Caveolin: A possible biomarker of degradable metallic materials toxicity in vascular cells

Iron-based materials could constitute an interesting option for cardiovascular biodegradable stent applications due to their appropriate ductility compared with their counterparts, magnesium alloys. However, the predicted degradation rate of pure iron is considered to be too slow for such applications. We explored manganese (35 wt.%) as an alloying element in combination with iron to circumvent this problem through powder metallurgical processing (Fe–35Mn). Manganese, on the other hand, is highly cytotoxic. We recently explored a new method to better characterize the safety of degradable metallic materials (DMMs) by establishing the gene expression profile (GEP) of cells (mouse 3T3 fibroblasts) exposed to Fe–35Mn degradation products in order to better understand their global response to a potentially cytotoxic DMM. We identified a number of up- and down-regulated genes and confirmed the regulation of a subset of them by quantitative real time polymerase chain reaction. Caveolin-1 (cav1), the structural protein of caveolae, small, smooth plasma membrane invaginations present in various differentiated cell types, was one of the most down-regulated genes in our GEPs. In the present study we further studied the potential of this 22 kDa protein to become a biomarker for cytotoxicity after exposure to degradable metallic elements. In order to better characterize cav1 expression in this context 3T3 mouse fibroblasts were exposed to either ferrous and manganese ions at cytostatic concentrations for 24 or 48 h. cav1 gene expression was not influenced by exposure to ferrous ions. On the other hand, exposure to manganese for 24 h reduced cav1 gene expression by about 30% and by >65% after 48 h compared with control 3T3 cells. The cav1 cellular protein content was reduced to the same extent. The same pattern of expression of cav3 (the muscle-specific caveolin subtype) was also observed in this study. This strong and reproducible pattern of regulation of caveolins thus indicates potential as a biomarker for the toxicity of DMM elements.     

In vitro cytotoxicity of porous silicon microparticles: Effect of the particle concentration,surface chemistry and size

We report here the in vitro cytotoxicity of mesoporous silicon (PSi) microparticles on the Caco-2 cells as a function of particle size fractions (1.2–75 μm), particle concentration (0.2–4 mg ml^?1) and incubation times (3, 11 and 24 h). The particle size (smaller PSi particles showed higher cytotoxicity) and the surface chemistry treatment of the PSi microparticles were considered to be the key factors regarding the toxicity aspects. These effects were significant after the 11 and 24 h exposure times, and were explained by cell–particle interactions involving mitochondrial disruption resulting from ATP depletion and reactive oxygen species production induced by the PSi surface. These events further induced an increase in cell apoptosis and consequent cell damage and cell death in a dose-dependent manner and as a function of the PSi particle size. These effects were, however, less pronounced with thermally oxidized PSi particles. Under the experimental conditions tested and at particle sizes >25 μm, the non-toxic threshold concentration for thermally hydrocarbonized and carbonized PSi particles was <2 mg ml^?1, and for thermally oxidized PSi microparticles was <4 mg ml^?1.     

Effects of hydroxyapatite on endothelial network formation in collagen/fibrin composite hydrogels in vitro and in vivo

Co-culture of endothelial cells (EC) and mesenchymal stem cells (MSC) results in robust vascular network formation in constrained 3-D collagen/fibrin (COL/FIB) composite hydrogels. However, the ability to form endothelial networks is lost when such gels are allowed to compact via cell-mediated remodeling. In this study, we created co-cultures of human EC and human MSC in both constrained and unconstrained COL/FIB matrices and systematically added nanoparticulate hydroxyapatite (HA, 0–20 mg ml⁻¹), a bone-like mineral that has been shown to have pro-vasculogenic effects. Constructs cultured for 7 days were assayed for gel compaction, vascular network formation, and mechanical properties. In vitro, robust endothelial network formation was observed in constrained COL/FIB constructs without HA, but this response was significantly inhibited by addition of 5, 10, or 20 mg ml⁻¹ HA. In unconstrained matrices, network formation was abolished in pure COL/FIB constructs but was rescued by 1.25 or 2.5 mg ml⁻¹ HA, while higher levels again inhibited vasculogenesis. HA inhibited gel compaction in a dose-dependent manner, which was not correlated to endothelial network formation. HA affected initial stiffness of the gels, but gel remodeling abrogated this effect. Subcutaneous implantation of COL/FIB with 0, 2.5 or 20 mg ml⁻¹ HA in the mouse resulted in increased perfusion at the implant site, with no significant differences between materials. Histology at day 7 showed both host and human CD31-stained vasculature infiltrating the implants. These findings are relevant to the design of materials and scaffolds for orthopedic tissue engineering, where both vasculogenesis and formation of a mineral phase are required for regeneration.     

