Release of Bacteriocins from Nanofibers Prepared with Combinations of Poly(d,l-lactide) (PDLLA) and Poly(Ethylene Oxide) (PEO)

Plantaricin 423, produced by Lactobacillus plantarum, and bacteriocin ST4SA produced by Enterococcus mundtii, were electrospun into nanofibers prepared from different combinations of poly(d,l-lactide) (PDLLA) and poly(ethylene oxide) (PEO) dissolved in N,N-dimethylformamide (DMF). Both peptides were released from the nanofibers with a high initial burst and retained 88% of their original antimicrobial activity at 37 °C. Nanofibers have the potential to serve as carrier matrix for bacteriocins and open a new field in developing controlled antimicrobial delivery systems for various applications

New biomaterial based on cotton with incorporated biomolecules

The aim of this study was to investigate a method of embedding l-cysteine (l-cys), an antimicrobial agent, between layers of chitosan (CH) and sodium alginate (ALG) onto cotton samples obtained via a layer-by-layer electrostatic deposition technique via several embedding methods. The results show that the best way to incorporate l-cys into the layers was the one that used the property of gelling ALG. To monitor the l-cys embedding into the CH/ALG multilayer film, different methods were used: energy-dispersive X-ray spectrometry analysis to assess the presence of sulfur on the sample, Ellman's reagent method to analyze l-cys release from the sample, and attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (FTIR) to compare the ATR–FTIR spectra of the pure l-cys and l-cys embedded in the CH/ALG multilayer film to study the interaction between the l-cys and the CH/ALG multilayer films. Functionalized CH/ALG cotton samples were also investigated for their antibacterial properties toward Staphylococcus aureus andKlebsiella pneumonia with the Japanese Industrial Standard method JIS L 1902:2002, and the results show an enhancement of the antibacterial effect due to the presence of l-cys.The authors thank Fundação para a Ciência e Tecnologia for the funding granted concerning the project PTDC/EBB-BIO/113671/ 2009 (FCOMP-01-0124-FEDER-014752). Also, they thank Fundo Europeu de Desenvolvimento Regional through COMPETE—Programa Operacional Factores de Competitividade for cofunding

Amphiphilic beads as depots for sustained drug release integrated into fibrillar scaffolds

Native extracellular matrix (ECM) is a complex fibrous structure loaded with bioactive cues that affects the surrounding cells. A promising strategy to mimicking native tissue architecture for tissue engineering applications is to engineer fibrous scaffolds using electrospinning. By loading appropriate bioactive cues within these fibrous scaffolds, various cellular functions such as cell adhesion, proliferation and differentiation can be regulated. Here, we report on the encapsulation and sustained release of a model hydrophobic drug (dexamethasone (Dex)) within beaded fibrillar scaffold of poly(ethylene oxide terephthalate)-poly(butylene terephthalate) (PEOT/PBT), a polyether-ester multiblock copolymer to direct differentiation of human mesenchymal stem cells (hMSCs). The amphiphilic beads act as depots for sustained drug release that is integrated into the fibrillar scaffolds. The entrapment of Dex within the beaded structure results in sustained release of the drug over the period of 28days. This is mainly attributed to the diffusion driven release of Dex from the amphiphilic electrospun scaffolds. In vitro results indicate that hMSCs cultured on Dex containing beaded fibrillar scaffolds exhibit an increase in osteogenic differentiation potential, as evidenced by increased alkaline phosphatase (ALP) activity, compared to the direct infusion of Dex in the culture medium. The formation of a mineralized matrix is also significantly enhanced due to the controlled Dex release from the fibrous scaffolds. This approach can be used to engineer scaffolds with appropriate chemical cues to direct tissue regenerationAKG, SMM, LM and AK conceived the idea and designed the experiments. AKG and SMM fabricated electrospun scaffolds and performed the structural (SEM, FTIR), mechanical, and in vitro studies. AAK and AKGperformedDex release study. AKGand AP performed thermal analysis. AKG analyzed experimental data. AKG, SMM, LMand AK wrote the manuscript. ADL and CvB provided the polymers and corrected the manuscript. AKK, AP, MG and RLR revised the paper. All authors discussed the results and commented on the manuscript. Authors would like to thank Shilpaa Mukundan, Poornima Kulkarni and Dr. Arghya Paul for help with image analysis, drug release modeling and technical discussion respectively. AKG would like to thank Prof. Robert Langer for access to equipment and acknowledge financial support from MIT Portugal Program (MPP-09Call-Langer-47). SMMthanks the Portuguese Foundation for Science and Technology (FCT) for the personal grant SFRH/BD/42968/2008 (MIT-Portugal Program). This research was funded by the office of Naval Research Young National Investigator Award (AK), the Presidential Early Career Award for Scientists and Engineers (PECASE) (AK), the NIH (EB009196; DE019024; EB007249; HL099073; AR057837), the National Science Foundation CAREER award (DMR 0847287; AK), and the Dutch Technology Foundation (STW # 11135; LM, CvB, and AD)

