Engineering vascularized soft tissue flaps in an animal model using human adipose–derived stem cells and VEGF+PLGA/PEG microspheres on a collagen-chitosan scaffold with a flow-through vascular pedicle

Insufficient neovascularization is associated with high levels of resorption and necrosis in autologous and engineered fat grafts. We tested the hypothesis that incorporating angiogenic growth factor into a scaffold–stem cell construct and implanting this construct around a vascular pedicle improves neovascularization and adipogenesis for engineering soft tissue flaps. Poly(lactic-co-glycolic-acid/polyethylene glycol (PLGA/PEG) microspheres containing vascular endothelial growth factor (VEGF) were impregnated into collagen-chitosan scaffolds seeded with human adipose-derived stem cells (hASCs). This setup was analyzed in vitro and then implanted into isolated chambers around a discrete vascular pedicle in nude rats. Engineered tissue samples within the chambers were harvested and analyzed for differences in vascularization and adipose tissue growth. In vitro testing showed that the collagen-chitosan scaffold provided a supportive environment for hASC integration and proliferation. PLGA/PEG microspheres with slow-release VEGF had no negative effect on cell survival in collagen-chitosan scaffolds. In vivo, the system resulted in a statistically significant increase in neovascularization that in turn led to a significant increase in adipose tissue persistence after 8 weeks versus control constructs. These data indicate that our model—hASCs integrated with a collagen-chitosan scaffold incorporated with VEGF-containing PLGA/PEG microspheres supported by a predominant vascular vessel inside a chamber—provides a promising, clinically translatable platform for engineering vascularized soft tissue flap. The engineered adipose tissue with a vascular pedicle could conceivably be transferred as a vascularized soft tissue pedicle flap or free flap to a recipient site for the repair of soft-tissue defects.     

脑源性神经营养因子缓释微球对周围神经损伤大鼠的神经保护作用

目的 观察脑源性营养因子缓释微球(BDNF-PLGA缓释微球)对大鼠周围神经损伤的保护作用。方法 采用复乳化溶剂挥发法制备BDNF-PLGA缓释微球。40只SD大鼠随机分为假手术组、模型组、BDNF组和BDNF-PLGA缓释微球组;除假手术外,其它30只SD大鼠,制备坐骨神经钳夹损伤模型。术后神经损伤局部注射BDNF-PLGA缓释微球,观察大鼠的大体形态、步态、关节活动等情况;术后4周进行神经行为学评分,检查神经传导速度(NCV)、波幅、潜伏期和复合肌肉动作电位(CMAP)的幅度,以及组织病理学观察。结果 BDNF-PLGA缓释微球组明显改善坐骨神经损伤大鼠的步态、关节活动等一般状况。BDNF-PLGA缓释微球可有效地改善大鼠损伤神经功能,到第4周时已基本恢复正常,对神经功能恢复明显快于BDNF组。与模型组比较,BDNF-PLGA缓释微球组显著提高大鼠NCV,增大电位波幅,缩短潜伏期(P<0.01)。术后4周,BDNF-PLGA缓释微球组的大鼠坐骨神经NCV、波幅和潜伏期,CMAP波幅及恢复率均显著优于BDNF组。BDNF-PLGA缓释微球明显改善坐骨神经髓鞘和轴突结构破坏,髓神经纤维的髓鞘肿胀、碎裂,神经纤维中的空泡及空泡变性等组织病理学改变。结论 BDNF-PLGA缓释微球对周围神经损伤具有显著的保护作用。     

Improved anticancer delivery of paclitaxel by albumin surface modification of PLGA nanoparticles

Background

Nanoparticles (NPs) play an important role in anticancer delivery systems. Surface modified NPs with hydrophilic polymers such as human serum albumin (HSA) have long half-life in the blood circulation system.

Methods

The method of modified nanoprecipitation was utilized for encapsulation of paclitaxel (PTX) in poly (lactic-co-glycolic acid) (PLGA). Para-maleimide benzoic hydrazide was conjugated to PLGA for the surface modifications of PLGA NPs, and then HSA was attached on the surface of prepared NPs by maleimide attachment to thiol groups (cysteines) of albumin. The application of HSA provides for the longer blood circulation of stealth NPs due to their escape from reticuloendothelial system (RES). Then the physicochemical properties of NPs like surface morphology, size, zeta potential, and in-vitro drug release were analyzed.

