OBJECTIVE: To explore the mechanism of antibiotic delivery system targeting bacterial biofilm with linezolid (LZD) based on ε-poly- L-lysine (ε-PLL) and cyclodextrin (CD) (ε-PLL-CD-LZD), aiming to enhance antibiotic bioavailability, effectively penetrate and disrupt biofilm structures, and thereby improve the treatment of bone and joint infections. METHODS: ε-PLL-CD-LZD was synthesized via chemical methods. The grafting rate of CD was characterized using nuclear magnetic resonance. In vitro biocompatibility was evaluated through live/dead cell staining after co-culturing with mouse embryonic osteoblast precursor cells (MC3T3-E1), human umbilical vein endothelial cells, and mouse embryonic fibroblast cells (3T3-L1). The biofilm-enrichment capacity of ε-PLL-CD-LZD was assessed using Staphylococcus aureus biofilms through enrichment studies. Its biofilm eradication efficacy was investigated via minimum inhibitory concentration (MIC) determination, scanning electron microscopy, and live/dead bacterial staining. A bone and joint infection model in male Sprague-Dawley rats was established to validate the antibacterial effects of ε-PLL-CD-LZD. RESULTS: In ε-PLL-CD-LZD, the average grafting rate of CD reached 9.88%. The cell viability exceeded 90% after co-culturing with three types cells. The strong biofilm enrichment capability was observed with a MIC of 2 mg/L. Scanning electron microscopy observations revealed the effective disruption of biofilm structure, indicating potent biofilm eradication capacity. In vivo rat experiments demonstrated that ε-PLL-CD-LZD significantly reduced bacterial load and infection positivity rate at the lesion site ( P<0.05). CONCLUSION The ε-PLL-CD antibiotic delivery system provides a treatment strategy for bone and joint infections with high clinical translational significance. By effectively enhancing antibiotic bioavailability, penetrating, and disrupting biofilms, it demonstrated significant anti-infection effects in animal models.
Biofilms/drug effects* ; Animals ; Anti-Bacterial Agents/pharmacology* ; Polylysine/chemistry* ; Cyclodextrins/administration & dosage* ; Humans ; Linezolid/pharmacology* ; Staphylococcus aureus/physiology* ; Rats, Sprague-Dawley ; Mice ; Rats ; Male ; Drug Delivery Systems ; Staphylococcal Infections/drug therapy* ; Microbial Sensitivity Tests ; Human Umbilical Vein Endothelial Cells ; Osteoblasts/cytology*

During the production of ε-poly-L-lysine (ε-PL) in fed-batch fermentation, the decline of ε-PL synthesis often occurs at middle or late phase of the fermentation. To solve the problem, we adopted two strategies, namely pH shift and feeding yeast extract, to improve the productivity of ε-PL. ε-PL productivity in fermentation by pH shift and feeding yeast extract achieved 4.62 g/(L x d) and 5.16 g/(L x d), which were increased by 27.3% and 42.2% compared with the control ε-PL fed-batch fermentation, respectively. Meanwhile, ε-PL production enhanced 36.95 g/L and 41.32 g/L in 192 h with these two strategies, increased by 27.4% and 42.48% compared to the control, respectively. ε-PL production could be improved at middle or late phase of fed-batch fermentation by pH shift or feeding yeast extract.
Batch Cell Culture Techniques ; Fermentation ; Industrial Microbiology ; Nitrogen ; chemistry ; Polylysine ; biosynthesis

To enhance the production of ε-poly-L-lysine (ε-PL) by improving dissolved oxygen level of the fermentation system, different oxygen-vectors were added to broth and n-dodecane was screened as the best oxygen-vector. The best amount of n-dodecane was 0.5% (V/V) and the best time was at start of the fermentation. In a fed-batch fermentation in a 5 L bioreactor, ε-PL concentration reached a maximum of (30.8 ± 0.46) g/L and the dry cell weight obtained was (33.8 ± 0.29) g/L, increasing by 31.6% and 20.7% compared with the control group, respectively. This improvement can be related to 0.5% n-dodecane could maintain dissolved oxygen concentration > 32% of air concentration compared with 23.8% in ε-PL production phase, and the production of a main by-product, poly-L-diaminopropionic acid, fell by 31%. These results indicated that the dissolved oxygen level in the broth was improved by adding n-dodecane, which can inhibit the by-product production and improve the biosynthesis of ε-PL.
Alkanes ; chemistry ; Batch Cell Culture Techniques ; Bioreactors ; Fermentation ; Oxygen ; chemistry ; Polylysine ; biosynthesis

