The Effect of Polybutylcyanoacrylate Nanoparticles as a Protos Delivery Vehicle on Dental Bone Formation.

BACKGROUND: Dental implants are commonly used for missing teeth, for which success depends heavily on the quality of the alveolar bone. The creation of an ideal implant site is a key component in shortening the treatment time, which remains clinically challenging. Strontium ranelate (Protos) is an anti-osteoporotic agent which has previously been used to promote bone formation, however the systemic use of Protos has been linked to serious cardiovascular and venous thromboembolic events, thus local delivery strategies may be better suited for this purpose. In this study, a biodegradable, and biocompatible nanocarrier "polybutylcyanoacrylate" (PBCA) loaded with strontium was constructed and its ability to promote bone formation was assessed. METHODOLOGY: PBCA nanoparticles loaded with strontium (PBCA-Sr NPs) were synthesized using the emulsion polymerization method, and their physical properties (zeta potential, size and shape) and entrapment efficiency were characterized. Committed MSCs (osteoblasts) were derived from the differentiation of cultured rat mesenchymal stem cells (MSC), which were tested with the PBCA-Sr NPs for cytotoxicity, inflammatory response, bone formation and mineralization. Scanning electron microscopy was performed following a 7-day treatment of PBCA-Sr NPs on decellularized procaine mandibular bone blocks grafted with osteoblasts. RESULTS: Spherical PBCA-Sr NPs of 166.7 ± 2.3 nm, zeta potential of -1.15 ± 0.28 mV with a strontium loading efficiency of 90.04 ± 3.27% were constructed. The presence of strontium was confirmed by energy-dispersive X-ray spectroscopy. Rat committed MSCs incubated in PBCA-Sr NPs for 24 hrs showed viabilities in excess of 90% for concentrations of up to 250 ug/mL, the cellular expression of osteocalcin and alkaline phosphatase were 1.4 and 1.3 times higher than the untreated control, and significantly higher than those treated with strontium alone. Bone formation was evident following osteoblast engraftment on the decellularized procaine mandibular bone block with PBCA-Sr NPs, which appeared superior to those treated with strontium alone. CONCLUSION: Treatment of committed MSCs with PBCA-Sr NPs showed higher expression of markers of bone formation when compared with strontium alone and which corresponded to greater degree of bone formation observed on the 3-dimensinal decellularized procaine mandibular bone block. Further quantitative analysis on the extent of new bone formation is warranted.

Characterization and Optimization of Chitosan-Coated Polybutylcyanoacrylate Nanoparticles for the Transfection-Guided Neural Differentiation of Mouse Induced Pluripotent Stem Cells.

Gene transfection is a valuable tool for analyzing gene regulation and function, and providing an avenue for the genetic engineering of cells for therapeutic purposes. Though efficient, the potential concerns over viral vectors for gene transfection has led to research in non-viral alternatives. Cationic polyplexes such as those synthesized from chitosan offer distinct advantages such as enhanced polyplex stability, cellular uptake, endo-lysosomal escape, and release, but are limited by the poor solubility and viscosity of chitosan. In this study, the easily synthesized biocompatible and biodegradable polymeric polysorbate 80 polybutylcyanoacrylate nanoparticles (PS80 PBCA NP) are utilized as the backbone for surface modification with chitosan, in order to address the synthetic issues faced when using chitosan alone as a carrier. Plasmid DNA (pDNA) containing the brain-derived neurotrophic factor (BDNF) gene coupled to a hypoxia-responsive element and the cytomegalovirus promotor gene was selected as the genetic cargo for the in vitro transfection-guided neural-lineage specification of mouse induced pluripotent stem cells (iPSCs), which were assessed by immunofluorescence staining. The chitosan-coated PS80 PBCA NP/BDNF pDNA polyplex measured 163.8 ± 1.8 nm and zeta potential measured -34.8 ± 1.8 mV with 0.01% (w/v) high molecular weight chitosan (HMWC); the pDNA loading efficiency reached 90% at a nanoparticle to pDNA weight ratio of 15, which also corresponded to enhanced polyplex stability on the DNA stability assay. The HMWC-PS80 PBCA NP/BDNF pDNA polyplex was non-toxic to mouse iPSCs for up to 80 µg/mL (weight ratio = 40) and enhanced the expression of BDNF when compared with PS80 PBCA NP/BDNF pDNA polyplex. Evidence for neural-lineage specification of mouse iPSCs was observed by an increased expression of nestin, neurofilament heavy polypeptide, and beta III tubulin, and the effects appeared superior when transfection was performed with the chitosan-coated formulation. This study illustrates the versatility of the PS80 PBCA NP and that surface decoration with chitosan enabled this delivery platform to be used for the transfection-guided differentiation of mouse iPSCs.

