How Do Amphiphilic Biopolymers Gel Blood? An Investigation Using Optical Microscopy.

Amphiphilic biopolymers such as hydrophobically modified chitosan (hmC) have been shown to convert liquid blood into elastic gels. This interesting property could make hmC useful as a hemostatic agent in treating severe bleeding. The mechanism for blood gelling by hmC is believed to involve polymer-cell self-assembly, i.e., insertion of hydrophobic side chains from the polymer into the lipid bilayers of blood cells, thereby creating a network of cells bridged by hmC. Here, we probe the above mechanism by studying dilute mixtures of blood cells and hmC in situ using optical microscopy. Our results show that the presence of hydrophobic side chains on hmC induces significant clustering of blood cells. The extent of clustering is quantified from the images in terms of the area occupied by the 10 largest clusters. Clustering increases as the fraction of hydrophobic side chains increases; conversely, clustering is negligible in the case of the parent chitosan that lacks hydrophobes. Moreover, the longer the hydrophobic side chains, the greater the clustering (i.e., C12 > C10 > C8 > C6). Clustering is negligible at low hmC concentrations but becomes substantial above a certain threshold. Finally, clustering due to hmC can be reversed by adding the supramolecule α-cyclodextrin, which is known to capture hydrophobes in its binding pocket. Overall, the results from this work are broadly consistent with the earlier mechanism, albeit with a few modifications.

Expanding Hydrophobically Modified Chitosan Foam for Internal Surgical Hemostasis: Safety Evaluation in a Murine Model.

BACKGROUND: A novel injectable expanding foam based on hydrophobically modified chitosan (HM-CS) was developed to improve hemostasis during surgeries. HM-CS is an amphiphilic derivative of the natural biopolymer chitosan (CS); HM-CS has been shown to improve the natural hemostatic characteristics of CS, but its internal safety has not been systematically evaluated. The goal of this study was to compare the long-term in vivo safety of HM-CS relative to a commonly used fibrin sealant (FS), TISSEEL (Baxter). METHODS: Sixty-four Sprague-Dawley rats (275-325 g obtained from Charles River Laboratories) were randomly assigned to control (n = 16) or experimental (n = 48) groups. Samples of the test materials (HM-CS [n = 16], CS [n = 16], and FS [n = 16]) applied to a nonlethal liver excision (0.4 ± 0.3 g of the medial lobe) in rats were left inside the abdomen to degrade. Animals were observed daily for signs of morbidity and mortality. Surviving animals were sacrificed at 1 and 6 wk; the explanted injury sites were microscopically assessed. RESULTS: All animals (64/64) survived both the 1- and 6-wk time points without signs of morbidity. Histological examination showed a comparable pattern of degradation for the various test materials. FS remnants and significant adhesions to neighboring tissues were observed at 6 wk. Residual CS and HM-CS were observed at the 6 wk with fatty deposits at the site of injury. Minimal adhesions were observed for CS and HM-CS. CONCLUSIONS: The internal safety observed in the HM-CS test group after abdominal implantation indicates that injectable HM-CS expanding foam may be an appropriate internal use hemostatic candidate.

Hydrophobically modified chitosan gauze: a novel topical hemostat.

BACKGROUND: Currently, the standard of care for treating severe hemorrhage in a military setting is Combat Gauze (CG). Previous work has shown that hydrophobically modified chitosan (hm-C) has significant hemostatic capability relative to its native chitosan counterpart. This work aims to evaluate gauze coated in hm-C relative to CG as well as ChitoGauze (ChG) in a lethal in vivo hemorrhage model. METHODS: Twelve Yorkshire swine were randomized to receive either hm-C gauze (n = 4), ChG (n = 4), or CG (n = 4). A standard hemorrhage model was used in which animals underwent a splenectomy before a 6-mm punch arterial puncture of the femoral artery. Thirty seconds of free bleeding was allowed before dressings were applied and compressed for 3 min. Baseline mean arterial pressure was preserved via fluid resuscitation. Experiments were conducted for 3 h after which any surviving animal was euthanized. RESULTS: hm-C gauze was found to be at least equivalent to both CG and ChG in terms of overall survival (100% versus 75%), number of dressing used (6 versus 7), and duration of hemostasis (3 h versus 2.25 h). Total post-treatment blood loss was lower in the hm-C gauze treatment group (4.7 mL/kg) when compared to CG (13.4 mL/kg) and ChG (12.1 mL/kg) groups. CONCLUSIONS: hm-C gauze outperformed both CG and ChG in a lethal hemorrhage model but without statistical significance for key endpoints. Future comparison of hm-C gauze to CG and ChG will be performed on a hypothermic, coagulopathic model that should allow for outcome significance to be differentiated under small treatment groups.

Hydrophobically-modified chitosan foam: description and hemostatic efficacy.

