our data show that prior administration of NQC and/or QC significantly blocked synthesis and secretion of matrix metalloproteinase (MMP)-9 as well as infiltration of inflammatory cells and oxidative damage in rat gastric tissues. As compared to free QC, NQC protected much better the mitochondrial integrity and size along with mitochondrial functions by controlling succinate dehydrogenase and NADH oxidase in rat gastric tissues. In addition, both free QC and NQC down regulated PARP-1 as well as apoptosis during protection against ethanol-induced gastric ulcer. Herein, the effect of NQC was greater than QC on expression of enzymes like cyclooxygenase and nitric oxidase synthase (NOS)-2. We conclude that NQC with greater bioavailability offers significantly higher potency in downregulating MMP-9 and NOS-2 as well as oxidative stress in blocking ethanol-induced gastric ulcer.
Keywords: Nanocapsulated quercetin; Gastric ulcer; Matrix metalloproteinase-9; Nitric oxide synthase; Oxidative stress; Atomic force microscopy;

Red blood cell (RBC) transfusions are an important clinical intervention. However, RBC express hundreds of non-ABO antigens making alloimmunization a significant risk. RhD expression is the most immunologically important non-ABO antigen. Availability of RhD blood, often problematic in North America and Europe, is a significant issue in Asia and Africa where RhD blood is uncommon (<0.5% of supply). The immunocamouflage of RhD is readily accomplished by the covalent grafting of methoxypoly(ethylene glycol) [mPEG] to the RBC membrane. To determine if RhD immunocamouflage would inhibit its immunologic recognition, an in vitro RhD-sensitized antigen presentation assay using PBMC and dendritic cells (DC) from RhD-sensitized women was used. The immunological effects of polymer grafting to an immunodominant RhD peptide, purified RhD protein and intact RhD+ RBC were examined via T cell proliferation and cytokine release assays. At Day 11, PEGylation significantly attenuated T cell proliferation arising from RhD peptide (∼80 → 5%), protein (36 → 0.2%) and intact RBC (33 → 1.4%). Cytokine secretion was similarly blunted following PEGylation of the purified protein or intact RBC. These data support the immunomodulatory effects of PEGylation and the potential utility of this technology in transfusion medicine - especially in situations where RhD blood is rare or in short supply.
Keywords: Immunomodulation; Methoxypoly(ethylene glycol); Allogeneic cell; Red blood cell; Antigenicity; RhD;

Magnetite-loaded fluorine-containing polymeric micelles for magnetic resonance imaging and drug delivery by Xiaolong Li; Huan Li; Guoqiang Liu; Ziwei Deng; Shuilin Wu; Penghui Li; Zushun Xu; Haibo Xu; Paul K. Chu (3013-3024).
Magnetite (Fe3O4) – loaded polymer micelles (denoted as “magnetomicelles”) are produced by self-assembly of fluorine-containing amphiphilic poly(HFMA-g-PEGMA) copolymers with oleic acid modified Fe3O4 nanoparticles in an aqueous medium. The oleic acid modified Fe3O4 nanoparticles form small clusters in the poly(HFMA-g-PEGMA) micelles with a mean diameter of 100 nm and the magnetomicelles show high stability in an aqueous medium due to the high hydrophobic fluorine segments in graft copolymers enhance the stability of the micelles. The magnetomicelles also show good cytocompatibility based on the MTT cytotoxicity assay and possess paramagnetic properties with saturation magnetization of 17.14 emu/g.Their good stability, cytocompatibility, and paramagnetic properties render the materials attractive in drug delivery and in vivo magnetic resonance imaging (MRI) applications. Controlled release of hydrophobic drug-5-fluorouracil is achieved from the magnetomicelles with a loading efficiency of 20.94 wt%. The magnetomicelles have transverse relaxivity rates (r 2 ) of 134.27 mM−1 s−1 and exhibit high efficacy as a negative MRI agent in T2-weighted imaging. In vivo MRI studies demonstrate that the contrast between liver and spleen is enhanced by the magnetomicelles. These favorable properties suggest clinical use as nanocarriers in drug delivery applications and contrast agents in MRI.
Keywords: Copolymers; Fluorine-fluoride; Micelle; MRI; Drug delivery;

Gene delivery using dendrimer-entrapped gold nanoparticles as nonviral vectors by Yuebin Shan; Ting Luo; Chen Peng; Ruilong Sheng; Amin Cao; Xueyan Cao; Mingwu Shen; Rui Guo; Helena Tomás; Xiangyang Shi (3025-3035).
Development of highly efficient nonviral gene delivery vectors still remains a great challenge. In this study, we report a new gene delivery vector based on dendrimer-entrapped gold nanoparticles (Au DENPs) with significantly higher gene transfection efficiency than that of dendrimers without AuNPs entrapped. Amine-terminated generation 5 poly(amidoamine) (PAMAM) dendrimers (G5.NH2) were utilized as templates to synthesize AuNPs with different Au atom/dendrimer molar ratios (25:1, 50:1, 75:1, and 100:1, respectively). The formed Au DENPs were used to complex two different pDNAs encoding luciferase (Luc) and enhanced green fluorescent protein (EGFP), respectively for gene transfection studies. The Au DENPs/pDNA polyplexes with different N/P ratios and compositions of Au DENPs were characterized by gel retardation assay, light scattering, zeta potential measurements, and atomic force microscopic imaging. We show that the Au DENPs can effectively compact the pDNA, allowing for highly efficient gene transfection into the selected cell lines as demonstrated by both Luc assay and fluorescence microscopic imaging of the EGFP expression. The transfection efficiency of Au DENPs with Au atom/dendrimer molar ratio of 25:1 was at least 100 times higher than that of G5.NH2 dendrimers without AuNPs entrapped at the N/P ratio of 2.5:1. The higher gene transfection efficiency of Au DENPs is primarily due to the fact that the entrapment of AuNPs helps preserve the 3-dimensional spherical morphology of dendrimers, allowing for more efficient interaction between dendrimers and DNA. With the less cytotoxicity than that of G5.NH2 dendrimers demonstrated by thiazoyl blue tetrazolium bromide assay and higher gene transfection efficiency, it is expected that Au DENPs may be used as a new gene delivery vector for highly efficient transfection of different genes for various biomedical applications.
Keywords: PAMAM dendrimers; Gold nanoparticles; Gene delivery; Transfection efficiency;