Valproic acid-induced amphiregulin secretion confers resistance to temozolomide treatment in human glioma cells.

BACKGROUND: Glioblastoma multiforme (GBM) is the most severe type of primary brain tumor with a high mortality rate. Although extensive treatments for GBM, including resection, irradiation, chemotherapy and immunotherapy, have been tried, the prognosis is still poor. Temozolomide (TMZ), an alkylating agent, is a front-line chemotherapeutic drug for the clinical treatment of GBM; however, its effects are very limited because of the chemoresistance. Valproic acid (VPA), an antiepileptic agent with histone deacetylase inhibitor activity, has been shown to have synergistic effects with TMZ against GBM. The mechanism of action of VPA on TMZ combination therapy is still unclear. Accumulating evidence has shown that secreted proteins are responsible for the cross talking among cells in the tumor microenvironment, which may play a critical role in the regulation of drug responses. METHODS: To understand the effect of VPA on secreted proteins in GBM cells, we first used the antibody array to analyze the cell culture supernatant from VPA-treated and untreated GBM cells. The results were further confirmed by lentivirus-mediated knockdown and exogenous recombinant administration. RESULTS: Our results showed that amphiregulin (AR) was highly secreted in VPA-treated cells. Knockdown of AR can sensitize GBM cells to TMZ. Furthermore, pretreatment of exogenous recombinant AR significantly increased EGFR activation and conferred resistance to TMZ. To further verify the effect of AR on TMZ resistance, cells pre-treated with AR neutralizing antibody markedly increased sensitivity to TMZ. In addition, we also observed that the expression of AR was positively correlated with the resistance of TMZ in different GBM cell lines. CONCLUSIONS: The present study aimed to identify the secreted proteins that contribute to the modulation of drug response. Understanding the full set of secreted proteins present in glial cells might help reveal potential therapeutic opportunities. The results indicated that AR may potentially serve as biomarker and therapeutic approach for chemotherapy regimens in GBM.

Comparison between Polybutylcyanoacrylate Nanoparticles with Either Surface-Adsorbed or Encapsulated Brain-Derived Neurotrophic Factor on the Neural Differentiation of iPSCs.

The brain-derived neurotrophic factor (BDNF) is vital in the neural differentiation of neural stem/progenitor cells, and together may have therapeutic potential for neural regeneration. In this study, a multiplexed polybutylcyanoacrylate nanoparticle (PBCA NP) delivery platform was constructed, incorporating either surface-adsorbed or encapsulated BDNF for the induction of neural differentiation in induced pleuripotent stem cells (iPSCs), where tween 80 (T80) and superparamagnetic iron oxide (SPIO) were added for central nervous system (CNS) targeting and magnetic resonance (MR) image tracking, respectively. Both methods by which the BDNF was carried resulted in loading efficiencies greater than 95%. The nanoparticle-mediated delivery of BDNF resulted in neural differentiation of iPSCs detected on immunofluorescence staining as early as 7 days, with enhanced differentiation efficiency by 1.3-fold compared to the control on flow cytometry; the delivery system of surface-adsorbed BDNF gave rise to cells that had the best neural development than the encapsulated formulation. T80-coating disrupted the in vitro blood⁻brain barrier model with a corresponding 1.5- to two-fold increase in permeability. SPIO-loaded PBCA NPs exhibited a concentration-dependent, rapid decay in signal intensity on the phantom MR experiment. This study demonstrates the versatility of the PBCA NP, and the surface-adsorption of BDNF is the preferred method of delivery for the differentiation of iPSCs.

