Glioma homing peptide-modified PEG-PCL nanoparticles for enhanced anti-glioma therapy

Background: Peptide-mediated drug delivery system (DDS) has been increasingly used to promote on-demand treatment efficacy of cancers. Herein, LTLRWVGLMS (LS10) peptide is selected as the functional ligand for specific glioma-targeting drug delivery. LS10 peptide selectively binds to NG2 proteins that are widely overexpressed in the glioma cells and restricted in normal tissue. LS10 peptide-decorated DDS is expected to hold vast promises in glioma therapy and decrease unwanted side effects.

Methods: LS10 peptide was conjugated on the surface of poly(ethylene glycol)-poly(?-caprolactone) (PEG-PCL) nanoparticles via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride/N-hydroxysulfosuccinimide coupling reaction. Using U87MG cells as the glioma cell model, cellular uptake, internalization mechanism, cellular cytotoxicity and apoptosis were investigated. 1,1′-Dioctadecyl-3,3,3′,3′-tetramethyl indotricarbocyanine iodide were used as fluorescence probes to investigated in vivo glioma targeting capability of LS10-NP. The glioma therapeutic efficacy of paclitaxel-loaded LS10-NP was studied on glioma-bearing nude mice.

Results: The LS10-NP with size of 119?nm enhanced cellular uptake on U87MG cells, increased cytotoxicity of the loaded paclitaxel (PTX), and improved penetration in 3D U87MG glioma spheres. In vivo biodistribution experiments showed that LS10-NP exhibited the enhanced drug localization at glioma site, which resulted in prolonged survival time of glioma-bearing mice.

Conclusion: Our results indicated that LS10 peptide-modified nanoparticulate DDS could significantly improve the anti-glioma efficacy.     

Pep-1&borneol–Bifunctionalized Carmustine-Loaded Micelles Enhance Anti-Glioma Efficacy Through Tumor-Targeting and BBB-Penetrating

Tumor-targeting and blood-brain barrier (BBB)-penetrating are highly desirable for the treatment of glioma. In this study, we developed Pep-1&borneol–bifunctionalized carmustine-loaded micelles (Pep-1/Bor/CMS-M) capable of targeting interleukin-13 receptor–overexpressed glioma and penetrating the brain microvascular endothelial cells–associated physiologic barriers. Pep-1/Bor/CMS-M were nearly spherical particles with a diameter of 32.6 ± 1.1 nm and zeta potential of ?21.3 ± 3.1 mV. Carmustine (CMS) released from Pep-1/Bor/CMS-M in pH 7.4 was significantly faster than in acidic environments. In human glioma BT325 cellular studies, Pep-1/Bor/CMS-M remarkably increased the cytotoxicity, notably improved the internalization, and effectively induced the cell apoptosis. Likewise, in human brain microvascular endothelial cells, Pep-1/Bor/CMS-M obviously promoted the cellular uptake, rapidly decreased the transepithelial electrical resistance, and thereby enhanced the ability of penetration. In orthotopic Luc-BT325 glioma tumor-bearing nude mouse models, the stronger fluorescence signal and longer retention were observed in brain tissues compared with other controls, after single administration of DiD-labeled Pep-1/Bor/M (DiD/Pep-1/Bor/M). Importantly, Pep-1/Bor/CMS-M displayed the strongest inhibition of tumor growth, the longest survival period, and low systemic toxicity in treating orthotopic glioma tumor-bearing nude mice. Simultaneous functionalization of Pep-1 and borneol offers a novel strategy for designing CMS-based nanomedicine and precisely treating glioma.     

Targeted gene delivery to glioblastoma using a C-end rule RGERPPR peptide-functionalised polyethylenimine complex

Safe and efficient systems capable of specifically targeting brain tumour cells represent a promising approach for the treatment glioblastoma multiforme. Neuropilin-1 (NRP-1) is over-expressed in U87 glioma cells. In the current study, the tumour specific peptide RGERPPR, which binds specifically to NRP-1, was used as a targeting ligand in a gene delivery strategy for glioblastoma. The RGERPPR peptide was coupled to branched polyethylenimine (PEI, 25 kDa) using heterobifunctional Mal–PEG–NHS, resulting in a novel gene delivery polymer. Polymer/plasmid DNA (pDNA) complexes were formed and their sizes and zeta potentials were measured. Compared with the unmodified mPEG–PEI/pDNA complexes, the RGERPPR–PEG–PEI/pDNA complex led to a significant enhancement in intracellular gene uptake and tumour spheroid penetration. Furthermore, the RGERPPR–PEG–PEI/pDNA complex facilitated enhanced transfection efficiency levels, as well as a reduction in cytotoxicity when tested in U87 glioma cells in vitro. Most significantly of all, when complexes formed with pDsRED-N1 were injected into the tail vein of intracranial U87 tumour-bearing nude mice, the RGERPPR–PEG–PEI complexes led to improved levels of red fluorescence protein expression in the brain tissue. Taken together, the results show that RGERPPR–PEG–PEI could be used as a safe and efficient gene delivery vehicle with potential applications in glioblastoma gene delivery.     