A large-scale in vitro 3D tumor model was generated to evaluate gene delivery procedures in vivo. This 3D tumor model consists of a “tissue-like” spheroid that provides a micro-environment supportive of melanoma proliferation, allowing cells to behave similarly to cells in vivo. This functional spheroid measures approximately 1 cm in diameter and can be used to effectively evaluate plasmid transfection when testing various electroporation (EP) electrode applicators. In this study, we identified EP conditions that efficiently transfect green fluorescent protein (GFP) and interleukin 15 (IL-15) plasmids into tumor cells residing in the 3D construct. We found that plasmids delivered using a 6-plate electrode applying 6 pulses with nominal electric field strength of 500 V/cm and pulse-length of 20 ms produced significant increase of GFP (7.3-fold) and IL-15 (3.0–fold) expression compared to controls. This in vitro 3D model demonstrates the predictability of cellular response toward delivery techniques, limits the numbers of animals employed for transfection studies, and may facilitate future developments of clinical trials for cancer therapies in vivo.
Keywords: 3D tumor model; Melanoma; Electroporation; Gene transfer; Bioreactor;

In vivo circulation, clearance, and biodistribution of polyglycerol grafted functional red blood cells by Rafi Chapanian; Iren Constantinescu; Donald E. Brooks; Mark D. Scott; Jayachandran N. Kizhakkedathu (3047-3057).
The in vivo circulation of hyperbranched polyglycerol (HPG) grafted red blood cells (RBCs) was investigated in mice. The number of HPG molecules grafted per RBC was measured using tritium labeled HPGs (3H-HPG) of different molecular weights; the values ranged from 1 × 105 to 2 × 106 molecules per RBC. HPG-grafted RBCs were characterized in vitro by measuring the electrophoretic mobility, complement mediated lysis, and osmotic fragility. Our results show that RBCs grafted with 1.5 × 105 HPG molecules per RBC having molecular weights 20 and 60 kDa have similar characteristics as that of control RBCs. The in vivo circulation of HPG-grafted RBCs was measured by a tail vain injection of 3H-HPG60K-RBC in mice. The radioactivity of isolated RBCs, whole blood, plasma, different organs, urine and feces was evaluated at different time intervals. The portion of 3H-HPG60K-RBC that survived the first day in mice (52%) remained in circulation for 50 days. Minimal accumulation radioactivity in organs other than liver and spleen was observed suggesting the normal clearance mechanism of modified RBCs. Animals gained normal weights and no abnormalities observed in necropsy analysis. The stability of the ester-amide linker between the RBC and HPG was evaluated by comparing the clearance rate of 3H-HPG60K-RBC and PKH-26 lipid fluorescent membrane marker labeled HPG60K-RBCs. HPG modified RBCs combine the many advantages of a dendritic polymer and RBCs, and hold great promise in systemic drug delivery and other applications of functional RBC.
Keywords: Polyglycerol; Functional red blood cells; In vivo circulation; Biodistribution;

Responsive fluorescent Bi2O3@PVA hybrid nanogels for temperature-sensing, dual-modal imaging, and drug delivery by Hongbo Zhu; Yaoxin Li; Runqi Qiu; Lei Shi; Weitai Wu; Shuiqin Zhou (3058-3069).
The polymer-inorganic hybrid nanogels with temperature-responsive characteristic are of considerable current interest to many fields ranging from fundamental biomaterials science to bionanomedicine. This paper reports the preparation of temperature-responsive hybrid nanogels by immobilization of Bi2O3 quantum dots (QDs) in the interior of a nanogel of poly(vinyl alcohol) (PVA). Unlike conventional temperature-responsive hybrid nanogels with the responsive features deriving from the temperature-responsive polymers (e.g. PNIPAMs or non-linear PEGs), we demonstrate that QDs can work cooperatively with the gel networks of PVA, an unconventional responsive polymer, to enable the temperature-induced volume phase transition of the designed Bi2O3@PVA hybrid nanogels. Building on the rationales, Bi2O3@PVA hybrid nanogels can adapt to a surrounding fluids of different temperatures over the physiologically important range of 37–40 °C, convert the disruptions in homeostasis of environmental temperature into high-sensitive fluorescent signals, enter into the mouse melanoma B16F10 cells for dark-field and fluorescence dual-modal imaging, and regulate the release of a model anticancer drug temozolomide. The unconventional strategy that can broaden the design scheme of temperature-responsive hybrid nanogels for theranostic action should enhance our ability to address the complexity of biological systems.
Keywords: Hybrid nanogels; Poly(vinyl alcohol); Quantum dots; Optical temperature-sensing; Dual-modal imaging; Drug delivery;