Hot off the presses! Aug 01 Biomat

The Aug 01 issue of the Biomat is now up on Pubget (About Biomat): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• Editorial board
  - Biomat 32(22):IFC (2011)
  
• An equivalent strain/Coffin–Manson approach to multiaxial fatigue and life prediction in superelastic Nitinol medical devices
  - Biomat 32(22):4987-4993 (2011)
  Medical devices, particularly endovascular stents, manufactured from superelastic Nitinol, a near-equiatomic alloy of Ni and Ti, are subjected to complex mixed-mode loading conditions in vivo, including axial tension and compression, radial compression, pulsatile, bending and torsion. Fatigue lifetime prediction methodologies for Nitinol, however, are invariably based on uniaxial loading and thus fall short of accurately predicting the safe lifetime of stents under the complex multiaxial loading conditions experienced physiologically. While there is a considerable body of research documented on the cyclic fatigue of Nitinol in uniaxial tension or bending, there remains an almost total lack of comprehensive fatigue lifetime data for other loading conditions, such as torsion and tension/torsion. In this work, thin-walled Nitinol tubes were cycled in torsion at various mean and alternating strains to investigate the fatigue life behavior of Nitinol and results compared to! equivalent fatigue data collected under uniaxial tensile/bending loads. Using these strain-life results for various loading modes and an equivalent referential (Lagrangian) strain approach, a strategy for normalizing these data is presented. Based on this strategy, a fatigue lifetime prediction model for the multiaxial loading of Nitinol is presented utilizing a modified Coffin–Manson approach where the number of cycles to failure is related to the equivalent alternating transformation strain.
• Intraocular degradation behavior of crosslinked and linear poly(trimethylene carbonate) and poly(d,l-lactic acid)
  - Biomat 32(22):4994-5002 (2011)
  The intraocular degradation behavior of poly(trimethylene carbonate) (PTMC) networks and poly(d,l-lactic acid) (PDLLA) networks and of linear high molecular weight PTMC and PDLLA was evaluated. PTMC is known to degrade by enzymatic surface erosion in vivo, whereas PDLLA degrades by hydrolytic bulk degradation. Rod shaped specimens were implanted in the vitreous of New Zealand white rabbits for 6 or 13 wk. All materials were well tolerated in the rabbit vitreous. The degradation of linear high molecular weight PTMC and PTMC networks was very slow and no significant mass loss was observed within 13 wk. Only some minor signs of macrophage mediated erosion were found. The fact that no significant enzymatic surface erosion occurs can be related to the avascularity of the vitreous and the limited number of cells it contains. PDLLA samples showed more evident signs of degradation. For linear PDLLA significant swelling and a large decrease in molecular weight in time was obser! ved and PDLLA network implants started to lose mass within 13 wk. Of the tested materials, PDLLA networks seem to be most promising for long term degradation controlled intravitreal drug delivery since this material degrades without significant swelling. Furthermore the preparation method of these networks allows easy and efficient incorporation of drugs.
• Microstructure design of biodegradable scaffold and its effect on tissue regeneration
  - Biomat 32(22):5003-5014 (2011)
  Biodegradable scaffolds play a critical role in therapeutic tissue engineering, in which the matrix degradation and tissue ingrowth are of particular importance for determining the ongoing performance of tissue-scaffold system during regenerative process. This paper aims to explore the mechanobiological process within biodegradable scaffolds, where the representative volume element (RVE) is extracted from periodic scaffold micro-architectures as a base-cell design model. The degradation of scaffold matrix is modeled in terms of a stochastic hydrolysis process enhanced by diffusion-controlled autocatalysis; and the tissue ingrowth is modeled through the mechano-regulatory theory. By using the finite element based homogenization technique and topology optimization approach, the effective properties of various periodic scaffold structures are obtained. To explore the effect of scaffold design on the mechanobiological evolutions of tissue-scaffold systems, different scaffo! ld architectures are considered for polymer degradation and tissue regeneration. It is found that the different tissues can grow into the degraded voids inside the polymer matrix. It is demonstrated that the design of scaffold architecture has a considerable impact on the tissue regeneration outcome, which exhibits the importance of implementing different criteria in scaffold micro-structural design, before being fabricated via rapid prototyping technique, e.g. solid free-form fabrication (SFF). This study models such an interactive process of scaffold degradation and tissue growth, thereby providing some new insights into design of biodegradable scaffold micro-architecture for tissue engineering.
• Array-based functional screening of growth factors toward optimizing neural stem cell microenvironments
  - Biomat 32(22):5015-5022 (2011)
  To gain insights into the effect of various growth factors on the behaviors of neural stem cells, cell culture assays were performed on the array that displayed five different growth factors including basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-1, brain-derived neurotrophic factor, and ciliary neurotrophic factor. These factors were expressed in Escherichia coli as fusion proteins with a hexahistidine sequence and arrayed on a nickel ion-functionalized chip as single factors or the combination of two factors. Neural stem cells obtained from the fetal rat brain were cultured on the array to investigate their proliferation and differentiation. It was shown that the five growth factors displayed as a single component had significant impacts on cell behaviors. These effects are overall in accordance with those reported previously. On the other hand, in the case that two different growth factors were co-displayed on a single spot, the! behaviors of neural stem cells could not be simply predicted from their individual effects. We performed a multivariate cluster analysis for the quantitative data on cell proliferation and differentiation. It was shown that the effect of two growth factors co-displayed was competitive, synergistic, or destructive depending on the combinations. In other peculiar cases, the effect of growth factors was totally different from those of individual factors.
