Enhanced of antibacterial activity of antibiotic-functionalized silver nanocomposites with good biocompatibility.

Antimicrobial resistance to traditional antibiotics leads to a serious concern for medical care owing to ineffective antibiotic therapies. This study focused on the preparation of silver nanocomposites (AgNPs@Tob&PAGA) by modifying AgNPs with tobramycin (Tob) and carbohydrate polymer of poly(2-(acrylamido) glucopyranose) (PAGA). The enhanced antibacterial activities of nanocomposites against common pathogens of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were explored. The introduction of PAGA onto silver nanocomposites improved both citocompatibility and antibacterial activity. Compared with nude Tob, AgNPs@Tob&PAGA showed more fascinating antimicrobial effect against E. coli and S. aureus with about 20-fold increase in the antibacterial activity, simultaneously no detectable resistance was observed. Consequently, the silver nanocomposite as an antimicrobial agent presents promising prospects in the treatment of bacterial infections caused by antimicrobial resistant bacteria.

Alendronate release from calcium phosphate cement for bone regeneration in osteoporotic conditions.

Osteoporosis represents a major health problem in terms of compromising bone strength and increasing the risk of bone fractures. It can be medically treated with bisphosphonates, which act systemically upon oral or venous administration. Further, bone regenerative treatments in osteoporotic conditions present a challenge. Here, we focused on the development of a synthetic bone substitute material with local diminishing effects on osteoporosis. Composites were created using calcium phosphate cement (CPC; 60 wt%) and polylactic-co-glycolic acid (PLGA; 40 wt%), which were loaded with alendronate (ALN). In vitro results showed that ALN-loaded CPC/PLGA composites presented clinically suitable properties, including setting times, appropriate compressive strength, and controlled release of ALN, the latter being dependent on composite degradation. Using a rat femoral condyle bone defect model in osteoporotic animals, ALN-loaded CPC/PLGA composites demonstrated stimulatory effects on bone formation both within and outside the defect region.

cmRNA/lipoplex encapsulation in PLGA microspheres enables transfection via calcium phosphate cement (CPC)/PLGA composites.

In this study lipoplexes containing chemically modified messenger RNA (cmRNA) were incorporated into poly (lactic-co-glycolic acid) (PLGA) microspheres via water-in-oil-in-water (W/O/W) double emulsion solvent evaporation technique. The nanoparticle encapsulation by microparticle formation was optimized to achieve lipoplex release and maximum transfection efficiency in surrounding cells. It was possible to adjust characteristic features in surface topology and size of the PLGA-microspheres by varying the extent of lipoplex loading into the polymer matrix. The partial release of lipids and mRNA out of the microparticle system, their accumulation in cells and the production of encoded protein were visualized via fluorescence microscopy. These bioactive microspheres, containing cmRNA bearing lipoplexes, were developed for the incorporation of a therapeutic component into injectable calcium phosphate cements (CPC). Due to the incorporation of PLGA/lipoplex microspheres as a degradable entity, the porosity of the cement phase could additionally be adjusted. This approach of complex nanoparticle incorporation into polymer/cement composites represents a promising example for combining transcript therapy with biomechanical engineering.

Improving bone repair of femoral and radial defects in rabbit by incorporating PRP into PLGA/CPC composite scaffold with unidirectional pore structure.

In this study, a platelet-rich plasma poly(lactic-co-glycolic acid) (PRP-PLGA)/calcium phosphate cement (CPC) composite scaffold was prepared by incorporating PRP into PLGA/CPC scaffold with unidirectional pore structure, which was fabricated by the unidirectional freeze casting of CPC slurry and the following infiltration of PLGA. The results from in vitro cell experiments and in vivo implantation in femoral defects manifested that incorporation of PRP into PLGA/CPC scaffold improved in vitro cell response (cell attachment, proliferation, and differentiation), and markedly boosted bone formation, angiogenesis and material degradation. The incorporation of PRP into scaffold showed more outstanding improvement in osteogenesis as the scaffolds were used to repair the segmental radial defects, especially at the early stage. The new bone tissues grew along the unidirectional lamellar pores of scaffold. At 12 weeks postimplantation, the segmental radial defects treated with PRP-PLGA/CPC scaffold had almost recuperated, whereas treated with the scaffold without PRP was far from healed. Taken together, the PRP-PLGA/CPC scaffold with unidirectional pore structure is a promising candidate to repair bone defects at various sites.

