用户名： 密码： 验证码：
Heparinized chitosan/poly(γ-glutamic acid) nanoparticles for multi-functional delivery of fibroblast growth factor and heparin
详细信息    查看全文
文摘
To improve blood supply following ischemic injury, angiogenic factors such as fibroblast growth factor (bFGF) that stimulate new blood vessel formation have been used for therapeutic angiogenesis in ischemic tissues. In this study, heparin-functionalized chitosan (CS)/poly(γ-glutamic acid) (γ-PGA) nanoparticles (HP-CS/γ-PGA nanoparticles) were prepared for multi-functional delivery of basic fibroblast growth factor (bFGF) and heparin. The mean particle sizes and bFGF loading efficiency increased with the increase of functionalized heparin contents. The HP-CS/γ-PGA nanoparticles were pH-sensitive that could sustain bFGF release at pH 6.7 (simulate the pH of ischemia tissue) and were rapidly disintegrated at pH 7.4 (simulate the pH of repaired tissue). Sustained release of bFGF from the nanoparticles enhanced the proliferation of human foreskin fibroblast cells (HFF) and angiogenic tube formation by human umbilical vein endothelial cells (HUVEC), suggesting the retaining of bFGF mitogenic activity. Heparin, a traditionally used anticoagulant, could release from the disintegrated nanoparticles to maintain the anti-factor Xa activity in blood plasma, after increasing the pH value from 6.6 to 7.4. The nanocarriers for mutil-functional delivery of bFGF and heparin developed in this study may be a potential therapeutic method for enhancing ischemic tissue regeneration and preventing blood vessel rethrombosis.Keywords: Heparin-functionalized; Chitosan; -PGA; Nanoparticles; bFGFArticle Outline1. Introduction2. Materials and methods2.1. Materials2.2. Preparation and characterization of heparinized CS/-PGA nanoparticles2.3. Stability of prepared nanoparticles2.4. Heparin loading and release2.5. bFGF loading and release2.6. Circular dichroism of released bFGF2.7. Proliferation assay of human foreskin fibroblasts2.8. CLSM visualization2.9. Anticoagulant study2.10. Matrigel tube formation assay2.11. Statistical analysis3. Result and discussion3.1. Characterization of CS/-PGA and HP-CS/-PGA nanoparticles3.2. pH-responsive characteristics of nanoparticles3.3. Characteristics of heparin loading and release3.4. Anticoagulant study3.5. Characteristics of bFGF release profiles3.6. Mitogenic activity of released bFGF3.7. Matrigel tube formation assay4. ConclusionAcknowledgementsAppendix. AppendixReferencesFig.1.FT-IR spectra of (A) CS, -PGA and CS/-PGA nanoparticles and (B) HP, CS/-PGA nanoparticles and HP-CS/-PGA nanoparticles functionalized with 0.5 or 1.0mg/ml heparin.Fig.2.Light transmittance of test nanoparticles in distinct pH solutions at 500nm, (A) CS/-PGA nanoparticles and (B) HP-CS/-PGA nanoparticles functionalized with 0.5mg/ml heparin.Fig.3.TEM micrography of HP-CS/-PGA nanoparticles at distinct pH value: (A) pH 6.0 (after 2h), (B) pH 7.4 (after 10min), (C) pH 7.4 (after 30min), (D) pH 7.4 (after 2h).Fig.4.(A) Scheme of polyelectrolyte self-assembly of the polyanions (-PGA and heparin) and polycation (chitosan) into heparinized CS/-PGA nanoparticles (HP-CS/-PGA NPs) and (B) release of bFGF or heparin from the smart nanoparticles, depending on the environmental pH variation.Fig.5.Cumulative releases of heparin from the HP-CS/-PGA nanoparticles at pH 6.0, 6.6 and 7.4, (A) dissolution medium without -GPT (B) dissolution medium containing with 2.9 units/ml of -GPT.Fig.6.Fluorescence images of HFF cell incubated with (A) FA-HP and (B) FA-HP-CS/-PGA nanoparticles.Fig.7.In vitro anti-factor Xa activity measured in blood plasma following addition of the heparin-containing medium into blood plasma.Fig.8.Cumulative releases of bFGF from CS/-PGA and HP-CS/-PGA nanoparticles at (A) pH 6.0 and (B) pH 7.4 (C) circular dichroism (CD) spectra of the standard bFGF and bFGF released from HP-CS/-PGA nanoparticles.Fig.9.(A) Cell viability of the stimulating effect of bFGF released from the CS/-PGA and HP-CS/-PGA nanoparticles, on the proliferation of human foreskin fibroblasts (HFF) within 96h of cell culture, HFF proliferated in basal culture medium with FBS (), basal culture medium without FBS (serum-free culture medium) (), serum-free culture medium containing HP-CS/-PGA nanoparticles without bFGF (), serum-free culture medium containing HP-CS/-PGA nanoparticles loaded with bFGF; heparin concentration: 0.1mg/ml (), 0.5mg/ml () and 1.0mg/ml () (B) The maximum HFF proliferation at 72h post-culture. HFF proliferated in basal culture medium with (1) FBS, (2) basal culture medium without FBS (serum-free culture medium), (3) serum-free culture medium containing HP-CS/-PGA nanoparticles without bFGF, and serum-free culture medium containing HP-CS/-PGA nanoparticles loaded with bFGF; heparin concentration: (4) 0.1mg/ml, (5) 0.5mg/ml and (6) 1.0mg/ml.Fig.10.bFGF and bFGF-loaded nanoparticles induces tube formation of HUVEC on growth factor-reduced Matrigel: (A) HUVEC without previously treated with growth factor (B) HUVEC pre-treated with bFGF alone (C) HUVEC pre-treated with bFGF and heparin mixture (D) HUVEC pre-treated with bFGF-loaded nanoparticles.Fig.11.(A) The schematic application and function of the nanoparticles for multi-functional bFGF and heparin delivery (B) Schematic brief description of the mechanism for multi-functional delivery of bFGF and heparin.Table 1. Mean particle sizes, zeta potential values and polydispersity indices of HP-CS/-PGA nanoparticles functionalized with different concentrations of heparin (n=5 batches).aHeparin Index (mg/ml)Mean particle size (nm)Zeat potential (mV)Polydispersity0.0206.38.722.11.90.260.020.1223.75.821.52.50.190.010.5247.15.219.51.30.220.051.0272.56.617.90.90.160.03aNanoparticles were prepared from CS solution (2.0mg/ml, 2ml) and distinct heparin/-PGA mixtures (0.1, 0.5 or 1.0mg/ml heparin, 0.5mg/ml -PGA, 2ml).Table 2. Mean particle sizes, zeta potential values and polydispersity indices of CS/-PGA nanoparticlesa and HP-CS/-PGA nanoparticlesb at distinct pH environments (n=5 batches).Mean Particle Size (nm)Zeta Potential (mV)Polydispersity IndexCS/-PGApH 6.0209.56.423.74.20.280.05pH 6.6225.77.115.13.20.170.03pH 7.4N/AcN/AN/AHP-CS/-PGApH 6.0243.56.817.82.50.210.02pH 6.6256.45.59.31.40.150.04pH 7.4N/AN/AN/AaCS/-PGA nanoparticles were prepared using 2ml, 0.5mg/ml -PGA and 2ml, 2.0mg/ml CS.bHP-CS/-PGA nanoparticles were prepared using 2ml, heparin/-PGA mixture (0.5mg/ml heparin and 0.5mg/ml -PGA) and 2ml, 2.0mg/ml CS.cN/A: Precipitation of aggregates was observed.Table 3. Heparin loading efficiency and loading contents of HP-CS/-PGA nanoparticles functionalized with different concentrations of heparin (n=5 batches).aHeparin (mg/ml)Heparin incorporated (g)Loading efficiency ( % )Loading content (g/mg)0.1194.87.197.43.536.10.5907.352.890.65.3153.51.01716.498.885.84.9255.4aNanoparticles were prepared by adding a -PGA/heparin/bFGF mixed solution (0.1, 0.5 or 1.0mg/ml heparin, 0.5mg/ml -PGA, 2.0g/ml bFGF, 2ml) into an aqueous CS (2.0mg/ml, 2ml).Table 4. bFGF loading efficiency and loading contents of HP-CS/-PGA nanoparticles functionalized with different concentrations of heparin (n=5 batches).aHeparin (mg/ml)bFGF incorporated (g)Loading efficiency ( % )Loading content (ng/mg)0.00.6510.06432.57.9130.38.20.11.1740.05458.74.6226.113.70.51.5240.08976.25.8257.99.51.01.7160.07685.84.4255.411.4aNanoparticles