Sustained release of melatonin from poly (lactic‐co‐glycolic acid) (PLGA) microspheres to induce osteogenesis of human mesenchymal stem cells in vitro

Abstract: Melatonin promotes bone formation and prevents bone degradation via receptor‐dependent or receptor‐independent actions. The aim of this study is to encapsulate melatonin into poly (lactic‐co‐glycolic acid) (PLGA) microspheres (PLGA‐MEL‐MS) and create a melatonin sustained release system, then to evaluate its effect on the osteogenesis of human mesenchymal stem cells (hMSCs) in vitro. PLGA‐MEL‐MS were prepared by single emulsion solvent evaporation technique. Scanning electron microscopy demonstrated the incorporation of melatonin did not disturb the conventional generation of PLGA microspheres in size and morphology. In vitro drug release assay showed that PLGA‐MEL‐MS exhibited a biphasic drug release pattern: a low initial burst release effect with approximately 40% drug release at the first 3 days and a relatively retarded and continuous release with about 85% drug release over the 25 days. Cell proliferation assay demonstrated that PLGA‐MEL‐MS had no apparent effect on proliferation of human MSCs. In an osteogenesis assay, PLGA‐MEL‐MS obviously enhanced alkaline phosphatase (ALP) mRNA expression and increased ALP activity compared to that in the control group. Meanwhile, several markers of osteoblast differentiation were also significantly upregulated, including runx2, osteopontin, and osteocalcin. Furthermore, quantificational alizarin red‐based assay demonstrated that PLGA‐MEL‐MS significantly enhanced calcium deposit of hMSCs compared to the controls. Therefore, this simple melatonin sustained release system can control released melatonin to generate a microenvironment with a relatively stable concentration of melatonin for a period of time to support osteogenic differentiation of hMSCs in vitro. This suggests that this system may be used as bone growth stimulator in bone healing in vivo.     

Recombinant Human Bone Morphogenetic Protein-2 Binding and Incorporation in PLGA Microsphere Delivery Systems

The objective of this research was to determine the binding capacity and kinetics, and total incorporation of recombinant human bone morphogenetic protein-2 (rhBMP-2) in microspheres made from hydrophilic and hydrophobic poly(lactide-co-glycolide) (PLGA). Polymers were characterized by molecular weight, polydispersity, and acid number. Microspheres were produced via a water-in-oil-in-water double emulsion system and characterized for bulk density, size, specific surface area, and porosity. Protein concentrations were determined by reversed phase HPLC. Protein was loaded by soaking microspheres in a buffered solution, pH 4.5, of rhBMP-2, decanting excess liquid, and vacuum drying the wetted particles. Total loading and binding were determined by comparing protein concentration remaining to non-microsphere containing samples. Polymer acid number was the dominant polymer feature affecting the binding. Higher acid values correlated with increased rhBMP-2 binding. The amount of non-bound incorporated rhBMP-2 linearly correlated with the concentration of protein used in binding. High rhBMP-2 concentrations inhibit binding to PLGA microspheres. Binding was also inhibited by increased lactide content in the PLGA polymer. The polymer characteristics controlling rhBMP-2 binding to PLGA microspheres are acid value foremost followed by molecular weight and lactide/glycolide ratio. The total amount of rhBMP-2 incorporated depends on the bound amount and on the amount of free protein present.     

Development of a single-dose recombinant CAMP factor entrapping poly(lactide-co-glycolide) microspheres-based vaccine against Streptococcus agalactiae

Streptococcus agalactiae is an important contagious bovine mastitis pathogen. Although it is well controlled and even eradicated in most Northern European and North American dairy herds, the prevalence of this pathogen remains very high in China. However, research on development of a vaccine against S. agalactiae mastitis is scarce. The aims of the present study were to: (1) develop a single-dose vaccine against S. agalactiae based on poly(lactic-co-glycolic acid) (PLGA) microspheres (MS) encapsulated CAMP factor, a conserved virulent protein encoded by S. agalactiae’s cfb gene; and (2) evaluate its immunogenicity and protective efficacy in a mouse model. The cfb gene was cloned and expressed in a recombinant Escherichia coli strain Trans1-T1. The CAMP factor was tested to determine a safe dose range and then encapsulated in MS of PLGA (50:50) to assess its release pattern in vitro and immune reaction in vivo. Furthermore, a mouse model and a histopathological assay were developed to evaluate bacterial burden and vaccine efficacy. In the low dosage range (<100 μg), CAMP factor had no obvious toxicity in mice. The release pattern in vitro was characterized by an initial burst release (44%), followed by a sustained and slower release over 7 wk. In mice immunized with either pure CAMP factor protein or PLGA-CAMP, increased antibody titers were detected in the first 2 wk, whereas only PLGA-CAMP immunization induced a sustained increase of antibody titers. In mice vaccinated with PLGA-CAMP, mortality and bacteria counts were lower (compared to a control group) after S. agalactiae challenge. Additionally, no pathological lesions were detected in the vaccinated group. Therefore, PLGA-CAMP conferred protective efficacy against S. agalactiae in our mouse model, indicating its potential as a vaccine against S. agalactiae mastitis. Furthermore, the slow-release kinetics of PLGA MS warranted optimism for development of a single-dose vaccine.     