Pluronic-poly[alpha-(4-aminobutyl)-1-glycolic acid] polymeric micelle-like nanoparticles as carrier for drug delivery.

Pluronic-poly[alpha-(4-aminobutyl)-1-glycolic acid] (Pluronic-PAGA) with different types of Pluronic, the different molecule weight of PAGA, and the different molar ratios of Pluronic to PAGA were synthesized. These materials were bio-degradable, amphiphilic, could be degraded into non-toxic small molecules and could be used to carry drugs. 5-Fluorouracil (5-Fu) loaded Pluronic-PAGA micelle-like nanoparticles (5-Fu loaded P-PAGA NPs) were prepared by a simple self-assembly method, and characterized by dynamic light scattering, transmission electron microscope. The degradation and release characteristics have also been studied in this paper. With the time passing, the 5-Fu loaded P-PAGA NPs degraded into smaller ones with the similar characteristics of the original NPs. Both the types of Pluronic and the molecule weight of the PAGA affected the releasing progresses. It was found that 5-Fu loaded P-PAGA NPs exhibited high growth inhibitory effect on human gastric cancer cells by MTT assay. The cellular uptake of Rhodamine B loaded P-PAGA NPs was higher than free Rhodamine B. This study suggested that the Pluronic-PAGA with acceptable drug entrapment efficiencies, drug loading efficiencies and tunable release profiles could offer an alternative carrier for 5-Fu delivery and have the potential for the delivery of other anti-tumour drug.

[Experimental study of tissue-engineered bone constructed with simvastatin carried by PLGA/CPC and bone marrow stromal cells].

PURPOSE: To study the feasibility of tissue engineered bone constructed with simvastatin carried by PLGA/CPC and bone marrow stromal cells (BMSCs) and screen the effective drug loading of simvastatin. METHODS: Solvent casting-particle leaching technology combined with the phase separation process was used to prepare the different concentrations (simvastatin mass: 0.1, 0.5, 1 mg) of simvastatin carried by PLGA/CPC composite scaffold materials. Scanning electron microscopy was used to observe the porosity and drug release curve was drawn; Alizarin red staining and type I collagen staining were applied to observe the effect of osteogenic medium and simvastatin on the role of BMSCs to the osteogenetic differentiation. The induced passage 3 cells after dil staining were mixed with the composite scaffold material to a complex. Scanning electron microscopy and laser confocal microscope were used to observe the adhesion on the complex. CCK-8 and alkaline phosphatase (ALP) were applied to observe the proliferation and differentiation. SPSS 18.0 software package was used for statistical analysis. RESULTS: The scaffold porosity was more than 90% with an average aperture of 200-300 µm. The drug released slowly. There was no obvious interpretation. Type I collagen showed positive expression. Alizarin red staining proofed the formation of mineralization nodules in group which was induced with the conditional medium and simvastatin. 0.5 mg group showed cells adhered to the inner surface of the scaffold material and could significantly promote the proliferation and differentiation of cells. CONCLUSIONS: Combination of simvastatin and osteogenic medium can effectively promote the differentiation of BMSCs. Simvastatin carried by PLGA/CPC scaffold materials is an ideal tissue engineering scaffold material. PLGA/CPC scaffold containing 0.5 mg simvastatin can effectively promote the proliferation and differentiation of BMSCs. Supported by Natural Science Foundation of Jilin Province (201215052).

A controlled release of antibiotics from calcium phosphate-coated poly(lactic-co-glycolic acid) particles and their in vitro efficacy against Staphylococcus aureus biofilm.

Ceramic-polymer hybrid particles, intended for osteomyelitis treatment, were fabricated by preparing poly(lactic-co-glycolic acid) particles through an emulsion solvent evaporation technique, followed by calcium phosphate (CaP) coating via a surface adsorption-nucleation method. The presence of CaP coating on the surface of the particles was confirmed by scanning electron microscopy, energy-dispersive X-ray spectroscopy, and X-ray photoelectron spectroscopy. Subsequently, two antibiotics for treating bone infection, nafcillin (hydrophilic) and levofloxacin (amphiphilic), were loaded into these hybrid particles and their in vitro drug release studies were investigated. The CaP coating was shown to reduce burst release, while providing sustained release of the antibiotics for up to 4 weeks. In vitro bacterial study against Staphylococcus aureus demonstrated the capability of these antibiotic-loaded hybrid particles to inhibit biofilm formation as well as deteriorate established biofilm, making this hybrid system a potential candidate for further investigation for osteomyelitis treatment.

