Optimized method for isolating highly purified and functional porcine aortic endothelial and smooth muscle cells.

Numerous protocols exist for isolating aortic endothelial and smooth muscle cells from small animals. However, establishing a protocol for isolating pure cell populations from large animal vessels that are more elastic has been challenging. We developed a simple sequential enzymatic approach to isolate highly purified populations of porcine aortic endothelial and smooth muscle cells. The lumen of a porcine aorta was filled with 25 U/ml dispase solution and incubated at 37°C to dissociate the endothelial cells. The smooth muscle cells were isolated by mincing the tunica media of the treated aorta and incubating the pieces in 0.2% and then 0.1% collagenase type I solution. The isolated endothelial cells stained positive for von Willebrand factor, and 97.2% of them expressed CD31. Early and late passage endothelial cells had a population doubling time of 38 hr and maintained a capacity to take up DiI-Ac-LDL and form tubes in Matrigel®. The isolated smooth muscle cells stained highly positive for alpha-smooth muscle actin, and an impurities assessment showed that only 1.8% were endothelial cells. Population doubling time for the smooth muscle cells was ∼70 hr at passages 3 and 7; and the cells positively responded to endothelin-1, as shown by a 66% increase in the intracellular calcium level. This simple protocol allows for the isolation of highly pure populations of endothelial and smooth muscle cells from porcine aorta that can survive continued passage in culture without losing functionality or becoming overgrown by fibroblasts.

Release-Modulated Antioxidant Activity of a Composite Curcumin-Chitosan Polymer.

Curcumin is known to have immense therapeutic potential but is hindered by poor solubility and rapid degradation in solution. To overcome these shortcomings, curcumin has been conjugated to chitosan through a pendant glutaric anhydride linker using amide bond coupling chemistry. The hybrid polymer has been characterized by UV-visible, fluorescence, and infrared spectroscopies as well as zeta potential measurements and SEM imaging. The conjugation reactivity was confirmed through gel permeation chromatography and quantification of unconjugated curcumin. An analogous reaction of curcumin with glucosamine, a small molecule analogue for chitosan, was performed and the purified product characterized by mass spectrometry, UV-visible, fluorescence, and infrared spectroscopies. Conjugation of curcumin to chitosan has greatly improved curcumin aqueous solubility and stability, with no significant curcumin degradation detected after one month in solution. The absorbance and fluorescence properties of curcumin are minimally perturbed (λmax shifts of 2 and 5 nm, respectively) by the conjugation reaction. This conjugation strategy required use of one out of two curcumin phenols (one of the main antioxidant functional groups) for covalent linkage to chitosan, thus temporarily attenuating its antioxidant capacity. Hydrolysis-based release of curcumin from the polymer, however, is accompanied by full restoration of curcumin's antioxidant potential. Antioxidant assays show that curcumin radical scavenging potential is reduced by 40% after conjugation, but that full antioxidant potential is restored upon hydrolytic release from chitosan. Release studies show that curcumin is released over 19 days from the polymer and maintains a concentration of 0.23 ± 0.12 µM curcumin/mg polymer/mL solution based on 1% curcumin loading on the polymer. Release studies in the presence of carbonic anhydrase, an enzyme with known phenolic esterase activity, show no significant difference from nonenzymatic release studies, implying that simple ester hydrolysis is the dominant release mechanism. Conjugation of curcumin to chitosan through a phenol ester modification provides improved stability and solubility to curcumin, with ester hydrolysis restoring the full antioxidant potential of curcumin.

Curcumin encapsulation in submicrometer spray-dried chitosan/Tween 20 particles.

Optimal curcumin delivery for medicinal applications requires a drug delivery system that both solubilizes curcumin and prevents degradation. To achieve this, curcumin has been encapsulated in submicrometer chitosan/Tween 20 particles via a benchtop spray-drying process. Spray-drying parameters have been optimized using a Taguchi statistical approach to minimize particle size and to favor spheroid particles with smooth surfaces, as evaluated with scanning electron microscopy (SEM) imaging. Nearly spherical particles with 285 ± 30 nm diameter and 1.21 axial ratio were achieved. Inclusion of curcumin in the spray-drying solution results in complete encapsulation of curcumin within the chitosan/Tween 20 particles. Release studies confirm that curcumin can be released completely from the particles over a 2 h period.

Biomimetic hydrogels with VEGF induce angiogenic processes in both hUVEC and hMEC.

