Shifeng Yan

Layer-by-layer assembly of poly(l-glutamic acid)/chitosan microcapsules for high loading and sustained release of 5-fluorouracil

Hollow polyelectrolyte microcapsules based on poly(l-glutamic acid) (PLGA) and chitosan (CS) with opposite charges were fabricated by layer-by-layer (LbL) assembly technique using melamine formaldehyde (MF) microparticles as sacrificial templates. The LbL assembly of polyelectrolytes and the resultant PLGA/CS microcapsules were characterized. A hydrophilic anticancer drug, 5-fluorouracil (5-FU), was chosen to investigate the loading and release properties of the microcapsules. The PLGA/CS microcapsules show high loading capacity of 5-FU under conditions of high drug concentration and salt adding. The high loading can be ascribed to spontaneous deposition of 5-FU induced by hydrogen bonding between 5-FU and PLGA/CS microcapsules. The PLGA/CS microcapsules show sustained release behavior. The release rate of 5-FU drastically slows down after loading in PLGA/CS microcapsules. The 5-FU release from PLGA/CS microcapsules can be best described using Ritger-Peppas or Baker-Londale models, indicating the diffusion mechanism of 5-FU release from the PLGA/CS microcapsules. In vitro cytotoxicity evaluation by the MTT assay shows good cell viability over the entire concentration range of PLGA/CS microcapsules. Therefore, the novel PLGA/CS microcapsules are expected to find application in drug delivery systems because of the properties of biodegradability, high loading, sustained release and cell compatibility.

Injectable In Situ Self-Cross-Linking Hydrogels Based on Poly(l-glutamic acid) and Alginate for Cartilage Tissue Engineering

Injectable hydrogels as an important biomaterial class have been widely used in regenerative medicine. A series of injectable poly(l-glutamic acid)/alginate (PLGA/ALG) hydrogels were fabricated by self-cross-linking of hydrazide-modified poly(l-glutamic acid) (PLGA-ADH) and aldehyde-modified alginate (ALG-CHO). Both the degree of PLGA modification and the oxidation degree of ALG-CHO could be adjusted by the amount of activators and sodium periodate, respectively. The effect of the solid content of the hydrogels and oxidation degree of ALG-CHO on the gelation time, equilibrium swelling, mechanical properties, microscopic morphology, and in vitro degradation of the hydrogels was examined. Encapsulation of rabbit chondrocytes within hydrogels showed viability of the entrapped cells and good biocompatibility of the injectable hydrogels. A preliminary study exhibited injectability and rapid in vivo gel formation, as well as mechanical stability, cell ingrowth, and ectopic cartilage formation. The injectable PLGA/ALG hydrogels demonstrated attractive properties for future application in a variety of pharmaceutical delivery and tissue engineering, especially in cartilage tissue engineering.

Self-Healing Supramolecular Self-Assembled Hydrogels Based on Poly(l-glutamic acid)

Self-healing polymeric hydrogels have the capability to recover their structures and functionalities upon injury, which are extremely attractive in emerging biomedical applications. This research reports a new kind of self-healing polypeptide hydrogels based on self-assembly between cholesterol (Chol)-modified triblock poly(L-glutamic acid)-block-poly(ethylene glycol)-block-poly(L-glutamic acid) ((PLGA-b-PEG-b-PLGA)-g-Chol) and β-cyclodextrin (β-CD)-modified poly(L-glutamic acid) (PLGA-g-β-CD). The hydrogel formation relied on the host and guest linkage between β-CD and Chol. This study demonstrates the influences of polymer concentration and β-CD/Chol molar ratio on viscoelastic behavior of the hydrogels. The results showed that storage modulus was highest at polymer concentration of 15% w/v and β-CD/Chol molar ratio of 1:1. The effect of the PLGA molecular weight in (PLGA-b-PEG-b-PLGA)-g-Chol on viscoelastic behavior, mechanical properties and in vitro degradation of the supramolecular hydrogels was also studied. The hydrogels showed outstanding self-healing capability and good cytocompatibility. The multilayer structure was constructed using hydrogels with self-healing ability. The developed hydrogels provide a fascinating glimpse for the applications in tissue engineering.