were prepared by adding a -PGA/heparin/bFGF mixed solution (0.1, 0.5 or 1.0mg/ml heparin, 0.5mg/ml -PGA, 1.0g/ml bFGF, 2ml) into an aqueous CS (2.0mg/ml, 2ml).1. IntroductionThe assembly and covalent production of polymeric nanoparticles has become a popular area of advanced and applied research with aims toward the production of drug and protein delivery systems 1. Compared with traditionally used drug carriers, nanoparticle drug delivery systems possess unique abilities to pass through the smallest capillary vessels with prolonged duration in blood stream and penetrate cells and tissue gap to arrive at target organs 2. A variety of synthetic and naturally derived polymers have been employed in investigations of the preparation of nanoparticle drug delivery systems, including diblock and multiblock copolymers, dendrimers and natural polymers such as chitosan 3, 4, 5, 6, 7 and 8. Manipulation of the bulk properties and stability of these nanoparticles are key issues in their effectiveness to release drugs in a controlled manner to induce desired biological responses 9.Chitosan (CS) is a nontoxic, antibacterial polymer obtained by N-deacetylation of chitin. In contrast to chitosan which is a cationic polysaccharide in nature, -PGA is an anionic polypeptide produced as capsular substance or as slime by members of the genus Bacillus. It is also a biodegradable and nontoxic polymer. In our previous studies, a nanoparticle delivery system, composed of chitosan (CS) and poly(-glutamic acid) (-PGA) was prepared to protect insulin from the GI environment and enhance absorption of insulin in the intestinal epithelium via the paracellular pathway 10, 11, 12 and 13. Additionally, CS/-PGA/DNA nanoparticles were used for transdermal DNA delivery using a low-pressure gene gun 14. As compared with traditionally studied, CS/-PGA/DNA nanoparticles improved their penetration depth into the mouse skin and enhanced gene expression 15 and 16.Basic fibroblast growth factors (bFGF) is a heparin-binding protein known to play a role in such diverse biological processes as wound healing, angiogenesis and the promotion of neuronal cell survival and human cranial osteogenesis 17. Strategies to integrate functional materials into bFGF delivery vehicles have remained an important candidate for pharmacological stimulation of therapeutic angiogenesis in limb, brain and cardiac ischemia 18 and 19.. Heparin-functionalized materials have been particularly widely used in biomedical applications, owing to the importance of the highly sulfated glycosaminoglycans (GAGs) heparin and heparan sulfate in many biological processes 20 and 21. The binding of bFGF to heparin not only stabilizes the molecule against denaturation or proteolysis but also enhances their binding with cellular receptors 22.Exogenous administration of growth factors has been identified as a potential therapeutic approach in accelerating the rate of healing of ischemic and chronic wounds 23. However, myocardial extracellular pH drops from 7.4 to less than 6.7 during severe cardiac ischemia 24. Brain tissue pH typically falls to 6.06.5 during ischemia under normoglycemic conditions and can fall below 6.0 during severe ischemia or under hyperglycemic conditions 25. Based on this knowledge, a smart, pH-sensitive heparin-functionalized CS/-PGA (HP-CS/-PGA) nanocarriers was prepared and characterized. The HP-CS/-PGA nanoparticles could stabilize the mitogenic activity of loaded bFGF. Additionally, the heparinized nanoparticles were sensitive to pH variation therefore could control bFGF release at the simulated pH of ischemia tissue (pH6.7) and were disintegrated to continuously release heparin at the simulated pH of repaired tissue (pH 7.4) to prevent blood vessel rethrombosis.In this study, mean particle sizes as well as zeta potential values of the nanoparticles were examined by dynamic light scattering. Evaluation of the stability of test nanoparticles in response to the pH value was investigated by light transmittance study. The release of bFGF from the nanoparticles was quantified by Enzyme-linked Immunosorbent Assay (ELISA). Human foreskin fibroblast (HFF) proliferation and human umbilical vein endothelial cells (HUVEC) tube formation were observed to examine the mitogenic activity of released bFGF. Additionally, anti-factor Xa activity of heparin releasing from the nanoparticles was also investigated.2. Materials and methods2.1. MaterialsCS (MW 60kDa) with a degree of deacetylation of approximately 85 % was acquired from Koyo Chemical Co. Ltd. (Japan). -PGA was purchased from Vedan (Taichung, Taiwan, MW 60kDa). Heparin, toluidine-blue and -glutamyl transpeptidase (-GPT) were obtained from Sigma Chemical Company (USA). Human recombinant bFGF (isoelectric point is 9.6) and enzyme-linked immunosorption assay (ELISA) kit were purchased from RD Ltd (Minneapolis, MN, USA). Fetal bovine serum (FBS) and DMEM were purchased from Gibco BRL (Grand Island, NY, USA). Growth factor-reduced Matrigelk was from Becton Dickinson Labware (Bedford, MA, USA). All other reagents and solvents used were of reagent grade.2.2. Preparation and characterization of heparinized CS/-PGA nanoparticlesCS/-PGA nanoparticles were prepared using a simple polyelectrolyte self-assembly method under magnetic stirring at room temperature. In brief, an aqueous -PGA (0.5mg/ml, 2ml, in deionized (DI) water) was added by flush mixing with a pipette tip into an aqueous CS 2.0mg/ml, 2ml, in deionized (DI) water under magnetic stirring at room temperature. The self-assembled nanoparticles were collected by centrifugation at 12000rpm for 20min. Supernatants were discarded and nanoparticles were resuspended in DI water for further studies. To prepare heparin-functionalized CS/-PGA nanoparticles, a sample of 10mg of heparin was dissolved in 5ml of DI water and diluted. The obtained heparin solutions (0.2mg/ml, 1.0mg/ml or 2.0mg/ml heparin, 1ml) were premixed with the aqueous -PGA (2.0mg/ml, 1ml) to obtain the final heparin/-PGA mixtures (0.1, 0.5 or 1.0mg/ml heparin, 0.5mg/ml -PGA, 2ml). The mixed solutions were respectively added into an aqueous CS (2.0mg/ml, 2ml) and the obtained HP-CS/-PGA nanoparticles were collected as described before.The mean particle sizes and zeta potential values of nanoparticles were measured using a Zetasizer (3000HS, Malvern Instruments Ltd., Worcestershire, UK). The chemical compositions of original and heparin-functionalized nanoparticles were analyzed by FT-IR (PerkinElmer Spectrum RX1 FT-IR System, Buckinghamshire, England).2.3. Stability of prepared nanoparticlesThe CS/-PGA and HP-CS/-PGA nanoparticles were dispersed in distinct dissolution media (pH 6.0, 6.6 and 7.4 of buffer solutions, simulating the pH environments in ischemia tissue, critical ischemic tissue and normal tissue respectively) 24 and 25, and the dispersive stabilities of the nanoparticle were evaluated by turbidity measurement using a UVVis spectrophotometer (Uvikon923, Kontron Instruments, Italy) at 500nm 26. The mean particle sizes and zeta potential values of nanoparticles were measured using a Zetasizer as abovementioned method. The TEM sample was prepared by placing a drop of the nanoparticle suspension onto a 400 mesh a carbon-coated copper grid. About 2min after deposition, the grid was tapped