Angiogenesis is the process by which new blood vessels arise from the pre-existing vasculature. Human endothelial cells are known to be involved in three key cellular processes during angiogenesis: increased cell proliferation, degradation of the extracellular matrix during cell migration, and the survival of apoptosis. The above processes depend upon the presence of growth factors, such as vascular endothelial growth factor isoform 165 (VEGF(165)) that is released from the extracellular matrix as it is being degraded or secreted from activated endothelial cells. Thus, the goal of the current study is to develop a system with a backbone of polyethylene glycol (PEG) and grafted angiogenic signals to compare the initial angiogenic response of human umbilical vein endothelial cells (hUVEC) or human microvascular endothelial cells (hMEC). Adhesion ligands (PEG-RGDS) for cell attachment and PEG-modified VEGF(165) (PEG-VEGF(165)) are grafted into the hydrogels to encourage the angiogenic response. Our data suggest that our biomimetic system is equally effective in stimulating proliferation, migration, and survival of apoptosis in hMEC as compared to the response to hUVEC.

Decellularized matrices in regenerative medicine.

Of all biologic matrices, decellularized extracellular matrix (dECM) has emerged as a promising tool used either alone or when combined with other biologics in the fields of tissue engineering or regenerative medicine - both preclinically and clinically. dECM provides a native cellular environment that combines its unique composition and architecture. It can be widely obtained from native organs of different species after being decellularized and is entitled to provide necessary cues to cells homing. In this review, the superiority of the macro- and micro-architecture of dECM is described as are methods by which these unique characteristics are being harnessed to aid in the repair and regeneration of organs and tissues. Finally, an overview of the state of research regarding the clinical use of different matrices and the common challenges faced in using dECM are provided, with possible solutions to help translate naturally derived dECM matrices into more robust clinical use. STATEMENT OF SIGNIFICANCE: Ideal scaffolds mimic nature and provide an environment recognized by cells as proper. Biologically derived matrices can provide biological cues, such as sites for cell adhesion, in addition to the mechanical support provided by synthetic matrices. Decellularized extracellular matrix is the closest scaffold to nature, combining unique micro- and macro-architectural characteristics with an equally unique complex composition. The decellularization process preserves structural integrity, ensuring an intact vasculature. As this multifunctional structure can also induce cell differentiation and maturation, it could become the gold standard for scaffolds.

Silk fibroin mediated delivery of liposomal emodin to breast cancer cells.

The efficacy of a drug is dependent on its mode of delivery and its potency at the tumor site. In this study, the drug delivery and efficacy of silk fibroin coated liposomes (SF-ELP), encapsulating a receptor tyrosine kinase inhibitor, emodin, on Her2/neu over-expressing breast cancer cells, was investigated. This study demonstrates that SF-ELP was more efficacious in suppressing the growth of Her2/neu over-expressing breast cancer cells MDA-MB-453 and BT-474 as compared to uncoated emodin loaded liposomes (ELP). Reduced levels of phosphorylated Her2/neu correlated with growth inhibition observed in the MDA-MB-453 cells, treated with both ELP and SF-ELP. ELP treatment of MDA-MB-453 breast cancer cells resulted in inhibition of the PI3K pathway whereas SF-ELP treatment inhibited both the PI3K and MAPK pathways, which contributed to the enhanced growth inhibitory effects of Her2/neu over-expressing breast cancer cells. Coating of ELP with silk fibroin did not alter the target specificity of emodin, on the other hand the emodin efficacy was enhanced. Higher uptake of emodin delivered by SF-ELP lead to increased cell death as compared to emodin delivery via ELP. Silk fibroin coating around the liposome imparts an extra layer that emodin has to extravasate in order to release from the encapsulating liposome. This increases retention of the drug in the cell for a longer time and protects emodin from quick release and metabolism. Longer intracellular retention may lead to the longer availability of emodin for down-modulation of various Her2/neu pathways. This study demonstrates that silk fibroin coating enhanced emodin delivery in Her2/neu over-expressing breast cancer cells thereby increasing the overall efficacy of the drug.

Inverted orientation improves decellularization of whole porcine hearts.