Results

The particle size of NPs ranged from 170 to 190 nm and increased about 20–30 nm after HSA conjugation. The zeta potential was about -6 mV and it decreased further after HSA conjugation. The HSA conjugation in prepared NPs was proved by Fourier transform infrared (FT-IR) spectroscopy, faster degradation of HSA in Differential scanning calorimetry (DSC) characterization, and other evidences such as the increasing in size and the decreasing in zeta potential. The PTX released in a biphasic mode for all colloidal suspensions. A sustained release profile for approximately 33 days was detected after a burst effect of the loaded drug. The in vitro cytotoxicity evaluation also indicated that the HSA NPs are more cytotoxic than plain NPs.

Conclusions

HSA decoration of PLGA NPs may be a suitable method for longer blood circulation of NPs.     

Investigation of the stabilizer elimination during the washing step of charged PLGA microparticles utilizing a novel HPLC-UV-ELSD method

Quantification of stabilizer content in microparticles and other products is of great importance for formulation development, drug product quality control as well as for reproducible manufacturing. A fast and sensitive HPLC method with evaporative light scattering detection (ELSD) capable of detecting docusate sodium (DOSS), poly (lactic-co-glycolic acid) (PLGA; Resomer RG 503 H) and R-1,2-dioleoyloxy-3-trimethylammonium-propane (DOTAP) in a single run was successfully developed. In contrast to previously described methods, hydrolysis of PLGA as pretreatment is not necessary, thereby enabling accurate quantification of stabilizer next to the intact matrix polymer.This method was used to investigate the impact of washing procedures of polymeric microparticles manufactured either with anionic stabilizer DOSS or with cationic stabilizer DOTAP. High amounts of DOSS were detected in the washing water. This finding was consistent with the result that no DOSS could be detected in the washed and dried microparticles (<limit of detection).In contrast, DOTAP was hardly measurable in the washing water during all washing cycles. However, DOTAP could be quantified in dried particles. The ratio of DOTAP to dry particle mass was approximately 1:10.This is most probably due to the different polymer surfactant interactions (e.g. charge) and the different hydrophilicity of the stabilizers used.     

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.     

In vivo biocompatibility of the PLGA microparticles in parotid gland

Poly(lactic-co-glycolic acid) (PLGA) microparticles are used in various disorders for the controlled or sustained release of drugs, with the management of salivary gland pathologies possible using this technology. There is no record of the response to such microparticles in the glandular parenchyma. The purpose of this study was to assess the morphological changes in the parotid gland when injected with a single dose of PLGA microparticles. We used 12 adult female Sprague Dawley rats (Rattus norvegicus) that were injected into their right parotid gland with sterile vehicle solution (G1, n=4), 0.5 mg PLGA microparticles (G2, n=4), and 0.75 mg PLGA microparticles (G3, n=4); the microparticles were dissolved in a sterile vehicle solution. The intercalar and striated ducts lumen, the thickness of the acini and the histology aspect in terms of the parenchyma organization, cell morphology of acini and duct system, the presence of polymeric residues, and inflammatory response were determined at 14 days post-injection. The administration of the compound in a single dose modified some of the morphometric parameters of parenchyma (intercalar duct lumen and thickness of the glandular acini) but did not induce tissue inflammatory response, despite the visible presence of polymer waste. This suggests that PLGA microparticles are biocompatible with the parotid tissue, making it possible to use intraglandular controlled drug administration.     

Polyethylene glycol as an alternative polymer solvent for nanoparticle preparation

Solvent toxicity is one of the major drawbacks in the preparation of polymeric nanoparticles today. Here, polyethylene glycols (PEGs) are proposed as non-toxic solvents for the preparation of polymeric nanoparticles. Based on a preparation process similar to the solvent displacement technique, several process parameters were examined for their effects on the properties of the prepared nanoparticles by this method to achieve the optimum preparation conditions. The investigated parameters included polymer type and concentration, volume and temperature of the dispersing phase, methods of dispersing the solvent phase into the non-solvent phase, duration and speed of stirring and washing by dialysis. Ammonio methacrylate copolymer (Eudragit RL), poly-lactide-co-glycolide (PLGA), and PEG-PLGA were found to be successful polymer candidates for the preparation of nanoparticles by this method. Nanoparticles with diameters ranging from 80 to 400 nm can be obtained. The encapsulation efficiencies of bovine serum albumin, and lysozyme as model proteins were ranging from 7.3 ± 2.2% to 69.3 ± 1.8% depending on the strength of polymer–protein interaction. Biological assays confirmed a full lysozyme activity after the preparation process. PEG proved to be a suitable non-toxic solvent for the preparation of polymeric protein-loaded nanoparticles, maintaining the integrity of protein.