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Herein, we demonstrate the reversible addition–fragmentation chain transfer (RAFT) synthesis of an adamantane-conjugated glycopolymer, poly(2-methacrylamido-2-deoxy glucopyranose) (Ad-pMAG), as a hydrophilic coating to promote colloidal stability of click cluster-pDNA complexes in biological media. The Ad-pMAG is assembled via noncovalent interactions through inclusion complex formation between adamantane (Ad) and the β-cyclodextrin (βCD) core of the click cluster/pDNA and then further assembled with plasmid DNA to form polyplexes. Ad-pMAG incorporation was favorable over Ad-poly(ethylene glycol) (Ad-PEG) due to the enhanced colloidal stability of the click cluster/pDNA polyplex under physiological salt conditions at high N/P ratios. Interestingly, the uptake and reporter gene expression with polyplexes coated with the Ad-pMAG was much lower in HeLa cells than that observed with two glioma cell lines (U87 and U251 cells) in vitro, possibly indicating some delivery specificity.

The improvement of nonviral gene therapies relies to a large extent on understanding many fundamental physical and biological properties of these systems. This includes interactions of synthetic delivery systems with the cell and mechanisms of trafficking delivery vehicles, which remain poorly understood on both the extra- and intracellular levels. In this study, the mechanisms of cellular internalization and trafficking of polymer-based nanoparticle complexes consisting of polycations and nucleic acids, termed polyplexes, have been observed in detail at the cellular level. For the first time evidence has been obtained that filopodia, actin projections that radiate out from the surface of cells, serve as a route for the direct endocytosis of polyplexes. Confocal microscopy images demonstrated that filopodia on HeLa cells detect external polyplexes and extend into the extracellular milieu to internalize these particles. Polyplexes are observed to be internalized into membrane-bound vesicles (i.e., clathrin-coated pits and caveolae) directly within filopodial projections and are subsequently transported along actin to the main cell body for potential delivery of the nucleic acids to the nucleus. The kinetics and speed of polyplex trafficking have also been measured. The polyplex-loaded vesicles were also discovered to traffic between two cells within filopodial bridges. These findings provide novel insight into the early events of cellular contact with polyplexes through filopodial-based interactions in addition to endocytic vesicle trafficking-an important fundamental discovery to enable advancement of nonviral gene editing, nucleic acid therapies, and biomedical materials.

The development and thorough characterization of nonviral delivery agents for nucleic acid and genome editing therapies are of high interest to the field of nanomedicine. Indeed, this vehicle class offers the ability to tune chemical architecture/biological activity and readily package nucleic acids of various sizes and morphologies for a variety of applications. Herein, we present the synthesis and characterization of a class of trehalose-based block copolycations designed to stabilize polyplex formulations for lyophilization and in vivo administration. A 6-methacrylamido-6-deoxy trehalose (MAT) monomer was synthesized from trehalose and polymerized via reversible addition–fragmentation chain transfer (RAFT) polymerization to yield pMAT43. The pMAT43 macro-chain transfer agent was then chain-extended with aminoethylmethacrylamide (AEMA) to yield three different pMAT-b-AEMA cationic-block copolymers, pMAT-b-AEMA-1 (21 AEMA repeats), -2 (44 AEMA repeats), and -3 (57 AEMA repeats). These polymers along with a series of controls were used to form polyplexes with plasmids encoding firefly luciferase behind a strong ubiquitous promoter. The trehalose-coated polyplexes were characterized in detail and found to be resistant to colloidal aggregation in culture media containing salt and serum. The trehalose-polyplexes also retained colloidal stability and promoted high gene expression following lyophilization and reconstitution. Cytotoxicity, cellular uptake, and transfection ability were assessed in vitro using both human glioblastoma (U87) and human liver carcinoma (HepG2) cell lines wherein pMAT-b-AEMA-2 was found to have the optimal combination of high gene expression and low toxicity. pMAT-b-AEMA-2 polyplexes were evaluated in mice via slow tail vein infusion. The vehicle displayed minimal toxicity and discouraged nonspecific internalization in the liver, kidney, spleen, and lungs as determined by quantitative polymerase chain reaction (qPCR) and fluorescence imaging experiments. Hydrodynamic infusion of the polyplexes, however, led to very specific localization of the polyplexes to the mouse liver and promoted excellent gene expression in vivo.