Predicting bulk mechanical properties of cellularized collagen gels using multiphoton microscopy

Cellularized collagen gels are a common model in tissue engineering, but the relationship between the microstructure and bulk mechanical properties is only partially understood. Multiphoton microscopy (MPM) is an ideal non-invasive tool for examining collagen microstructure, cellularity and crosslink content in these gels. In order to identify robust image parameters that characterize microstructural determinants of the bulk elastic modulus, we performed serial MPM and mechanical tests on acellular and cellularized (normal human lung fibroblasts) collagen hydrogels, before and after glutaraldehyde crosslinking. Following gel contraction over 16 days, cellularized collagen gel content approached that of native connective tissues (～200 mg ml^–1). Young’s modulus (E) measurements from acellular collagen gels (range 0.5–12 kPa) exhibited a power-law concentration dependence (range 3–9 mg ml^–1) with exponents from 2.1 to 2.2, similar to other semiflexible biopolymer networks such as fibrin and actin. In contrast, cellularized collagen gel stiffness (range 0.5–27 kPa) produced concentration-dependent exponents of 0.7 uncrosslinked and 1.1 crosslinked (range ～5–200 mg ml^–1). The variation in E of cellularized collagen hydrogels can be explained by a power-law dependence on robust image parameters: either the second harmonic generation (SHG) and two-photon fluorescence (TPF) (matrix component) skewness (R² = 0.75, exponents of -1.0 and -0.6, respectively); or alternatively the SHG and TPF (matrix component) speckle contrast (R² = 0.83, exponents of ?0.7 and ?1.8, respectively). Image parameters based on the cellular component of TPF signal did not improve the fits. The concentration dependence of E suggests enhanced stress relaxation in cellularized vs. acellular gels. SHG and TPF image skewness and speckle contrast from cellularized collagen gels can predict E by capturing mechanically relevant information on collagen fiber, cell and crosslink density.     

Enhanced insulin secretion of physically crosslinked pancreatic β-cells by using a poly(ethylene glycol) derivative with oleyl groups

A polymeric crosslinker was developed to promote the formation of cellular spheroids. Our approach was based on the crosslinking of cell membrane using a polymeric crosslinker that worked via hydrophobic interaction. The crosslinker, a poly(ethylene glycol) derivative with oleyl groups as a hydrophobic group at both ends, was synthesized and characterized by gel permeation chromatography and Fourier-transform infrared spectroscopy. Cell culture experiments were then performed to confirm spheroid formation. The rat pancreatic islet β-cell line RIN, which possesses the ability to secrete insulin, was cultured with the crosslinker in a round-bottomed 96-well plate. The formation of a spheroid was achieved when the crosslinker was added to the cell suspension, especially in the absence of serum. The size of the spheroid decreased with time and with increasing crosslinker concentration, and depended on the number of cells plated in each well. The number of cells cultured with crosslinker was almost constant during 7 days and hardly proliferated in crosslinker concentrations of 0–2.5 mg ml^?1, while the number of cells showed a decrease in the 25 mg ml^?1 crosslinker concentration. It was shown that the insulin protein secretion in the spheroid cultured with crosslinker for 1 week was enhanced. The cell adhesion protein E-cadherin mRNA expression of the resulting spheroid was also enhanced. These results indicate that the promoted cell function was due to the cell–cell and cell–matrix interactions in the spheroid, suggesting that this polymeric crosslinker was useful for the formation of cell spheroids.     

Toxico-pathological changes induced by cypermethrin in broiler chicks: Their attenuation with Vitamin E and selenium

