Nanotextured polymer substrates show enhanced cancer cell isolation and cell culture.

Detection of circulating tumor cells (CTCs) in the early stages of cancer is a great challenge because of their exceedingly small concentration. There are only a few approaches sensitive enough to differentiate tumor cells from the plethora of other cells in a sample like blood. In order to detect CTCs, several antibodies and aptamers have already shown high affinity. Nanotexture can be used to mimic basement membrane to further enhance this affinity. This article reports an approach to fabricate nanotextured polydimethylsiloxane (PDMS) substrates using micro reactive ion etching (micro-RIE). Three recipes were used to prepare nanotextured PDMS using oxygen and carbon tetrafluoride. Micro-RIE provided better control on surface properties. Nanotexturing improved the affinity of PDMS surfaces to capture cancer cells using surface immobilized aptamers against cell membrane overexpressed with epidermal growth factor receptors. In all cases, nanotexture of PDMS increased the effective surface area by creating nanoscale roughness on the surface. Nanotexture also enhanced the growth rate of cultured cells compared to plain surfaces. A comparison among the three nanotextured surfaces demonstrated an almost linear relationship between the surface roughness and density of captured tumor cells. The nanotextured PDMS mimicked biophysical environments for cells to grow faster. This can have many implications in microfluidic platforms used for cell handling.

Nanotextured substrates with immobilized aptamers for cancer cell isolation and cytology.

BACKGROUND: The detection of a small number of circulating tumor cells (CTCs) is important, especially in the early stages of cancer. Small numbers of CTCs are hard to detect, because very few approaches are sensitive enough to differentiate these from the pool of other cells. Improving the affinity of a selective, surface-functionalized molecule is important given the scarcity of CTCs in vivo. There are several proteins and aptamers that provide such high affinity; however, using surface nanotexturing increases this affinity even further. METHODS: The authors report an approach to improve the affinity of tumor cell capture by using novel aptamers against cell membrane overexpressed epidermal growth factor receptors (EGFRs) on a nanotextured polydimethylsiloxane (PDMS) substrate. Surface-immobilized aptamers were used to specifically capture tumor cells from physiologic samples. RESULTS: The nanotexturing of PDMS increased surface roughness at the nanoscale. This increased the effective surface area and resulted in a significantly higher degree of surface functionalization. The phenomenon resulted in increased density of immobilized EGFR-specific RNA aptamer molecules and provided significantly higher efficiency to capture cancer cells from a mixture. The data indicated that CTCs could be captured and enriched, leading to higher yield yet higher background. CONCLUSIONS: A comparison between glass slides, plain PDMS, and nanotextured PDMS functionalized with aptamers demonstrated that a 2-fold approach of using aptamers on nanotextured PDMS can be important for cancer cytology devices, and especially for the idea of a "lab-on-chip," toward higher yield in capture efficiency.

Synthesis of nano-textured biocompatible scaffolds from chicken eggshells.

Cell adhesion, morphology and growth are influenced by surface topography at nano and micrometer scales. Nano-textured surfaces are prepared using photolithography, plasma etching and long polymer chemical etching which are cost prohibitive and require specialized equipment. This article demonstrates a simple approach to synthesize nano-textured scaffolds from chicken eggshells. Varieties of pattern are made on the eggshells like micro-needle forests and nanopores, giving very uniform nano-textures to the surfaces. The surfaces are characterized for chemical composition and crystal phase. The novel patterns are transferred to PDMS surfaces and the nano-textured PDMS surfaces are used to study the effect of texturing on human fibroblast cell growth and attachment. The effects of surface topographies, along with laminin coating on cell cultures, are also studied. We find an exciting phenomenon that the initial seeding density of the fibroblast cells affects the influence of the nano-texturing on cell growth. These nano-textured surfaces give 16 times more fibroblast growth when compared to flat PDMS surfaces. The novel nano-textured patterns also double the laminin adsorption on PDMS.

Sustained VEGF delivery via PLGA nanoparticles promotes vascular growth.