Brain-Derived Neurotrophic Factor Loaded PS80 PBCA Nanocarrier for In Vitro Neural Differentiation of Mouse Induced Pluripotent Stem Cells.

Brain derived neurotrophic factor (BDNF) can induce neural differentiation in stem cells and has the potential for repair of the nervous system. In this study, a polysorbate 80-coated polybutylcyanoacrylate nanocarrier (PS80 PBCA NC) was constructed to deliver plasmid DNAs (pDNAs) containing BDNF gene attached to a hypoxia-responsive element (HRE-cmvBDNF). The hypoxia-sensing mechanism of BDNF expression and inductiveness of the nano-formulation on mouse induced pluripotent stem cells (iPSCs) to differentiate into neurons following hypoxia was tested in vitro with immunofluorescent staining and Western blotting. The HRE-cmvBDNF appeared to adsorb onto the surface of PS80 PBCA NC, with a resultant mean diameter of 92.6 ± 1.0 nm and zeta potential of -14.1 ± 1.1 mV. HIF-1α level in iPSCs was significantly higher in hypoxia, which resulted in a 51% greater BDNF expression when transfected with PS80 PBCA NC/HRE-cmvBDNF than those without hypoxia. TrkB and phospho-Akt were also elevated which correlated with neural differentiation. The findings suggest that PS80 PBCA NC too can be endocytosed to serve as an efficient vector for genes coupled to the HRE in hypoxia-sensitive cells, and activation of the PI3/Akt pathway in iPSCs by BDNF is capable of neural lineage specification.

Dexamethasone reduces brain cell apoptosis and inhibits inflammatory response in rats with intracerebral hemorrhage.

Spontaneous intracerebral hemorrhage (ICH) is associated with high rates of mortality and morbidity. Thus, the identification of novel therapeutic agents for preventing strokes and attenuating poststroke brain damage is crucial. Dexamethasone (DEX) is used clinically to reduce edema formation in patients with spinal cord injury and brain tumors. In this study, we sought to elucidate the effects of DEX treatment on apoptosis and inflammation following ICH in rats. A high dose of DEX (15 mg/kg) was administered immediately following ICH induction and again 3 days later. The inflammatory and apoptotic responses in the rat brains were evaluated by using hematoxylin-eosin, terminal deoxynucleotidyl transferase dUTP nick end labeling, Nissl, and neurofilament-H staining. Levels of phosphorylated neurofilaments and apoptosis-related proteins such as B-cell lymphoma 2 (Bcl-2), Bcl-2 associated X protein (Bax), caspase-3, and P53 were analyzed by Western blotting. This study shows that rats without ICH that received DEX treatment had a fourfold higher expression of Bcl-2 than sham-operated rats. ICH causes an increase in Bax, cleaved caspase-3, and P53 proteins from 4 hr to 7 days following ICH induction. In comparison with the ICH rats, the ICH/DEX rats showed significantly decreased apoptotic cell death and increased neuron survival and maintained neurofilament integrity in the perihematomal region. DEX increased the Bcl-2/Bax ratio and lowered the expression of cleaved caspase-3 at 12 hr and 5 days. The ICH rats were accompanied by activation of the inflammatory response, and DEX treatment modulated the expression of a variety of cell types and then decreased ICH-induced apoptosis.