with a filter paper to remove surface water, followed by air-drying. The dried samples were observed by TEM (Hitachi H-600, Japan) 12.2.4. Heparin loading and releaseThe loading efficiency of heparin in HP-CS/-PGA nanoparticles were determined by assaying the amounts of free heparin left in supernatants using a toluidine-blue colorimetric method 27, after centrifugation of the self-assembled HP-CS/-PG nanoparticles. The in vitro release of heparin from test nanoparticles was evaluated in dissolution medium at distinct pH value (pH 6.0, 6.6, and 7.4). At particular time intervals, the samples were taken out and centrifuged then the supernatants were used for a toluidine-blue colorimetric analysis. The amount of heparin released was expressed as a percentage of the total heparin associated with test nanoparticles as calculated from the loading efficiency. Additionally, the dissolution medium containing 2.9units/ml of -glutamyl transpeptidase (-GPT) was used for the heparin release study 28.2.5. bFGF loading and releaseA sample of 2.0g of bFGF was dissolved in 1ml of DI water and the prepared bFGF stock solution was blended into the aqueous heparin/-PGA mixed solutions with thoroughly stirring. The mixed solution (0.1, 0.5 or 1.0mg/ml heparin, 0.5mg/ml -PGA, 1.0g/ml bFGF, 2ml) was added into an aqueous CS (2.0mg/ml, 2ml) under magnetic stirring at room temperature and the obtained bFGF-containing HP-CS/-PGA nanoparticles were collected as described before.To determine their bFGF loading efficiency, nanoparticles were collected by centrifugation at 12000rpm, 4C for 20min and the bFGF concentration in the supernatant was assayed by enzyme-linked immunosorption assay (ELISA) method 17. ELISA plates (Elisa-PS-96A-F-H, Advangene Consumables Inc., USA) were coated with capture monoclonal antibodies, and blocked with 1 % BSA (w/v) for 1h. After adding the appropriately diluted samples to the ELISA plates, bFGF in the supernatant was detected using anti-human bFGF polyclonal antibodies. Then, streptavidin-conjugated horseradish peroxidase was added to the plates. The enzyme substrate (tetramethylbenzidine and peroxide) was added and incubated for color development for 20min. The enzyme reaction was stopped by adding an acidic solution. The absorbance of the samples was read at 450nm using PowerWave X340 (Bio-TEK Instrument, Inc., USA) plate reader. The amount of bFGF was determined from a calibration curve based on known concentrations of bFGF. All of the experiments were repeated for five times. The bFGF loading efficiency and loading content of prepared nanoparticles were calculated as previous studies 10 and 12.The bFGF release profiles of CS/-PGA and HP-CS/-PGA nanoparticles were determined to study the effect of heparin functionalization on the bFGF release. The samples were incubated at 37C under continuous agitation, in 10ml of buffer solution (pH 6.0 and 7.4). At various time points, the supernatant was withdrawn and fresh buffer was replenished. The amounts of bFGF in the supernatants were determined with abovementioned method of ELISA assay. All of the experiments were repeated for five times.2.6. Circular dichroism of released bFGFAll circular dichroism (CD) measurements were carried out at room temperature (298K) on a Jasco J720 spectropolarimeter using a quartz cell of 0.02cm path length. Each spectrum was an average of 10 scans. Individual CD spectrum was collected from 200 to 300nm with a resolution of 0.2nm and a scanning speed of 20nm/min. Spectra are presented as an average of three consecutive measurements with a buffer spectrum subtracted as the reference. The CD signals of native bFGF and bFGF released from the HP-CS/-PGA nanoparticles were converted into mean residual molar ellipticity (y) for comparative purpose.2.7. Proliferation assay of human foreskin fibroblastsHuman foreskin fibroblasts (HFF) incubated in 1:1 Mixture of Dulbeccos modified Eagles medium and DMEM medium containing 10 % (v/v) FBS served as a positive control. The bioactivity of bFGF released from CS/-PGA and HP-CS/-PGA nanoparticles were assessed in vitro by determining its mitogenic activity to stimulate the proliferation of human fibroblasts cultured in a basal medium (a 2:1 mixture of DMEM medium and PBS) without FBS. HFF (5104cells/ml) were seeded in each well containing the nanoparticles. On day 2, 3 and 4, viable cell densities in all wells were measured by MTT assay. HFF culture in the basal medium without bFGF and FBS served as a blank control.2.8. CLSM visualizationFluoresceinamine isomer I-labeled heparin (FA-HP) was synthesized according to the methods described in the literature 27 and used to prepare fluorescent nanoparticles for the CLSM study. After addition of FA-HP (used as a control group) or the FA-HP functionalized CS/-PGA fluorescent nanoparticles (FA-HP-CS/-PGA nanoparticles) in HFF cell and incubation for 120min at 37C, test samples were aspirated. Cells were washed twice with pre-warmed phosphate buffered saline (PBS) before they were fixed in 3.7 % paraformaldehyde. Subsequently, the cells were washed three times with PBS. Finally, the fixed cells were examined with excitations at 488nm, respectively, using CLSM (TCS SL, Leica, Germany).2.9. Anticoagulant study15ml of fasting venous blood samples were collected from 5 healthy volunteers. The anticoagulant activity of HP-CS/-PGA nanoparticles was determined in vitro by characterizing their ability to accelerate the inhibition of Xa factor. Heparin releasing from test nanoparticles in dissolution medium at distinct pH value (pH 6.0, 6.6, and 7.4) were collected at particular time intervals. After addition of the released heparin medium (100L) to human blood plasma (5mL), the antithrombin activity of heparin was determined by measuring the anti-factor Xa activity in blood plasma. The anti-factor Xa activity in blood plasma was determined with a chromogenic substrate assay using a standard kit (Stachrom anti-Xa, Diagnostica Stago, France) 27.2.10. Matrigel tube formation assayHuman umbilical vein endothelial cells (HUVEC) growth factor-reduced Matrigel plates (48-well) were prepared by adding 150l/well of thawed Matrigel (10mg/ml) to a refrigerated plate. The gel was allowed to solidify for 1h at 37C. Previously untreated HUVEC and HUVEC treated with bFGF or bFGF-loaded nanoparticles for 72h were suspended in medium containing only 5 % FBS (without bFGF) and seeded on Matrigel (3104cells/well). Cell culture was carried out at 37C in humidified air supplemented with 5 % CO2 for 18h. The cells were then fixed with 1.1 % glutaraldehyde for 15min. The Matrigel was dehydrated with 75 % ethanol at 20C for 1h, then with 96 % ethanol for 3min at room temperature. The cells were stained with Giemsa for 3min. Tube formation was examined by phase-contrast microscopy. The length of the tube structures was examined and quantified in the whole surface of each well by using a microscope equipped with a monochrome CCD camera connected to a computer equipped with the Biocom image analysis system. Tube formation rate was calculated using the following formula: Tube formation rate=(tube lengthsample/tube lengthcontrol)100 % 29.2.11. Statistical analysisComparison between two groups was analyzed by the one-tailed Students t-test using statistical software (SPSS, Chicago, Ill, USA). Data