Long-term variations in mechanical properties and in vivo degradability of CPC/PLGA composite.

A new type of bone cement composite was successfully achieved by mixing degradable biosecure polylactic-co-glycolic acid (PLGA) fibers with high initial strength calcium phosphate cement (CPC). Its higher initial strength was mainly responsible for the in situ reinforcing effect of residual tetra-calcium phosphate monoxide (r-TTCP) particles reported in our previous work. So this bone cement composite containing fibers and the controlling group could be termed as CPC/PLGA composite and pure CPC or fiber-free group, respectively. In this study, we had investigated mechanical properties and microstructures of the CPC/PLGA composite immersed in 0.9% saline solution for different time and its in vivo degradation behaviors after implanting in rabbit muscle and femur bone, respectively. Results showed that the incorporation of the degradable fibers not only greatly increased the initial toughness and flexural strength of the CPC/PLGA composite but also significantly improved its later osteo-conduction as well as degradation rate. The rabbit muscle implant tests showed that the weight loss ratio of the CPC/PLGA composite increased by 41.03% as compared to the pure CPC. And the rabbit femur implant tests showed that the composite exhibits outstanding biocompatibility and bioactivity and more excellent osteoconduction and degradability than the pure CPC.

Minute changes in composition of polymer substrates produce amplified differences in cell adhesion and motility via optimal ligand conditioning.

We explored the interplay between substratum chemistry of polymeric materials and surface-adsorbed ligand concentration (human plasma fibronectin) in the control of cell adhesion and cell motility. We found that small changes in the chemical composition of a polymeric substratum had different effects on cellular motility--depending on the concentration of preadsorbed fibronectin. We used two tyrosine-derived polyarylates, poly(DTD diglycolate) and poly(DTD glutarate), as substrata for the seeding of NIH-3T3 fibroblasts. The only compositional difference between the two test polymers was that one single oxygen atom in the polymer backbone of poly(DTD diglycolate) had been substituted by a methylene group in the backbone of poly(DTD glutarate), The two polymers had closely matched hydrophobicity and physical properties. Flat, spin-coated surfaces of these polymers were pretreated with different concentrations of human plasma fibronectin (0-20 microg/ml). After seeding with NIH-3T3 fibroblasts, we examined the adhesion and motility behavior of these cells. We found that NIH-3T3 fibroblasts migrated significantly faster on poly(DTD diglycolate), but only when the polymer surfaces were pretreated with intermediate concentrations of fibronectin. Only at these intermediate levels of ligand conditioning, did the presence of an extra oxygen atom in the backbone of poly(DTD diglycolate) relative to poly(DTD glutarate) (i) alter the overall organization/concentration of the fibronectin; (ii) weaken cell attachment strength and inhibited excessive cell spreading; and (iii) promote cell motility kinetics. These findings indicate that the biological effect of minute changes in substratum chemistry is critically dependent on the level of surface-adsorbed cell-binding ligands.

Prevention of autoimmune insulitis by delivery of a chimeric plasmid encoding interleukin-4 and interleukin-10.

The combined administration of interleukin-4 (IL-4) and interleukin-10 (IL-10) expression plasmids has demonstrated synergistic effects on the prevention of autoimmune diabetes. To this end, we constructed a co-expression 'chimeric' plasmid, pCMV-IL4-IL10, in which the expression of IL-4 and IL-10 was driven by two separate CMV immediate early promoters by using the biodegradable polymer, poly[alpha-(4-aminobutyl)-L-glycolic acid] (PAGA) as a gene carrier to optimize gene delivery. In vitro transfection assays of the chimeric plasmid in 293T cells showed higher expression levels as well as dose dependence than the single gene expression plasmids. To evaluate the in vivo efficacy of the chimeric plasmid, the pCMV-IL4-IL10/PAGA complex was intravenously injected into 4-week-old non-obese diabetic (NOD) mice and compared to the co-administration group. While both groups had persistent gene expression longer than 5 weeks, the IL-4 and IL-10 serum levels of the chimeric group were higher than those in the co-administration group. Furthermore, the degree of insulitis in the chimeric group was improved over both the co-administration and non-injected control groups. These results suggest that the chimeric IL-4 and IL-10 expression plasmid can effectively reduce the incidence of autoimmune insulitis.