Technologies to increase tissue vascularity are critically important to the fields of tissue engineering and cardiovascular medicine. Currently, limited technologies exist to encourage angiogenesis and arteriogenesis in a controlled manner. In the present study, we describe an injectable controlled release system consisting of VEGF encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs). The majority of VEGF was released gradually over 2-4 days from the NPs as determined by an ELISA release kinetics experiment. An in vitro aortic ring bioassay was used to verify the bioactivity of VEGF-NPs compared with empty NPs and no treatment. A mouse femoral artery ischemia model was then used to measure revascularization in VEGF-NP-treated limbs compared with limbs treated with naked VEGF and saline. 129/Sv mice were anesthetized with isoflurane, and a region of the common femoral artery and vein was ligated and excised. Mice were then injected with VEGF-NPs, naked VEGF, or saline. After 4 days, three-dimensional microcomputed tomography angiography was used to quantify vessel growth and morphology. Mice that received VEGF-NP treatment showed a significant increase in total vessel volume and vessel connectivity compared with 5 microg VEGF, 2.5 microg VEGF, and saline treatment (all P < 0.001). When the yield of the fabrication process was taken into account, VEGF-NPs were over an order of magnitude more potent than naked VEGF in increasing blood vessel volume. Differences between the VEGF-NP group and all other groups were even greater when only small-sized vessels under 300 mum diameter were analyzed. In conclusion, sustained VEGF delivery via PLGA NPs shows promise for encouraging blood vessel growth in tissue engineering and cardiovascular medicine applications.

The role of aligned polymer fiber-based constructs in the bridging of long peripheral nerve gaps.

Peripheral nerve regeneration across long nerve gaps is clinically challenging. Autografts, the standard of therapy, are limited by availability and other complications. Here, using rigorous anatomical and functional measures, we report that aligned polymer fiber-based constructs present topographical cues that facilitate the regeneration of peripheral nerves across long nerve gaps. Significantly, aligned but not randomly oriented fibers elicit regeneration, establishing that topographical cues can influence endogenous nerve repair mechanisms in the absence of exogenous growth promoting proteins. Axons regenerated across a 17 mm nerve gap, reinnervated muscles, and reformed neuromuscular junctions. Electrophysiological and behavioral analyses revealed that aligned but not randomly oriented constructs facilitated both sensory and motor nerve regeneration, significantly improved functional outcomes. Additionally, a quantitative comparison of DRG outgrowth in vitro and nerve regeneration in vivo on aligned and randomly oriented fiber films clearly demonstrated the significant role of sub-micron scale topographical cues in stimulating endogenous nerve repair mechanisms.

An Open-Label, Randomized, Parallel, Phase II Trial to Evaluate the Efficacy and Safety of a Cremophor-Free Polymeric Micelle Formulation of Paclitaxel as First-Line Treatment for Ovarian Cancer: A Korean Gynecologic Oncology Group Study (KGOG-3021).

PURPOSE: Genexol-PM is a biodegradable cremophor EL-free polymeric micelle formulation of paclitaxel. Here,we compared efficacy and safety of Genexol-PM plus carboplatin versus Genexol plus carboplatin for ovarian cancer treatment. MATERIALS AND METHODS: In this multicenter, randomized, phase II study, patients with International Federation of Gynecology and Obstetrics IC-IV epithelial ovarian cancer were randomly assigned (1:1) to receive Genexol-PM 260 mg/m2 or Genexol 175 mg/m2 with 5 area under the curve carboplatin every 3weeks (6 cycles). The primary endpointwas the carbohydrate antigen 125 and Response Evaluation Criteria In Solid Tumor composite overall response rate (ORR). RESULTS: Of 131 enrolled patients, 98 were included in intention-to-treat analysis. Mean dosages were 260.00±0.00 mg/m2 Genexol-PM or 174.24±3.81 mg/m2 Genexol. Median followup was 18.0 months (range, 6.1 to 33.8 months). ORR was 88.0% (95% confidence interval [CI], 80.4 to 95.6) with Genexol-PM, and 77.1% (95% CI, 67.1 to 87.1) with Genexol (noninferiority threshold, 16.3%). Median time to progression was 14.8 months (95% CI, 11.3 to 20.2) with Genexol-PM and 15.4 months (95% CI, 13.2 to 29.6) with Genexol (p=0.550). Overall, six patients died. Neutropenia was the most common toxicity (incidences of 86.0% vs. 77.1%, p=0.120). Peripheral neuropathy incidences were 84.0% versus 64.6% (p= 0.148). Peripheral neuropathy of ≥ grade 3 occurred in one patient receiving Genexol. All toxicities were manageable. CONCLUSION: Genexol-PM plus carboplatin as first-line treatment in patients with epithelial ovarian cancer demonstrated non-inferior efficacy and well-tolerated toxicities compared with the standard paclitaxel regimen. Further studies are warranted to optimize the dose and schedule, and to investigate long-term outcomes.