In structurally heterogeneous organs, such as heart, it is challenging to retain extracellular matrix integrity in the thinnest regions (eg, valves) during perfusion decellularization and completely remove cellular debris from thicker areas. The high inflow rates necessary to maintain physiologic pressure can distend or damage thin tissues, but lower pressures prolong the process and increase the likelihood of contamination. We examined two novel retrograde decellularization methods for porcine hearts: inverting the heart or venting the apex to decrease inflow rate. We measured flow dynamics through the aorta (Ao) and pulmonary artery (PA) at different Ao pressures and assessed the heart's appearance, turbidity of the outflow solutions, and coronary perfusion efficiency. We used rectangle image fitting of decellularized heart images to obtain a heart shape index. Using nonlinear optical microscopy, we determined the microstructure of collagen and elastin fibers of the aortic valve cusps. DNA, glycosaminoglycan, and residual detergent levels were compared. The inverted method was superior to the vented method, as shown by a higher coronary perfusion efficiency, more cell debris outflow, higher collagen and elastin content inside the aortic valve, lower DNA content, and better retention of the heart shape after decellularization. To our knowledge, this is the first study to use flow dynamics in a whole heart throughout the decellularization procedure to provide real-time information about the success of the process and the integrity of the vulnerable regions of the matrix. Heart orientation was important in optimizing decellularization efficiency and maintaining extracellular matrix integrity. STATEMENT OF SIGNIFICANCE: The use of decellularized tissue as a suitable scaffold for engineered tissue has emerged over the past decade as one of the most promising biofabrication platforms. The decellularization process removes all native cells, leaving the natural biopolymers, extracellular matrix materials and native architecture intact. This manuscript describes heart orientation as important in optimizing decellularization efficiency and maintaining extracellular matrix integrity. To our knowledge, this is the first study to assess flow dynamics in a whole heart throughout the decellularization procedure. Our findings compared to currently published methods demonstrate that continuous complex real-time measurements and analyses are required to produce an optimal scaffold for cardiac regeneration.

Repair and regeneration of the abdominal wall musculofascial defect using silk fibroin-chitosan blend.

Reconstructive surgery with synthetic or biological materials is commonly performed to repair abdominal wall musculofascial defects that result from ventral hernias. A study was conducted to investigate the feasibility of using silk fibroin and chitosan blend (SFCS) scaffolds for ventral hernia repair in guinea pigs. We compared SFCS with biodegradable human acellular dermal matrix (HADM) and nonbiodegradable polypropylene mesh by implanting each to repair an incisionally created ventral hernia in the abdominal wall using an inlay technique. At 4 weeks, both HADM and SFCS underwent remodeling by host tissue, but polypropylene mesh resulted in extensive bowel adhesions and scarring. Abdominal wall repairs with SFCS showed tissue remodeling in all 3 dimensions, with seamless integration at the interface with adjacent native tissue. The SFCS repair sites remained intact, and their mechanical strength was similar to that of the native abdominal wall despite greater degradation and remodeling of SFCS than of HADM. The deposition of new extracellular matrix consisting of collagen and ground substance, uniform vascularization, and cellular infiltration in SFCS repair sites contributed to the increase in mechanical strength of the regenerated tissue. Thus, SFCS is a potentially useful material for clinical abdominal wall reconstruction, since it becomes remodeled and integrated into the surrounding abdominal wall and maintains adequate tensile strength.

Poly(ethylene glycol) hydrogel system supports preadipocyte viability, adhesion, and proliferation.

The ultimate goal of this research is to develop an injectable cell-scaffold system capable of permitting adipogenesis to abrogate soft tissue deficiencies resulting from trauma, tumor resection, and congenital abnormalities. The present work compares the efficacy of photopolymerizable poly(ethylene glycol) and specific derivatives as a scaffold for preadipocyte (adipocyte precursor cell) viability, adhesion, and proliferation. Four variations of a poly(ethylene glycol) scaffold are prepared and examined. The first scaffold consists of poly(ethylene glycol) diacrylate, which is not susceptible to hydrolysis or enzymatic degradation. Preadipocyte death is observed over 1 week in this hydrogel configuration. Adhesion sites, specifically the laminin-binding peptide sequence YIGSR, were incorporated into the second scaffold to promote cellular adhesion as a prerequisite for preadipocyte proliferation. Preadipocytes remain viable in this scaffold system, but do not proliferate in this nondegradable hydrogel. The third scaffold system studied consists of poly(ethylene glycol) modified with the peptide sequence LGPA to permit polymer degradation by cell-secreted collagenase. No adhesion peptide is incorporated into this scaffold system. Cellular proliferation is initially observed, followed by cell death. The previous three scaffold configurations do not permit preadipocyte adhesion and proliferation. In contrast, the fourth system studied, poly(ethylene glycol) modified to incorporate both LGPA and YIGSR, permits preadipocyte adherence and proliferation subsequent to polymer degradation. Our results indicate that a scaffold system containing specific degradation sites and cell adhesion ligands permits cells to adhere and proliferate, thus providing a potential cell-scaffold system for adipogenesis.