are presented as meanSD. A difference of P0.05 was considered statistically significant.3. Result and discussion3.1. Characterization of CS/-PGA and HP-CS/-PGA nanoparticlesThe selection of chitosan and -PGA as nanoparticles components was based on their good biocompatibility and their safe use in biomedical application and drug delivery 30. bFGF has angiogenic and mitogenic properties, and has demonstrated its stimulative effects in accelerating wound healing processes 31. However, it would be necessary to deliver the growth factor in a controlled manner that increases the therapeutic efficacy of bFGF 19., 20, 21 and 32. CS/-PGA nanoparticles after functionalized with heparin will be able to effectively bind with bFGF and could release the bound bFGF in a sustained way to accelerate cell proliferation and wound repairing.Polyelectrolyte self-assembled nanoparticles were prepared by adding a -PGA aqueous solution into a CS aqueous solution under magnetic stirring at room temperature. Fig.1A shows the FT-IR spectra of the CS, -PGA and the CS/-PGA nanoparticles. As shown, the characteristic peaks observed at 1560 and 1590cm1 were the protonated amino groups (NH3+) on CS and the carboxylic ions (COO) on -PGA, respectively. In the spectrum of CS/-PGA nanoparticles, the characteristic peak at 1590cm1 for COO on -PGA shifted to 1595cm1, while the characteristic peak of NH3+ deformation on CS at 1560cm1 disappeared (possibly was overlapped by the band of carboxylic ions). These observations can be attributed to the electrostatic interaction between the negatively charged carboxylic acid salts (COO) on -PGA and the positively charged amino groups (NH3+) on CS. The electrostatic interaction between -PGA and CS induced the self-assembly of two polyelectrolytes.Fig.1.FT-IR spectra of (A) CS, -PGA and CS/-PGA nanoparticles and (B) HP, CS/-PGA nanoparticles and HP-CS/-PGA nanoparticles functionalized with 0.5 or 1.0mg/ml heparin.The FT-IR spectra of heparin, CS/-PGA and HP-CS/-PGA nanoparticles are also shown in Fig.1B. The characteristic absorption bands (SO asymmetric stretch) in the area of 11601260cm1 for the associated sulfate groups (2-O, 6-O-sulfation and N-sulfation) for the heparinized nanoparticles was observed 33 and 34. Additionally, the characteristic peak observed at 800cm1 representing the COS stretch, was assigned to the 2-O, and 6-O-sulfation on heparin. The nanoparticles functionalized with higher concentration of heparin (1mg/ml) showed stronger intensity of heparin characteristic bands, suggesting that heparin was successfully incorporated in the CS/-PGA nanoparticles.In the study, heparinized nanoparticles were self-assembled instantaneously upon addition of an aqueous -PGA/heparin into an aqueous CS under magnetic stirring at room temperature. The amount of positively charged CS significantly exceeded that of negatively charged -PGA//heparin; some of excessive CS molecules were entangled onto the surfaces of nanoparticles, thus displaying a positive surface charge (Table 1). The content of incorporated heparin in the nanoparticles had significant effects on the mean particle size and zeta potential of nanoparticles. The negatively charged heparin could be assembled into CS/-PGA nanoparticles to increase the mean particle sizes and decrease the zeta potential values.Table 1. Mean particle sizes, zeta potential values and polydispersity indices of HP-CS/-PGA nanoparticles functionalized with different concentrations of heparin (n=5 batches).aHeparin Index (mg/ml)Mean particle size (nm)Zeat potential (mV)Polydispersity0.0206.38.722.11.90.260.020.1223.75.821.52.50.190.010.5247.15.219.51.30.220.051.0272.56.617.90.90.160.03aNanoparticles were prepared from CS solution (2.0mg/ml, 2ml) and distinct heparin/-PGA mixtures (0.1, 0.5 or 1.0mg/ml heparin, 0.5mg/ml -PGA, 2ml).3.2. pH-responsive characteristics of nanoparticlesIt has been reported that the ischemic and inflamed areas in the body have a lower pH (6.7) than the surrounding tissues and blood (pH=7.4) 24 and 25. If a nanoparticle can release bFGF in a sustained way in the ischemic and inflamed areas (pH6.7), and then rapidly disintegrate after repairing the tissue (pH=7.4), this nanoparticle might be useful as an intelligent, controlled release material. Therefore, characterization of the heparinized nanoparticle in response to distinct pH environments (pH 6.0, 6.6 and 7.4) must be investigated.Fig.2A shows the variation of light transmittance (=500nm) of the CS/-PGA nanoparticles in distinct pH solutions. The turbidity change took place in the nanoparticles-containing aqueous solutions. The instability of nanoparticles defined as the increased transmittance (T % ) of the solutions. It is known that the pKa values of CS (amine groups) and -PGA (carboxylic groups) are 6.5 and 2.9, respectively 10. At pH 6.0, the CS/-PGA nanoparticles were stable because most carboxylic groups on -PGA were in the form of COO and the amine groups on CS were in the form of NH3+. Therefore, CS and -PGA molecular chains in their self-assembled complex were tightly entangled to each other. The mean particle sizes and zeta potential values were similar to their original counterparts (Table 2). In contrast, the nanoparticles quickly disintegrated at pH 7.4 (T % increased obviously). There was little electrostatic interaction between CS and -PGA because the free amine groups on CS were deprotonated at pH values above 6.5. At pH 6.6, near the critical pH for CS deprotonation, the nanoparticles gradually swelled but not disintegrated, resulted in the increase of mean particle sizes and the decrease of zeta potential values (Table 2).Fig.2.Light transmittance of test nanoparticles in distinct pH solutions at 500nm, (A) CS/-PGA nanoparticles and (B) HP-CS/-PGA nanoparticles functionalized with 0.5mg/ml heparin.Table 2. Mean particle sizes, zeta potential values and polydispersity indices of CS/-PGA nanoparticlesa and HP-CS/-PGA nanoparticlesb at distinct pH environments (n=5 batches).Mean Particle Size (nm)Zeta Potential (mV)Polydispersity IndexCS/-PGApH 6.0209.56.423.74.20.280.05pH 6.6225.77.115.13.20.170.03pH 7.4N/AcN/AN/AHP-CS/-PGApH 6.0243.56.817.82.50.210.02pH 6.6256.45.59.31.40.150.04pH 7.4N/AN/AN/AaCS/-PGA nanoparticles were prepared using 2ml, 0.5mg/ml -PGA and 2ml, 2.0mg/ml CS.bHP-CS/-PGA nanoparticles were prepared using 2ml, heparin/-PGA mixture (0.5mg/ml heparin and 0.5mg/ml -PGA) and 2ml, 2.0mg/ml CS.cN/A: Precipitation of aggregates was observed.After heparinization, the stability of nanoparticles at pH 6.0 and 6.6 was similar to the original CS/-PGA nanoparticles (Fig.2B). The increase of mean particle sizes and the decrease of zeta potential values were also observed from HP-CS/-PGA nanoparticles at pH 6.6. However, at pH 7.4, rapid disintegration of the nanoparticles resulted in the increase of light transmittance. Fig.4 shows the TEM micrography of the disintegration process of the HP-CS/-PGA nanoparticle at distinct pH value. At pH 6.0 the simulated pH of ischemia tissue, the nanoparticles were still kept stable (Fig.4A). In the simulated normal tissue fluid (pH 7.4), the nanoparticles began swelled after 10min (Fig.3B) and the outer layer of the HP-CS/-PGA nanoparticles became gradually bubbled (after 30min), suggesting that the nanoparticles started on disintegration (Fig.3C). After 2h, the nanoparticles were disintegrated into fragments and the resulting fragments aggregates were observed from the TEM micrography (Fig.4D). Fig.4A shows the scheme for polyelectrolyte self-assembly of the polyanions (-PGA and heparin) and polycation (chitosan) into heparinized CS/-PGA nanoparticles (HP-CS/-PGA NPs). The smart nanoparticles could release bFGF or heparin, depending on the environmental pH variation (Fig.4B).Fig.3.TEM micrography of HP-CS/-PGA nanoparticles at distinct pH value: (A) pH 6.0 (after 2h), (B) pH 7.4 (after 10min), (C) pH 7.4 (after 30min), (D) pH 7.4 (after 2h).Fig.4.