Biodegradable polymer-based interleukin-12 gene delivery: role of induced cytokines, tumor infiltrating cells and nitric oxide in anti-tumor activity.

The objective of this study was to investigate the role of induced cytokines, tumor infiltrating cells and nitric oxide (NO) in anti-tumor activity upon intratumoral injection of free and condensed plasmid DNA encoding murine interleukin-12 (pmIL-12) into BALB/c mice bearing subcutaneous tumors. Poly[alpha-(4-aminobutyl)-L-glycolic acid] (PAGA) was used for complex formation with pmIL-12 in presence of 5% (w/v) glucose. Upon characterization, PAGA/pmIL-12 (3/1, +/-) complexes were found to be most effective in gene transfer and were used consistently throughout this study. The levels of mIL-12 p70 and induced cytokines were determined by ELISA in the supernatant of the cultured tumors of the CT-26 subcutaneous tumor bearing BALB/c female mice 48 h after intratumoral injection of PAGA/pmIL-12 complexes and naked pmIL-12. The levels of IL-12, IFN-gamma, TNF-alpha and NO were higher for the PAGA/pmIL-12 complexes than those for the naked pmIL-12, PAGA alone and 5% glucose injected groups. The relative presence of natural killer (NK) cells, CD4(+) T cells, and antigen presenting cells, such as macrophages and dendritic cells determined using immunohistochemistry was higher for PAGA/pmIL-12 complexes compared with naked pmIL-12. The presence of CMV promoter in plasmid encoding IL-12 cDNAs did not induce any type I interferon response. There was a significant improvement in the survival rate and the inhibition of tumor growth after repeated injections of PAGA/pmIL-12 complexes.

Prevention of autoimmune insulitis by delivery of interleukin-4 plasmid using a soluble and biodegradable polymeric carrier.

PURPOSE: We delivered interleukin-4 (IL-4) plasmid (pCAGGS-IL-4) using the biodegradable polymer, poly[alpha-(4-aminobutyl)-L-glycolic acid] (PAGA), to prevent autoimmune insulitis in NOD mice. METHODS: The pCAGGS-IL-4/PAGA complex was transfected to 293T cells. The expression level of IL-4 was measured by ELISA. The pCAGGS IL-4/PAGA complex was injected once to NOD mice intravenously at the age of 4 weeks. RT-PCR was performed to evaluate the level of the IL-4 mRNA in the liver. At 6 weeks after the injection, the grade of insulitis of the mice was evaluated by double blind methods. RESULTS: In vitro transfecton assays showed that PAGA enhanced the expression of IL-4 in 293T cells. RT-PCR of the liver showed that IL-4 was expressed highest in the complex injected group. In the plasmid/PAGA complex injected group, the prevalence of severe insulitis in NOD mice was markedly improved, suggesting that PAGA enhanced the delivery of IL-4 plasmid. CONCLUSION: The pCAGGS-IL-4/PAGA complex is an effective system to prevent autoimmune insulitis in NOD mice and applicable for the prevention of autoimmune diabetes.

Biodegradable polyester, poly[alpha-(4-aminobutyl)-L-glycolic acid], as a non-toxic gene carrier.

PURPOSE: The aim of this study was to develop a non-toxic polymeric gene carrier. For this purpose, biodegradable cationic polymer, poly[alpha-(4-aminobutyl)-L-glycolic acid] (PAGA) was synthesized. PAGA was designed to have ester linkage because polyesters usually show biodegradability. METHODS: Degradation of PAGA in an aqueous solution was followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). PAGA/DNA complexes were characterized by gel electrophoresis, atomic force microscopy (AFM), dynamic light scattering (DLS). The transfection was measured by using the beta-galactosidase reporter gene. RESULTS: PAGA was degraded in aqueous solution very quickly and the final degradation product was a monomer (L-oxylysine). Formation of self-assembling biodegradable complexes between PAGA and DNA at a charge ratio 1:1 (+/-) was confirmed by gel band shift assay and AFM. In these studies, controlled release of DNA from the complexes could be seen. The complexes showed about 2-fold higher transfection efficiency than DNA complexes of poly-L-lysine (PLL), a structural analogue of PAGA, which is the most commonly used poly-cation for gene delivery. The polymer did not show cytotoxicity, possibly because of its degradability and the biocompatibility of the monomer. CONCLUSIONS: The use of the biodegradable poly-cation, PAGA, as a DNA condensing agent will be useful in safe gene delivery.