Repair of an articular cartilage defect using adipose-derived stem cells loaded on a polyelectrolyte complex scaffold based on poly(L-glutamic acid) and chitosan

As a synthetic polypeptide water-soluble poly(l-glutamic acid) (PLGA) was designed to fabricate scaffolds for cartilage tissue engineering. Chitosan (CHI) has been employed as a physical cross-linking component in the construction of scaffolds. PLGA/CHI scaffolds act as sponges with a swelling ratio of 760±45% (mass%), showing promising biocompatibility and biodegradation. Autologous adipose-derived stem cells (ASCs) were expanded and seeded on PLGA/CHI scaffolds, ASC/scaffold constructs were then subjected to chondrogenic induction in vitro for 2weeks. The results showed that PLGA/CHI scaffolds could effectively support ASC adherence, proliferation and chondrogenic differentiation. The ASCs/scaffold constructs were then transplanted to repair full thickness articular cartilage defects (4mm in diameter, to the depth of subchondral bone) created in rabbit femur trochlea. Histological observations found that articular defects were covered with newly formed cartilage 6weeks post-implantation. After 12weeks the regenerated cartilage had integrated well with the surrounding native cartilage and subchondral bone. Toluidine blue and immunohistochemical staining confirmed similar accumulation of glycosaminoglycans and type II collagen in engineered cartilage as in native cartilage 12weeks post-implantation. The result was further supported by quantitative analysis of extracellular matrix deposition. The compressive modulus of the engineered cartilage increased significantly from 30% of that of normal cartilage at 6weeks to 83% at 12weeks. Cyto-nanoindentation also showed analogous biomechanical behavior of the engineered cartilage to that of native cartilage. The results of the present study thus demonstrate the potentiality of PLGA/CHI scaffolds in cartilage tissue engineering.

Layer‐by‐Layer Buildup of Poly(L‐glutamic acid)/Chitosan Film for Biologically Active Coating

A new biocompatible film based on chitosan and poly(L-glutamic acid) (CS/PGA), created by alternate deposition of CS and PGA, was investigated. FT-IR spectroscopy, UV-vis spectroscopy and QCM were used to analyze the build-up process. The growth of CS and PGA deposition are both exponential to the deposition steps at first. After about 9 (CS/PGA) depositions, the exponential to linear transition takes place. QCM measurements combined with UV-vis spectra revealed the increase in the multilayer film growth at different pH (4.4, 5.0 and 5.5). The build-up of the multilayer stops after a few depositions at pH = 6.5. A muscle myoblast cell (C2C12) assay showed that (CS/PGA)(n) multilayer films obviously promote C2C12 attachment and growth.

Poly(l-glutamic acid)/chitosan polyelectrolyte complex porous microspheres as cell microcarriers for cartilage regeneration

In this study a novel kind of porous poly(l-glutamic acid) (PLGA)/chitosan polyelectrolyte complex (PEC) microsphere was developed through electrostatic interaction between PLGA and chitosan. By adjusting the formula parameters chitosan microspheres with an average pore size of 47.5 ± 5.4 μm were first developed at a concentration of 2 wt.% and freeze temperature of -20 °C. For self-assembly of the PEC microspheres porous chitosan microspheres were then incubated in PLGA solution at 37 °C. Due to electrostatic interaction a large amount of PLGA (110.3 μg mg(-1)) was homogeneously absorbed within the chitosan microspheres. The developed PEC microspheres retained their original size, pore diameters and interconnected porous structure. Fourier transform infrared spectroscopy, thermal gravimetric analysis and zeta potential analysis revealed that the PEC microspheres were successfully prepared through electrostatic interaction. Compared with microspheres fabricated from chitosan, the porous PEC microspheres were shown to efficiently promote chondrocyte attachment and proliferation. After injection subcutaneously for 8 weeks PEC microspheres loaded with chondrocytes were found to produce significant more cartilaginous matrix than chitosan microspheres. These results indicate that these novel fabricated porous PLGA/chitosan PEC microspheres could be used as injectable cell carriers for cartilage tissue engineering.

Fabrication of poly(L-glutamic acid)/chitosan polyelectrolyte complex porous scaffolds for tissue engineering

Porous scaffolds composed of polypeptides and polysaccharides have remarkable biocompatibility and potential to mimic an extracellular matrix for tissue engineering. This study presented a novel design of polyelectrolyte complex porous scaffolds of a synthetic polypeptide poly(l-glutamic acid) (PLGA) and a natural polysaccharide chitosan (CS) using a freeze drying method. The microstructure of the porous scaffolds could be adjusted by changing the freezing temperature and solid content of the reacting polymer. PLGA/CS scaffolds fabricated from 2% solid content and at a freezing temperature of -20 °C exhibited an interconnected porous structure with average pore size between 150 and 200 μm. The contact angle of less than 75° and high swelling ratio of more than 700% showed the excellent hydrophilic performance of these scaffolds. Degradation of the PLGA/CS composite scaffolds could be modified and more CS content contributed a higher resistance to biodegradation. The mechanical properties of the scaffolds could be controlled by varying the PLGA/CS molar ratio and solid content. The scaffolds exhibited good elastic behavior in wet state. In vitro culture of rabbit adipose-derived stem cells (ASCs) indicated that the selected PLGA/CS porous scaffolds supported cell attachment and growth. In summary, the PLGA/CS porous scaffolds show excellent properties, such as an interconnected porous structure, mechanical strength, hydrophilicity, biodegradability and biocompatibility. The successful repair of articular cartilage defects showed the potentiality of using PLGA/CS scaffolds in cartilage tissue engineering.