• Induced pluripotent stem cells for neural tissue engineering
  - Biomat 32(22):5023-5032 (2011)
  Induced pluripotent stem cells (iPSCs) hold great promise for cell therapies and tissue engineering. Neural crest stem cells (NCSCs) are multipotent and represent a valuable system to investigate iPSC differentiation and therapeutic potential. Here we derived NCSCs from human iPSCs and embryonic stem cells (ESCs), and investigated the potential of NCSCs for neural tissue engineering. The differentiation of iPSCs and the expansion of derived NCSCs varied in different cell lines, but all NCSC lines were capable of differentiating into mesodermal and ectodermal lineages, including neural cells. Tissue-engineered nerve conduits were fabricated by seeding NCSCs into nanofibrous tubular scaffolds, and used as a bridge for transected sciatic nerves in a rat model. Electrophysiological analysis showed that only NCSC-engrafted nerve conduits resulted in an accelerated regeneration of sciatic nerves at 1 month. Histological analysis demonstrated that NCSC transplantation promote! d axonal myelination. Furthermore, NCSCs differentiated into Schwann cells and were integrated into the myelin sheath around axons. No teratoma formation was observed for up to 1 year after NCSC transplantation in vivo. This study demonstrates that iPSC-derived multipotent NCSCs can be directly used for tissue engineering and that the approach that combines stem cells and scaffolds has tremendous potential for regenerative medicine applications.
• Tissue-engineered bone formation using periosteal-derived cells and polydioxanone/pluronic F127 scaffold with pre-seeded adipose tissue-derived CD146 positive endothelial-like cells
  - Biomat 32(22):5033-5045 (2011)
  The aim of this study was to generate tissue-engineered bone formation using periosteal-derived cells seeded into a polydioxanone/pluronic F127 (PDO/Pluronic F127) scaffold with adipose tissue-derived CD146 positive endothelial-like cells. Considering the hematopoietic and mesenchymal phenotypes of adipose tissue-derived cells cultured in EBM-2 medium, CD146 positive adipose tissue-derived cells was sorted to purify more endothelial cells in characterization. These sorted cells were referred to as adipose tissue-derived CD146 positive endothelial-like cells. Periosteum is a good source of osteogenic cells for tissue-engineered bone formation. Periosteal-derived cells were found to have good osteogenic capacity in a PDO/Pluronic F127 scaffold, which could provide a suitable environment for the osteoblastic differentiation of these cells. Through the investigation of capillary-like tube formation on matrigel and the cellular proliferation of adipose tissue-derived CD146 ! positive endothelial-like cells cultured in different media conditions, we examined these cells could be cultured in EBM-2 with osteogenic induction factors. We also observed that the osteogenic activity of periosteal-derived cells could be good in EBM-2 with osteogenic induction factors, in the early period of culture. The experimental results obtained in the miniature pig model suggest that tissue-engineered bone formation using periosteal-derived cells and PDO/Pluronic F127 scaffold with pre-seeded adipose tissue-derived CD146 positive endothelial-like cells can be used to restore the bony defects of the maxillofacial region when used in clinics.
• A phosphorylcholine-modified chitosan polymer as an endothelial progenitor cell supporting matrix
  - Biomat 32(22):5046-5055 (2011)
  The aim of the present study was to develop a new biopolymer to increase endothelial progenitor cells (EPC) survival and amplification. As a cell culture platform, bone marrow-derived cells (BMDC) were used to investigate the biocompatibility of chitosan–phosphorylcholine (CH–PC). On CH–PC, BMDC were found in colonies with a mortality rate similar to that of fibronectin (FN), the control matrix. Adhesion/proliferation assays demonstrated a greater number of BMDC on CH–PC after 7 days with an amplification phase occurring during the second week. Difference in adhesion mechanisms between (CH–PC) and the control FN matrix suggest distinctive cell retention ability. Confocal microscopy analyses confirmed that (CH–PC) supported the survival/differentiation of endothelial cells. Moreover, flow cytometry analyses demonstrated that, (CH–PC) increased the percentage of progenitor cells (CD117+CD34+) (7.1 ± 0.8%, FN: 4.1 ± 0.8%) and EPC (CD117+CD34+VEGFR-2+CD31+)! (2.33 ± 0.6%, FN: 0.25 ± 0.1%), while the mesenchymal stem cell fraction (CD44+CD106+CD90+) was decreased (0.07 ± 0.01%, FN: 0.55 ± 0.22%). Polymeric substrate CH–PC might provide a suitable surface to promote the amplification of EPC for future vascular therapeutic applications.
• The role of substratum compliance of hydrogels on vascular endothelial cell behavior
  - Biomat 32(22):5056-5064 (2011)
  Cardiovascular disease (CVD) remains a leading cause of death both within the United States (US) as well as globally. In 2006 alone, over one-third of all deaths in the US were attributable to CVD. The high prevalence, mortality, morbidity, and socioeconomic impact of CVD has motivated a significant research effort; however, there remain significant knowledge gaps regarding disease onset and progression as well as pressing needs for improved therapeutic approaches. One critical area of research that has received limited attention is the role of biophysical cues on the modulation of endothelial cell behaviors; specifically, the impact of local compliance, or the stiffness, of the surrounding vascular endothelial extracellular matrix. In this study, the impact of substratum compliance on the modulation of cell behaviors of several human primary endothelial cell types, representing different anatomic sites and differentiation states in vivo, were investigated. Substrates ! used within our studies span the range of compliance that has been reported for the vascular endothelial basement membrane. Differences in substratum compliance had a profound impact on cell attachment, spreading, elongation, proliferation, and migration. In addition, each cell population responded differentially to changes in substratum compliance, documenting endothelial heterogeneity in the response to biophysical cues. These results demonstrate the importance of incorporating substratum compliance in the design of in vitro experiments as well as future prosthetic design. Alterations in vascular substratum compliance directly influence endothelial cell behavior and may participate in the onset and/or progression of CVDs.