In vivo pain relief effectiveness of an analgesic-anesthetic carrying biodegradable controlled release rod systems

Pain is the most common and feared symptom for patients, especially those with cancer. Treatment of chronic pain with conventional ways of medication usually fails with increasing severity of the pain. New approaches enabling the prolonged provision of pain relievers are required. We designed a controlled release system of pain relievers, mainly for opioids (morphine, M, codeine, C, and hydromorphone, HM), and a local anesthetic (bupivacaine, BP) in the form of poly(L-lactide-coglycolide) (PLGA) rods. The efficacy of these rods implanted alone or in combination in relieving chronic pain in rats caused by the ligation of the sciatic nerve of their right hind limbs was studied. The two most common tests for measuring analgesia, i.e. tail-flick tests, that show analgesia at sites other than the site of injury, were used to study the degree of systemic distribution of the drugs and paw-withdrawal tests were used to study the analgesia at the site of injury. Alleviation of this chronic and severe neuropathic pain could be obtained for about 3-4 days when rods for two drugs, 'dual drug' (analgesic-anesthetic), were used. This duration is decreased by half (2 days) with the single-drug rods. Also the dual-drug rods, though at half the dose of each single drug application, enhanced the degree of analgesia of the first day. These in vivo results are also consistent with the previous in vitro results as in the case with codeine which had a higher first-day analgesia than morphine, despite a lower potency due to the faster in vitro release rate. Similarly, slower release of hydromorphone from PLGA (85 :15) rods resulted in less systemic analgesia than the more rapidly eroding PLGA (50 : 50) rods of the same drug.     

Aqueous N,N-diethylnicotinamide (DENA) solution as a medium for accelerated release study of paclitaxel

N,N-Diethylnicotinamide (DENA) was identified as an excellent hydrotropic agent for paclitaxel (PTX) in our previous studies. The aqueous solubility of PTX was increased by several orders of magnitude in the presence of DENA. Because of such a high hydrotropic property, DENA was used as a release medium providing a sink condition for the release of PTX from poly(lactic-co-glycolic acid) (PLGA) matrices. The release profiles of PTX from PLGA matrices into DENA, serum and phosphate-buffered saline (PBS) were compared. The stability of PTX in DENA and the degradation of PLGA molecules in DENA were examined. The degradation rate constant of PTX in 2 M DENA was similar to those in other aqueous solutions. The use of 2 M DENA as a release medium allowed differentiation of the release profiles of PTX from PLGA matrices made of different PLGA compositions. The PTX release from PLGA matrices was much faster in DENA solution than in serum or PBS, and the concentration of DENA affected the PTX release rate. The presence of DENA in the release medium increased the hydrolysis rate of PLGA polymers. The faster release of PTX from PLGA matrices in DENA solution may be due to the high PTX solubility and faster degradation of PLGA polymers in the presence of DENA. Our study suggests that the aqueous DENA solution can be used for the accelerated release study of PTX from PLGA matrices.     

Paclitaxel and etoposide-loaded Poly (lactic-co-glycolic acid) microspheres fabricated by coaxial electrospraying for dual drug delivery

Abstract

In this study, we fabricated paclitaxel (PTX) and etoposide (ETP) loaded Poly (lactic-co-glycolic acid) (PLGA) microspheres with core–shell structures and particle sizes ranging from 1 to 4?µm by coaxial electrospraying. The microspheres were analyzed by scanning electron microscopy (SEM), transmission electron microscopy (TEM). The drug loading rate and entrapment efficiency of the microspheres were detected by high performance liquid chromatograph (HPLC). Moreover, the drug release profiles and degradation of drug-loaded PLGA microspheres in vitro were investigated, respectively. The distinct layered structure that existed in the manufactured core–shell microspheres can be observed by TEM. The in vitro release profiles indicated that the PLGA/PTX?+?ETP (PLGA/PE) microspheres exhibited the controlled release of two drugs in a sequential manner. Cell Counting Kit-8 was used to detect the toxic and side effects of the microspheres on bone tumor cells. PTX and ETP for combination drug therapy loaded microspheres had more cytotoxic effect on saos-2 osteosarcoma cells than the individual drugs. In conclusion, core–shell PLGA microspheres by electrospraying for combination drug therapy is promising for medicine applications, the PLGA/PE microspheres have some potential for osteosarcoma treatment.     