Chronic cigarette smoke exposure enhances brain-derived neurotrophic factor expression in rats with traumatic brain injury.

The involvement of brain-derived neurotrophic factor (BDNF) in regulating neuronal survival during neuron differentiation, growth, and maturation, and during the regeneration of injured nerve cells, has already been documented. In experimental Parkinson's disease, chronic exposure to cigarette smoke increased BDNF levels and survival of dopaminergic neurons. BDNF is also elevated in traumatic brain injury (TBI), where it is potentially involved in post-injury repair and regeneration. The aim of this study was to investigate the effects of chronic exposure to cigarette smoke on BDNF expression and apoptosis in rats with TBI. Three groups of rats were compared: rats with TBI after chronic exposure to cigarette smoke, rats with TBI and no exposure to cigarette smoke, and sham-operated rats. BDNF mRNA expression in the hippocampus increased from 2 to 24 h after TBI, and chronic exposure to cigarette smoke upregulated TBI-induced BDNF mRNA elevation at 0, 2, 4, 12, and 24 h after head injury. The BDNF protein levels generally corresponded to the mRNA levels in the hippocampal region. Compared to the TBI group without smoke exposure, chronic cigarette smoke exposure in rats inhibited the decrease of the Bcl-2/Bax ratio and reduced P53 expression and apoptosis 24 h after TBI. In addition, neuronal damage in the parietal and cingulate cortex 7 days after TBI was less extensive in rats exposed to cigarette smoke. In conclusion, although chronic exposure to cigarette smoke is a risk factor for myocardial and pulmonary disease, cigarette smoke exposure increases BDNF expression after TBI and thereby can play a neuroprotective role.

Dexamethasone inhibits ICAM-1 and MMP-9 expression and reduces brain edema in intracerebral hemorrhagic rats.

BACKGROUND: The molecular mechanism of hemorrhagic stroke is unclear, and the identification of therapeutic agents for attenuating post-stroke brain damage remains an unresolved challenge. Dexamethasone (DEX) is used clinically to treat spinal cord injury and brain tumor patients by reducing edema formation, but has produced conflicting results in stroke management. METHODS: In this study, intracerebral hemorrhage (ICH) was induced in rats by intracranial stereotactic injection of collagenase into the caudate nucleus. DEX was given immediately and 3 days after ICH. The expression of intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase-9 (MMP-9), nuclear factor (NF)-κB, and IκB were analyzed by Western blotting, and perihematomal edema formation was evaluated by magnetic resonance imaging. RESULTS: The results showed that ICH caused an increase of ICAM-1 and MMP-9 expression from 4 h to 7 days, which was inhibited following the administration of DEX. The perihematomal edema volume in ICH rats was high, with two peak periods at 12 h and 3 days, which was also reduced in DEX-treated groups. Furthermore, the administration of DEX not only maintained IκB in cytoplasm, but also decreased NF-κB elevation in the nucleus at 3 and 5 days in ICH rats. CONCLUSIONS: In conclusion, these data show that DEX successfully reduced post-stroke brain edema by decreasing MMP-9 and ICAM-1 levels, partially through the IκB/NF-κB signaling pathway. The timing of DEX administration in relation to the onset of brain injury may be critical.

Induced pluripotent stem cells can improve thrombolytic effect of low-dose rt-PA after acute carotid thrombosis in rat.