BACKGROUND: Trauma represents a significant public health burden, and hemorrhage alone is responsible for 40% of deaths within the first 24 h after injury. Noncompressible hemorrhage accounts for the majority of hemorrhage-related deaths. Thus, materials which can arrest bleeding rapidly are necessary for improved clinical outcomes. This preliminary study evaluated several self-expanding hydrophobically modified chitosan (HM-CS) foams to determine their efficacy on a noncompressible severe liver injury under resuscitation. METHODS: Six HM-CS foam formulations (HM-CS1, HM-CS2, HM-CS3, HM-CS4, HM-CS5, and HM-CS6) of different graft types and densities were synthesized, characterized, and packaged into spray canisters using dimethyl ether as the propellant. Expansion profiles of the foams were evaluated in bench testing. Foams were then evaluated in vitro, interaction with blood cells was determined via microscopy, and cytotoxicity was assessed via live-dead cell assay on MCF7 breast cancer cells. For in vivo evaluation, rats underwent a 14 ± 3% hepatectomy. The animals were treated with either: (1) an HM-CS foam formulation, (2) CS foam, and (3) no treatment (NT). All animals were resuscitated with lactated Ringer solution. Survival, total blood loss, mean arterial pressures (MAP), and resuscitation volume were recorded for 60 min. RESULTS: Microscopy showed blood cells immobilizing into colonies within tight groups of adjacent foam bubbles. HM-CS foam did not display any toxic effects in vitro on MCF7 cells over a 72 h period studied. Application of HM-CS foam after hepatectomy decreased total blood loss (29.3 ± 7.8 mL/kg in HM-CS5 group versus 90.9 ± 20.3 mL/kg in the control group; P <0.001) and improved survival from 0% in controls to 100% in the HM-CS5 group (P <0.001). CONCLUSIONS: In this model of severe liver injury, spraying HM-CS foams directly on the injured liver surface decreased blood loss and increased survival. HM-CS formulations with the highest levels of hydrophobic modification (HM-CS4 and HM-CS5) resulted in the lowest total blood loss and highest survival rates. This pilot study suggests HM-CS foam may be useful as a hemostatic adjunct or solitary hemostatic intervention.

Encapsulated fusion protein confers "sense and respond" activity to chitosan-alginate capsules to manipulate bacterial quorum sensing.

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.

Self-destructing "mothership" capsules for timed release of encapsulated contents.

We describe a new class of hierarchical containers that are formed via single-step assembly and, at a later time, self-destruct because of their packaged contents. These containers are spherical capsules formed by electrostatic complexation of the anionic biopolymer, gellan gum, with the cationic biopolymer, chitosan. The capsules are termed "motherships" and are engineered to carry a cargo of much smaller containers (e.g., nanoscale liposomes ("babyships")), within their lumen. Additionally, we package an enzyme, chitosanase, in the capsule that is capable of degrading polymeric chitosan into short oligomers. Thereby, we create motherships that self-destruct, liberating their cargo of babyships into the external solution. The time scale for self-destruction can be engineered based on the internal concentration of enzyme. The motherships are stable when stored in a freeze-dried form and can be readily dispersed into water or buffer solutions at a later time, whereupon their "internal clock" for self-destruction is initiated. The above concept could be useful for the triggered release of a variety of payloads including drugs, biological therapeutics, cosmetics, and flavor ingredients.

Foams with Enhanced Rheology for Stopping Bleeding.

Bleeding from injuries to the torso region is a leading cause of fatalities in the military and in young adults. Such bleeding cannot be stopped by applying direct pressure (compression) of a bandage. An alternative is to introduce a foam at the injury site, with the expansion of the foam counteracting the bleeding. Foams with an active hemostatic agent have been tested for this purpose, but the barrier created by these foams is generally not strong enough to resist blood flow. In this paper, we introduce a new class of foams with enhanced rheological properties that enable them to form a more effective barrier to blood loss. These aqueous foams are delivered out of a double-barrelled syringe by combining precursors that produce bubbles of gas (CO2) in situ. In addition, one barrel contains a cationic polymer (hydrophobically modified chitosan, hmC) and the other an anionic polymer (hydrophobically modified alginate, hmA). Both these polymers function as hemostatic agents due to their ability to connect blood cells into networks. The amphiphilic nature of these polymers also enables them to stabilize gas bubbles without the need for additional surfactants. hmC-hmA foams have a mousse-like texture and exhibit a high modulus and yield stress. Their properties are attributed to the binding of hmC and hmA chains (via electrostatic and hydrophobic interactions) to form a coacervate around the gas bubbles. Rheological studies are used to contrast the improved rheology of hmC-hmA foams (where a coacervate arises) with those formed by hmC alone (where there is no such coacervate). Studies with animal wound models also confirm that the hmC-hmA foams are more effective at curtailing bleeding than the hmC foams due to their greater mechanical integrity.

Multiphoton-absorption-induced-luminescence (MAIL) imaging of tumor-targeted gold nanoparticles.

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)ß3 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)ß3 integrin-mediated endocytosis being an important uptake mechanism.

pH-responsive jello: gelatin gels containing fatty acid vesicles.

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.