Ninety 1-day old broiler chicks of mixed gender (as hatched) procured from a local hatchery were randomly divided into five equal groups. All the treatments were given through crop tubing. Groups 1–4 received cypermethrin (CY) (600 mg kg^?1 b. wt.) daily for 30 days. In addition to CY (group 1), groups 2–4 received Vit E (150 mg kg^?1 b. wt.), Se (0.25 mg kg^?1 b. wt.), and Vit E (150 mg kg^?1 b. wt.)+Se (0.25 mg kg^?1 b. wt.), respectively. Group 5 served as control andreceived normal saline (2 ml kg^?1 b. wt.) for 30 days. Randomly selected six broiler chicks from each group were slaughtered at experimental days 10, 20 and 30 for the collection of serum/plasma and morbid tissues. Absolute organ weights were recorded. Total plasma proteins, fibrinogen and creatinine were significantly (P<0.05) increased while alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and urea decreased significantly (P<0.05) in CY-treated group when compared with the control group. Kidneys were swollen grossly in treated broiler chicks. In liver, necrosis of hepatocytes, cytoplasmic vacuolation, bile duct hyperplasia and mononuclear cellular infiltration were observed. In kidneys, necrosis of tubular epithelial cells, cytoplasmic vacuolation, cellular infiltration and atrophy of glomeruli were observed. Sub-arachnoid space was much dilated in CY-treated broiler chicks. It can be concluded that CY induces biochemical and histopathological alterations in broilers chicks; however, these toxic effects can be ameliorated by Vit E or Se. Combination of Vit E and Se was more effective in ameliorating toxic effects of cypermethrin in broilers chicks.     

High density type I collagen gels for tissue engineering of whole menisci

This study investigates the potential of high density type I collagen gels as an injectable scaffold for tissue engineering of whole menisci, and compares these results with previous strategies using alginate as an injectable scaffold. Bovine meniscal fibrochondrocytes were mixed with collagen and injected into micro-computed tomography-based molds to create 10 and 20 mg ml^?1 menisci that were cultured for up to 4 weeks and compared with cultured alginate menisci. Contraction, histological, confocal microscopy, biochemical and mechanical analysis were performed to determine tissue development. After 4 weeks culture, collagen menisci had preserved their shape and significantly improved their biochemical and mechanical properties. Both 10 and 20 mg ml^?1 menisci maintained their DNA content while significantly improving the glycosaminoglycan and collagen content, at values significantly higher than the alginate controls. Collagen menisci matched the alginate control in terms of the equilibrium modulus, and developed a 3- to 6-fold higher tensile modulus than alginate by 4 weeks. Further fibrochondrocytes were able to reorganize the collagen gels into a more fibrous appearance similar to native menisci.     

Clinico-hematological and micronuclear changes induced by cypermethrin in broiler chicks: Their attenuation with vitamin E and selenium

This study was carried out on 90 one-day-old broiler chicks to know clinico-hematological alterations, DNA damage caused by cypermethrin (CY), and attenuation of toxic effects by vitamin E (Vit E) and selenium (Se). Birds were randomly divided into five equal groups. Groups 1–4 received CY (600 ml kg^?1 b.wt) daily for 30 days by crop tubing. In addition to CY, groups 2, 3 and 4 received Vit E (150 mg kg^?1 b.wt), Se (0.25 mg kg^?1 b.wt), and Vit E (150 mg kg^?1 b.wt)+Se (0.25 mg kg^?1 b.wt), respectively. Group 5 served as control. Birds were monitored twice daily for clinical signs. They were weighed and blood samples were collected at experimental days 10, 20 and 30 for hematological studies. CY-treated birds showed more prominent signs of toxicity compared to CY+Vit E, CY+Se and CY+Vit E+Se birds. Body weight in groups 1–3 was significantly (P<0.05) smaller at days 20 and 30 when compared with the control group. Significantly (P<0.001) higher numbers of micronuclei appeared in chicks treated with CY compared to CY+Vit E- and CY+Se-treated birds. Significantly decreased total erythrocyte counts (TEC), hemoglobin (Hb) concentration and packed cell volume (PCV) in all treated groups were recorded. Treated birds suffered from macrocytic hypochromic anemia. Leukocytosis in early stage and later leucopenia was seen in treated birds. It can be concluded that CY induces toxic effects in broilers chicks; however, these toxic effects can be ameliorated by Vit E or Se. Combination of Vit E and Se was more effective to ameliorate toxic effects of cypermethrin.     