BACKGROUND: Intravenous thrombolysis using recombinant tissue plasminogen activator (rt-PA) is the standard treatment for acute ischemic stroke. Standard-dose rt-PA (0.9 mg/kg) is known to achieve good recanalization but carries a high bleeding risk. Lower dose of rt-PA has less bleeding risk but carries a high re-occlusion rate. We investigate if induced pluripotent stem cells (iPSCs) can improve the thrombolytic effect of low-dose rt-PA (0.45 mg/kg). METHODS: Single irradiation with 6 mW/cm2 light-emitting diode (LED) for 4 h at rat common carotid artery was used as thrombosis model according to our previous report. Endothelin-1 (ET-1), intercellular adhesion molecule-1 (ICAM-1), and interleukin 1 beta (IL-1 beta) were used as the inflammatory markers for artery endothelial injury. Angiopoietin-2 (AP-2), brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) were examined in artery wall and iPSCs culture. Animal ultrasound was used to evaluate the stenosis degree of common carotid artery before and at 2 h, 24 h, 4 days and 7 days after LED irradiation. RESULTS: After LED irradiation alone, there was a persistent occlusion from 2 h to 7 days. Standard-dose rt-PA alone could recanalize the occluded artery from 24 h to 7 days to stenotic degree ≤ 50%. Low-dose rt-PA or 1 × 106 mouse iPSCs alone could not recanalize the occluded arteries from 2 h to 7 days. Combination use of low-dose rt-PA plus 1 × 106 mouse iPSCs caused better recanalization from 24 h to 7 days. ET-1, ICAM-1 and IL-1 beta were strongly expressed after LED irradiation but reduced after iPSCs treatment. AP-2, BDNF and VEGF were rarely induced after LED irradiation but strongly expressed after iPSCs treatment. In vitro study showed iPSCs could express AP-2, BDNF and VEGF. CONCLUSION: The adjuvant use of iPSCs may help improving the thrombolytic effect of low-dose rt-PA by suppressing inflammatory factors and inducing angiogenic trophic factors. Stem cells could be a potential regimen in acute thrombolytic therapy to improve recanalization and reduce complications.

Photochemical Internalization of Etoposide Using Dendrimer Nanospheres Loaded with Etoposide and Protoporphyrin IX on a Glioblastoma Cell Line.

Glioblastoma multiforme (GBM) is the most common malignant primary neoplasm of the adult central nervous system originating from glial cells. The prognosis of those affected by GBM has remained poor despite advances in surgery, chemotherapy, and radiotherapy. Photochemical internalization (PCI) is a release mechanism of endocytosed therapeutics into the cytoplasm, which relies on the membrane disruptive effect of light-activated photosensitizers. In this study, phototherapy by PCI was performed on a human GBM cell-line using the topoisomerase II inhibitor etoposide (Etop) and the photosensitizer protoporphyrin IX (PpIX) loaded in nanospheres (Ns) made from generation-5 polyamidoamine dendrimers (PAMAM(G5)). The resultant formulation, Etop/PpIX-PAMAM(G5) Ns, measured 217.4 ± 2.9 nm in diameter and 40.5 ± 1.3 mV in charge. Confocal microscopy demonstrated PpIX fluorescence within the endo-lysosomal compartment, and an almost twofold increase in cellular uptake compared to free PpIX by flow cytometry. Phototherapy with 3 min and 5 min light illumination resulted in a greater extent of synergism than with co-administered Etop and PpIX; notably, antagonism was observed without light illumination. Mechanistically, significant increases in oxidative stress and apoptosis were observed with Etop/PpIX-PAMAM(G5) Ns upon 5 min of light illumination in comparison to treatment with either of the agents alone. In conclusion, simultaneous delivery and endo-lysosomal co-localization of Etop and PpIX by PAMAM(G5) Ns leads to a synergistic effect by phototherapy; in addition, the finding of antagonism without light illumination can be advantageous in lowering the dark toxicity and improving photo-selectivity.

Cigarette smoking decreases neurotrophin-3 expression in rat hippocampus after transient forebrain ischemia.