Determination of efficacy of a novel alginate dressing in a lethal arterial injury model in swine.

INTRODUCTION: Alginate is a biocompatible polysaccharide that is commonly used in the pharmaceutical, biomedical, cosmetic, and food industries. Though solid dressings composed of alginate can absorb water and promote wound healing, they are not effective hemostatic materials, particularly against massive hemorrhage. The purpose of this study is to attempt to increase the hemostatic capabilities of alginate by means of hydrophobic modification. Previous studies have illustrated that modifying a different polysaccharide, chitosan, in this way enhances its hemostatic efficacy as well as its adhesion to tissue. Here, it was hypothesized that modifying alginate with hydrophobic groups would demonstrate analogous effects. METHODS: Fifteen Yorkshire swine were randomized to receive hydrophobically-modified (hm) alginate lyophilized sponges (n=5), unmodified alginate lyophilized sponges (n=5), or standard Kerlix™ gauze dressing (n=5) for hemostatic control. Following a splenectomy, arterial puncture (6mm punch) of the femoral artery was made. Wounds were allowed to freely bleed for 30s, at which time dressings were applied and compressed for 3min in a randomized fashion. Fluid resuscitation was given to preserve the baseline mean arterial pressure. Wounds were monitored for 180min after arterial puncture, and surviving animals were euthanized. RESULTS: Blood loss for the hm-alginate group was significantly less than the two control groups of (1) alginate and (2) Kerlix™ gauze (p=<0.0001). Furthermore, 80% of hm-alginate sponges were able to sustain hemostasis for the full 180min, whereas 0% of dressings from the control groups were able to achieve initial hemostasis. CONCLUSIONS: Hm-alginate demonstrates a greatly superior efficacy, relative to unmodified alginate and Kerlix™ gauze dressings, in achieving hemostasis from a lethal femoral artery puncture in swine. This is a similar result as has been previously described when performing hydrophobic modification to chitosan. The current study further suggests that hydrophobic modification of a hydrophilic biopolymer backbone can significantly increase the hemostatic capabilities relative to the native biopolymer.

Sprayable Foams Based on an Amphiphilic Biopolymer for Control of Hemorrhage Without Compression.

Hemorrhage (severe blood loss) from traumatic injury is a leading cause of death for soldiers in combat and for young civilians. In some cases, hemorrhage can be stopped by applying compression of a tourniquet or bandage at the injury site. However, the majority of hemorrhages that prove fatal are "non-compressible", such as those due to an internal injury in the truncal region. Currently, there is no effective way to treat such injuries. In this initial study, we demonstrate that a sprayable polymer-based foam can be effective at treating bleeding from soft tissue without the need for compression. When the foam is sprayed into an open cavity created by injury, it expands and forms a self-supporting barrier that counteracts the expulsion of blood from the cavity. The active material in this foam is the amphiphilic biopolymer, hydrophobically modified chitosan (hmC), which physically connects blood cells into clusters via hydrophobic interactions (the hemostatic mechanism of hmC is thus distinct from the natural clotting cascade, and it works even with heparinized or citrated blood). The amphiphilic nature of hmC also allows it to serve as a stabilizer for the bubbles in the foam. We tested the hmC-based hemostatic foam for its ability to arrest bleeding from an injury to the liver in pigs. Hemostasis was achieved within minutes after application of the hmC foams (without the need for external compression). The total blood loss was 90% lower with the hmC foam relative to controls.

Reversible gelation of cells using self-assembling hydrophobically-modified biopolymers: towards self-assembly of tissue.

Polymer hydrogels have long been used to hold and culture biological cells within their three-dimensional (3-D) matrices. Typically, in such cases, the cells are passively entrapped in a mesh of polymer chains. Here, we demonstrate an alternate approach where cells serve as active structural elements (crosslinks) within a polymer gel network. The polymers used in this context are hydrophobically modified (hm) derivatives of common biopolymers such as chitosan and alginate. We show that hm-polymers rapidly transform a liquid suspension of cells into an elastic gel. In contrast, the native biopolymers (without hydrophobes) do not cause such gelation. Gelation occurs because the hydrophobes on the polymer get embedded within the hydrophobic interiors of cell bilayer membranes. The polymer chains thus connect the cells into a 3-D sample-spanning network, with the cells serving as the junctions in the network. We demonstrate that a variety of cell types, including blood cells, endothelial cells, and breast cancer cells can be gelled by this approach. Cells gelled by hm-alginate are shown to remain viable within the network. Also, since the crosslinking mechanism is based on hydrophobic interactions, we show that the addition of supramolecules with hydrophobic binding pockets can reverse the gelation and release the cells. Cell-gels can be employed as injectable biomaterials since the connections in the network are susceptible to shear, but recover rapidly once shear is stopped. The overall approach provides a simple route towards the directed assembly of cell clusters and potentially to living tissue.

Determination of efficacy of novel modified chitosan sponge dressing in a lethal arterial injury model in swine.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

A self-assembling hydrophobically modified chitosan capable of reversible hemostatic action.

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.