Investigation of size–dependent cell adhesion on nanostructured interfaces

BACKGROUND: Cells explore the surfaces of materials through membrane-bound receptors, such as the integrins, and use them to interact with extracellular matrix molecules adsorbed on the substrate surfaces, resulting in the formation of focal adhesions. With recent advances in nanotechnology, biosensors and bioelectronics are being fabricated with ever decreasing feature sizes. The performances of these devices depend on how cells interact with nanostructures on the device surfaces. However, the behavior of cells on nanostructures is not yet fully understood. Here we present a systematic study of cell-nanostructure interaction using polymeric nanopillars with various diameters. RESULTS: We first checked the viability of cells grown on nanopillars with diameters ranging from 200 nm to 700 nm. It was observed that when cells were cultured on the nanopillars, the apoptosis rate slightly increased as the size of the nanopillar decreased. We then calculated the average size of the focal adhesions and the cell-spreading area for focal adhesions using confocal microscopy. The size of focal adhesions formed on the nanopillars was found to decrease as the size of the nanopillars decreased, resembling the formations of nascent focal complexes. However, when the size of nanopillars decreased to 200 nm, the size of the focal adhesions increased. Further study revealed that cells interacted very strongly with the nanopillars with a diameter of 200 nm and exerted sufficient forces to bend the nanopillars together, resulting in the formation of larger focal adhesions. CONCLUSIONS: We have developed a simple approach to systematically study cell-substrate interactions on physically well-defined substrates using size-tunable polymeric nanopillars. From this study, we conclude that cells can survive on nanostructures with a slight increase in apoptosis rate and that cells interact very strongly with smaller nanostructures. In contrast to previous observations on flat substrates that cells interacted weakly with softer substrates, we observed strong cell-substrate interactions on the softer nanopillars with smaller diameters. Our results indicate that in addition to substrate rigidity, nanostructure dimensions are additional important physical parameters that can be used to regulate behaviour of cells

Nanofiber-mediated controlled release of siRNA complexes for long term gene-silencing applications