Stroke is a common cause of death and severe disability among adults in developed countries. Cigarette smoking adversely affects human health in many ways and is considered to be a risk factor for a stroke. However, the mechanism that determines the relative importance of neurotrophins in this process remains unclear. To study the effect of chronic cigarette smoking on ischemic stroke, in situ hybridization and immunohistochemistry were employed to detect the mRNA and protein expression of neurotrophin-3 (NT-3), respectively, which is thought to play a critical role in protection against neuronal death in brain ischemia. Rats, with or without chronic cigarette smoking, were subjected to 20 min of transient forebrain ischemia. Distribution and quantification of mRNA and protein of NT-3 in the whole hippocampus and the cell death in the hippocampal CA1-CA3 regions were determined in these rats. Experimental results show that chronic cigarette smoking produces a significantly delay and persistent down-regulation of ischemia-induced NT-3 mRNA and protein changes at 6-24h post-ischemia, and seemingly increases neuron death 7 days after reperfusion. These experimental results indicate that by influencing NT-3 expression, directly or indirectly, chronic cigarette smoking has a potentially harmful effect when acute brain ischemia attacks.

Correction: Evaluation of a novel biodegradable thermosensitive keto-hydrogel for improving postoperative pain in a rat model.

[This corrects the article DOI: 10.1371/journal.pone.0186784.].

Propyl Gallate Exerts an Antimigration Effect on Temozolomide-Treated Malignant Glioma Cells through Inhibition of ROS and the NF-κB Pathway.

In this study, we demonstrated that temozolomide (TMZ) and propyl gallate (PG) combination enhanced the inhibition of migration in human U87MG glioma cells. PG inhibited the TMZ-induced reactive oxygen species (ROS) generation. The mitochondrial complex III and NADPH oxidase are two critical sites that can be considered to regulate antimigration in TMZ-treated U87MG cells. PG can enhance the antimigration effect of TMZ through suppression of metalloproteinase-2 and metalloproteinase-9 activities, ROS generation, and the NF-κB pathway and possibly provide a novel prospective strategy for treating malignant glioma.

Evaluation of a novel biodegradable thermosensitive keto-hydrogel for improving postoperative pain in a rat model.

This study evaluates the sustained analgesic effect of ketorolac-eluting thermosensitive biodegradable hydrogel in the plantar incisional pain model of the rat hind-paw. A ketorolac-embedded 2, 2'-Bis (2-oxazolin) (BOX) linking methoxy-poly(ethylene glycol) and poly(lactide-co-glycolide) (mPEG-PLGA) diblock copolymer (BOX copolymer) was synthesized as keto-hydrogel based on optimal sol-gel phase transition and in vitro drug release profile. The effect of keto-hydrogel on postoperative pain (POP) was assessed using the established plantar incisional pain model in hind-paw of rats and compared to that of ketorolac solution. Pain and sensory threshold, as well as pain scoring, were evaluated with behavioral tests by means of anesthesiometer and incapacitance apparatus, respectively. Pro-inflammatory cytokine levels (TNF-α, IL-6, VEGF, and IL-1ß) around incisional wounds were measured by ELISA. Tissue histology was assessed using hematoxylin and eosin and Masson's trichrome staining. Ten mg/mL (25 wt%) keto-hydrogel showed a sol-gel transition at 26.4°C with a 10-day sustained drug release profile in vitro. Compared to ketorolac solution group, the concentration of ketorolac in tissue fluid was higher in the keto-hydrogel group during the first 18 h of application. Keto-hydrogel elevated pain and sensory threshold, increased weight-bearing capacity, and significantly reduced the levels of TNF-α, IL-6, and IL-1ß while enhanced VEGF in tissue fluid. Histologic analysis reveals greater epithelialization and collagen deposition around wound treated with keto-hydrogel. In conclusion, our study suggests that keto-hydrogel is an ideal compound to treat POP with a secondary gain of improved incisional wound healing.

Application of bovine pituitary extract and polyglycolide/poly(lactide-co-glycolide) scaffold to the cultivation of bovine knee chondrocytes.