• Critical-size calvarial bone defects healing in a mouse model with silk scaffolds and SATB2-modified iPSCs
  - Biomat 32(22):5065-5076 (2011)
  Induced pluripotent stem cells (iPSCs) can differentiate into mineralizing cells and thus have a great potential in application in engineered bone substitutes with bioactive scaffolds in regeneration medicine. In the current study we characterized and demonstrated the pluripotency and osteogenic differentiation of mouse iPSCs. To enhance the osteogenic differentiation of iPSCs, we then transduced the iPSCs with the potent transcription factor, nuclear matrix protein SATB2. We observed that in SATB2-overexpressing iPSCs there were increased mineral nodule formation and elevated mRNA levels of key osteogenic genes, osterix (OSX), Runx2, bone sialoprotein (BSP) and osteocalcin (OCN). Moreover, the mRNA levels of HoxA2 was reduced after SATB2 overexpression in iPSCs. The SATB2-overexpressing iPSCs were then combined with silk scaffolds and transplanted into critical-size calvarial bone defects created in nude mice. Five weeks post-surgery, radiological and micro-CT analysi! s revealed enhanced new bone formation in calvarial defects in SATB2 group. Histological analysis also showed increased new bone formation and mineralization in the SATB2 group. In conclusion, the results demonstrate that SATB2 facilitates the differentiation of iPSCs towards osteoblast-lineage cells by repressing HoxA2 and augmenting the functions of the osteoblast determinants Runx2, BSP and OCN.
• The promotion of cerebral ischemia recovery in rats by laminin-binding BDNF
  - Biomat 32(22):5077-5085 (2011)
  Brain-derived neurotrophic factor (BDNF) has been shown to have therapeutic effects on cerebral ischemia. However, the delivery approach limits its application. Laminin is a rich extra cellular matrix in the central nervous system, and is highly expressed in the ischemic region after cerebral ischemia. We reported here by fusing with laminin-binding domain (LBD) to BDNF to construct laminin-binding BDNF (LBD-BDNF). LBD-BDNF could target accumulated laminin in the ischemic region and exert targeting therapy of injured neurons after ischemia. We examined the laminin-binding ability and neurotrophic bioactivity of LBD-BDNF in vitro, and assessed its targeting therapy using a rat permanent middle cerebral artery occlusion (MCAO) model in vivo. It was found that LBD-BDNF could specifically bind to laminin and maintain BDNF activity both in vitro and in vivo. LBD-BDNF treatment attenuated neural-degeneration after MCAO, and also resulted in a reduction of infarct volume that! is associated with a parallel improvement in neurological functional outcome and neurogenesis in the dentate gyrus of hippocamp.
• Human corneal epithelial equivalents constructed on Bombyx mori silk fibroin membranes
  - Biomat 32(22):5086-5091 (2011)
  Membranes prepared from a protein, fibroin, isolated from domesticated silkworm (Bombyx mori) silk, support the cultivation of human limbal epithelial (HLE) cells and thus display significant potential as biomaterials for ocular surface reconstruction. We presently extend this promising avenue of research by directly comparing the attachment, morphology and phenotype of primary HLE cell cultures grown on fibroin to that observed on donor amniotic membrane (AM), the current clinical standard substrate for HLE transplantation. Fibroin membranes measuring 6.3 ± 0.5 μm (mean ± sd) in thickness and permeable to FITC dextran of a molecular weight up to 70 kDa, were used. Attachment of HLE cells to fibroin was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. Nevertheless, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (K3/K12 expression) and displayed a comparable number and dist! ribution of ΔNp63+ progenitor cells to that seen in cultures grown on AM. These results support the suitability of membranes constructed from Bombyx mori silk fibroin as substrata for HLE cultivation and encourage progression to studies of efficacy in preclinical models.
• Gene therapy vectors with enhanced transfection based on hydrogels modified with affinity peptides
  - Biomat 32(22):5092-5099 (2011)
  Regenerative strategies for damaged tissue aim to present biochemical cues that recruit and direct progenitor cell migration and differentiation. Hydrogels capable of localized gene delivery are being developed to provide a support for tissue growth, and as a versatile method to induce the expression of inductive proteins; however, the duration, level, and localization of expression is often insufficient for regeneration. We thus investigated the modification of hydrogels with affinity peptides to enhance vector retention and increase transfection within the matrix. PEG hydrogels were modified with lysine-based repeats (K4, K8), which retained approximately 25% more vector than control peptides. Transfection increased 5- to 15-fold with K8 and K4 respectively, over the RDG control peptide. K8- and K4-modified hydrogels bound similar quantities of vector, yet the vector dissociation rate was reduced for K8, suggesting excessive binding that limited transfection. These h! ydrogels were subsequently applied to an in vitro co-culture model to induce NGF expression and promote neurite outgrowth. K4-modified hydrogels promoted maximal neurite outgrowth, likely due to retention of both the vector and the NGF. Thus, hydrogels modified with affinity peptides enhanced vector retention and increased gene delivery, and these hydrogels may provide a versatile scaffold for numerous regenerative medicine applications.
• Binding of the cell adhesive protein tropoelastin to PTFE through plasma immersion ion implantation treatment
  - Biomat 32(22):5100-5111 (2011)
  The interaction of proteins and cells with polymers is critical to their use in scientific and medical applications. In this study, plasma immersion ion implantation (PIII) was used to modify the surface of polytetrafluorethylene (PTFE), enabling the covalent binding of a cell adhesive protein, tropoelastin, without employing chemical linking molecules. Tropoelastin coating of untreated or PIII treated PFTE simultaneously promoted and blocked cell interactions respectively, i.e. PIII treatment of the PTFE surface completely inverses the cell interactive properties of bound tropoelastin. This activity persisted over long term storage of the PIII treated surfaces. The integrin binding C-terminus of tropoelastin was markedly less solvent exposed when bound to PIII treated PTFE than untreated PTFE, accounting for the modulation of cell adhesive activity. This presents a new methodology to specifically modulate cell behavior on a polymer surface using a simple one step trea! tment process, by adjusting the adhesive activity of a single extracellular matrix protein.