Soluble biodegradable polymer-based cytokine gene delivery for cancer treatment.

Transgene expression and tumor regression after direct injection of plasmid DNA encoding cytokine genes, such as mIL-12 and mIFN-gamma, remain very low. The objective of this study is to develop nontoxic biodegradable polymer-based cytokine gene delivery systems, which should enhance mIL-12 expression, increasing the likelihood of complete tumor elimination. We synthesized poly[alpha-(4-aminobutyl)-l-glycolic acid] (PAGA), a biodegradable nontoxic polymer, by melting condensation. Plasmids used in this study encoded luciferase (pLuc) and murine interleukin-12 (pmIL-12) genes. PAGA/plasmid complexes were prepared at different (+/-) charge ratios and characterized in terms of particle size, zeta potential, osmolality, surface morphology, and cytotoxicity. Polyplexes prepared by complexing PAGA with pmIL-12 as well as pLuc were used for transfection into cultured CT-26 colon adenocarcinoma cells as well as into CT-26 tumor-bearing BALB/c mice. The in vitro and in vivo transfection efficiency was determined by luciferase assay (for pLuc), enzyme-linked immunosorbent assay (for mIL-12, p70, and p40), and reverse transcriptase-polymerase chain reaction (RT-PCR) (for Luc and mIL-12 p35). PAGA condensed and protected plasmids from nuclease degradation. The mean particle size and zeta potential of the polyplexes prepared in 5% (w/v) glucose at 3:1 (+/-) charge ratio were approximately 100 nm and 20 mV, respectively. The surface characterization of polyplexes as determined by atomic force microscopy showed complete condensation of DNA with an ellipsoidal structure in Z direction. The levels of mIL-12 p40, mIL-12 p70, and mIFN-gamma were significantly higher for PAGA/pmIL-12 complexes compared to that of naked pmIL-12. This is in good agreement with RT-PCR data, which showed significant levels of mIL-12 p35 expression. The PAGA/pmIL-12 complexes did not induce any cytotoxicity in CT-26 cells as evidenced by 3-¿4, 5-dimethylthiazol-2-yl¿-2,5-diphenyltetrazolium bromide assay and showed enhanced antitumor activity in vivo compared to naked pmIL-12. PAGA/pmIL-12 complexes are nontoxic and significantly enhance mIL-12 expression at mRNA and protein levels both in vitro and in vivo.

Degradable polymeric carrier for the delivery of IL-10 plasmid DNA to prevent autoimmune insulitis of NOD mice.

Recently, we have reported that biodegradable poly [alpha-(4-aminobutyl)-L-glycolic acid] (PAGA) can condense and protect plasmid DNA from DNase I. In this study, we investigated whether the systemic administration of pCAGGS mouse IL-10 (mIL-10) expression plasmid complexed with PAGA can reduce the development of insulitis in non-obese diabetic (NOD) mice. PAGA/mIL-10 plasmid complexes were stable for more than 60 min, but the naked DNA was destroyed within 10 min by DNase I. The PAGA/DNA complexes were injected into the tail vein of 3-week-old NOD mice. Serum mIL-10 level peaked at 5 days after injection, and could be detected for more than 9 weeks. The prevalence of severe insulitis on 12-week-old NOD mice was markedly reduced by the intravenous injection of PAGA/DNA complex (15.7%) compared with that of naked DNA injection (34.5%) and non-treated controls (90.9%). In conclusion, systemic administration of pCAGGS mIL-10 plasmid/PAGA complexes can reduce the severity of insulitis in NOD mice. This study shows that the PAGA/DNA complex has the potential for the prevention of autoimmune diabetes mellitus. Gene Therapy (2000) 7, 2099-2104.