While nucleic acids such as small interfering RNA (siRNA) and plasmid DNA (pDNA) are promising research tools and therapeutic modalities, their potential in medical applications is limited by a fundamental mechanistic understanding and inadequate efficiency. Herein, two series of carbohydrate-based polycations were synthesized and examined that varied in the degree of polymerization (n)—one containing trehalose [Tr4(n) series: Tr4(23), Tr4(55), Tr4(77)] and the other containing beta-cyclodextrin [CD4(n) series: CD4(10), CD4(26), CD4(39), CD4(143), CD4(239)]. In addition, two monosaccharide models were examined for comparison that contain tartaramidoamine (T4) and galactaramidoamine (G4 or Glycofect) repeats. Delivery profiles for pDNA were compared with those obtained for siRNA delivery and reveal that efficacy differs significantly as a function of carbohydrate type, nucleic acid type and dose, polymer length, and presence of excess polymer in the formulation. The Tr4 polymers yielded higher efficacy for pDNA delivery, yet, the CD4 polymers achieved higher siRNA delivery and gene down regulation. The T4 and Glycofect derivatives, while efficient for pDNA delivery, were completely ineffective for siRNA delivery. A strong polymer length and dose dependence on target gene knockdown was observed for all polymers tested. Also, free polymer in solution (uncomplexed) was demonstrated to be a key factor in promoting siRNA uptake and gene down regulation.

Synthetic polymers are ubiquitous in the development of drug and polynucleotide delivery vehicles, offering promise for personalized medicine. However, the polymer structure plays a central yet elusive role in dictating the efficacy, safety, mechanisms, and kinetics of therapeutic transport in a spatial and temporal manner. Here, we decipher the intracellular evolutionary pathways pertaining to shape, size, location, and mechanism of four structurally-divergent polymer vehicles (Tr455, Tr477, jetPEI™ and Glycofect™) that create colloidal nanoparticles (polyplexes) when complexed with fluorescently-labeled plasmid DNA (pDNA). Multiple high resolution tomographic images of whole HeLa (human cervical adenocarcinoma) cells were captured via confocal microscopy at 4, 8, 12 and 24 hours. The images were reconstructed to visualize and quantify trends in situ in a four-dimensional spatio-temporal manner. The data revealed heretofore-unseen images of polyplexes in situ and structure-function relationships, i.e., Glycofect™ polyplexes are trafficked as the smallest polyplex complexes and Tr455 polyplexes have expedited translocation to the perinuclear region. Also, all of the polyplex types appeared to be preferentially internalized and trafficked via early endosomes affiliated with caveolae, a Rab-5-dependent pathway, actin, and microtubules.

We demonstrate a highly efficient method for gene delivery into clinically relevant human cell types, such as induced pluripotent stem cells (iPSCs) and fibroblasts, reducing the protocol time by one full day. To preserve cell physiology during gene transfer, we designed a microfluidic strategy, which facilitates significant gene delivery in a short transfection time (

Current surgical therapy for diseased vessels less than 6mm in diameter involves bypass grafting with autologous arteries or veins. Although this surgical practice is common, it has significant limitations and complications, such as occlusion, intimal hyperplasia and compliance mismatch. As a result, cardiovascular biomaterials research has been motivated to develop tissue-engineered blood vessel substitutes. In this study, vascular tissue engineering scaffolds were fabricated using two different approaches, namely melt spinning and electrospinning. Small diameter tubes were fabricated from an elastomeric bioresorbable 50:50 poly(l-lactide-co-epsilon-caprolactone) copolymer having dimensions of 5mm in diameter and porosity of over 75%. Scaffolds electrospun from two different solvents, acetone and 1,1,1,3,3,3-hexafluoro-2-propanol were compared in terms of their morphology, mechanical properties and cell viability. Overall, the mechanical properties of the prototype tubes exceeded the transverse tensile values of natural arteries of similar caliber. In addition to spinning the polymer separately into melt-spun and electrospun constructs, the approach in this study has successfully demonstrated that these two techniques can be combined to produce double-layered tubular scaffolds containing both melt-spun macrofibers (200microm in diameter) and electrospun submicron fibers (400nm in diameter). Since the vascular wall has a complex multilayered architecture and unique mechanical properties, there remain several significant challenges before a successful tissue-engineered artery is achieved.