An open-label, multicenter, phase I trial of a cremophor-free, polymeric micelle formulation of paclitaxel combined with carboplatin as a first-line treatment for advanced ovarian cancer: a Korean Gynecologic Oncology Group study (KGOG-3016).

OBJECTIVE: This phase I study aimed to determine the maximum tolerated dose (MTD) of Genexol-PM, when combined with carboplatin, as a first-line treatment in patients with advanced ovarian cancer. METHODS: This open-label, multicenter, phase I, dose-escalation study included 18 patients (median age: 59.0 years, range: 40-75 years) diagnosed with advanced epithelial ovarian cancer. All patients had measurable residual disease after debulking surgery. Patients were assigned to groups (n=6 each group) that received different doses of Genexol-PM (220, 260, and 300 mg/m², once every 3 weeks) and 5 area under the curve (AUC) carboplatin. Safety and efficacy were analyzed for each dose group. RESULTS: In this intention-to-treat population, 3 out of 18 patients dropped out of the study: 1 due to dose-limiting toxicity (DLT), 1 due to hypersensitivity, and 1 was lost during follow-up. DLTs were not reported at 220 mg/m² or 260 mg/m², but at 300 mg/m², 1 patient experienced DLT (grade 3 general pain). The MTD of Genexol-PM was not determined, but a dose of 300 mg/m² or less could be recommended for the phase II study. Most patients (73.9%) with adverse events recovered without sequelae, and no death occurred that was related to the disease or treatment. The best overall response rate was 94.1%. CONCLUSION: Genexol-PM combined with carboplatin was well tolerated as a first-line treatment, and good responses were observed in patients with advanced ovarian cancer. Based on these results, we recommended a dose of 300 mg/m² or less for a phase II study.

A cell encapsulation device for studying soluble factor release from cells transplanted in the rat brain.

The transplantation of a variety of naturally occurring and genetically modified cell types has been shown to be an effective experimental method to achieve sustained delivery of therapeutic molecules to specific target areas in the brain. To acquire a better understanding of dosing, implant mechanism of action, and how certain cell types affect remodeling of central nervous system (CNS) tissue, a refillable cell encapsulation device was developed for introducing cells into the brain while keeping them physically isolated from contact with brain tissue with a semipermeable membrane. The stereotactically placed device consists of a hollow fiber membrane (HFM), a polyurethane grommet with watertight cap that snaps into a precisely drilled hole in the rat skull, and a removable cell-containing insert. The cell-containing insert can be introduced or removed in a time-dependent manner to study the influence of soluble factors released from transplanted cells. The study describes the device design and validates its utility using a well-established cell transplantation model of Parkinson's disease.

Chronic response of adult rat brain tissue to implants anchored to the skull.