Biocompatibility and biodegradability of Mg–Sr alloys: The formation of Sr-substituted hydroxyapatite

Magnesium is an attractive material for use in biodegradable implants due to its low density, non-toxicity and mechanical properties similar to those of human tissue such as bone. Its biocompatibility makes it amenable for use in a wide range of applications from bone to cardiovascular implants. Here we investigated the corrosion rate in simulated body fluid (SBF) of a series of Mg–Sr alloys, with Sr in the range of 0.3–2.5%, and found that the Mg–0.5 Sr alloy showed the slowest corrosion rate. The degradation rate from this alloy indicated that the daily Sr intake from a typical stent would be 0.01–0.02 mg day^?1, which is well below the maximum daily Sr intake levels of 4 mg day^?1. Indirect cytotoxicity assays using human umbilical vascular endothelial cells indicated that Mg–0.5 Sr extraction medium did not cause any toxicity or detrimental effect on the viability of the cells. Finally, a tubular Mg–0.5 Sr stent sample, along with a WE43 control stent, was implanted into the right and left dog femoral artery. No thrombosis effect was observed in the Mg–0.5 Sr stent after 3 weeks of implantation while the WE43 stent thrombosed. X-ray diffraction demonstrated the formation of hydroxyapatite and Mg(OH)₂ as a result of the degradation of Mg–0.5 Sr alloy after 3 days in SBF. X-ray photoelectron spectroscopy further showed the possibility of the formation of a hydroxyapatite Sr-substituted layer that presents as a thin layer at the interface between the Mg–0.5 Sr alloy and the corrosion products. We believe that this interfacial layer stabilizes the surface of the Mg–0.5 Sr alloy, and slows down its degradation rate over time.     

An explanation of the mineralization mechanism in osteoblasts induced by calcium hydroxide

Calcium hydroxide (Ca(OH)₂) has been broadly used in endodontics, including apexification to obtain apical closure by mineralization. However, the detailed mechanism of mineralization induced by Ca(OH)₂ is still unclear. This study focuses on the function of calcium and hydroxyl ions which dissociate from Ca(OH)₂ during the mineralization process. Though primary osteoblasts cultured in the medium without or with 0.025 mg ml^?1 Ca(OH)₂ did not show mineralization, they did exhibit mineralization when they were cultured with a higher concentration of Ca(OH)₂ (0.25 mg ml^?1). Mineralization induced in the presence of 0.25 mg ml^?1 Ca(OH)₂ was greater at pH 7.4 than at pH 8.5. The high mineralization activity observed under neutral conditions was caused by the prolonged activation of p38 and JNK. Hydroxyl ions did not have any effect on the mineralization. The results demonstrate that calcium ions dissociated from Ca(OH)₂ are critical for inducing the mineralization of osteoblasts.     

Biodegradable Fe-based alloys for use in osteosynthesis: Outcome of an in vivo study after 52 weeks

This study investigates the degradation performance of three Fe-based materials in a growing rat skeleton over a period of 1 year. Pins of pure Fe and two Fe-based alloys (Fe–10Mn–1Pd and Fe–21Mn–0.7C–1Pd, in wt.%) were implanted transcortically into the femur of 38 Sprague–Dawley rats and inspected after 4, 12, 24 and 52 weeks. The assessment was performed by ex vivo microfocus computed tomography, weight-loss determination, surface analysis of the explanted pins and histological examination. The materials investigated showed signs of degradation; however, the degradation proceeded rather slowly and no significant differences between the materials were detected. We discuss these unexpected findings on the basis of fundamental considerations regarding iron corrosion. Dense layers of degradation products were formed on the implants’ surfaces, and act as barriers against oxygen transport. For the degradation of iron, however, the presence of oxygen is an indispensable prerequisite. Its availability is generally a critical factor in bony tissue and rather limited there, i.e. in the vicinity of our implants. Because of the relatively slow degradation of both pure Fe and the Fe-based alloys, their suitability for bulk temporary implants such as those in osteosynthesis applications appears questionable.     

Control of cellular adhesiveness in an alginate-based hydrogel by varying peroxidase and H2O2 concentrations during gelation

An aqueous solution of alginate possessing phenolic hydroxyl (Alg-Ph) groups is gellable via a horseradish peroxidase (HRP)-catalyzed oxidative crosslinking reaction between Ph groups, consuming H₂O₂ as an electron acceptor. This study evaluates the effect of H₂O₂ and HRP concentrations on cellular adhesiveness and proliferation on the resultant enzymatically crosslinked Alg-Ph gels. After 4 h of seeding, 81.1% of L929 fibroblast cells adhere to an Alg-Ph hydrogel prepared with 1 U ml^?1 HRP and 1 mM H₂O₂. Increasing the concentration of H₂O₂ to 15 mM decreases the percentage of adhering cells to 28.4%. The cellular adhesion at this H₂O₂ concentration is increased to 82.6% by increasing the HRP concentration to 10 U ml^?1. The cells adhering to the Alg-Ph hydrogels with higher cellular adhesiveness establish a confluent monolayer during 168 h of culture. A cell sheet can then be harvested within 5 min of immersion in a medium containing alginate lyase at 1.0 mg ml^?1. The harvested cell sheet re-adhere, and the cells contained in the sheet proliferate after being transferred to another cell culture dish.     