(A) Scheme of polyelectrolyte self-assembly of the polyanions (-PGA and heparin) and polycation (chitosan) into heparinized CS/-PGA nanoparticles (HP-CS/-PGA NPs) and (B) release of bFGF or heparin from the smart nanoparticles, depending on the environmental pH variation.3.3. Characteristics of heparin loading and releaseThe amount of the positively charged CS used in the preparation of nanoparticles significantly exceeded that of the negatively charged heparin, resulting in a high loading efficiency for heparin (Table 3). Fig.5A shows the results of the in vitro heparin release from HP-CS/-PGA nanoparticles at distinct pH values. The release of heparin from test nanoparticles was inhibited at pH 6.0, due to the high stability of nanoparticles and strong electrostatic interaction between the ionized CS and heparin. The nanoparticles remained relatively stable when the environmental pH was increased to 6.6. Heparin molecules released slowly from test nanoparticles at pH 6.6, while the release of heparin became significant at pH 7.4 due to the disintegration of nanoparticles. These results indicated that the stability of nanoparticles in response to pH had a significant effect on their release of heparin. The release of heparin from test nanoparticles in the medium simulating the pH value of repaired tissues became much more prominent (P0.05).Table 3. Heparin loading efficiency and loading contents of HP-CS/-PGA nanoparticles functionalized with different concentrations of heparin (n=5 batches).aHeparin (mg/ml)Heparin incorporated (g)Loading efficiency ( % )Loading content (g/mg)0.1194.87.197.43.536.10.5907.352.890.65.3153.51.01716.498.885.84.9255.4aNanoparticles were prepared by adding a -PGA/heparin/bFGF mixed solution (0.1, 0.5 or 1.0mg/ml heparin, 0.5mg/ml -PGA, 2.0g/ml bFGF, 2ml) into an aqueous CS (2.0mg/ml, 2ml).Fig.5.Cumulative releases of heparin from the HP-CS/-PGA nanoparticles at pH 6.0, 6.6 and 7.4, (A) dissolution medium without -GPT (B) dissolution medium containing with 2.9 units/ml of -GPT.Taking into account enzymatic degradation of nanoparticles, the release studies were performed by incorporating -GPT in the dissolution medium. -GPT is an enzyme present in the cell membranes of many tissues, including the kidneys, bile duct, pancreas, liver, spleen, heart, brain, and seminal vesicles that transfer -glutamyl functional groups. Enzymatic degradation of nanoparticles by -glutamyl transpeptidase (-GPT) has been reported 28. The heparin release kinetics of the nanoparticles in the enzyme-containing dissolution medium was shown in Fig.5B. At pH 6.0, in the dissolution medium containing -GPT, heparin release from the nanoparticles was slow with final release of approximately 12 % of the incorporated heparin within 24h of incubation. Considering expected strong electrostatic interaction between CS and -PGA, enzymatic degradation of -PGA by -GPT was inhibited, resulting in slow heparin release. In contrast, at pH 7.4, heparin was released in a faster, continuous manner form the HP-CS/-PGA nanoparticles in the enzyme-containing dissolution medium, with a final release of approximately 49 % in the same period. The faster heparin release rate could be attributed the fact that -GPT can more easily hydrolyzed the glutamyl moieties of -PGA and its implication in the generation of the smaller molecular weight of -PGA, which allows better diffusion of heparin from the disintegrated nanoparticles.A further examination of heparin release at the simulated pH of repaired tissue (pH 7.4), heparin was labeled with fluoresceinamine isomer I and incubated with HFF cell at pH 7.4. Fig.6 shows the fluorescence images of HFF cell 120min after incubation with fluoresceinamine isomer I-labeled heparin (FA-HP) and FA-HP functionalized CS/-PGA nanoparticles (FA-HP-CS/-PGA nanoparticles) as examined by CLSM. As shown, intense green fluorescent signals were observed from the HFF cell incubated together with FA-HP (Fig.6A). Moreover, HFF cell cultured with FA-HP-CS/-PGA nanoparticles demonstrated the same fluorescent signals (Fig.6B). These results suggested that nanoparticles were disintegrated after cell uptake to allow the release of FA-HP. These findings confirmed the results observed in our heparin release experiments (Fig.5A).Fig.6.Fluorescence images of HFF cell incubated with (A) FA-HP and (B) FA-HP-CS/-PGA nanoparticles.3.4. Anticoagulant studyHeparin binds to the enzyme inhibitor antithrombin causing a conformational change that results in its activation through an increase in the flexibility of its reactive site loop 35. The activated antithrombin then inactivates thrombin and serine proteases involved in blood clotting, most notably factor Xa 35. Fig.7 shows the in vitro anti-factor Xa activity measured in blood plasma following addition of the heparin-containing medium into blood plasma. As shown, no significant anti-factor Xa activity was detected after adding the heparin release medium (at pH 6.0) from HP-CS/-PGA nanoparticles (functionalized with 0.5mg/ml heparin), indicating the poor anticoagulant activity of the nanoparticles in the absence of released heparin. Instead, at pH 7.4, significant release of heparin from the nanoparticles can maintain the anti-factor Xa activity in blood plasma of 0.090.22IU/mL (Fig.7). The minimum effective concentration for the treatment of deep vein thrombosis and pulmonary embolism was reported to be 0.10.2IU/mL 36. Therefore, at pH 7.4, administration of HP-CS/-PGA nanoparticles would be effective in the delivery of heparin into the blood stream to prevent blood vessel rethrombosis.Fig.7.In vitro anti-factor Xa activity measured in blood plasma following addition of the heparin-containing medium into blood plasma.3.5. Characteristics of bFGF release profilesThe loading efficiency of bFGF in CS/-PGA nanoparticles was only 32.57.9 % . To increase the amount of bFGF-loaded, bFGF was blended with the premixed aqueous heparin/-PGA solution for the preparation of bFGF-loaded nanoparticles. As shown in the literatures, heparin molecules in solution can bind bFGF depending on its molecular weight 37. As shown in Table 4, HP-CS/-PGA nanoparticles had a greater ability to bind bFGF than its CS/-PGA nanoparticles counterpart. The bFGF loading efficiency increases significantly with the increase of incorporated heparin in the nanoparticles.Table 4. bFGF loading efficiency and loading contents of HP-CS/-PGA nanoparticles functionalized with different concentrations of heparin (n=5 batches).aHeparin (mg/ml)bFGF incorporated (g)Loading efficiency ( % )Loading content (ng/mg)0.00.6510.06432.57.9130.38.20.11.1740.05458.74.6226.113.70.51.5240.08976.25.8257.99.51.01.7160.07685.84.4255.411.4aNanoparticles were