This study aimed to characterize and magnetic resonance imaging (MRI) track the mesenchymal stem cells labeled with polylysine-coated superparamagnetic iron oxide (PLL-SPIO). Rat bone marrow derived mesenchymal stem cells (rMSCs) were labeled with 25, 50 and 100 microg/mL PLL-SPIO for 24 hours. The labeling efficiency was assessed by iron content, Prussian blue staining, electron microscopy and in vitro MR imaging. The labeled cells were also analyzed for cytotoxicity and differentiation potential. Electron microscopic observations and Prussian blue staining revealed that 75% -100% of cells were labeled with iron particles. PLL-SPIO did not show any cytotoxicity up to 100 microg/mL concentration. Both 25 microg/mL and 50 microg/mL PLL-SPIO labeled stem cells did not exhibit any significant alterations in the adipo/osteo/chondrogenic differentiation potential compared to unlabeled control cells. The lower concentration of 25 microg/mL iron labeled cells emitted an obvious dark signal in T1W, T2WI and T2 * WI MR image. The novel PLL-SPIO enables to label and track rMSCs for in vitro MRI without cellular alteration. Therefore PLL-SPIO may potentially become a better MR contrast agent especially in tracking the transplanted stem cells and other cells without compromising cell functional quality.
Animals ; Bone Marrow Cells ; Cell Differentiation ; Dextrans ; chemistry ; Magnetic Resonance Imaging ; Magnetite Nanoparticles ; chemistry ; Mesenchymal Stromal Cells ; cytology ; Polylysine ; chemistry ; Rats ; Staining and Labeling

OBJECTIVETo overcome the deficiency in the current therapies for erectile dysfunction (ED), we designed and synthesized a novel high-efficiency polymer/gene compound drug controlled release system and discussed the feasibility of pH and temperature dually sensitive injectable hydrogel in ED gene therapy.

METHODSWe synthesized optimal siRNA gene nanoparticles by characterizing the zeta potential of polylysine (PLL)/siRNA gene compounds, and established a pH and temperature dually sensitive injectable gene compound drug controlled release system via Schiffs reaction between glycol chitosan (GC) and benzaldehyde capped OHC-PEO-PPO-PEO-CHO. Then we demonstrated the sustained release of the system at different temperatures.

RESULTSWhen the mass ratio of PLL to siRNA was 20:1, the zeta potential of the PLL/siRNA gene compound reached the peak (+23.5 mV) and the siRNA was encapsulated by PLL in the maximal degree. GC and OHC-PEO-PPO-PEO-CHO was crosslinked via benzoicimine reaction when environmental pH was changed from 5.5 to 7.4. The reslease of the siRNA encapsulated in this system kept at a low rate at 37 degrees C, significantly enhanced with the increase of the temperature to 60 degrees C, rising to (122.5 +/- 5.3) microg at 1 000 minutes as compared with (23.8 +/- 6.0) microg at 37 degrees C (P < 0.05).

CONCLUSIONThe polymer/gene compound drug controlled release system was successfully synthesized, which improved the stability and capacity of gene carriers and achieved siRNA release at different temperatures, promising to be a new approach to the gene therapy of ED.

Delayed-Action Preparations ; pharmacology ; Drug Delivery Systems ; Erectile Dysfunction ; drug therapy ; Genetic Therapy ; Humans ; Male ; Nanoparticles ; chemistry ; Polylysine ; chemistry ; Polymers ; RNA, Small Interfering ; pharmacology

OBJECTIVETo prepare polyelectrolyte multilayer film-coated microbubble ultrasound contrast agent (UCA) and evaluate its effects in contrast imaging on normal rabbit's liver parenchyma.