• Hydroxyapatite nanoparticle-containing scaffolds for the study of breast cancer bone metastasis
  - Biomat 32(22):5112-5122 (2011)
  Breast cancer frequently metastasizes to bone, where it leads to secondary tumor growth, osteolytic bone degradation, and poor clinical prognosis. Hydroxyapatite Ca10(PO4)6(OH)2 (HA), a mineral closely related to the inorganic component of bone, may be implicated in these processes. However, it is currently unclear how the nanoscale materials properties of bone mineral, such as particle size and crystallinity, which change as a result of osteolytic bone remodeling, affect metastatic breast cancer. We have developed a two-step hydrothermal synthesis method to obtain HA nanoparticles with narrow size distributions and varying crystallinity. These nanoparticles were incorporated into gas-foamed/particulate leached poly(lactide-co-glycolide) scaffolds, which were seeded with metastatic breast cancer cells to create mineral-containing scaffolds for the study of breast cancer bone metastasis. Our results suggest that smaller, poorly-crystalline HA nanoparticles promote great! er adsorption of adhesive serum proteins and enhance breast tumor cell adhesion and growth relative to larger, more crystalline nanoparticles. Conversely, the larger, more crystalline HA nanoparticles stimulate enhanced expression of the osteolytic factor interleukin-8 (IL-8). Our data suggest an important role for nanoscale HA properties in the vicious cycle of bone metastasis and indicate that mineral-containing tumor models may be excellent tools to study cancer biology and to define design parameters for non-tumorigenic mineral-containing or mineralized matrices for bone regeneration.
• Decoration of polymeric micelles with cancer-specific peptide ligands for active targeting of paclitaxel
  - Biomat 32(22):5123-5133 (2011)
  Polymeric micelles based on poly(ethylene oxide)-b-poly(ε-caprolactone) PEO-b-PCL or poly(ethylene oxide)-b-poly(α-benzyl carboxylate-ε-caprolactone) PEO-b-PBCL block copolymers were prepared and decorated with either c(RGDfK) or p160, a cancer cell-specific peptide ligand, on their surface. The cellular uptake of p160-decorated PEO-b-PBCL micelles containing DiI fluorescent label by MDA-MB-435 cancer cells was assessed and compared to that for c(RGDfK)-decorated micelles. The hydrophobic anticancer drug paclitaxel (PTX) was physically encapsulated into PEO-b-PCL or PEO-b-PBCL micelles (with and without peptide ligands) using a dialysis technique. The effect of the micellar formulation on the specificity of encapsulated PTX against cancer cells was assessed by investigating the in vitro cytotoxicity of free and encapsulated PTX against MDA-MB-435 cancer cell line versus two normal cells, Human Umbilical Vein Endothelial Cells (HUVEC) and MCF10A cells, using the MTT ! assay. Our results showed both peptide ligands to facilitate the association of micelles with MDA-MB-435 cells. The p160-micelles, however showed better binding and internalizing in MDA-MB-435 cells than c(RGDfK)-micelles. In general, peptide decoration enhanced the selective cytotoxicity of encapsulated PTX against MDA-MB-435 cells over normal HUVEC and MCF10A cells. The extent of this increase in cancer cell specificity for encapsulated PTX was more for p160-decorated micelles than c(RGDfK)-decorated ones.
• Induction of potent anti-tumor responses while eliminating systemic side effects via liposome-anchored combinatorial immunotherapy
  - Biomat 32(22):5134-5147 (2011)
  Immunostimulatory therapies that activate immune response pathways are of great interest for overcoming the immunosuppression present in advanced tumors. Agonistic anti-CD40 antibodies and CpG oligonucleotides have previously demonstrated potent, synergistic anti-tumor effects, but their clinical use even as monotherapies is hampered by dose-limiting inflammatory toxicity provoked upon systemic exposure. We hypothesized that by anchoring immuno-agonist compounds to lipid nanoparticles we could retain the bioactivity of therapeutics in the local tumor tissue and tumor-draining lymph node, but limit systemic exposure to these potent molecules. We prepared PEGylated liposomes bearing surface-conjugated anti-CD40 and CpG and assessed their therapeutic efficacy and systemic toxicity compared to soluble versions of the same immuno-agonists, injected intratumorally in the B16F10 murine model of melanoma. Anti-CD40/CpG-liposomes significantly inhibited tumor growth and induced! a survival benefit similar to locally injected soluble anti-CD40 + CpG. Biodistribution analyses following local delivery showed that the liposomal carriers successfully sequestered anti-CD40 and CpG in vivo, reducing leakage into systemic circulation while allowing draining to the tumor-proximal lymph node. Contrary to locally-administered soluble immunotherapy, anti-CD40/CpG-liposomes did not elicit significant increases in serum levels of ALT enzyme, systemic inflammatory cytokines, or overall weight loss, confirming that off-target inflammatory effects had been minimized. The development of a delivery strategy capable of inducing robust anti-tumor responses concurrent with minimal systemic side effects is crucial for the continued progress of potent immunotherapies toward widespread clinical translation.
• Role of cellular uptake in the reversal of multidrug resistance by PEG-b-PLA polymeric micelles
  - Biomat 32(22):5148-5157 (2011)
  Understanding the processes involved in the cellular uptake of nanoparticles is critical for developing effective nano drug delivery systems. In this paper we found that PEG-b-PLA polymeric micelles firstly interacted with cell membrane using atomic force microscopy (AFM) and then released their core-loaded agents into the cell membrane by fluorescence resonance energy transfer (FRET). The released agents were internalized into the cells via lipid raft/caveolae-mediated endocytosis using total internal reflection fluorescence microscopy (TIRFM) and endocytic inhibitors. Further studies revealed that paclitaxel (PTX)-loaded PEG-b-PLA micelles (M-PTX) increased the cellular accumulation of PTX in PTX-resistant human ovarian cell line A2780/T which resulted in more apoptosis as measured by flow cytometry and the cleavage of poly (ADP-ribose) polymerase (PARP) compared with free PTX. PEG-b-PLA micelles inhibited P-glycoprotein (Pgp) function and Pgp ATPase activity but had! no effect on Pgp protein expression. The membrane microenvironment studies showed that PEG-b-PLA micelles induced cell membrane depolarization and enhanced membrane microviscosity. These results suggested that PEG-b-PLA micelles might inhibit Pgp function to reverse multidrug resistance (MDR) via interaction with cell membrane to affect the membrane microenvironment. This study provides a foundation for understanding the mechanism of reversing MDR by nanoparticles better and designing more effective nano drug carriers.