prepared by adding a -PGA/heparin/bFGF mixed solution (0.1, 0.5 or 1.0mg/ml heparin, 0.5mg/ml -PGA, 1.0g/ml bFGF, 2ml) into an aqueous CS (2.0mg/ml, 2ml).CS/-PGA nanoparticles and the HP-CS/-PGA nanoparticles functionalized with 0.5mg/ml heparin were chosen for the in vitro release study. The in vitro release of growth factor was determined by ELISA assay. Fig.8A and B respectively shows the release profiles of bFGF from CS/-PGA and HP-CS/-PGA nanoparticles at pH 6.0 and 7.4, simulating the pH environments in the ischemic tissues and repaired tissues, respectively. At pH 7.4 (simulating the pH value in the repaired tissues), CS/-PGA and HP-CS/-PGA nanoparticles became disintegrated, resulting in a rapid release of bFGF, which failed to provide an adequate retention of loaded bFGF (more than 80 % of loaded bFGF was released within 24h) (Fig.8B). In contrast, at pH 6.0 (simulating the pH value in the ischemic tissues), the release profiles of bFGF from both test nanoparticles (CS/-PGA and HP-CS/-PGA nanoparticles) were different. For heparinized nanoparticles, the cumulative amount of released bFGF was significantly reduced (approximately 73 % and 52 % of the loaded bFGF was released within 24h respectively for 0.5mg/ml and 1.0mg/ml heparin-functionalized HP-CS/-PGA nanoparticles) (Fig.8A). This result could not only be attributed to the relative stability of HP-CS/-PGA nanoparticles at pH 6.0 but also the high affinity of bFGF to heparin.Fig.8.Cumulative releases of bFGF from CS/-PGA and HP-CS/-PGA nanoparticles at (A) pH 6.0 and (B) pH 7.4 (C) circular dichroism (CD) spectra of the standard bFGF and bFGF released from HP-CS/-PGA nanoparticles.It is noted that binding of bFGF and heparin is mediated by ionic interaction between both 2-O-sulfate groups and N-sulfate groups of heparin molecules and certain lysine (Lys+) and arginine (Arg+) cations in proteins and peptides 38 and 39. Multiple clusters of basic amino acids in bFGF that has the proper spacing could result in much stronger binding force 40. Attributed to the reasons, the release rate of bFGF from the HP-CS/-PGA nanoparticles was reduced, and no burst effect was observed. As indicated by the circular dichroism spectra (Fig.8C), no significant conformation change was observed for the bFGF released from HP-CS/-PGA nanoparticles at pH 7.4 as compared to the standard bFGF. The CD spectrum of original and released bFGF revealed a prominent negative CD band centered at around 235nm corresponding to the D2 domain (a -sheet protein structure) that are crucial for ligand (FGF)-receptor binding 41. This result indicated that HP-CS/-PGA nanoparticles might effectively protected the loaded bFGF from denaturizing.3.6. Mitogenic activity of released bFGFFig.9A showed the cell viability of the stimulating effect of bFGF released from the bFGF-containing CS/-PGA and HP-CS/-PGA nanoparticles, on the growth of human foreskin fibroblasts (HFF) within 96h of cell culture. The mitogenic activity of released bFGF was examined by measuring its ability to stimulate the proliferation of HFF cultured in medium containing the test nanoparticles. HFF demonstrated significant proliferation in the basal medium with FBS (positive control). In contrast, HFF showed nearly no additional cell growth in the basal medium without FBS (blank control). Fig.9B showed the maximum HFF proliferation at 72h post-culture. The growth of HFF, in the basal medium without FBS, was obviously enhanced by the released bFGF from HP-CS/-PGA nanoparticles, in comparison to the blank control (p0.05). Especially, bFGF released from the nanoparticles functionalized with 0.5mg/ml heparin showed the best efficiency for the enhancement of cell proliferation. Interestingly, it was found that bFGF releasing from HP-CS/-PGA nanoparticles functionalized with the highest concentration of heparin (1.0mg/ml) was less effective than those functionalized with 0.5mg/ml heparin for the enhancement of cell proliferation.Fig.9.(A) Cell viability of the stimulating effect of bFGF released from the CS/-PGA and HP-CS/-PGA nanoparticles, on the proliferation of human foreskin fibroblasts (HFF) within 96h of cell culture, HFF proliferated in basal culture medium with FBS (), basal culture medium without FBS (serum-free culture medium) (), serum-free culture medium containing HP-CS/-PGA nanoparticles without bFGF (), serum-free culture medium containing HP-CS/-PGA nanoparticles loaded with bFGF; heparin concentration: 0.1mg/ml (), 0.5mg/ml () and 1.0mg/ml () (B) The maximum HFF proliferation at 72h post-culture. HFF proliferated in basal culture medium with (1) FBS, (2) basal culture medium without FBS (serum-free culture medium), (3) serum-free culture medium containing HP-CS/-PGA nanoparticles without bFGF, and serum-free culture medium containing HP-CS/-PGA nanoparticles loaded with bFGF; heparin concentration: (4) 0.1mg/ml, (5) 0.5mg/ml and (6) 1.0mg/ml.bFGF is known as a heparin-binding growth factor, because of its high affinity for heparin 42. At physiological pH and temperature, the in vitro half-life time of bFGF activity is approximately 12h 43. Heparin binds bFGF, and this interaction has been demonstrated to protect bFGF against physical denaturation and protease degradation 44. In this work, the heparin-bFGF complex was mixed with -PGA and assembled with chitosan into nanoparticles, leading to the encapsulation of bFGF within the HP-CS/-PGA nanoparticles. The result from the cell proliferation study indicated that bFGF released from the heparinized nanoparticles retained its mitogenic activity and the incorporated heparin mediated the activation of bFGF receptors, therefore enhanced the proliferation of human fibroblasts.In contrast to the promoting effects relating to the protection of growth factors from proteolytic degradation 45 and presentation of growth factor to its receptor forming a ternary complex 38, suppression of cell proliferation in the presence of heparin was reported to be related to inhibition of the first cell cycle traverse 45. In vivo, bFGF is stored bound to low-affinity sites provided by heparan sulfate proteoglycans and is released from heparin sulfate chains by heparanase or by competitive binding of soluble heparin-like molecules 46. However, at the cellular level, the bFGF release pathway is still unclear. Although it is clear that heparin can regulate bFGF dimerization and FGF receptor activation, an over functionalized nanoparticles (1.0mg/ml heparin) may release excess heparin therefore suppress the cell proliferation. This is a possible explanation why HP-CS/-PGA nanoparticles functionalized with the highest concentration of heparin (1.0mg/ml) demonstrated less mitogenic activity than its lower heparin-containing counterpart (0.5mg/ml).3.7. Matrigel tube formation assaybFGF induces many endothelial cell modifications involved in angiogenesis. Because migration, proliferation, and tube formation of vascular endothelial cells are important process during angiogenesis, the effect of heparinized nanoparticles on the bFGF-induced migration of HUVEC was examined. HUVEC without previously treated with bFGF-loaded nanoparticles did not form tubes after 24h on growth factor-reduced Matrigel (Fig.10A). Additionally, vascular tubes were formed in a partially organized capillary-like network when HUVEC were