Endocytotic uptake of iron oxide nanoparticles by cultured brain microglial cells

Microglia are the phagocytotic cells of the brain that respond rapidly to alterations in brain homeostasis. Since iron oxide nanoparticles (IONPs) are used for diagnostic and therapeutic applications in the brain, the consequences of an exposure of microglial cells to IONPs are of particular interest. To address this topic we have synthesized and characterized fluorescent BODIPY®-labelled IONPs (BP-IONPs). The average hydrodynamic diameter and the ζ-potential of BP-IONPs in water were ～65 nm and ?49 mV, respectively. Both values increased after dispersion of the particles in serum containing incubation medium to ～130 nm and ?8 mV. Exposure of cultured rat microglial cells with BP-IONPs caused a time-, concentration- and temperature-dependent uptake of the particles, as demonstrated by strong increases in cellular iron contents and cellular fluorescence. Incubation for 3 h with 150 and 450 μM iron as BP-IONPs increased the cellular iron content from a low basal level of ～50 nmol iron mg^?1 to 219 ± 52 and 481 ± 28 nmol iron (mg protein)^?1, respectively. These conditions did not affect cell viability, but exposure to higher concentrations of BP-IONPs or for longer incubation periods severely compromised cell viability. The BP-IONP fluorescence in viable microglial cells was co-localized with lysosomes. In addition, BP-IONP accumulation was lowered by 60% in the presence of the endocytosis inhibitors 5-(N-ethyl-N-isopropyl)amiloride, tyrphostin 23 and chlorpromazin. These results suggest that the rapid accumulation of BP-IONPs by microglial cells is predominantly mediated by macropinocytosis and clathrin-mediated endocytosis, which direct the accumulated particles into the lysosomal compartment.     

Poly-l-lysine-coated albumin nanoparticles: Stability,mechanism for increasing in vitro enzymatic resilience,and siRNA release characteristics

Enzymatic degradation of nanoparticle (NP)-based drug delivery vehicles is a major factor influencing the administration routes as well as the site-specific delivery of NPs. To understand the stability of albumin NPs in an aggressive proteolytic environment, bovine serum albumin (BSA) NPs were fabricated via a coacervation technique and stabilized by coating using different molecular weights (MWs: 0.9–24 kDa) and concentrations (0.1–1.0 mg ml^?1) of the cationic polymer, poly-l-lysine (PLL). A short interfering ribonucleic acid (siRNA) was used as a model drug for encapsulation in the BSA NPs. The generated NPs were characterized for morphology (with atomic force microscopy), size (with photon correlation spectroscopy) and charge (zeta-potential). The size range of formed BSA particles (155 ± 11 to 3800 ± 1600 nm) was effectively controlled by the MW and concentration of the PLL used for coating. The aqueous solution stability of NPs increased with an increasing MW and PLL concentration. However, in the presence of trypsin, NPs coated with higher MW PLL were not as stable as those formed using lower MW PLL. This trend was also confirmed based on the release pattern of siRNA in the presence of trypsin. We conclude that, when designing stabilizing coatings for soft protein-based NPs, smaller molecules may be more suitable for particle coating if enhanced proteolytic resistance and more stable NPs are desired for targeted drug delivery applications.     

Hemocompatibility evaluation of different silver nanoparticle concentrations employing a modified Chandler-loop in vitro assay on human blood

Due to their antibacterial effects, the use of silver nanoparticles (AgNPs) in a great variety of medical applications like coatings of medical devices has increased markedly in the last few years. However, blood in contact with AgNPs may induce adverse effects, thereby altering hemostatic functions. The objective of this study was to investigate the hemocompatibility of AgNPs in whole blood. Human whole blood (n = 6) was treated with different AgNPs concentrations (1, 3 and 30 mg l^?1) or with saline/blank solutions as controls before being circulated in an in vitro Chandler-loop model for 60 min at 37 °C. Before and after circulation, various hematologic markers were investigated. Based on the hematologic parameters measured, no profound changes were observed in the groups treated with AgNP concentrations of 1 or 3 mg l^?1. AgNP concentrations of 30 mg l^?1 induced hemolysis of erythrocytes and α-granule secretion in platelets, increased CD11b expression on granulocytes, increased coagulation markers thrombin–antithrombin-III complex, kallikrein-like and FXIIa-like activities as well as complementing cascade activation. Overall, we provide for the first time a comprehensive evaluation including all hematologic parameters required to reliably assess the hemocompatibility of AgNPs. We strongly recommend integrating these hemocompatibility tests to preclinical test procedures prior to in vivo application of new AgNP-based therapies.     