• Active targeting of RGD-conjugated bioreducible polymer for delivery of oncolytic adenovirus expressing shRNA against IL-8 mRNA
  - Biomat 32(22):5158-5166 (2011)
  Even though oncolytic adenovirus (Ad) has been highlighted in the field of cancer gene therapy, transductional targeting and immune privilege still remain difficult challenges. The recent reports have noted the increasing tendency of adenoviral surface shielding with polymer to overcome the limits of its practical application. We previously reported the potential of the biodegradable polymer, poly(CBA-DAH) (CD) as a promising candidate for efficient gene delivery. To endow the selective-targeting moiety of tumor vasculature to CD, cRGDfC well-known as a ligand for cell-surface integrins on tumor endothelium was conjugated to CD using hetero-bifunctional cross-linker SM (PEG)n. The cytopathic effects of oncolytic Ad coated with the polymers were much more enhanced dose-dependently when compared with that of naked Ad in cancer cells selectively. Above all, the most potent oncolytic effect was assessed with the treatment of Ad/CD-PEG500-RGD in all cancer cells. The enhanc! ed cytopathic effect of Ad/RGD-conjugated polymer was specifically inhibited by blocking antibodies to integrins, but not by blocking antibody to CAR. HT1080 cells treated with Ad/CD-PEG500-RGD showed strong induction of apoptosis and suppression of IL-8 and VEGF expression as well. These results suggest that RGD-conjugated bioreducible polymer might be used to deliver oncolytic Ad safely and efficiently for tumor therapy.
• Gadolinium-loaded polymeric nanoparticles modified with Anti-VEGF as multifunctional MRI contrast agents for the diagnosis of liver cancer
  - Biomat 32(22):5167-5176 (2011)
  Molecular imaging is essential to increase the sensitivity and selectivity of cancer diagnosis especially in the early stage of tumor. Here, we designed a novel multifunctional polymeric nanoparticle contrast agent (Anti-VEGF PLA-PEG-PLL-Gd NP) simultaneously modified with Gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) and anti-vascular endothelial growth factor (VEGF) antibody to deliver Gd-DTPA to the tumor area and achieve the early diagnosis of hepatocellular carcinoma (HCC). The Anti-VEGF PLA-PEG-PLL-Gd NPs exhibited high T1 relaxivity and no obvious cytotoxicity under the experimental concentrations in human hepatocellular carcinoma (HepG2) cells. The results of in vitro cell uptake experiments demonstrated that the uptake process of NPs was both concentration and time depended. Compared with non-targeted NPs, the Anti-VEGF antibody modified NPs showed much higher cell uptake in the HepG2 cells. During in vivo studies, the targeted NPs showed significan! tly signal intensity enhancement at the tumor site (mouse hepatocarcinoma tumor, H22) compared with non-targeted NPs and Gd-DTPA injection in tumor-bearing mice and the imaging time was significantly prolonged from less than an hour (Gd-DTPA injection group) to 12 h. These results demonstrated that this novel MRI contrast agent Anti-VEGF PLA-PEG-PLL-Gd NPs showed great potential in the early diagnosis of liver tumors.
• Chlorotoxin-modified macromolecular contrast agent for MRI tumor diagnosis
  - Biomat 32(22):5177-5186 (2011)
  Clinical diagnosis of cancers using magnetic resonance imaging (MRI) is highly dependent on contrast agents, especially for brain tumors which contain blood–brain barrier (BBB) at the early stage. However, currently mostly used low molecular weight contrast agents such as Gd-DTPA suffer from rapid renal clearance, non-specificity, and low contrast efficiency. The aim of this paper is to investigate the potential of a macromolecular MRI contrast agent based on dendrigraft poly-l-lysines (DGLs), using chlorotoxin (CTX) as a tumor-specific ligand. The contrast agent using CTX-modified conjugate as the main scaffold and Gd-DTPA as the payload was successfully synthesized. The results of fluorescent microscopy showed that the modification of CTX could markedly enhance the cellular uptake in C6 glioma and liver tumor cell lines, but not in normal cell line. Significantly increased accumulation of CTX-modified conjugate within glioma and liver tumor was further demonstrated! in tumor-bearing nude mice using in vivo imaging system. The MRI results showed that the signal enhancement of mice treated with CTX-modified contrast reached peak level at 5 min for both glioma and liver tumor, 144.97% ± 19.54% and 158.69% ± 12.41%, respectively, significantly higher than that of unmodified counterpart and commercial control. And most importantly, the signal enhancement of CTX-modified contrast agent maintained much longer compared to that of controls, which might be useful for more exact diagnosis for tumors. CTX-modified dendrimer-based conjugate might be applied as an efficient MRI contrast agent for targeted and accurate tumor diagnosis. This finding is especially important for tumors such as brain glioma which is known hard to be diagnosed due to the presence of BBB.
• The performance of gadolinium diethylene triamine pentaacetate-pullulan hepatocyte-specific T1 contrast agent for MRI
  - Biomat 32(22):5187-5194 (2011)
  The magnetic resonance (MR) functionalities of pullulan-conjugated gadolinium diethylene triamine pentaacetate (Gd-DTPA-Pullulan) as a new hepatocyte-specific contrast agent were evaluated. Pullulan, which specifically accumulates on hepatocytes via asialoglycoprotein receptors, was chemically linked with Gd-DTPA. Gd-DTPA-Pullulan displayed three times greater contrast enhancement than Gd-DTPA-BMA (Omniscan®) in delayed MR imaging (MRI) on orthotopic rat hepatocarcinoma (HCC). This contrast effect lasted up to 24 h. In particular, Gd-DTPA-Pullulan displayed a discriminative MR contrast on the regenerative and malignant hepatic nodules sequentially observed during the progress of cirrhotic HCC. Approximately 50% of injected Gd-DTPA-Pullulan was eliminated via the hepato-biliary system. IC50 of Gd-DTPA-Pullulan on Chang liver cells was much higher than Gd-DTPA and Gd-DTPA-BMA (309.1 ± 11.2, 173.5 ± 15.5 and 49.4 ± 8.9 μM, respectively). Any significant toxicities of! Gd-DTPA-Pullulan at the conventional dose on the rats weren't detected on histology studies. Gd-DTPA-Pullulan worked as a hepatocyte-specific MR contrast agent with increased MR functionalities and an acceptable safety profile setting the scene for future clinical trials.