METHODSPerfluorocarbon (PFC) -containing microbubble UCA (ST68-PFC) were prepared by sonication-based on surfactants (Span 60 and Tween 80). Subsequently, the resulting ST68-PFC microbubbles were coated using oppositely charged polylysine (PLL) and alginate (Alg) by microbubble-templated layer-by-layer self-assembly technique via electrostatic interaction. The enhancement effects in contrast imaging on normal rabbit's liver parenchyma were assessed.

RESULTSThe obtained microbubble UCA exhibited a narrow size distribution. The polyelectrolytes were successfully assembled onto the surface of ST68-PFC microbubbles. In vivo experiment showed that polyelectrolyte multilayer film-coated UCA effectively enhanced the imaging of rabbit's liver parenchyma.

CONCLUSIONSThe novel microbubble UCA obtained via layer-by-layer self-assembly, when enabling more functions, has no obvious difference in enhancement effects compared with the premodified microbubbles. The polymers with chemically active groups (such as amino group and carboxyl group) can be used as the outermost layer for the attachment of targeting ligands to microbubbles, which allows the selective targeting of the microbubbles to desired sites.

Alginates ; chemistry ; Animals ; Contrast Media ; administration & dosage ; chemistry ; Fluorocarbons ; chemistry ; Glucuronic Acid ; chemistry ; Hexuronic Acids ; chemistry ; Liver ; diagnostic imaging ; Microbubbles ; Polylysine ; chemistry ; Rabbits ; Ultrasonography

In this work, a surface biological and chemical modification method was used for improving the biological behavior of endothelial cells onto titanium-oxide films. The titanium-oxide films were first activated by HCl and H2O2 to produce hydroxyl group, then coated with poly-L-lysine and further immobilized with fibronectin. The surface characteristics of samples were analyzed by Fourier Transfer Infrared Spectrum, X-ray Photoelectron Spectroscopy and contact angle method. The biological behavior of cultured human umbilical vein endothelial cell (HUVEC) seeding onto different samples surface was evaluated by the in vitro HUVEC original cultured experiment. The results showed that the method of coating with poly-L-lysine and immobilizing with fibronectin can promote the adhesion and growth of endothelial cells onto titanium-oxide film.
Cell Adhesion ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cells, Immobilized ; drug effects ; Coated Materials, Biocompatible ; pharmacology ; Endothelial Cells ; cytology ; Fibronectins ; pharmacology ; Humans ; Polylysine ; chemistry ; Titanium ; chemistry ; Umbilical Veins ; cytology

Nerve growth factor (NGF) can promote the regeneration of peripheral nerve as well as contraction and reepithelization of wound. We constructed a bioengineered dermis containing microencapsulated NGF-expressing NIH-3T3 cells and study the effect of the microencapsule to the bioengineered dermis and seed cells. NGF gene was transfected into NIH-3T3 cells and enclosed into alginate-poly-L-lysine-alginate (APA) microencapsulation and cultivated in vitro. Content of NGF in microencapsules culturing supernatant was measured by enzyme linked immunosorbent assay (ELISA) method. These microencapsules were co-cultured with epidermic cells and fibroblast cells. Bioengineered dermis was constructed with NGF-expressing micorencapsules as seed cells using tissue engineering method. NIH-3T3 microencapsules, empty microencapsules, normal culture media were control groups. After one week culture, the characteristics of the dermis were described by MTT test, the content of hydroxyproline (HP), HE staining and ultrastructure photograph. We found the NGF-expressing microencapsulates can secret NGF steadly after cultured 8w in vitro, promot the proliferation of epidermic cells and secret collagen of fibroblast cells. These functions can maintaine in bioengineered dermis. So NGF-expressing NIH-3T3 microencapsulates can promote the quality of bioengineered dermis.
Alginates ; chemistry ; Animals ; Biocompatible Materials ; chemistry ; Cell Proliferation ; Dermis ; cytology ; Gene Expression Regulation ; Mice ; NIH 3T3 Cells ; Nerve Growth Factor ; genetics ; Polylysine ; analogs & derivatives ; chemistry ; Skin Physiological Phenomena ; Tissue Engineering ; methods ; Transfection ; methods