Using quantitative immunohistological methods, we examined the brain tissue response to hollow fiber membranes (HFMs) that were either implanted intraparenchymally, as in a cell encapsulation application, or were attached to the skull as in a biosensor application (transcranially). We found that the reaction surrounding transcranially implanted HFMs was significantly greater than that observed with intraparenchymally implanted materials including increases in immunoreactivity against GFAP, vimentin, ED-1 labeled macrophages and microglia, and several extracellular matrix proteins including collagen, fibronectin, and laminin. In general, these markers were elevated along the entire length of transcranially implanted HFMs extending into the adjacent parenchyma up to 0.5 mm from the implant interface. Intraparenchymal implants did not appear to have significant involvement of a fibroblastic component as suggested by a decreased expression of vimentin, fibronectin and collagen-type I at the implant tissue interface. The increase in tissue reactivity observed with transcranially implanted HFMs may be influenced by several mechanisms including chronic contact with the meninges and possibly motion of the device within brain tissue. Broadly speaking, our results suggest that any biomaterial, biosensor or device that is anchored to the skull and in chronic contact with meningeal tissue will have a higher level of tissue reactivity than the same material completely implanted within brain tissue.

Capture, isolation and release of cancer cells with aptamer-functionalized glass bead array.

Early detection and isolation of circulating tumor cells (CTC) can enable better prognosis for cancer patients. A Hele-Shaw device with aptamer functionalized glass beads is designed, modeled, and fabricated to efficiently isolate cancer cells from a cellular mixture. The glass beads are functionalized with anti-epidermal growth factor receptor (EGFR) aptamer and sit in ordered array of pits in polydimethylsiloxane (PDMS) channel. A PDMS encapsulation is then used to cover the channel and to flow through cell solution. The beads capture cancer cells from flowing solution depicting high selectivity. The cell-bound glass beads are then re-suspended from the device surface followed by the release of 92% cells from glass beads using combination of soft shaking and anti-sense RNA. This approach ensures that the cells remain in native state and undisturbed during capture, isolation and elution for post-analysis. The use of highly selective anti-EGFR aptamer with the glass beads in an array and subsequent release of cells with antisense molecules provide multiple levels of binding and release opportunities that can help in defining new classes of CTC enumeration devices.

An optimal micropatterned end-effecter for enhancing frictional force on large intestinal surface.

We present a simple surface modification method for enhancing the frictional properties on soft, viscoelastic tissue of large intestine by integrating micropatterned structures with controlled shape and geometry. The micropatterned end-effecter (EE) was fabricated onto micromachined EE body (20 mm long, 2 mm diameter cylinders) in the forms of line, box, pyramid, and bottle shape by utilizing capillary molding technique with UV-curable poly(urethane acrylate) (PUA) polymer. To evaluate the frictional behavior of micropatterned EE, we employed a biotribotester, for easy loading and test of a biological organ specimen. It was found that the frictional properties of micropatterned EE are heavily dependent upon the shape of microstructure. The patterned EE with parallel lines (to the direction of locomotion) showed better frictional performance (average frictional coefficient approximately 1.53 and maximum approximately 3.98) compared with other micropatterned EEs (average frictional coefficient 0.72-0.94 and maximum 1.78-2.49) and nonpatterned EE (average frictional coefficient approximately 0.58 and maximum approximately 1.51). In addition, various geometric parameters (e.g., height, width, and space) as well as operating conditions (e.g., contact load and sliding speed) were systematically investigated for probing optimal anchoring function of the parallel line patterned EE.

Slow-release nanoparticle-encapsulated delivery system for laryngeal injection.

OBJECTIVES/HYPOTHESIS: There is a need for a slow-release system for local delivery of therapeutics to the larynx. Most therapeutic substances, such as steroids or chemotherapeutic agents that are injected into the larynx are cleared rapidly. Repeated laryngeal injection of these substances at short intervals is impractical. Injectable encapsulated poly(lactide-co-glycolide) (PLGA) nanoparticles offer a potential slow-release delivery system for biologically active substances in the larynx. STUDY DESIGN: Controlled animal study. METHODS: PLGA nanoparticles were fabricated using a double emulsion method and were loaded with Texas Red-dextran (NPTR), hepatocyte growth factor (NPHGF), and bovine serum albumin (NPBSA). In vitro release of NPTR, NPBSA, and NPHGF was determined over approximately 2 weeks to assess potential duration of PLGA nanoparticle delivery. In vivo release of NPTR was assessed in a murine vocal fold injection model. The transcriptional effect of NPHGF on procollagen was measured in vitro to assess whether released growth factor retained functionality. RESULTS: In vitro release kinetics demonstrated slow release of NPTR, NPBSA, and NPHGF over 12 to 14 days. In vitro NPTR release correlated with in vivo results. In vivo presence of NPTR occurred up to 7 days compared to 1 day for Texas Red control. In addition, NPHGF ameliorated transforming growth factor-beta induced procollagen in vitro in 3T3 fibroblast cells. CONCLUSIONS: The results demonstrate the potential utility of nanoparticle encapsulation as an effective method for long-term delivery of specific drugs and biologically active substances to the larynx.