• Quantum dot labeling using positive charged peptides in human hematopoetic and mesenchymal stem cells
  - Biomat 32(22):5195-5205 (2011)
  Quantum dots (QDs), as new and promising fluorescent probes, hold great potential in long term non-invasive bio-imaging, however there are many uncovered issues regarding their competency. In the present study, different QDs (525, 585 and 800 nm) were used to label CD133, CD34, CD14 and mesenchymal stem cells (MSCs) using positively charged peptides. Results demonstrated highly efficient internalization with the possible involvement of macropinocytosis. As indicated by LDH release and the TUNEL assay, no measurable effects on cell viability were detected at a concentration of 10 nM. QDs did not have any deleterious effects on normal cell functionality where both labeled CD133+ cells and MSCs remarkably differentiated along multiple lineages with the use of the colony forming assay and adipo/osteo induction, respectively. Our results regarding QD maintenance revealed that these nano-particles are not properly stable and various excretion times have been observed depending on particle size and cell type. In vitro co-culture system and transplantation of labeled cells to an animal model showed that QDs leaked out from labeled cells and the released nano-particles! were able to re-enter adjacent cells over time. These data suggest that before any utilization of QDs in bio-imaging and related applications, an efficient intra-cellular delivery technique should be considered to preserve QDs for a prolonged time as well as eliminating their leakage.
• The induction of innate and adaptive immunity by biodegradable poly(γ-glutamic acid) nanoparticles via a TLR4 and MyD88 signaling pathway
  - Biomat 32(22):5206-5212 (2011)
  The induction of adaptive immunity through the activation of innate immunity is indispensable for vaccine development. Although strategies for particulate antigen delivery are widely investigated, their immunological mechanisms are unclear. We describe in this study that biodegradable nanoparticles (NPs) elaborated with poly(γ-glutamic acid) (γ-PGA) are able to induce potent innate and adaptive immune responses through Toll-like receptor 4 (TLR4) and MyD88 signaling pathways. The production of inflammatory cytokines from macrophages and the maturation of dendritic cells were impaired in MyD88-knockout and TLR4-deficient mice compared with their wild-types, when the cells were stimulated with γ-PGA NPs. The immunization of these mice with antigen-carrying γ-PGA NPs also resulted in diminished induction of antigen-specific cellular immune responses. These results suggest that γ-PGA NPs have not only an antigen-carrying capacity but also a potent adjuvant function of! eliciting adaptive immune responses to the carrying antigen through recognition of the first-line host-sensor system.
• Cell penetrating peptide conjugated bioreducible polymer for siRNA delivery
  - Biomat 32(22):5213-5222 (2011)
  The primary cardiomyocyte–specific peptide (PCM) and the cell-penetrating peptide (CPP), HIV-Tat (49–57), were incorporated into the polymer, cystamine bisacrylamide-diaminohexane (CBA-DAH), to increase the delivery of RNAi to target cells, specifically cardiomyocytes. Interestingly, the impact of PCM and Tat conjugation on cellular uptake and transfection efficiency was greater in H9C2 rat cardiomyocytes than in NIH 3T3 cells. We examined the potential for siRNA targeting SHP-1 or Fas to inhibit the apoptosis of cardiomyocytes under hypoxic conditions using PCM and Tat-modified poly(CBA-DAH), (PCM-CD-Tat). To evaluate for efficacy in inhibiting apoptosis, either Fas siRNA/polymer or SHP-1 siRNA/polymer were transfected into cardiomyocytes treated under hypoxic and serum-deprived conditions. After incubation under hypoxic conditions, treatment with either the SHP-1 siRNA complex or the Fas siRNA complex resulted in an increase in cell viability and a reduction in L! DH-cytotoxicity. The cells transfected with either of the siRNA polyplexes had a lower incidence of apoptosis as demonstrated by Annexin V–FITC/PI staining. Both the SHP-1 siRNA/PCM-CD-Tat complex and the Fas siRNA/PCM-CD-Tat complex warrant further investigation as therapeutic agents to inhibit the apoptosis of cardiomyocytes.
• Redox-responsive nanocapsules for intracellular protein delivery
  - Biomat 32(22):5223-5230 (2011)
  Direct delivery of proteins to the cytosol of cells holds tremendous potential in biological and medical applications. Engineering vehicles for escorting proteins to the cytosol in a controlled release fashion has thus generated considerable interest. We report here the preparation of redox-responsive single-protein nanocapsules for intracellular protein delivery. Through in situ interfacial polymerization, the target protein is noncovalently encapsulated into a positively-charged polymeric shell interconnected by disulfide-containing crosslinkers. The dissociation of the polymeric shell under reducing conditions and the subsequent release of protein were confirmed using cell-free assays in the presence of glutathione (GSH). The nanocapsules were demonstrated to be efficiently internalized into the cells and to release the protein in the reducing cytosol. Using the nanocapsule as a vehicle, we showed that active caspase 3 (CP-3) can be delivered and can induce apoptosi! s in a variety of human cancer cell lines, including HeLa, MCF-7 and U-87 MG. Our approach therefore presents an effective intracellular protein delivery strategy for therapeutic, diagnostic and reprogramming applications.