OBJECTIVE: We wanted to compare the human neural stem cell (hNSC) labeling efficacy of different superparamagnetic iron oxide nanoparticles (SPIONs), namely, ferumoxides, monocrystalline iron oxide (MION), cross-linked iron oxide (CLIO)-NH2 and tat-CLIO. MATERIALS AND METHODS: The hNSCs (5x105 HB1F3 cells/ml) were incubated for 24 hr in cell culture media that contained 25 microgram/ml of ferumoxides, MION or CLIO-NH2, and with or without poly-L-lysine (PLL) and tat-CLIO. The cellular iron uptake was analyzed qualitatively with using a light microscope and this was quantified via atomic absorption spectrophotometry. The visibility of the labeled cells was assessed with MR imaging. RESULTS: The incorporation of SPIONs into the hNSCs did not affect the cellular proliferations and viabilities. The hNSCs labeled with tat-CLIO showed the longest retention, up to 72 hr, and they contained 2.15+/-0.3 pg iron/cell, which are 59 fold, 430 fold and six fold more incorporated iron than that of the hNSCs labeled with ferumoxides, MION or CLIO-NH2, respectively. However, when PLL was added, the incorporation of ferumoxides, MION or CLIO-NH2 into the hNSCs was comparable to that of tat-CLIO. CONCLUSION: For MR imaging, hNSCs can be efficiently labeled with tat-CLIO alone or with a combination of ferumoxides, MION, CLIO-NH2 and the transfection agent PLL.
Cells, Cultured ; Contrast Media/chemical synthesis/pharmacokinetics ; Cross-Linking Reagents/chemistry ; Ferric Compounds/chemistry/*pharmacokinetics ; Ferrosoferric Oxide/chemical synthesis/pharmacokinetics ; Gene Products, tat/chemistry ; Humans ; Iron/*pharmacokinetics ; Magnetic Resonance Imaging/methods ; Nanoparticles ; Neural Tube ; Oxides/*pharmacokinetics ; Phantoms, Imaging ; Polylysine/pharmacokinetics ; Spectrophotometry, Atomic ; Staining and Labeling/*methods ; Stem Cells/cytology/*drug effects/metabolism ; Time Factors ; Transfection

OBJECTIVETo induce the hair follicle regeneration in mice ear by microencapsulated dermal papillae cells (DPs) and to investigate the permeability of fluorescein in APA microencapsulation to search the ideal diameter of microencapsulation.

METHODSThe DPs were encapsulated with alginate-polylysine-alginate by a high-voltage electric field droplet generator. The microencapsulated dermal papilla cells were xenotransplanted into the mice ears. After 6 week, the histological examination was made by microscopy. The diffusion way and speed of fluorescein into the microencapsulations were observed by confocal laser scanning microscopy. The comparison of fluorescein intensity was made in APA microencapsulations with different diameters.

RESULTSFully developed hair follicles could be easily identified in the skin of implanted site following xenotransplantation of microencapsulation DPs, which were different from the control groups in configuration, number, size and differentiation degree. The fluorescein was diffused gradually into the microencapsulations with a shape of concentric circularity. The fluorescein intensity inside three groups of APA microencapsulations was: small > middle > big.

CONCLUSIONSThe microencapsulated DPs retain the physiological function to induce the follicle regeneration. The APA microencapsulations with 400um diameter could ensure the nutrition and metabolite to pass in and out freely, and isolate the immunocompetent substance absolutely.

Alginates ; chemistry ; Animals ; Cell Differentiation ; Cell Transplantation ; Cells, Cultured ; Ear ; Female ; Fluorescein ; chemistry ; Hair Follicle ; cytology ; physiology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Polylysine ; analogs & derivatives ; chemistry ; Scalp ; cytology ; transplantation