柚皮素-PLGA纳米粒的制备及其体内药动学研究

目的 制备柚皮素-PLGA纳米粒,并考察其体内药动学.方法 纳米沉淀法制备PLGA纳米粒,在单因素试验基础上采用正交试验优化处方,测定包封率、载药量、粒径、Zeta电位、体外释药.大鼠分别灌胃给予柚皮素及其PLGA纳米粒混悬液(40 mg/kg)后采血,HPLC法测定柚皮素血药浓度,计算主要药动学参数.结果 最佳处方为...     

PLGA/HA/生物活性玻璃复合膜的制备及抗菌性能研究

目的　通过对PLGA/HA/生物活性玻璃复合膜的制备和检测,探索一种便于牙周临床使用的缓释杀菌体系。方法：采用溶剂挥发法制备PLGA/HA/生物活性玻璃复合膜。实验中各组PLGA/HA载体相同,向其中添加不同质量的BAG粉末,测量复合膜的厚度和表面微观结构变化情况。提取复合膜以及与复合膜中BAG等量的粉末的浸提液,测量浸提过程中离子释放、复合膜质量损失、浸提液酸碱性变化情况,以牙周膜成纤维细胞为实验对象测试浸提液的细胞毒性水平;并以MTT法测试浸提液对牙龈卟啉单胞菌（P.g.）和伴放线放线杆菌（A.a.）的杀菌作用。结果：在复合膜体系中,BAG的释放速度受到控制;较之BAG粉末,复合膜对P.g.和A.a.的杀灭作用更持久。结论：PLGA/HA/生物活性玻璃复合膜有望成为一种局部使用的牙周炎缓释药物。     

三氧化二砷磁性微球的磁性能

目的:制备包裹三氧化二砷的磁性微球,分别测量磁性微球中三氧化二砷和四氧化三铁的含量,并分析其磁性能。讨论包裹三氧化二砷的磁性微球的磁靶向性效果和临床应用的可能性。 方法:采用化学共沉淀法制备四氧化三铁纳米铁磁粒子,采用w/o/w复乳法将纳米铁磁粒子和三氧化二砷包裹于聚D,L乳酸-羟基乙酸共聚物（PLGA）高分子材料中制备成磁性微球,利用全谱直读等离子体原子发射光谱仪测量磁性微球的载药量,分别测量纳米铁磁粒子和磁性微球的磁滞回线,并检验磁性微球的磁分离效果。 结果:纳米铁磁粒子和磁性微球的矫顽力和剩余磁化强度均接近零,但磁性微球的饱和磁化强度大大低于纳米铁磁粒子的饱和磁化强度。磁性微球的磁分离效果明显。 结论:合成的磁性微球外壳由PLGA高分子材料组成,纳米级四氧化三铁分散于PLGA中,壳内是三氧化二砷水溶液。磁性微球具有超顺磁性和磁靶向性。     

Degradation and Characterisation of Electrospun Polycaprolactone (PCL) and Poly(lactic-co-glycolic acid) (PLGA) Scaffolds for Vascular Tissue Engineering

The current study aimed to evaluate the characteristics and the effects of degradation on the structural properties of Poly(lactic-co-glycolic acid) (PLGA)- and polycaprolactone (PCL)-based nanofibrous scaffolds. Six scaffolds were prepared by electrospinning, three with PCL 15% (w/v) and three with PLGA 10% (w/v), with electrospinning processing times of 30, 60 and 90 min. Both types of scaffolds displayed more robust mechanical properties with increased spinning times. The tensile strength of both scaffolds with 90-min electrospun membranes did not show a significant difference in their strengths, as the PCL and PLGA scaffolds measured at 1.492 MPa ± 0.378 SD and 1.764 MPa ± 0.7982 SD, respectively. All membranes were shown to be hydrophobic under a wettability test. A degradation behaviour study was performed by immersing all scaffolds in phosphate-buffered saline (PBS) solution at room temperature for 12 weeks and for 4 weeks at 37 °C. The effects of degradation were monitored by taking each sample out of the PBS solution every week, and the structural changes were investigated under a scanning electron microscope (SEM). The PCL and PLGA scaffolds showed excellent fibre structure with adequate degradation, and the fibre diameter, measured over time, showed slight increase in size. Therefore, as an example of fibre water intake and progressive degradation, the scaffold’s percentage weight loss increased each week, further supporting the porous membrane’s degradability. The pore size and the porosity percentage of all scaffolds decreased substantially over the degradation period. The conclusion drawn from this experiment is that PCL and PLGA hold great promise for tissue engineering and regenerative medicine applications.