In vitro cultivation of primary bovine knee chondrocytes (BKCs), using bovine pituitary extract (BPE) and porous scaffolds composed of polyglycolide (PGA) and 85/15 poly(lactide-co-glycolide) (PLGA), was investigated. Here, BPE was prepared from fresh bovine pituitaries, and cylindrical PGA/PLGA scaffolds with various chemical compositions were fabricated by solvent merging/particulate leaching method. Experimental results showed that in microcarrier systems, the rate of BKC growth on PGA surfaces is faster than that on PLGA surfaces, and the decrease in the medium pH value of BKCs-adsorbed PGA particles is faster than that of BKCs-adsorbed PLGA particles. After 28-day construct cultivation, the BKC amount and the content of glycosaminoglycans and collagen per construct increased with BPE protein concentration. For a constant BPE protein concentration, a higher PGA percentage in scaffold leads to a better biological environment for the growth of BKCs and the synthesis of extracellar matrices.

Polybutylcyanoacrylate nanoparticle-mediated neurotrophin-3 gene delivery for differentiating iPS cells into neurons.

Guided neuronal differentiation of induced pluripotent stem cells (iPSCs) with genetic regulation is an important issue in biomedical research and in clinical practice for nervous regeneration and repair. To enhance the intracellular delivery of plasmid DNA (pDNA), polybutylcyanoacrylate (PBCA) nanoparticles (NPs) were employed to mediate the transport of neurotrophin-3 (NT-3) into iPSCs. The ability of iPSCs to differentiate into neuronal lineages was shown by immunofluorescent staining, western blotting, and flow cytometry. By transmission electron microscopy, we found that PBCA NPs could efficiently grasp pDNA, thereby increasing the particle size and conferring a negative surface charge. In addition, the treatments with PBCA NP/NT-3 complexes enhanced the expression of NT-3, TrkC, NH-H, NSE, and PSD95 by differentiating iPSCs. Neurons produced from iPSCs were incapable of returning to pluripotency, demonstrating with a series of differentiation scheme for adipogenesis and osteogenesis. The pretreatment with PBCA NP/NT-3 complexes can be one of critical biotechnologies and effective delivery systems in gene transfection to accelerate the differentiation of iPSCs into neurons.

Polybutylcyanoacrylate nanoparticles for delivering hormone response element-conjugated neurotrophin-3 to the brain of intracerebral hemorrhagic rats.

Hypertensive intracerebral hemorrhage (ICH) is a rapidly evolutional pathology, inducing necrotic cell death followed by apoptosis, and alters gene expression levels in surrounding tissue of an injured brain. For ICH therapy by controlled gene release, the development of intravenously administrable delivery vectors to promote the penetration across the blood-brain barrier (BBB) is a critical challenge. To enhance transfer efficiency of genetic materials under hypoxic conditions, polybutylcyanoacrylate (PBCA) nanoparticles (NPs) were used to mediate the intracellular transport of plasmid neurotrophin-3 (NT-3) containing hormone response element (HRE) with a cytomegalovirus (cmv) promoter and to differentiate induced pluripotent stem cells (iPSCs). The differentiation ability of iPSCs to neurons was justified by various immunological stains for protein fluorescence. The effect of PBCA NP/cmvNT-3-HRE complexes on treating ICH rats was studied by immunostaining, western blotting and Nissl staining. We found that the treatments with PBCA NP/cmvNT-3-HRE complexes increased the capability of differentiating iPSCs to express NT-3, TrkC and MAP-2. Moreover, PBCA NPs could protect cmvNT-3-HRE against degradation with EcoRI/PstI and DNase I in vitro and raise the delivery across the BBB in vivo. The administration of PBCA NP/cmvNT-3-HRE complexes increased the expression of NT-3, inhibited the expression of apoptosis-inducing factor, cleaved caspase-3 and DNA fragmentation, and reduced the cell death rate after ICH in vivo. PBCA NPs are demonstrated as an appropriate delivery system for carrying cmvNT-3-HRE to the brain for ICH therapy.