Cell migration through defined, synthetic ECM analogs.

We have developed synthetic hydrogel extracellular matrix (ECM) analogues that can be used to study mechanisms involved in cell migration, such as receptor-ligand interactions and proteolysis. The biomimetic hydrogels consist of bioinert polyethylene glycol diacrylate derivatives with proteolytically degradable peptide sequences included in the backbone of the polymer and adhesive peptide sequences grafted to the network. Hydrogels have been developed that degrade as cells secrete proteolytic enzymes. Adhesive peptide sequences grafted to the hydrogel provide ligands that can interact with receptors on the cell surface to mediate adhesion and spreading. In this study, we have characterized the effects of adhesive ligand density on fibroblast migration through collagenase-degradable and plasmin-degradable hydrogels and on smooth muscle cell migration through elastase-degradable hydrogels. In all three cases, we found that cell migration has a biphasic dependence on adhesion ligand concentration, with optimal migration at intermediate ligand levels. Furthermore, both adhesive and proteolytically degradable sequences were required for cell migration to occur. These synthetic ECM analogues may be useful for 3-D mechanistic studies of many aspects of cell migration

Covalent immobilization of RGDS on hydrogel surfaces to direct cell alignment and migration.

This study extends the capability for directing cell behavior using PEG-based hydrogels in tissue-engineering applications to include control over the spatial distribution of the adhesive peptide, RGDS. A continuous linear gradient was formed by simultaneously using a gradient maker to combine precursor solutions and using photopolymerization to lock the RGDS gradient in place. Hydrogels containing entrapped gradients of bovine serum albumin (BSA) were characterized using Coomassie brilliant blue stain, which indicated that BSA concentration increases along the hydrogel's length and that the steepness of the gradient's slope can be varied by changing the relative BSA concentrations in the precursor solutions. Human dermal fibroblasts responded to covalently immobilized RGDS gradients by changing their morphology to align in the direction of increasing RGDS concentration. After 24 h, approximately 46% of fibroblasts were aligned with the RGDS-gradient axis. This proportion of cells further increased to approximately 53% (p < 0.05) and approximately 58% after 48 and 96 h, respectively. Also, fibroblasts migrated differentially depending on the concentration of RGDS. Fibroblasts migrated approximately 48% further going up the concentration gradient (0 to 6 micromol/ml PEG-RGDS) than going down the concentration gradient. Migration up the concentration gradient was also approximately 33% greater than migration on control surfaces with a constant concentration of RGDS (2 micromol/ml), while migration down the gradient was reduced approximately 12% relative to the control surface. In addition, directed migration was further enhanced by increasing the RGDS gradient's slope. This hydrogel system is expected to be useful for directing cell migration to enhance the formation of engineered tissues.

Structural and mechanical characteristics of silk fibroin and chitosan blend scaffolds for tissue regeneration.

The expanding field of tissue engineering has required the necessity of developing biomaterials that are tissue compatible, biodegradable, and comparable in mechanical properties to that of native tissue. We propose that the blending of two natural polymers, silk fibroin (SF) and chitosan (CS), into a 3D scaffold will provide unique chemical, structural, and mechanical properties that can be utilized for in vivo tissue regeneration. SF is an attractive material for biomedical applications because it is a fibrous protein that has high permeability to oxygen and water, relatively low thrombogenicity, low inflammatory response, protease susceptibility, supports cell adhesion and growth, and, foremost, high tensile strength with flexibility. CS is a crystalline polysaccharide, with structure similar to glycosaminoglycans, that has good wound healing properties, is nontoxic, and has minimal foreign body reactions. We hypothesized that increasing the SF-to-CS ratio would increase the ultimate tensile strength and elastic modulus and decrease the water capacity of the SFCS scaffolds. With increasing content of silk fibroin, it is observed that the ultimate tensile strength and elastic modulus increase significantly. The ultimate tensile strength and the elastic modulus were significantly higher in the short axis direction for 25:75 and 50:50 SFCS blends as compared to the long axis (p<0.05), while they were similar for the 75:25 SFCS blend. However, no differences were observed in the strain at failure among blends or due to directionality of applied strain. Increasing the chitosan content resulted in an increased water capacity of SFCS blends.