Thin-film enhanced nerve guidance channels for peripheral nerve repair.

It has been demonstrated that nerve guidance channels containing stacked thin-films of aligned poly(acrylonitrile-co-methylacrylate) fibers support peripheral nerve regeneration across critical sized nerve gaps, without the aid of exogenous cells or proteins. Here, we explore the ability of tubular channels minimally supplemented with aligned nanofiber-based thin-films to promote endogenous nerve repair. We describe a technique for fabricating guidance channels in which individual thin-films are fixed into place within the lumen of a polysulfone tube. Because each thin-film is <10 microm thick, this technique allows fine control over the positioning of aligned scaffolding substrate. We evaluated nerve regeneration through a 1-film guidance channel--containing a single continuous thin-film of aligned fibers--in comparison to a 3-film channel that provided two additional thin-film tracks. Thirty rats were implanted with one of the two channel types, and regeneration across a 14 mm tibial nerve gap was evaluated after 6 weeks and 13 weeks, using a range of morphological and functional measures. Both the 1-film and the 3-film channels supported regeneration across the nerve gap resulting in functional muscular reinnervation. Each channel type characteristically influenced the morphology of the regeneration cable. Interestingly, the 1-film channels supported enhanced regeneration compared to the 3-film channels in terms of regenerated axon profile counts and measures of nerve conduction velocity. These results suggest that minimal levels of appropriately positioned topographical cues significantly enhance guidance channel function by modulating endogenous repair mechanisms, resulting in effective bridging of critically sized peripheral nerve gaps.

Diagnostic challenge of fetal ontogeny and its application on the ovarian teratomas.

Although neuroepithelial tubules (NET) often are a component of immature teratoma (IT), they are not always required for diagnosis. Other somatic elements are sufficient and often verified with immunohistochemical stain. This study was designed to determine the definition of immaturity versus fetal ontogeny, using several molecular markers in IT. It is our contention that IT is equivalent to an embryonic stage less than a fertilization age (FA) of 8 weeks, and a mature teratoma (MT) to a fetal stage later than a FA of 8 weeks, whereas an embryonal carcinoma (Eca) matches a pre-embryonic stage earlier than a FA of 2 weeks. The teratomatous components used as a roadmap to evaluate maturity included: a lobular structure of primitive endodermal tubules (FA 4 to 6 weeks), a ventricle-lined cortical plate (FA 9 weeks), a complex papillary choroid plexus (FA 10 weeks), melanin deposition in hair follicles (FA 15 weeks), and the bell stage of odontogenesis (FA 19 weeks). The teratomatous components of 25 resected ovarian solid teratoma samples were compared with fetal ontogeny. For an immunohistochemical analysis, the CD30, CD34, CD99, bcl-2, alpha-fetoprotein (AFP), and placenta-like alkaline phosphatase (PLAP) were assessed. The AFP and Ki-1 were positive in the embryoid body, which was identified at a FA less than 4 weeks in Eca. The AFP was positive in the primitive endodermal components and some of the squamous epithelium in IT. The CD99 and bcl-2 were selectively stained in the primitive NET, which was detected no later than a FA of 6 weeks. The CD34 and bcl-2 were positive in the immature-looking precartilage blastomatous components, which proved useful for detecting immature cartilage, corresponding to a FA of 5 to 6 weeks. The ontogeny of IT was found to correspond to the embryonic stage at a FA of 2 to 8 weeks, and CD99, CD34, bcl-2, AFP, CD30, and PLAP could be used as supportive tools to define IT. This new grading system could be more scientific and more reproducible in any spectra of teratoma.