Neurovirulence, viscerotropism and immunogenicity of live attenuated yellow fever 17D vaccine virus in non-human primates.

Yellow fever vaccine associated neurovirulence and viscerotropism have been reported by various countries. In this study, the neurovirulence, viscerotropism and immunogenicity of yellow fever vaccine seed lots (master and working) and final product manufactured at Serum Institute of India (SII) were evaluated in cynomolgus monkeys. WHO reference virus 168-73 and Stamaril™ as a control vaccine was used for comparison. Neurovirulence and viscerotropism scores of the seed lots and final product were lower than Stamaril™. The SII seed virus and vaccine complies to the WHO requirement for neurovirulence, viscerotropism and immunogenicity, when tested in comparison to WHO reference seed virus 168/73. All challenged animals showed 100 % seroconversion as early as day 14 and neutralizing antibody titers were sustainable at day 30 in all animals.

Evaluation of manufacturing feasibility and safety of an MDCK cell-based live attenuated influenza vaccine (LAIV) platform.

Cell culture based live attenuated influenza vaccines (LAIV) as an alternative to egg-based LAIV have been explored because of lack of easy access to SPF eggs for large scale production. In this study, feasibility of MDCK platform was assessed by including multiple LAIV strains covering both type A (H1 and H3) and type B seasonal strains as well as the candidate pandemic potential strains like A/H5 and A/H7 for the growth in MDCK cells. A risk assessment study was conducted on the cell banks to evaluate safety concerns related to tumorigenicity with a regulatory perspective. Tumorigenic potential of the MDCK cells was evaluated in nude mice (107cells/mouse) model system. The 50% tumor producing dose (TPD50) of MDCK cells was studied in SCID mice to determine the amount of cells required for induction of tumors. Further, we conducted an oncogenicity study in three sensitive rodent species as per the requirements specified in the WHO guidelines. We determined TPD50 value of 1.9 X 104 cells/mice through subcutaneous route. Our results suggest that, the intranasal route of administration of the cell culture based LAIV pose minimal to no risk of tumorigenicity associated with the host cells. Also, non-oncogenic nature of MDCK cells was demonstrated. Host cell DNA in the vaccine formulations was < 10 ng/dose which ensures vaccine safety. Production efficiency and consistency were characterized and the observed titer values of the viral harvest and the processed bulk were comparable to the expansion in embryonated eggs. The present study clearly establishes the suitability of MDCK cells as a substrate for the manufacture of a safe and viable LAIV.

Polyplexes Are Endocytosed by and Trafficked within Filopodia.

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.

Detection of human parvovirus 4 DNA in the patients with acute encephalitis syndrome during seasonal outbreaks of the disease in Gorakhpur, India.

Seasonal outbreaks of acute encephalitis syndrome (AES) at Gorakhpur, India have been recognized since 2006. So far, the causative agent has not been identified. Use of next generation sequencing identified human parvovirus 4 (HPARV4) sequences in a CSF/plasma pool. These sequences showed highest identity with sequences earlier identified in similar patients from south India. Real-time PCR detected HPARV4 DNA in 20/78 (25.6%) CSF and 6/31 (19.3%) plasma of AES patients. Phylogenetic analysis classified three almost complete genomes and 24 partial NS1 sequences as genotype 2A. The observed association of HPARV4 with AES needs further evaluation. ELISAs for the detection of IgM and IgG antibodies against scrub typhus (Orientia tsutsugamushi, OT) showed ∼70% IgM/IgG positivity suggestive of etiologic association. Prospective, comprehensive studies are needed to confirm association of these agents, singly or in combination with AES in Gorakhpur region.

Immunogenicity and efficacy of the monovalent, trivalent and quadrivalent intranasal live attenuated influenza vaccines containing different pdmH1N1 strains.

A ferret challenge study was conducted to address the efficacy of the egg-based and Madin-Darby canine kidney (MDCK)-based live attenuated influenza vaccine (LAIV) strains. Vaccines derived as 6:2 reassortants from the A/Leningrad/134/17/57 master donor strain and the HA and NA components from the A/California/07/2009 (A/Cal)- and A/Michigan/45/2015 (A/Mich)-like strains of type A H1N1 influenza virus were used in the study. Monovalent, trivalent and quadrivalent formulations of the LAIV containing either of the two H1N1 strains were analysed. A total of ten groups of six animals each were immunised intranasally (i.n.) with a single dose of 0.5-ml vaccine formulation or placebo and challenged on day 28 with the homologous wild-type A/Cal or A/Mich strain. Immune response post immunisation and virus replication post challenge were studied. Both the strains derived from embryonated eggs or MDCK cells, irrespective of the vaccine valency, were capable of rendering complete protection from virus replication in the lung. The A/Mich vaccine strain showed higher immune titres and efficacy than the A/Cal vaccine strain in all the vaccine formulations. The haemagglutination inhibition and virus neutralisation antibody titres were induced, and the reduction in the virus load in the respiratory tract was observed to be higher in animals treated with the monovalent formulation compared to the trivalent and quadrivalent formulations. Overall, it appears that the monovalent formulations render better protection from infection and would therefore be the best candidate during a pandemic.