• The influence of the structural orientation of amide linkers on the serum compatibility and lung transfection properties of cationic amphiphiles
  - Biomat 32(22):5231-5240 (2011)
  Understanding the structural parameters of cationic amphiphiles which can influence gene transfer efficiencies of cationic amphiphiles continues to remain important for designing efficient liposomal gene delivery reagents. Previously we demonstrated the influence of structural orientation of the ester linker (widely used in covalently tethering the polar head and the non-polar tails) in modulating in vitro gene transfer efficiencies of cationic amphiphiles. However, our previously described cationic amphiphiles with ester linkers failed to deliver genes under in vivo conditions. Herein we report on the development of a highly serum compatible cationic amphiphile with circulation stable amide linker which shows remarkable selectivity in transfecting mouse lung. We also demonstrate that reversing structural orientation of the amide linker adversely affects both serum compatibility and the lung selective gene transfer property. Dynamic laser light scattering and atomic fo! rce microscopic studies revealed smaller average hydrodynamic sizes of the liposomes of transfection efficient lipid than those for the liposomes of transfection incompetent analog (148 ± 1 nm vs 214 ± 4 nm). Average surface potential of the liposomes of transfection competent amphiphiles were found to be significantly higher than that for the liposomes of transfection incompetent analog (10.7 ± 5.4 mV vs 2.8 ± 1.3 mV, respectively). Findings in fluorescence resonance energy transfer and dye entrapment experiments support lower rigidity and higher biomembrane fusogenicity of the liposomes of the transfection efficient amphiphiles. Importantly, cationic lipoplexes of the novel amide-linker based amphiphile exhibited higher mouse lung selective gene transfer properties than DOTAP, one of the widely used commercially available liposomal lung transfection kits. In summary, the present findings demonstrate for the first time that amide linker structural orientation profoundl! y influences the serum compatibility and lung transfection eff! iciencies of cationic amphiphiles.
• Effects of protein dose and delivery system on BMP-mediated bone regeneration
  - Biomat 32(22):5241-5251 (2011)
  Delivery of recombinant proteins is a proven therapeutic strategy to promote endogenous repair mechanisms and tissue regeneration. Bone morphogenetic protein-2 (rhBMP-2) has been used to promote spinal fusion and repair of challenging bone defects; however, the current clinically-used carrier, absorbable collagen sponge, requires high doses and has been associated with adverse complications. We evaluated the hypothesis that the relationship between protein dose and regenerative efficacy depends on delivery system. First, we determined the dose-response relationship for rhBMP-2 delivered to 8-mm rat bone defects in a hybrid nanofiber mesh/alginate delivery system at six doses ranging from 0 to 5 μg. Next, we directly compared the hybrid delivery system to the collagen sponge at 0.1 and 1.0 μg. Finally, we compared the in vivo protein release properties of the two delivery methods. In the hybrid delivery system, bone volume, connectivity and mechanical properties incre! ased in a dose-dependent manner to rhBMP-2. Consistent bridging of the defect was observed for doses of 1.0 μg and greater. Compared to collagen sponge delivery at the same 1.0 μg dose, the hybrid system yielded greater connectivity by week 4 and 2.5-fold greater bone volume by week 12. These differences may be explained by the significantly greater protein retention in the hybrid system compared to collagen sponge. This study demonstrates a clear dose-dependent effect of rhBMP-2 delivered using a hybrid nanofiber mesh/alginate delivery system. Furthermore, the effective dose was found to vary with delivery system, demonstrating the importance of biomaterial carrier properties in the delivery of recombinant proteins.
• Real-time and non-invasive optical imaging of tumor-targeting glycol chitosan nanoparticles in various tumor models
  - Biomat 32(22):5252-5261 (2011)
  Recently, various nanoparticle systems have been developed for tumor-targeted delivery of imaging agents or drugs. However, large amount of them still have insufficient tumor accumulation and this limits their further clinical applications. Moreover, the in vivo characteristics of nanoparticles have been largely unknown, because there are few proper technologies to achieve the direct and non-invasive characterization of nanoparticles in live animals. In this paper, we determined the key factors of nanoparticles for in vivo tumor-targeting using our glycol chitosan nanoparticles (CNPs) which have proved their tumor-targeting ability in many previous papers. For this study, CNPs were labeled with near-infrared fluorescence (NIRF) dye, Cy5.5 for in vivo analysis by non-invasive optical imaging techniques. With these Cy5.5-CNPs, the factors such as in vitro/in vivo stability, deformability, and rapid uptake into target tumor cells and their effects on in vivo tumor-targeti! ng were evaluated in various tumor-bearing mice models. In flank tumor models, Cy5.5-CNPs were selectively localized in tumor tissue than other organs, and the real-time intravascular tracking of CNPs proved the enhanced permeation and retention (EPR) effect of nanoparticles in tumor vasculature. Importantly, tumor-targeting CNPs showed an excellent tumor-specificity in brain tumors, liver tumors, and metastasis tumor models, indicating their great potential in both cancer imaging and therapy.
• The cell penetrating ability of the proapoptotic peptide, KLAKLAKKLAKLAK fused to the N-terminal protein transduction domain of translationally controlled tumor protein, MIIYRDLISH
  - Biomat 32(22):5262-5268 (2011)
  We show here, that the proapoptotic peptide, KLAKLAKKLAKLAK (KLA), which by itself does not penetrate cell membranes, can do so when fused to a protein transduction domain derived from NH2-terminus of translationally controlled tumor protein (TCTP-PTD, MIIYRDLISH). Once inside the cell, the conjugated KLA exerts its proapoptotic activity to inhibit tumor growth. We evaluated the cellular uptake of KLA fused to TCTP-PTD (hereafter called TCTP-KLA) and its effect on cancer cell viability. The IC50 of TCTP-KLA was between 7 and 10 μmol/L. We also evaluated its anti-tumor activity in vivo by injecting it into xenografts of lung carcinoma in Balb/c nude mice. Tumor growth inhibition resulting from treatment with TCTP-KLA was better than that of TAT-KLA. These results suggest that TCTP-KLA can be applied to design cancer therapeutics.