Encapsulation of fibroblasts causes accelerated alginate hydrogel degradation

Calcium-alginate hydrogel has been widely studied as a material for cell encapsulation for tissue engineering. At present, the effect that cells have on the degradation of alginate hydrogel is largely unknown. We have shown that fibroblasts encapsulated at a density of 7.5 × 10⁵ cells ml^?1 in both 2% and 5% w/v alginate remain viable for at least 60 days. Rheological analysis was used to study how the mechanical properties exhibited by alginate hydrogel changed during 28 days in vitro culture. Alginate degradation was shown to occur throughout the study but was greatest within the first 7 days of culture for all samples, which correlated with a sharp release of calcium ions from the construct. Fibroblasts were shown to increase the rate of degradation during the first 7 days when compared with acellular samples in both 2% and 5% w/v gels, but after 28 days both acellular and cell-encapsulating samples retained disc-shaped morphologies and gel-like spectra. The results demonstrate that although at an early stage cells influence the mechanical properties of encapsulating alginate, over a longer period of culture, the hydrogels retain sufficient mechanical integrity to exhibit gel-like properties. This allows sustained immobilization of the cells at the desired location in vivo where they can produce extracellular matrix and growth factors to expedite the healing process.     

Synthesis and characterization of hyaluronic acid–poly(ethylene glycol) hydrogels via Michael addition: An injectable biomaterial for cartilage repair

Injectable hydrogels based on hyaluronic acid (HA) and poly(ethylene glycol) (PEG) were designed as biodegradable matrices for cartilage tissue engineering. Solutions of HA conjugates containing thiol functional groups (HA-SH) and PEG vinylsulfone (PEG-VS) macromers were cross-linked via Michael addition to form a three-dimensional network under physiological conditions. Gelation times varied from 14 min to less than 1 min, depending on the molecular weights of HA-SH and PEG-VS, degree of substitution (DS) of HA-SH and total polymer concentration. When the polymer concentration was increased from 2% to 6% (w/v) in the presence of 100 U ml^?1 hyaluronidase the degradation time increased from 3 to 15 days. Hydrogels with a homogeneous distribution of cells were obtained when chondrocytes were mixed with the precursor solutions. Culturing cell–hydrogel constructs prepared from HA185k-SH with a DS of 28 and cross-linked with PEG5k-4VS for 3 weeks in vitro revealed that the cells were viable and that cell division took place. Gel–cell matrices degraded in approximately 3 weeks, as shown by a significant decrease in dry gel mass. At day 21 glycosaminoglycans and collagen type II were found to have accumulated in hydrogels. These results indicate that these injectable hydrogels have a high potential for cartilage tissue engineering.     

Glycyrrhetinic acid-modified poly(ethylene glycol)–b-poly(γ-benzyl l-glutamate) micelles for liver targeting therapy

Liver targeted micelles were successfully constructed via self-assembly of glycyrrhetinic acid (GA)-modified poly(ethylene glycol)–b-poly(γ-benzyl l-glutamate) (GA–PEG–PBLG) block co-polymers, which were fabricated via ring opening polymerization of γ-benzyl l-glutamate N-carboxyanhydride monomer initiated by GA-modified PEG. The in vivo biodistribution and the in vitro cellular uptake of these micelles were investigated. The results showed that the relative uptake of doxorubicin (DOX)-loaded micelles (DOX/GA–PEG–PBLG) in liver was much higher than in other tissues, and the resulting DOX concentration in liver was about 2.2-fold higher than that from the micelles without modification by GA. Moreover, the cellular uptake study demonstrated that the introduction of GA to the micelles could significantly increase the affinity for human hepatic carcinoma 7703 cells, which induced a 3.7-fold higher endocytosis than unmodified ones. The cytotoxicity of DOX/GA–PEG–PBLG micelles (IC₅₀ 47 ng ml^?1) was much higher than that of free DOX (IC₅₀ 90 ng ml^?1). These results indicate that GA-modified micelles have great potential in liver targeting therapy.