Nanofiber scaffold-mediated delivery of small-interfering RNA (siRNA) holds great potential in regenerative medicine by providing biomimicking topographical signals and enhanced gene silencing effects to seeded cells. While the delivery of naked siRNA was demonstrated previously using poly (ε-caprolactone) (PCL) nanofibers, the resulting siRNA release kinetics and gene knockdown efficiencies were sub-optimal. In this study, we investigated the feasibility of encapsulating siRNA and transfection reagent (TKO) complexes within nanofibers comprising of a copolymer of caprolactone and ethyl ethylene phosphate (PCLEEP, diameter ∼ 400 nm). Sustained release of bioactive naked siRNA and siRNA/TKO complexes were obtained for at least 28 days. By copolymerizing EEP with caprolactone, siRNA release was significantly enhanced (total siRNA that was released by day 49 was ∼ 89.3–97.2% as compared to previously reported 3% by plain PCL nanofiber delivery). Using GAPDH as the model protein, bioactivity analyses by supernatant transfection revealed the partial retention of bioactivity of naked siRNA and siRNA/TKO complexes for at least 30 days. In particular, GAPDH siRNA/TKO supernatant alone induced significant gene silencing (∼40%), indicating the feasibility of co-encapsulating siRNA and transfection reagent within a single scaffold construct for sustained delivery. Direct culture of cells on siRNA incorporated scaffolds for scaffold-mediated gene transfection revealed significant gene knockdown even in the absence of transfection reagent (21.3% knockdown efficiency by scaffolds incorporating naked siRNA only). By encapsulating siRNA/TKO complexes, more significant gene knockdown was obtained (30.9% knockdown efficiency as compared to previously reported 18% by plain PCL scaffold-mediated transfection). Taken together, the results demonstrated the feasibility of co-encapsulating siRNA-transfection reagent complexes within a single nanofiber construct for sustained siRNA delivery and enhanced gene knockdown efficiency. The study also highlights the potential of PCLEEP as a platform for tailoring siRNA release kinetics for long-term gene silencing applications

Directing stem cell fate by controlled RNA interference

Directing stem cell fate remains a major area of interest and also a hurdle to many, particularly in the field of regenerative medicine. Unfortunately, conventional methods of over-expressing inductive factors through the use of biochemical induction cocktails have led to sub-optimal outcomes. A potential alternative may be to adopt the opposite by selectively silencing genes or pathways that are pivotal to stem cell differentiation. Indeed, over recent years, there have been an increasing number of studies on directing stem cell fate through gene knockdown via RNA interference (RNAi). While the effectiveness of RNAi in controlling stem cell differentiation is evident from the myriad of studies, a chaotically vast collection of gene silencing targets have also been identified. Meanwhile, variations in methods of transfecting stem cells have also affected silencing efficiencies and the subsequent extent of stem cell differentiation. This review serves to unite the pioneers who have ventured into the emerging field of RNAi-enhanced stem cell differentiation by summarizing and evaluating the current approaches adopted in utilizing gene silencing to direct stem cell fate and their corresponding outcomes

Nanofiber-mediated release of retinoic acid and brain-derived neurotrophic factor for enhanced neuronal differentiation of neural progenitor cells

The treatment of an injured central nervous system using stem-cell-based regenerative medicine still faces considerable hurdles that need to be overcome. Chief among which is the lack of efficient strategies to generate functional neurons from stem cells. The sustained delivery of biochemical cues and synergistic topographical signaling from electrospun nanofibrous scaffolds may be a potential strategy to enhance neuronal differentiation of stem cells for therapeutic purposes. In this study, retinoic acid (RA) and brain-derived neurotrophic factor (BDNF) were encapsulated into a copolymer of ε-caprolactone and ethyl ethylene phosphate to form a multifunctional, electrospun nanofibrous scaffold. Sustained release of RA and BDNF was achieved for at least 7 and 14 days, respectively. Despite lower cumulative release of drugs as compared to bolus delivery to plain nanofibers (at least 2× and 50× lower for RA and BDNF, respectively), nanofiber-mediated delivery of RA and/or BDNF resulted in similar capacity for neuronal differentiation of mouse neural progenitor cells (NPCs). In addition, nanofiber topography significantly increased neuronal differentiation (with BDNF, 47.4 % Map2+ cells on 2D vs. 53.4 to 56.5 % on nanofibers, p < 0.05) and reduced glial cell differentiation. BDNF was a more potent inducer of neuronal differentiation than RA. RA supplementation alone resulted in minimal effect on NPC differentiation, and dual supplementation of RA and BDNF did not further enhance the neuronal differentiation of NPCs. Collectively, the results suggest that synergistic effects of nanofiber topography and sustained delivery of RA and/or BDNF may contribute towards the design of a multifunctional artificial stem cell niche for NPC neuronal differentiation.NMRC (Natl Medical Research Council, S’pore