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ABSTRACT: We demonstrate that "nanofactory"-loaded biopolymer capsules placed in the midst of a bacterial population can direct bacterial communication. Quorum sensing (QS) is a process by which bacteria communicate through small-molecules, such as autoinducer-2 (AI-2), leading to collective behaviors such as virulence and biofilm formation. In our approach, a "nanofactory" construct is created, which comprises an antibody complexed with a fusion protein that produces AI-2. These nanofactories are entrapped within capsules formed by electrostatic complexation of cationic (chitosan) and anionic (sodium alginate) biopolymers. The chitosan capsule shell is crosslinked by tripolyphosphate (TPP) to confer structural integrity. The capsule shell is impermeable to the encapsulated nanofactories, but freely permeable to small molecules. In turn, the capsules are able to take in substrates from the external medium via diffusion, and convert these via the nanofactories into AI-2, which then diffuses out. The exported AI-2 is shown to stimulate QS responses in vicinal Escherichia coli. Directing bacterial population behavior has potential applications in next-generation antimicrobial therapy and pathogen detection. We also envision such capsules to be akin to artificial "cells" that can participate in native biological signaling and communicate in real-time with the human microbiome. Through such interaction capabilities, these "cells" may sense the health of the microbiome, and direct its function in a desired, host-friendly manner. Biotechnol. Bioeng. © 2012 Wiley Periodicals, Inc.
Biotechnology and Bioengineering 08/2012; · 3.95 Impact Factor
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ABSTRACT: Chitosan is a functional biopolymer that has been widely used as a hemostat. Recently, its efficacy has been questioned due to clinical failures as a result of poor adhesiveness. The purpose of this study was to compare, in a severe groin injury model in swine, the hemostatic properties of an unmodified standard chitosan sponge with standard gauze dressing and a novel hydrophobically modified (hm) chitosan sponge. Previous studies have demonstrated that hm-chitosan provides greatly enhanced cellular adhesion and hemostatic effect via noncovalent insertion of hydrophobic pendant groups into cell membranes.
Twenty-four Yorkshire swine were randomized to receive hm-chitosan (n = 8), unmodified chitosan (n = 8), or standard Accu-Sorb gauze dressing (n = 8) for hemostatic control. A complex groin injury involving arterial puncture (4.4-mm punch) of the femoral artery was made after splenectomy. After 30 seconds of uncontrolled hemorrhage, the randomized dressing was applied and compression was held for 3 minutes. Fluid resuscitation was initiated to achieve and maintain the baseline mean arterial pressure and the wound was inspected for bleeding. Failure of hemostasis was defined as pooling of blood outside the wound. Animals were then monitored for 180 minutes and surviving animals were killed.
Blood loss before treatment was similar between groups (p < 0.1). Compared with the hm-chitosan sponge group, which had no failures, the unmodified chitosan sponge group and the standard gauze group each had eight failures over the 180-minute observation period. For the unmodified chitosan sponge failures, six of which provided initial hemostasis, secondary rebleeding was observed 44 minutes ± 28 minutes after application. Standard gauze provided no initial hemostasis after the 3-minute compression interval.
Hm-chitosan is superior to unmodified chitosan sponges (p < 0.001) or standard gauze for controlling bleeding from a lethal arterial injury. The hm-chitosan technology may provide an advantage over native chitosan-based dressings for control of active hemorrhage.
The journal of trauma and acute care surgery. 04/2012; 72(4):899-907.
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ABSTRACT: Blood loss at the site of a wound in mammals is curtailed by the rapid formation of a hemostatic plug, i.e., a self-assembled network of the protein, fibrin that locally transforms liquid blood into a gelled clot. Here, we report an amphiphilic biopolymer that exhibits a similar ability to rapidly gel blood; moreover, the self-assembly underlying the gelation readily allows for reversibility back into the liquid state via introduction of a sugar-based supramolecule. The biopolymer is a hydrophobically modified (hm) derivative of the polysaccharide, chitosan. When hm-chitosan is contacted with heparinized human blood, it rapidly transforms the liquid into an elastic gel. In contrast, the native chitosan (without hydrophobes) does not gel blood. Gelation occurs because the hydrophobes on hm-chitosan insert into the membranes of blood cells and thereby connect the cells into a sample-spanning network. Gelation is reversed by the addition of α-cyclodextrin, a supramolecule having an inner hydrophobic pocket: polymer hydrophobes unbind from blood cells and embed within the cyclodextrins, thereby disrupting the cell network. We believe that hm-chitosan has the potential to serve as an effective, yet low-cost hemostatic dressing for use by trauma centers and the military. Preliminary tests with small and large animal injury models show its increased efficacy at achieving hemostasis - e.g., a 90% reduction in bleeding time over controls for femoral vein transections in a rat model.