Integrin interactions with immobilized peptides in polyethylene glycol diacrylate hydrogels.

This study employs tissue-engineering technologies to evaluate neutrophil interactions with extracellular matrix (ECM)-mimetic peptides. We have used a polyethylene glycol (PEG) diacrylate derivative to form a hydrogel as a biologically inert surface. Covalent attachment of bioactive moieties to the hydrogel makes it bioactive. The goal is to define the mechanisms by which these moieties influence the interactions of neutrophils with this bioactive hydrogel, and thus understand the likely effects of similar ligands in the ECM. The current experiments analyze the interactions of isolated human neutrophils with PEG hydrogels modified with Arg-Gly-Asp-Ser (RGDS), a known ligand for some beta(1) and beta(3) integrins, and Thr-Met-Lys-Ile-Ile-Pro-Phe-Asn-Arg-Leu-Thr-Ile-Gly-Gly (TMKIIPFNRLTIGG), a ligand for Mac-1, a beta(2) integrin. Our results demonstrate that neutrophils, independent of chemotactic stimulation, show little ability to adhere to unmodified PEG hydrogels. However, cell adhesion and spreading are robust on peptide-modified hydrogels. Incorporating distinct bioactive peptides, either alone or in combination, has enabled recognition of differential functions of alpha(v)beta(3), beta(1), and beta(2) integrins on neutrophil adhesion and spreading. Combined interactions result in activity that differs markedly from that seen with either integrin independently engaged. This model allows investigation of specific ligand-induced leukocyte functions and the development of engineered matrices with defined bioactive properties.

Effects of epidermal growth factor on fibroblast migration through biomimetic hydrogels.

We have previously reported on the development and use of synthetic hydrogel extracellular matrix (ECM) analogues that can be used to study the mechanisms of migration. These biomimetic hydrogels consist of bioinert poly(ethylene glycol) diacrylate derivatives with proteolytically degradable peptide sequences included in the backbone of the polymer and adhesion peptide sequences grafted into the network. Cells adhere to the hydrogel via interaction between the grafted adhesion ligands and receptors on the cell surface. The cells migrate through the three-dimensional system by secreting the appropriate proteolytic enzymes, which are involved in cell migration and are targeted to the peptide sequences incorporated in the backbone of the polymer. It was observed that cell migration has a biphasic dependence on adhesion ligand concentration, with optimal migration at intermediate ligand levels. In this study, we demonstrate that we can covalently attach epidermal growth factor (EGF) to PEG and graft them into the hydrogels. It was observed that EGF when tethered maintained mitogenic activity. It was also observed that fibroblast migration significantly increased in the presence of the grafted EGF through the collagenase-sensitive hydrogels. In addition, the increase in migration was found to be independent from the proliferative response of the cells. These synthetic ECM analogues allow one to systematically control identities and concentrations of biomolecules and are useful tools to study mechanisms of cell migration.

Val-ala-pro-gly, an elastin-derived non-integrin ligand: smooth muscle cell adhesion and specificity.

The elastin-derived peptide val-ala-pro-gly (VAPG) may be useful as a biospecific cell adhesion ligand for smooth muscle cells. By grafting the peptide sequence into a hydrogel material, we were able to assess its effects on smooth muscle cell adhesion and spreading. These materials are photopolymerizable hydrogels based on acrylate-terminated derivatives of polyethylene glycol (PEG). Because of their high PEG content, these materials are highly resistant to protein adsorption and cell adhesion. However, PEG diacrylate derivatives can be mixed with adhesive peptide-modified PEG monoacrylate derivatives to facilitate cell adhesion. Following photopolymerization, PEG monoacrylate derivatives are grafted into the hydrogel network formed by the PEG diacrylate. This results in covalent immobilization of adhesive peptides to the hydrogel via a flexible linker chain. The resistance of PEG to protein adsorption makes it an ideal material for this model system since cell-material interactions are limited to biomolecules that are covalently incorporated into the material. In this case we were able to demonstrate that VAPG is specific for adhesion of smooth muscle cells. It also was shown that fibroblasts, endothelial cells, and platelets cannot adhere to VAPG. In addition, not only was smooth muscle cell adhesion dependent on ligand concentration, but also cell spreading increased with increasing ligand concentration.

Tissue engineered small-diameter vascular grafts.