Evaluation of Liposome, Heat-Killed Mycobacterium w, and Alum Adjuvants in the Protection Offered by Different Combinations of Recombinant HA, NP proteins, and M2e Against Homologous H5N1 Virus.

Continued evolution of highly pathogenic H5N1 viruses causing high mortality in humans obviates need for broadly cross-reactive vaccines. For this, hemagglutinin (HA) inducing specific protective antibodies, highly conserved nucleoprotein (NP), and ectodomain of matrix (M2e) protein, either singly or in combination, were evaluated in BALB/c mice. Recombinant HA and NP (baculovirus system) and M2e (synthetic peptide) and 3 adjuvants, that is, liposomes, Mw (heat killed Mycobacterium w), and alum were utilized for the homologous virus challenge. Additional immunogens included liposome-encapsulated HA/NP proteins and corresponding DNAs. Mice groups received two doses of respective formulations given at 3-week intervals and challenged intranasally with 100LD50 of H5N1 virus strain. Dynamics of weight loss, lung viral load, titres of IgG-anti-HA, NP, and M2e antibodies (ELISA), and IgG-subtype analysis was done. Two doses of all the formulations led to 100% seroconversion against the immunogens evaluated (100% seroconversion after the first dose in majority). Antibody titres against the components were dependent on the adjuvant and combination. HA-driven Th2 response with all the adjuvants, balanced Th1/Th2 response to NP protein, and Th2-bias with alum were noted. Low anti-M2e antibody titres did not allow subtype analysis. On challenge, complete protection was observed with Mw-HA, alum-HA+NP, Lipo-HA+NP+M2e, alum-HA+NP+M2e, and HA-DP formulations with 12-fold, 8-fold, 720-fold, 17-fold, and no reduction, respectively, in lung viral load. In conclusion, the results identify several adjuvant-immunogen combinations conferring 100% protection in mice that need further evaluation in higher animals.

Fast, Efficient, and Gentle Transfection of Human Adherent Cells in Suspension.

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 (<1 min) for several human cell types. This fast, optimized and generally applicable cell transfection method can be used for rapid screening of different delivery systems and has significant potential for high-throughput cell therapy applications.

Trehalose-Based Block Copolycations Promote Polyplex Stabilization for Lyophilization and in Vivo pDNA Delivery.

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.

Inter-Clade Protection Offered by Mw-Adjuvanted Recombinant HA, NP Proteins, and M2e Peptide Combination Vaccine in Mice Correlates with Cellular Immune Response.

We documented earlier that Mw (heat-killed suspension of Mycobacterium indicus pranii) adjuvant when used with conserved antigens, nucleoprotein (NP), and ectodomain of matrix (M2) protein (M2e) provided complete protection against homologous (clade 2.2) virus challenge in mice. The present study extends these observations to inter-clade challenge (clade 2.3.2.1) H5N1 virus and attempts to understand preliminary immunologic basis for the observed protection. Female BALB/c mice immunized with a single or two doses of vaccine formulations (clade 2.2 antigens) were challenged with 100LD50 homologous or heterologous (clade 2.3.2.1) virus. To understand the preliminary immunologic mechanism, we studied proportions of selected immune cell types, immune response gene expression, and Th1/Th2 cytokines induced by antigen-stimulated splenocytes from immunized mice, at different time points. Complete protection was conferred by Mw-HA, Mw-HA + NP, and Mw-HA + NP + M2e against homologous challenge. The protection correlated with IgG2a antibody titers indicating important role of Th1 response. Despite high inter-cladal antigenic differences, complete protection against the heterologous strain was achieved with Mw-HA + NP + M2e. Of note, a single dose with higher antigen concentrations (50 µg HA + 50 µg NP + 50 µg M2e) led to 80% protection against clade 2.3.2.1 strain. The protection conferred by Mw-HNM correlated with induction of IFN-γ, CD8+ T cytotoxic cells, and CD4+ T helper cells. Mw-adjuvanted HA + NP + M2e combination represents a promising vaccine candidate deserving further evaluation.

Clinical study to evaluate the Brinhaniya effect of Vidarikandadi Yog to enhance the sport performance in children.