previously treated with bFGF alone or bFGF/heparin mixtures (Fig.10B, C). Heparin modulated mitogenic activity of bFGF, released from HP-CS/-PGA nanoparticles (functionalized with 0.5mg/ml heparin), therefore increase the tube length of the capillary-like HUVEC network (Fig.10D). The tube formation rates were increased 1.5 fold (p0.05) compared to bFGF alone (Fig.10B). These results suggested that bFGF released from HP-CS/-PGA nanoparticles can effectively stimulate tube formation of HUVEC which involved in the angiogenesis process. However, HUVEC pre-treated with bFGF/heparin mixture, tube formation rates was 1.1 fold compared to bFGF alone (p0.05) (Fig.10C). This result suggested that bFGF directly mixed with heparin couldnt obviously enhance capillary-like tube formation of HUVEC. Excessive heparin might inhibit capillary-like tube formation induced by proangiogenic factors, i.e.: VEGF, bFGF and TNF-alpha.Fig.10.bFGF and bFGF-loaded nanoparticles induces tube formation of HUVEC on growth factor-reduced Matrigel: (A) HUVEC without previously treated with growth factor (B) HUVEC pre-treated with bFGF alone (C) HUVEC pre-treated with bFGF and heparin mixture (D) HUVEC pre-treated with bFGF-loaded nanoparticles.The schematic application and function of the multi-functional growth factor delivery system was shown in Fig.11. The HP-CS/-PGA nanoparticles can sustained release bFGF in the ischemic and inflamed areas of damaged tissues (pH6.7) to enhance the proliferation of HFF cell and angiogenic tube formation by HUVEC, and then rapidly disintegrated in repaired tissue (pH=7.4) to release heparin (Fig.11). Disintegration of the nanoparticles will continuously release heparin, a traditionally used anticoagulant, to increase the anti-factor Xa activity in blood plasma therefore prevent harmful clots from forming in the blood vessels. Brief description of the drawing was shown in Fig.11B. Such a smart, mutil-functional bFGF and heparin delivery system might be helpful to repair the ischemic tissue and preventing blood vessel rethrombosis.Fig.11.(A) The schematic application and function of the nanoparticles for multi-functional bFGF and heparin delivery (B) Schematic brief description of the mechanism for multi-functional delivery of bFGF and heparin.4. ConclusionThis study demonstrates the preparation of heparinized chitosan/poly(-glutamic acid) nanoparticle as a multi-functional carrier for fibroblast growth factor and heparin delivery. The heparinized nanoparticles could sustain bFGF release at the pH of ischemia tissue and be rapidly degraded at the pH of repaired tissue to release heparin. The released bFGF from the nanoparticles enhanced the proliferation of HFF cells and angiogenic tube formation by HUVEC, suggesting the retaining of bFGF mitogenic activity. The released heparin, a traditionally used anticoagulant, maintained the anti-factor Xa activity in blood plasma. The mutil-functional, controlled delivery system for bFGF developed in this study may be a potential therapeutic method for regeneration of ischemic tissue and prevention of harmful clots formation.AcknowledgmentsThis work was supported by a grant from the National Science Council (NSC 97-2221-E-238-002-MY2), Taiwan, Republic of China.#ACC#13795897# #dtd510# #0BKD# #xa03053b#01429612#00310035#10010963#2010-10-15T10:59:48# #conversion_date#10/15/2010# #SECTIONTITLE# #HIQAW# #GRANT#References1 T.M. Allen and P.R. Cullis, Drug delivery systems: entering the mainstream, Science 303 (2004), pp. 18181822.#Allen#2004#1818#1822#T2 S. Sengupta, D. Eavarone, I. Capila, G. Zhao, N. Watson and T. Kiziltepe et al., Temporal targeting of tumor cells and neovasculature with a nanoscale delivery system, Nature 436 (2005), pp. 568572.#Sengupta#2005#568#572#S3 S.F. Peng, C.J. Su, M.C. Wei, C.Y. Chen, Z.X. Liao and P.W. Lee et al., Effects of the nanostructure of dendrimer/DNA complexes on their endocytosis and gene expression, Biomaterials 31 (2010), pp. 56605670.#Peng#2010#5660#5670#S4 E.A. Murphy, B.K. Majeti, L.A. Barnes, M. Makale, S.M. Weis and K. Lutu-Fuga et al., Nanoparticle-mediated drug delivery to tumor vasculature suppresses metastasis, PNAS 105 (2008), pp. 93439348.#Murphy#2008#9343#9348#E5 K.H. Lo, M.C. Chen, R.M. Ho and H.W. Sung, Pore-filling nanoporous templates from degradable block copolymers for nanoscale drug delivery, ACS Nano 3 (2009), pp. 26602666.#Lo#2009#2660#2666#K6 T.W. Chung, S.S. Wang and W.J. Tsai, Accelerating thrombolysis with chitosan-coated plasminogen activators encapsulated in poly(lactide-co-glycolide) (PLGA) nanoparticles, Biomaterials 29 (2008), pp. 228237.#Chung#2008#228#237#T7 H.F. Liang, S.C. Chen, M.C. Chen, P.W. Lee, C.T. Chen and H.W. Sung, Paclitaxel-loaded poly(gamma-glutamic acid)-poly(lactide) nanoparticles as a targeted drug delivery system against cultured HepG2 cells, Bioconjug Chem 17 (2006), pp. 291299.#Liang#2006#291#299#H8 Y.L. Chiu, Y.C. Ho, Y.M. Chen, S.F. Peng, C.J. Ke and K.J. Chen et al., The characteristics, cellular uptake and intracellular trafficking of nanoparticles made of hydrophobically-modified chitosan, J Control Release (2010) 10.1016/j. jconrel.2010.05.023.#Chiu#2010###Y9 S.D. Li and L. Huang, Pharmacokinetics and biodistribution of nanoparticles, Mol Pharmacol 5 (2008), pp. 496504.#Li#2008#496#504#S10 Y.H. Lin, F.L. Mi, C.T. Chen, W.C. Chang, S.F. Peng and H.F. Liang et al., Preparation and characterization of nanoparticles shelled with chitosan for oral insulin delivery, Biomacromolecules 8 (2007), pp. 146152.#Lin#2007#146#152#Y11 Y.H. Lin, K. Sonaje, K.M. Lin, J.H. Juang, F.L. Mi and H.W. Yang et al., Multi-ion-crosslinked nanoparticles with pH-responsive characteristics for oral delivery of protein drugs, J Control Release 132 (2008), pp. 141149.#Lin#2008#141#149#Y12 F.L. Mi, Y.Y. Wu, Y.H. Lin, K. Sonaje, Y.C. Ho and C.T. Chen et al., Nanoparticles self-assembled by N-trimethylchitosan and poly(-glutamic acid) for oral delivery of protein drugs, Bioconjug Chem 19 (2008), pp. 12481255.#Mi#2008#1248#1255#F13 K. Sonaje, Y.J. Chen, H.L. Chen, S.P. Wey, J.H. Juang and H.N. Nguyen et al., Enteric-coated capsules filled with freeze-dried chitosan/poly(gamma-glutamic acid) nanoparticles for oral insulin delivery, Biomaterials 31 (2010), pp. 33843394.#Sonaje#2010#3384#3394#K14 P.W. Lee, S.F. Peng, C.J. Su, F.L. Mi, H.L. Chen and M.C. Wei et al., The use of biodegradable polymeric nanoparticles in combination with a low-pressure gene gun for transdermal DNA delivery, Biomaterials 29 (2008), pp. 742751.#Lee#2008#742#751#P15 S.F. Peng, M.J. Yang, C.J. Su, H.L. Chen, P.W. Lee and M.C. Wei et al., Effects of incorporation of poly(gamma-glutamic acid) in chitosan/DNA complex nanoparticles on cellular uptake and transfection efficiency, Biomaterials 30 (2009), pp. 17971808.#Peng#2009#1797#1808#S16 P.W. Lee, S.H. Hsu, J.S. Tsai, F.R. Chen, P.J. Huang and C.J. Ke et al., Multifunctional core-shell polymeric nanoparticles for transdermal DNA delivery and epidermal Langerhans cells tracking, Biomaterials 31 (2010), pp. 24252434.#Lee#2010#2425#2434#P17 A.C. Rapraeger, A. Krufka and B.B. Olwin, Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation, Science 252 (1991), pp. 17051708.#Rapraeger#1991#1705#1708#A18 A. Yanagisawa-Miwa, Y. Uchida, F. Nakamura, T. Tomaru, H. Kido and T. Kamijo et al., Salvage of infarcted myocardium by angiogenic action of basic fibroblast growth factor, Science 257 (1992), pp. 14011403.