The focus of the current work was to study how the geometric design of a single barbed monofilament suture effects its biomechanical behavior. Different cut angles and cut depths of barbs were prepared and tested in vitro for their tensile and tissue anchoring properties by means of a novel suture/tissue pullout test. Experiments were also performed using bovine tendon and porcine skin tissues. The experimental results revealed that since tendon tissue has a higher modulus than skin it needs a more rigid barb to penetrate and anchor the surrounding tissue. A cut angle of 150 degrees and a cut depth of 0.18 mm are therefore recommended. On the other hand, for the softer skin tissue, a cut angle of 170 degrees and a cut depth of 0.18 mm provides a more flexible barb that gives superior skin tissue anchoring. These findings confirm that the future development of barbed suture technology requires a detailed understanding of the biomechanical properties of the tissue in which they are to be used. This will lead to the future development of a range of tissue-specific barbed sutures.

Sunnhemp is a coarse, strong and stiff bast fiber similar to jute fiber but not considered by textile technologists for the purpose of spinning into yarn, which can be further woven into fabric. The objective of this study was to spin 100% Sunnhemp fibers in to different yarns of same count on a jute spinning system by using different number of passages of carding and drawing to find how the structural changes in the fiber after each stage mechanical processing. To observe structural changes in the fiber, random sampling method was used to collect the fiber samples at various stages of processing and analysis of each fiber was done using microscope. It was found that a lot of changes occur in the fiber structure due to mechanical processing. The structural details about the fiber entity are depicted in the article. Further, correlation was established between various fiber parameters such as fiber length, fiber diameter and fiber number of branches. Finally, an exponential frequency distribution of the fiber lengths for various stages in the spinning process was calculated. This curve explains how the degree of individualization increases as the fiber length decreases and makes the fiber spinnable into better quality yarn.

This study was an attempt to spin 100% sunnhemp fibers, grown in Akola district of Maharashtra, India, on Jute spinning system. The sunnhemp fibers were first sprayed with oil and then softened. These fibers were then processed through various machine sequence by varying the number of carding and drawing machines. The yarn was spun in each case and tested for Count Strength Product and Evenness. This yarn was then used as a weft yarn to weave a 3/1-drill fabric with a cotton warp and tested for tensile strength. It was concluded that the set III with three carding and three drawing sequence gave an even yarn. The optimum twist per inch in the yarn was found to be 6.0.

The development and thorough characterization of nonviral delivery agents for nucleic acid and genome editing therapies are of high interest to the field of nanomedicine. Indeed, this vehicle class offers the ability to tune chemical architecture/biological activity and readily package nucleic acids of various sizes and morphologies for a variety of applications. Herein, we present the synthesis and characterization of a class of trehalose-based block copolycations designed to stabilize polyplex formulations for lyophilization and in vivo administration. A 6-methacrylamido-6-deoxy trehalose (MAT) monomer was synthesized from trehalose and polymerized via reversible addition-fragmentation chain transfer (RAFT) polymerization to yield pMAT