• The optimization of polymalic acid peptide copolymers for endosomolytic drug delivery
  - Biomat 32(22):5269-5278 (2011)
  Membranolytic macromolecules are promising vehicles for cytoplasmic drug delivery, but their efficiency and safety remains primary concerns. To address those concerns, membranolytic properties of various poly(β-l-malic acid) (PMLA) copolymers were extensively investigated as a function of concentration and pH. PMLA, a naturally occurring biodegradable polymer, acquires membranolytic activities after substitution of pendent carboxylates with hydrophobic amino acid derivatives. Ruled by hydrophobization and charge neutralization, membranolysis of PMLA copolymers increased as a function of polymer molecular weight and demonstrated a maximum with 50% substitution of carboxylates. Charge neutralization was achieved either conditionally by pH-dependent protonation or permanently by masking carboxylates. Membranolysis of PMLA copolymers containing tripeptides of leucine, tryptophan and phenylalanine were pH-dependent in contrast to pH-independent copolymers of Leucine ethyl ! ester and Leu-Leu-Leu-NH2 with permanent charge neutralization. PMLA and tripeptides seemed a unique combination for pH-dependent membranolysis. In contrast to nontoxic pH-dependent PMLA copolymers, pH-independent copolymers were found toxic at high concentration, which is ascribed to their nonspecific disruption of plasma membrane at physiological pH. pH-Dependent copolymers were membranolytically active only at acidic pH typical of maturating endosomes, and are thus devoid of cytotoxicity. The PMLA tripeptide copolymers are useful for safe and efficient cytoplasmic delivery routed through endosome.
• The effect of polymer architecture, composition, and molecular weight on the properties of glycopolymer-based non-viral gene delivery systems
  - Biomat 32(22):5279-5290 (2011)
  Although a variety of non-viral gene delivery vectors has been synthesized and used for gene delivery purposes, well-defined glycopolymer-based gene delivery carriers is not well explored. Reversible Addition-Fragmentation Chain Transfer (RAFT) polymerization technique allows successful and facile synthesis of cationic glycopolymers containing pendant sugar moieties in the absence of protecting group chemistry. A library of cationic glycopolymers of pre-determined molar masses and narrow polydispersities ranging from 3 to 30 kDa has been synthesized using RAFT polymerization technique. These polymers differ from each other in their architectures (block versus random), molecular weights, and monomer ratios (carbohydrate to cationic segment). It is shown that the above-mentioned parameters can largely affect the toxicity, DNA condensation ability and gene delivery efficacy of these polymers. Statistical copolymers of high degree of polymerization are found to be the idea! l vector for gene delivery purposes. These statistical copolymers show lower toxicity and higher gene expression in the presence and absence of serum, as compared to the corresponding diblock copolymers. This is the first example of well-defined synthetic glycopolymers as DNA carriers that works both in the presence and absence of serum proteins. The critical composition of carbohydrate segment in copolymers for enhanced gene delivery and low toxicity was determined and an increase in carbohydrate residues in copolymers resulted in a decrease in transfection efficiencies of these polymers. The effect of serum proteins on statistical and diblock copolymer based polyplexes and hence gene delivery efficacy was studied. The results showed that the diblock copolymer-based polyplexes showed lower interactions with serum proteins, lower cellular uptake and very low gene expression in both Hep G2 and Hela cells in comparison to statistical copolymers.
• Formation and characterization of DNA-polymer-condensates based on poly(2-methyl-2-oxazoline) grafted poly(l-lysine) for non-viral delivery of therapeutic DNA
  - Biomat 32(22):5291-5303 (2011)
  Successful gene delivery systems deliver DNA in a controlled manner combined with minimal toxicity and high transfection efficiency. Here we investigated 15 different copolymers of poly(l-lysine)-graft-poly(2-methyl-2-oxazoline) (PLL-g-PMOXA) of variable grafting densities and PMOXA molecular weights for their potential to complex and deliver plasmid DNA. PLL20g7PMOXA4 formed at N/P charge ratio of 3.125 was found to transfect 9 ± 1.6% of COS-7 cells without impairment of cell viability. Furthermore these PLL-g-PMOXA-DNA condensates were internalized 2 h after transfection and localized in the perinuclear region after 6 h. The condensates displayed a hydrodynamic diameter of 100 nm and were found to be stable in serum and after 70 °C heat treatment, moreover the condensates protected DNA against DNase-I digestion. The findings suggest that DNA-PMOXA-g-PLL condensate formation for efficient DNA-delivery strongly depends on PMOXA grafting density and molecular weight s! howing an optimum at low grafting density between 7 and 14% and medium N/P charge ratio (3.125–6.25). Thus, PLL20g7PMOXA4 copolymers might be promising as alternative to PLL-g-PEG-DNA condensates for delivery of therapeutic DNA.
• The in vivo performance of an enzyme-assisted self-assembled peptide/protein hydrogel
  - Biomat 32(22):5304-5310 (2011)
  We demonstrate the distribution of the important extracellular matrix protein laminin in a novel biomaterial consisting of a hydrogel underpinned by nanofibrillar networks. These are formed by the immobilised enzyme mediated self-assembly of fmoc-L3 (9-fluorenylmethoxycarbonyl-tri-leucine). The peptide assembly yields nanofibrils formed of β-sheets that are locked together via π-stacking interactions. This ordering allows the localisation of the peptide sidechains on the surface, creating a hydrophobic environment. This induces the formation of bundles of these nanofibrils producing a clear hydrogel. This mechanism enables the three dimensional distribution of laminin throughout the network via supramolecular interactions. These forces favour the formation and improve the order of the network itself, as observed by spectroscopic and mechanical testing. In order to test the stability and suitability of this class of material for in vivo applications, we utilise microi! njection to deliver the biomaterial under fine spatial control into a dystrophic zebrafish model organism, which lacks laminin as a result of a genetic mutation. Using confocal and transmission electron microscopy, we confirm that the biomaterial remains stable structurally, and is confined spatially to the site of injection.