#YanagisawaMiwa#1992#1401#1403#A19. H. Layman, M. Sacasa, A.E. Murphy, A.M. Murphy, S.M. Pham and F.M. Andreopoulos, Co-delivery of FGF-2 and G-CSF from gelatin-based hydrogels as angiogenic therapy in a murine critical limb ischemic model, Acta Biomaterialia 5 (2009), pp. 230239.#Layman#2009#230#239#H20 Y.I. Chung, G. Tae and S.H. Yuk, A facile method to prepare heparin-functionalized nanoparticles for controlled release of growth factors, Biomaterials 27 (2006), pp. 26212626.#Chung#2006#2621#2626#Y21 S.T.M. Nillesen, P.J. Geutjes, R. Wismans, J. Schalkwijk, W.F. Daamen and T.H. van Kuppevelt, Increased angiogenesis and blood vessel maturation in acellular collagenheparin scaffolds containing both FGF2 and VEGF, Biomaterials 28 (2007), pp. 11231131.#Nillesen#2007#1123#1131#S22 E. Ruoslahti and Y. Yamaguchi, Proteoglycans as modulators of growth factor activities, Cell 64 (1991), pp. 867869.#Ruoslahti#1991#867#869#E23 Y. Chang, P.H. Lai, H.J. Wei, W.W. Lin, S.C. Chen and H.W. Sung et al., Tissue regeneration observed in a basic fibroblast growth factorloaded porous acellular bovine pericardium populated with mesenchymal stem cells, J Thorac Cardiovasc Surg 134 (2007), pp. 6573.#Chang#2007#65#73#Y24 S.P. Sutherland, C.J. Benson, J.P. Adelman and E.W. McCleskey, From the Cover: acid-sensing ion channel 3 matches the acid-gated current in cardiac ischemia-sensing neurons, PNAS 98 (2001), pp. 711716.#Sutherland#2001#711#716#S25 Z.G. Xiong, X.M. Zhu, X.P. Chu, M. Minami, J. Hey and W.L. Wei et al., Neuroprotection in ischemia: blocking calcium-permeable acid-sensing ion channels, Cell 118 (2004), pp. 687698.#Xiong#2004#687#698#Z26 Y. Tachibana, M. Kurisawa, H. Uyama and S. Kobayashi, Thermo- and pH-responsive biodegradable poly(alpha-N-substituted gamma-glutamine)s, Biomacromolecules 4 (2003), pp. 11321134.#Tachibana#2003#1132#1134#Y27 M.C. Chen, H.S. Wong, K.J. Lin, H.L. Chen, S.P. Wey and K. Sonaje et al., The characteristics, biodistribution and bioavailability of a chitosan-based nanoparticulate system for the oral delivery of heparin, Biomaterials 30 (2009), pp. 66296637.#Chen#2009#6629#6637#M28 T. Akagi, M. Higashi, T. Kaneko, T. Kida and M. Akashi, Hydrolytic and enzymatic degradation of nanoparticles based on amphiphilic Poly(-glutamic acid)-graft-l-phenylalanine copolymers, Biomacromolecules 7 (2006), pp. 297303.#Akagi#2006#297#303#T29 D.H. Yu, Q. Lu, J. Xie, C. Fang and H.Z. Chen, Peptide- conjugated biodegradable nanoparticles as a carrier to target paclitaxel to tumor neovasculature, Biomaterials 31 (2010), pp. 22782292.#Yu#2010#2278#2292#D30 F.L. Mi, Y.C. Tan, H.F. Liang and H.W. Sung, In vivo biocompatibility and degradability of a novel injectable-chitosan-based implant, Biomaterials 23 (2002), pp. 181191.#Mi#2002#181#191#F31 C.R. Martin, A.M. Thomas and K.H. Thomas, The future of recombinant growth factors in wound healing, Am J Surg 176 (1998), pp. 80S82S.#Martin#1998#80S#82S#C32 D.B. Pike, S. Cai, K.R. Pomraning, M.A. Firpo, R.J. Fisher and X.Z. Shu et al., Heparin-regulated release of growth factors in vitro and angiogenic response in vivo to implanted hyaluronan hydrogels containing VEGF and bFGF, Biomaterials 27 (2006), pp. 52425251.#Pike#2006#5242#5251#D33 Y.C. Ho, S.J. Wu, F.L. Mi, Y.L. Chiu, S.H. Yu and N. Panda et al., Thiol-modified chitosan sulfate nanoparticles for protection and release of basic fibroblast growth factor, Bioconjug Chem 21 (2010), pp. 2838.#Ho#2010#28#38#Y34 Y.C. Ho, F.L. Mi, H.W. Sung and P.L. Kuo, Heparin-functionalized chitosanalginate polyelectrolyte complex scaffolds for the stabilization and sustained release of basic fibroblast growth factor, Int J Pharm 376 (2009), pp. 6975.#Ho#2009#69#75#Y35 Y.J. Chuang, R. Swanson, S.M. Raja and S.T. Olson, Heparin enhances the specificity of antithrombin for thrombin and factor Xa independent of the reactive center loop sequence. Evidence for an exosite determinant of factor Xa specificity in heparin-activated antithrombin, J Biol Chem 276 (2001), pp. 1496114971.#Chuang#2001#14961#14971#Y36 S.K. Kim, B. Vaishali, E. Lee, S. Lee, Y.K. Lee and T.S. Kumar et al., Oral delivery of chemical conjugates of heparin and deoxycholic acid in aqueous formulation, Thromb Res 117 (2006), pp. 419427.#Kim#2006#419#427#S37 N.J. Harmer, L.L. Ilag, B. Mulloy, L. Pellegrini, C.V. Robinson and T.L. Blundell, Towards a resolution of the stoichiometry of the fibroblast growth factor (FGF)FGF receptorheparin complex, J Mol Biol 339 (2004), pp. 821834.#Harmer#2004#821#834#N38 M. Rusnati, D. Coltrini, P. Caccia, P. DellEra, G. Zoppetti and P. Oreste et al., Distinct role of 2-O-, N- and 6-O-sulfate groups of heparin in the formation of the ternary complex with basic fibroblast growth factor and soluble FGF receptor-1, Biochim Biophys Res Commun 203 (1994), pp. 450458.#Rusnati#1994#450#458#M39 A.E. Erikson, L.S. Cousens, L.H. Weaver and B.W. Matthews, Three dimensional structure of human basic fibroblast growth factor, PNAS 88 (1991), pp. 34413445.#Erikson#1991#3441#3445#A40 J.R. Fromm, R.E. Hileman, E.E.O. Caldwell, J.M. Weiler and J.R. Linhardt, Pattern and spacing of basic amino acids in heparin binding sites, Arch Biochem Biophys 343 (1997), pp. 92100.#Fromm#1997#92#100#J41 K.W. Hung, T.K.S. Kumar, Y.H. Chi, I.M. Chiu and C. Yu, Molecular cloning, overexpression, and characterization of the ligand-binding D2 domain of fibroblast growth factor receptor, Biochem Biophys Res Commun 317 (2004), pp. 253258.#Hung#2004#253#258#K42 T. Spivak-Kroizman, M.A. Lemmon, I. Dikic, J.E. Ladbury, D. Pinchasi and J. Huang et al., Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation, Cell 79 (1994), pp. 10151024.#SpivakKroizman#1994#1015#1024#T43 M. Ishihara, P.N. Shaklee, Z.C. Yang, W.S. Liang, Z. Wei and R.J. Stack et al., Structural features in heparin which modulate specific biological activities mediated by basic fibroblast growth factor, Glycobiology 4 (1994), pp. 451458.#Ishihara#1994#451#458#M44 A. Walker, J.E. Turnbull and J.T. Gallagher, Specific heparin sulfate saccharides mediated the activity of basic fibroblast growth factor, J Biol Chem 269 (1994), pp. 931935.#Walker#1994#931#935#A45 D. Peschel, K. Zhang, N. Aggarwal, E. Brendler, S. Fischer and T. Groth, Synthesis of novel celluloses derivatives and investigation of their mitogenic activity in the presence and absence of FGF2, Acta Biomaterialia 6 (2010), pp. 21162125.#Peschel#2010#2116#2125#D46 A. Yayon, M. Klagsbrun, J.D. Esko, P. Leder and D.M. Ornitz, Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor, Cell 64 (1991), pp. 841848.#Yayon#1991#841#848#AAppendix. Figures with essential color discrimination. Fig.1, Fig.2, Fig.4, Fig.6, Fig.7, Fig.8, Fig.9 and Fig.10 in this article are difficult to interpret in black and white. The full color images can be found in the on-line version, at doi:10.1016/j.biomaterials.2010.08.058.Corresponding author. Department of Biotechnology, Vanung University, Chung-Li 30320, Taiwan, ROC. Fax: +886 3 4333063.Corresponding author.1The contributions by the two collaborating parties are equal.1-s1.0-512T-FSM1-JB58-600C-00000-00December 201020

© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号

地址：北京市海淀区学院路29号 邮编：100083

电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700