When nanoparticles interact with their environment, the nature of that interaction is governed largely by the properties of its outermost surface layer. Here, we exploit the exceptional properties of a common disaccharide, trehalose, which is well-known for its unique biological stabilization effects. To this end, we have developed a synthetic procedure that readily affords a polymer of this disaccharide, poly(methacrylamidotrehalose) or "poly(trehalose)" and diblock copolycations containing this polymer with 51 repeat units chain extended with aminoethylmethacrylamide (AEMA) at three degrees of polymerization (n = 34, 65, and 84). Two series of experiments were conducted to study these diblock copolymers in detail and to compare their properties to two control polymers [PEG-P(AEMA) and P(AEMA)]. First, we demonstrate that the poly(trehalose) coating ensures colloidal stability of polyplexes containing siRNA in the presence of high salt concentrations and serum proteins. Poly(trehalose) retains the ability of trehalose to lower the phase transition energy associated with water freezing and can protect siRNA polyplexes during freeze-drying, allowing complete nanoparticle resuspension without loss of biological function. Second, we show that siRNA polyplexes coated with poly(trehalose) have exceptional cellular internalization into glioblastoma cells that proceeds with zero-order kinetics. Moreover, the amount of siRNA delivered by poly(trehalose) block copolycations can be controlled by the siRNA concentration in cell culture media. Using confocal microscopy we show that trehalose-coated polyplexes undergo active trafficking in cytoplasm upon internalization and significant siRNA-induced target gene down-regulation was achieved with an IC50 of 19 nM. These findings coupled with a negligible cytotoxicity suggests that poly(trehalose) has the potential to serve as an important component of therapeutic nanoparticle formulations of nucleic acids and has great promise to be extended as a new coating for other nanobased technologies and macromolecules, in particular, those related to nanomedicine applications.

This review summarizes barbed suture technology. It discusses their development, materials used, design and manufacture, testing and properties as well as applications.

Electrospinning using synthetic and natural polymers is a promising technique for the fabrication of scaffolds for tissue engineering. Numerous synthetic polymers are available to maximize durability and mechanical properties (polyurethane) versus degradability and cell adhesion (polycaprolactone). In this study, we explored the feasibility of creating scaffolds made of bicomponent nanofibers from both polymers using a coaxial electrospinning system. We used a core of poly(urethane) and a sheath of a mixture of poly(e-caprolactone) and gelatin, all dissolved in 1,1,1,3,3,3-hexafluror-2-propanol. These nanofibrous scaffolds were then evaluated to confirm their core-sheath nature and characterize their morphology and mechanical properties under static and dynamic conditions. Furthermore, the antigenicity of the scaffolds was studied to confirm that there is no significant foreign body response to the scaffold itself that would preclude its use in vivo. The results show the advantages of combining both natural and synethic polymers to create a coaxial scaffold capable of withstanding dynamic culture conditions and encourage cellular migration to the interior of the scaffold for tissue-engineering applications. Also, the results show that there is no significant immunoreactivity in vivo to the components of the scaffolds.

Poly(ethylenimine) (PEI) and PEI-based systems have been widely studied for use as nucleic acid delivery vehicles. However, many of these vehicles display high cytotoxicity, rendering them unfit for therapeutic use. By exploring the mechanisms that cause cytotoxicity, and through understanding structure-function relationships between polymers and intracellular interactions, nucleic acid delivery vehicles with precise intracellular properties can be tailored for specific function. Previous research has shown that PEI is able to depolarize mitochondria, but the exact mechanism as to how depolarization is induced remains elusive and therefore is the focus of the current study. Potential mechanisms for mitochondrial depolarization include direct mitochondrial membrane permeabilization by PEI or PEI polyplexes, activation of the mitochondrial permeability transition pore, and interference with mitochondrial membrane proton pumps, specifically Complex I of the electron transport chain and F(0)F(1)-ATPase. Herein, confocal microscopy and live cell imaging showed that PEI polyplexes do colocalize to some degree with mitochondria early in transfection, and the degree of colocalization increases over time. Cyclosporin a was used to prevent activation of the mitochondrial membrane permeability transition pore, and it was found that early in transfection cyclosporin a was unable to prevent the loss of mitochondrial membrane potential. Further studies done using rotenone and oligomycin to inhibit Complex I of the electron transport chain and F(0)F(1)-ATPase, respectively, indicate that both of these mitochondrial proton pumps are functioning during PEI transfection. Overall, we conclude that direct interaction between polyplexes and mitochondria may be the reason why mitochondrial function is impaired during PEI transfection.