BACKGROUND: Sport science studies applications of scientific principles and techniques with the aim of improving sports performance. OBJECTIVE: Present research work was carried out with the aim to enhance the sport performance of children. MATERIALS AND METHODS: Randomized double blind placebo controlled study was conducted in children involved in sports to assess the efficacy of trial drug "Vidarikandadi Yog". Total of 72 healthy students were selected for the study after screening 412 students. Out of them, 60 students completed the study. The students were randomly divided into two groups. Group A (Vidarikandadi Yog) comprising of 38 and Group B (placebo) of 34 students. The trial drug "Vidarikandadi Yog" was given in the dose of 200 mg/kg/day in two divided doses for 2 months with milk and follow up was conducted fortnightly. RESULTS: The study revealed the statistically significant results for weight and chest circumference, whereas highly significant results were obtained for muscular strength and endurance assessment parameters (Push-up Test, Sit-up Test, and Hand Grip Strength Test). Change in Ruler Drop Test was not significant. Results were significant for cardio-respiratory parameters (Resting Heart Rate, Resting Respiratory Rate, and Harvard Step Test). CONCLUSION: Vidarikandadi Yog is a potential drug for enhancing the sport performance due to its Brinhaneeya effect.

Poly(trehalose): sugar-coated nanocomplexes promote stabilization and effective polyplex-mediated siRNA delivery.

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.

Spatiotemporal cellular imaging of polymer-pDNA nanocomplexes affords in situ morphology and trafficking trends.

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 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 h. The images were reconstructed to visualize and quantify trends in situ in a four-dimensional spatiotemporal 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.

Highlighting the role of polymer length, carbohydrate size, and nucleic acid type in potency of glycopolycation agents for pDNA and siRNA delivery.

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 ß-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.

Polymeric nucleic acid vehicles exploit active interorganelle trafficking mechanisms.

Materials that self-assemble with nucleic acids into nanocomplexes (e.g. polyplexes) are widely used in many fundamental biological and biomedical experiments. However, understanding the intracellular transport mechanisms of these vehicles remains a major hurdle in their effective usage. Here, we investigate two polycation models, Glycofect (which slowly degrades via hydrolysis) and linear polyethyleneimine (PEI) (which does not rapidly hydrolyze), to determine the impact of polymeric structure on intracellular trafficking. Cells transfected using Glycofect underwent increasing transgene expression over the course of 40 h and remained benign over the course of 7 days. Transgene expression in cells transfected with PEI peaked at 16 h post-transfection and resulted in less than 10% survival after 7 days. While saccharide-containing Glycofect has a higher buffering capacity than PEI, polyplexes created with Glycofect demonstrate more sustained endosomal release, possibly suggesting an additional or alternative delivery mechanism to the classical "proton sponge mechanism". PEI appeared to promote release of DNA from acidic organelles more than Glycofect. Immunofluorescence images indicate that both Glycofect and linear PEI traffic oligodeoxynucleotides to the Golgi and endoplasmic reticulum, which may be a route towards nuclear delivery. However, Glycofect polyplexes demonstrated higher co-localization with the ER than PEI polyplexes, and co-localization experiments indicate the retrograde transport of polyplexes via COP I vesicles from the Golgi to the ER. We conclude that slow release and unique trafficking behaviors of Glycofect polyplexes may be due to the presence of saccharide units and the degradable nature of the polymer, allowing more efficacious and benign delivery.

MAG versus PEG: Incorporating a Poly(MAG) Layer to Promote Colloidal Stability of Nucleic Acid/"Click Cluster" Complexes.

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 ß-cyclodextrin (ßCD) 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.

Hybrid coaxial electrospun nanofibrous scaffolds with limited immunological response created for tissue engineering.

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(ε-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.

Interaction of poly(ethylenimine)-DNA polyplexes with mitochondria: implications for a mechanism of cytotoxicity.

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.

Poly(glycoamidoamine)s: a broad class of carbohydrate-containing polycations for nucleic acid delivery.

In the era of nucleic acid therapeutics, there is an urgent need for non-viral delivery vehicles that can cross the extracellular and intracellular barriers and deliver nucleic acids to specific intracellular regions. This paper reviews the development of a subclass of polymer-based delivery vehicles termed poly(glycoamidoamine)s (PGAAs). The general design of this family consists of carbohydrate residues copolymerized with oligoethyleneamine units, which have proven to be an effective motif that promotes polyplex formation, efficient cellular internalization, high gene expression and low cytotoxicity with cultured cell lines and primary cell types. We then discuss the structure-property relationships of the PGAA class of delivery vehicles and studies aimed at understanding the mechanisms involved in cellular internalization and trafficking.

Bioresorbable elastomeric vascular tissue engineering scaffolds via melt spinning and electrospinning.

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.