Biomaterials 02/2011; 32(13):3351-7. · 7.40 Impact Factor
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ABSTRACT: We demonstrate that multiphoton-absorption-induced luminescence (MAIL) is an effective means of monitoring the uptake of targeted nanoparticles into cells. Gold nanoparticles (AuNPs) with diameters of 4.5 and 16 nm were surface-functionalized with monocyclic RGDfK, an RGD peptide analogue that specifically targets the α(v)β₃ integrin, a membrane protein that is highly overexpressed in activated endothelial cells during tumor angiogenesis. To determine whether cyclic RGD can enhance the uptake of the functionalized AuNPs into activated endothelium, human umbilical vein endothelial cells (HUVECs) were used as a model system. MAIL imaging of HUVECs incubated with AuNPs demonstrates differential uptake of AuNPs functionalized with RGD analogues: RGDfK-modified nanoparticles are taken up by the HUVECs preferentially compared to AuNPs modified with linear RGD (GRGDSP) conjugates or with no surface conjugates. The luminescence counts observed for the AuNP-RGDfK conjugates are an order of magnitude greater than for AuNP-GRGDSP conjugates. Transmission electron microscopy shows that, once internalized, the AuNP-RGDfK conjugates remain primarily within endosomal and lysosomal vesicles in the cytoplasm of the cells. Significant aggregation of these particles was observed within the cells. MAIL imaging studies in the presence of specific uptake inhibitors indicate that AuNP-RGDfK conjugate uptake involves a specific binding event, with α(v)β₃ integrin-mediated endocytosis being an important uptake mechanism.
Bioconjugate Chemistry 10/2010; 21(11):1968-77. · 4.93 Impact Factor
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ABSTRACT: We describe a new way to impart pH-responsive properties to gels of biopolymers such as gelatin. This approach involves the embedding of pH-sensitive nanosized vesicles within the gel. The vesicles employed here are those of sodium oleate (NaOA), a fatty-acid-based amphiphile with a single C18 tail. In aqueous solution, NaOA undergoes a transition from vesicles at a pH approximately 8 to micelles at a pH higher than approximately 10. Here, we combine NaOA and gelatin at pH 8.3 to create a vesicle-loaded gel and then bring the gel in contact with a pH 10 buffer solution. As the buffer diffuses into the gel, the vesicles within the gel get transformed into micelles. Accordingly, a vesicle-micelle front moves through the gel, and this can be visually identified by the difference in turbidity between the two regions. Vesicle disruption can also be done in a spatially selective manner to create micelle-rich domains within a vesicle-loaded gel. A possible application of the above approach is in the area of pH-dependent controlled release. A vesicle-to-micelle transition releases hydrophilic solutes encapsulated within the vesicles into the bulk gel, and in turn these solutes can rapidly diffuse out of the gel into the external bath. Experiments with calcein dye confirm this concept and show that we can indeed use the pH in the bath to tune the release rate of solutes from vesicle-loaded gels.
Langmuir 04/2009; 25(15):8519-25. · 4.19 Impact Factor
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ABSTRACT: We describe a simple way to create patterns of ''soft'' biomolecular nanostructures such as vesicles on ''hard'' surfaces such as gold. The key to our approach is the use of an amphiphilic biopolymer as an ''interconnect'' or tether. The polymer is hydrophobically modified chitosan (hm-chitosan), which is obtained by covalently attaching alkyl tails to the backbone of chitosan. We electrodeposit films of hm-chitosan onto microscale gold cathodes formed by lithography on a silicon wafer. Subsequently, the hm-chitosan films are used to spontaneously capture vesicles from solution; this is demonstrated both for surfactant as well as lipid vesicles (liposomes). Vesicles remain strongly bound to the hm-chitosan to a much greater extent than to native chitosan. This suggests that the mechanism for vesicle capture involves non-covalent binding of hydrophobes from hm-chitosan chains to the hydrophobic portions of vesicle bilayers. Importantly, the vesicles captured by hm-chitosan films are intact—this is shown both by direct visualization of captured vesicles (via optical and cryo-transmission electron microscopy) as well as through the capture and subsequent disruption of dye-filled vesicles. Various microscale patterns of immobilized vesicles are created and the vesicles are demonstrated to be capable of sensing a reporter molecule from the external solution.