Synthetic polymers or naturally-derived extracellular matrix (ECM) proteins have been used to create tissue engineering scaffolds; however, the need for surface modification in order to achieve polymer biocompatibility and the lack of biomechanical strength of constructs built using proteins alone remain major limitations. To overcome these obstacles, we developed novel hybrid constructs composed of both strong biosynthetic materials and natural human ECM proteins. Taking advantage of the ability of cells to produce their own ECM, human foreskin fibroblasts were grown on silicon-based nanostructures exhibiting various surface topographies that significantly enhanced ECM protein production. After 4 weeks, cell-derived sheets were harvested and histology, immunochemistry, biochemistry and multiphoton imaging revealed the presence of collagens, tropoelastin, fibronectin and glycosaminoglycans. Following decellularization, purified sheet-derived ECM proteins were mixed with poly(epsilon-caprolactone) to create fibrous scaffolds using electrospinning. These hybrid scaffolds exhibited excellent biomechanical properties with fiber and pore sizes that allowed attachment and migration of adipose tissue-derived stem cells. Our study represents an innovative approach to generate strong, non-cytotoxic scaffolds that could have broad applications in tissue regeneration strategies.

Barbed surgical sutures are a new type of knotless suture that are currently being used clinically in cosmetic and plastic surgery procedures for faster healing and better cosmesis. Clinical studies are also underway to evaluate their performance in other deep tissue applications. However, little is known about their intrinsic mechanical behavior and their interactions with surrounding tissues. The primary objective of the current study was to analyze the mechanical behavior of barbed sutures using a finite element analysis approach. First, the effect of applying a point-pressure load to the tip of the barb and measuring its effect on barb displacement was studied. Second, the effect of an applied displacement to a barb anchored either in skin or tendon material for both the suture and the surrounding tissue. The results indicate that the flexibility of the barb can be increased or decreased by changing the barb geometry. It was concluded that the barb geometry and design need to be modified for use with different types of tissue. For example, in order to achieve the best mechanical anchoring with skin tissue the barb should be more flexible compared to the one designed to work with tendon tissue. The uniqueness of this study is that it is the first to establish a virtual prototyping and designing method for barbed sutures. For example, a new and improved virtual design of barb geometry is proposed and validated. It also provides the first report on how to develop a virtual bench top suture/tissue pullout testing environment.

Numerous endovascular stent grafts to treat intrarenal aortic aneurysms are now commercially available, and many new concepts are currently in development worldwide. In order to objectively quantify their outcomes, we propose a detailed protocol to examine a reference device that was harvested from a patient who died a few hours after endovascular stent-graft deployment for an abdominal aortic aneurysm according to the 3Bs rule (biocompatibility, biofunctionality, and biodurability). Relevant patient history of this 63-year-old man included radiotherapy treatment for lung cancer. Following the patient's death, the device was harvested en bloc together with the aneurysmal sac. The analysis of the device was conducted using nondestructive testing (X-rays, CT scan, magnetic resonance imaging [MRI], and endoscopy) and destructive testing (dissection, histology, and fabric and wire component analyses). Results from the gross examination demonstrated that the outer layer of the aneurysm sac was white, stiff, and continuous without any disruption. The Xray analysis, CT scan, and MRI confirmed that the device together with its modular segments was properly deployed at implantation. Endoscopy showed that the device was deployed securely immediately distal to the renal arteries. As anticipated, thin scattered mural thrombi at the blood/foreign material interface were observed on the blood tight flow surface. There were no tears in the fabric, and the dimensions and textile structure were well preserved. The metallic wires were intact. This fatality had no association with the stent graft as the patient's death was caused by the rupture of the pulmonary artery following intensive radiotherapy. In conclusion, autopsy, nondestructive testing, and destructive testing are therefore the necessary steps to validate any explanted endovascular stent graft in terms of biocompatibility, biofunctionality, and biodurability. In this specific case, the endovascular device fulfills the 3Bs rule. The authors recommend this protocol to investigate explanted endovascular devices.