In situ preparation of magnetic Fe3O4 nanoparticles inside nanoporous poly(l-glutamic acid)/chitosan microcapsules for drug delivery

The magnetic polymer microcapsules, as a promising environmental stimuli-responsive delivery vehicle, have been increasingly exploited to tackle the problem of remotely navigated delivery. This study presented a novel design and fabrication of magnetic poly(L-glutamic acid)/chitosan (PGA/CS) microcapsules. Magnetic Fe3O4 nanoparticles were in situ synthesized inside nanoporous PGA/CS microcapsules and resultant magnetic PGA/CS microcapsules were characterized. Mitoxantrone (MTX), an antineoplastic drug, was chosen as a water-soluble model drug to research the loading and release properties of the microcapsules. The results showed the carboxylate groups of PGA within polyelectrolyte walls could be used as binding sites for the absorption of iron ions and reaction sites for the synthesis of magnetic nanoparticles. Magnetic PGA/CS microcapsules were dissected using a dual-beam scanning electron microscope/focused ion beam (SEM/FIB) for morphological and microstructural examination. It was found that Fe3O4 nanoparticles with size of about 10nm were homogeneously dispersed in the polymer matrix and adhered to the pore walls of the microcapsules. Increasing the concentration of iron ions led to an increasing loading content of Fe3O4 nanoparticles and an increase in the resultant magnetization. The magnetic PGA/CS microcapsules could be easily manipulated by an external magnetic field. The MTX loading capacity depended on loading time and MTX concentration. The high loading could be ascribed to spontaneous deposition of MTX induced by electrostatic interaction. The microcapsules exhibited sustained release behavior. The MTX release from microcapsules could be best described using Korsmeyer-Peppas and Baker-Lonsdale models, indicating the diffusion mechanism of drug release from both PGA/CS microcapsules and magnetic PGA/CS microcapsules. Therefore, the novel magnetic PGA/CS microcapsules are expected to find application in drug delivery systems because of the properties of magnetic sensitivity, high drug loading and sustained release.

Porous scaffolds based on cross-linking of poly(L-glutamic acid).

Porous scaffolds based on water-soluble PLGA and CS were prepared. The pores were verified to be alveolate, uniform and continuous. The effects of freezing temperature, freeze-drying time, solid content and molecular weight of reactants on the pore structure of the scaffolds were studied. The scaffold morphology could be adjusted by changing the freezing temperature and solid content of reacting polymer. Their degradation rate can be adjusted by changing the proportion of PLGA and CS. The porosity of scaffolds was higher than 90% and the high swelling ratio showed that these scaffolds had excellent hydrophilic performance. The in vitro culture of chondrocytes indicates that the obtained PLGA/CS porous scaffolds are very promising biomaterials for tissue engineering applications.

Nanoporous multilayer poly(l-glutamic acid)/chitosan microcapsules for drug delivery

Nanoporous poly(L-glutamic acid)/chitosan (PLGA/CS) multilayer microcapsules were fabricated by layer-by-layer (LbL) assembly using the porous silica particles as sacrificial templates. The LbL assembled nanoporous PLGA/CS microcapsules were characterized by Zeta-potential analyzer, FTIR, TGA, SEM, TEM and CLSM. 5-Fluorouracil (5-FU) was chosen as model drug. The drug loading content of PLGA/CS microcapsules depends on loading time, loading temperature, pH value and NaCl concentration. High loading capacity of microcapsules can be achieved by simply adjusting pH value and salt concentration. Moreover, 5-Fu loaded microcapsules take on a sustained release behavior, especially in an acid solution, in contrast to burst release of bare 5-Fu. The kinetics of 5-Fu release from PLGA/CS microcapsules conforms to Korsmeyer-Peppas and Baker-Lonsdale models, the mechanism of which can be ascribed to priority of drug diffusion and subordination of polymer degradation. The MTT cytotoxicity assay in vitro reveals the satisfactory anticancer activity of the drug-loaded PLGA/CS microcapsules. Therefore, the novel nanoporous PLGA/CS microcapsules is expected to find application in drug delivery systems.