Transcytosis of CRM197-grafted polybutylcyanoacrylate nanoparticles for delivering zidovudine across human brain-microvascular endothelial cells.

This study investigates the capability of CRM197-grafted polybutylcyanoacrylate (PBCA) nanoparticles (NPs) (CRM197/PBCA NPs) to carry zidovudine (AZT) across the blood-brain barrier (BBB). AZT was loaded on CRM197/PBCA NPs to traverse the monolayer of human brain-microvascular endothelial cells (HBMECs) regulated by human astrocytes. The particle size distribution of AZT-loaded CRM197/PBCA NPs was quite uniform. In addition, AZT-loaded CRM197/PBCA NPs displayed a spherical shape with slightly fluffy exterior. The deposited thin film of AZT-loaded CRM197/PBCA NPs exhibited a hexagonal lattice-like geometry. When the diameter of AZT-loaded CRM197/PBCA NPs decreased, the loading efficiency of AZT on the drug carriers and the permeability coefficient of AZT across the BBB enhanced. An increase in the grafting quantity of CRM197 enhanced the permeability coefficient of AZT across the BBB and the uptake quantity of AZT-loaded CRM197/PBCA NPs by HBMECs. CRM197/PBCA NPs can be promising brain-targeting carriers for delivering AZT across the BBB.

TATVHL peptide-grafted alginate/poly(γ-glutamic acid) scaffolds with inverted colloidal crystal topology for neuronal differentiation of iPS cells.

The neuronal differentiation of induced pluripotent stem (iPS) cells in scaffolding biomaterials is an emerging issue in nervous regeneration and repair. This study presents the production of neuron-lineage cells from iPS cells in inverted colloidal crystal (ICC) scaffolds comprising alginate, poly(γ-glutamic acid) (γ-PGA), and TATVHL peptide. The ability of iPS cells to differentiate toward neurons in the constructs was demonstrated by flow-cytometeric sorting and immunochemical staining. The results revealed that hexagonally arrayed microspheres molded alginate/γ-PGA hydrogel into ICC topology with adequate interconnected pores. An increase in the quantity of surface TATVHL peptide enhanced the atomic ratio of nitrogen and the adhesion efficiency of iPS cells in constructs. However, the effect of TATVHL peptide on the viability of iPS cells was insignificant. The adhesion and viability of iPS cells in ICC constructs was higher than those in freeform ones. TATVHL peptide raised the percentage of ß III tubulin-identified cells differentiating from iPS cells, indicating that TATVHL peptide stimulated the neuronal development in alginate/γ-PGA ICC constructs. TATVHL peptide-grafted alginate/γ-PGA ICC scaffolds can be promising for establishing nerve tissue from iPS cells.

Chondrogenesis in scaffolds with surface modification of elastin and poly-L-lysine.

A tissue engineering cartilage is of great importance in the current diarthrodial surgery. This study presents the formation of neocartilage by cultivating chondrocytes in elastin- and poly-L-lysine-modified scaffolds. The hybrid bulk biomaterials used contained polyethylene oxide, chitin, and chitosan and were fabricated by crosslinking, pre-freezing, and lyophilization. Bovine knee chondrocytes were seeded in the scaffolds and cultured in a spinner-flask bioreactor over 4 weeks. Surface elastin showed a better efficiency in the adhesion and proliferation of bovine knee chondrocytes in the scaffolds than surface poly-L-lysine. In addition, elastin-modified constructs yielded higher quantities of secreted glycosaminoglycans and produced collagen than poly-L-lysine-modified constructs. The surface morphology demonstrated a thriving chondrogenesis in the two kinds of constructs. The staining images revealed that elastin induced larger amounts of regenerated bovine knee chondrocytes, glycosaminoglycans, and type II collagen in the constructs than poly-L-lysine. Elastin- and poly-L-lysine-grafted polyethylene oxide/chitin/chitosan scaffolds are effective in producing cartilaginous components.