Arterial occlusive disease remains the leading cause of death in western countries and often requires vascular reconstructive surgery. The limited supply of suitable small-diameter vascular grafts has led to the development of tissue engineered blood vessel substitutes. Many different approaches have been examined, including natural scaffolds containing one or more ECM proteins and degradable polymeric scaffolds. For optimal graft development, many efforts have modified the culture environment to enhance ECM synthesis and organization using bioreactors under physiologic conditions and biochemical supplements. In the past couple of decades, a great deal of progress on TEVGs has been made. Many challenges remain and are being addressed, particularly with regard to the prevention of thrombosis and the improvement of graft mechanical properties. To develop a patent TEVG that grossly resembles native tissue, required culture times in most studies exceed 8 weeks. Even with further advances in the field, TEVGs will likely not be used in emergency situations because of the time necessary to allow for cell expansion, ECM production and organization, and attainment of desired mechanical strength. Furthermore, TEVGs will probably require the use of autologous tissue to prevent an immunogenic response, unless advances in immune acceptance render allogenic and xenogenic tissue use feasible. TEVGs have not yet been subjected to clinical trials, which will determine the efficacy of such grafts in the long term. Finally, off-the-shelf availability and cost will become the biggest hurdles in the development of a feasible TEVG product. Although many obstacles exist in the effort to develop a small-diameter TEVG, the potential benefits of such an achievement are exciting. In the near future, a nonthrombogenic TEVG with sufficient mechanical strength may be developed for clinical trials. Such a graft will have the minimum characteristics of biological tissue necessary to remain patent over a period comparable to current vein graft therapies. As science and technology advance, TEVGs may evolve into complex blood vessel substitutes. TEVGs may become living grafts, capable of growing, remodeling, and responding to mechanical and biochemical stimuli in the surrounding environment. These blood vessel substitutes will closely resemble native vessels in almost every way, including structure, composition, mechanical properties, and function. They will possess vasoactive properties and be able to dilate and constrict in response to stimuli. Close mimicry of native blood vessels may aid in the engineering of other tissues dependent upon vasculature to sustain function. With further understanding of the factors involved in cardiovascular development and function combined with the foundation of knowledge already in place, the development of TEVGs should one day lead to improved quality of life for those with vascular disease and other life-threatening conditions.

Endothelial cell scaffolds generated by 3D direct writing of biodegradable polymer microfibers.

The engineering of large (thickness > 100 µm) tissues requires a microvascular network to supply nutrients and remove waste. To produce microvasculature in vitro, a scaffold is required to mechanically support and stimulate endothelial cell (EC) adhesion and growth. Scaffolds for ECs are currently produced by patterning polymers or other biomaterials into configurations which often possess isotropic morphologies such as porous films and fibrous mats. We propose a new "direct-write" process for fabricating scaffolds composed of suspended polymer microfibers that are precisely oriented in 3D, providing directional architecture for selectively guiding cell growth along a desired pathway. The diameters of the fibers produced with this process were predictably and repeatably controlled through modulation of the system parameters, enabling production of fibers with microvascular-scale diameters (5-20 µm) from a variety of biodegradable polymers. These scaffolds were successfully seeded with ECs, which conformed to the geometry of the fibers and proliferated over the course of one week.

Silk-fibroin-coated liposomes for long-term and targeted drug delivery.

Many barriers to drug delivery into a tumor site require careful consideration when designing a new drug. In this study, the adhesive targeting and drug specificity of modified liposomal vesicles on human-scar-producing cells, keloid fibroblasts, were investigated. Keloids express abundant levels of mucopolysaccharides and receptor tyrosine kinase (RTK). In this report, the structural properties, drug release kinetics, and therapeutic availability of silk-fibroin-coated, emodin-loaded liposomes (SF-ELP), compared with uncoated, emodin-loaded liposomes (ELP), were investigated. SF-ELP had a highly organized lamellae structure, which contributed to 55% of the liposomal diameter. This modified liposomal structure decreased emodin release rates by changing the release kinetics from a swelling and diffusional process to a purely diffusional process, probably due to steric hindrance. SF-ELP also increased adhesion targeting to keloid fibroblasts. Increased retention of SF-ELP is most likely due to the interaction of the fibrous protein coating around the ELP with the pericellular molecules around the cell. SF-ELP also decreased survival rate of keloids that expressed high levels of RTK. These results demonstrated that SF-ELP enhanced emodin delivery by improved diffusion kinetics and specific cell targeting.