Elucidating the cellular uptake mechanisms of heptamethine cyanine dye analogues for their use as an anticancer drug-carrier molecule for the treatment of glioblastoma

The development of chemotherapies for glioblastoma is hindered by their limited bioavailability and toxicity on normal brain function. To overcome these limitations, we investigated the structure-dependent activity of heptamethine cyanine dyes (HMCD), a group of tumour-specific and BBB permeable near-infrared fluorescent dyes, in both commercial (U87MG) and patient-derived GBM cell lines. HMCD analogues with strongly ionisable sulphonic acid groups were not taken up by patient-derived GBM cells, but were taken up by the U87MG cell line. HMCD uptake relies on a combination of transporter uptake through organic anion-transporting polypeptides (OATPs) and endocytosis into GBM cells. The uptake of HMCDs was not affected by p-glycoprotein efflux in GBM cells. Finally, we demonstrate structure-dependent cytotoxic activity at high concentrations (EC₅₀: 1–100 μM), likely due to mitochondrial damage-induced apoptosis. An in vivo orthotopic glioblastoma model highlights tumour-specific accumulation of our lead HMCD, MHI-148, for up to 7 days following a single intraperitoneal injection. These studies suggest that strongly ionisable groups like sulphonic acids hamper the cellular uptake of HMCDs in patient-derived GBM cell lines, highlighting cell line-specific differences in HMCD uptake. We envisage these findings will help in the design and structural modifications of HMCDs for drug-delivery applications for glioblastoma.     

Medicarpin suppresses proliferation and triggeres apoptosis by upregulation of BID,BAX, CASP3, CASP8, and CYCS in glioblastoma

Glioblastoma (GBM) is the most malignant brain tumor and incurable. Medicarpin (MED), a flavonoid compound from the legume family, has multiple targets and anticancer properties. However, the role of MED in GBM remains unclear. The objective of this study was to explore the effects of MED on the apoptosis of GBM and to explain the potential molecular mechanisms. We found that the IC₅₀ values of U251 and U-87 MG cells treated with MED for 24 h were 271 μg/mL and 175 μg/mL, and the IC₅₀ values for 48 h were 154 μg/mL and 161 μg/mL, respectively. Additionally, the cell cycle of U251 and U-87 MG cells were arrested at the G2/M phase. Furthermore, the apoptosis rate of U251 and U-87 MG cells increased from 6.26% to 18.36% and 12.46% to 31.33% for 48 h, respectively. The migration rate of U251 and U-87 MG decreased from 20% to 5% and 25% to 15% for 12 h and these of U251 and U-87 MG decreased from 50% to 28% and 60% to 25% for 24 h. MED suppressed GBM tumorigenesis, and improved survival rate of tumor-bearing mice. Taken together, MED triggered GBM apoptosis through upregulation of pro-apoptotic proteins (BID, BAX, CASP3, CASP8, and CYCS), showed strong inhibitory effects on cell proliferation and cell migration, and displayed anti-tumor activity in nude mice.     

EGFR siRNA lipid nanocapsules efficiently transfect glioma cells in vitro

Glioma are the most common malignant tumors of the central nervous system and remain associated with poor prognosis, despite the combination of chemotherapy and radiotherapy. EGFR targeting represents an interesting strategy to treat glioma. Indeed, a high level of endothelial growth factor receptors expression (EGFR), involved in the malignancy of the tumor, has been observed in glioma. Our strategy consisted in using EGFR siRNA entrapped into lipid nanocapsules (LNCs) via cationic liposomes. In vitro analyses on U87MG human glioma cells were performed to evaluate firstly the capacity of LNCs to efficiently deliver the siRNA and secondly the effect of EGFR siRNA targeting on U87MG proliferation. Then, the complement protein consumption was evaluated by CH50 assays to verify the suitability of the siRNA LNCs for systemic administration. The EGFR siRNA LNCs exhibited an adequate size lower than 150 nm as well as a neutral surface charge. The IC50 profile together with the 63% of protein extinction demonstrated the significant action of EGFR siRNA LNCs compared to scrambled LNCs. Dose and time-dependent survival assays showed a decrease of U87MG growth evaluated at 38%. Finally, low complement consumption demonstrated the suitability of EGFR siRNA LNCs for intravenous injection. In conclusion, EGFR siRNA LNCs demonstrated their capacity to efficiently encapsulate and deliver siRNA into U87MG human glioma cells, and will therefore be usable in the future for in vivo evaluation.     

Enhanced delivery of etoposide across the blood–brain barrier to restrain brain tumor growth using melanotransferrin antibody- and tamoxifen-conjugated solid lipid nanoparticles

Melanotransferrin antibody (MA) and tamoxifen (TX) were conjugated on etoposide (ETP)-entrapped solid lipid nanoparticles (ETP-SLNs) to target the blood–brain barrier (BBB) and glioblastom multiforme (GBM). MA- and TX-conjugated ETP-SLNs (MA–TX–ETP–SLNs) were used to infiltrate the BBB comprising a monolayer of human astrocyte-regulated human brain-microvascular endothelial cells (HBMECs) and to restrain the proliferation of malignant U87MG cells. TX-grafted ETP-SLNs (TX–ETP–SLNs) significantly enhanced the BBB permeability coefficient for ETP and raised the fluorescent intensity of calcein-AM when compared with ETP-SLNs. In addition, surface MA could increase the BBB permeability coefficient for ETP about twofold. The viability of HBMECs was higher than 86%, suggesting a high biocompatibility of MA–TX–ETP-SLNs. Moreover, the efficiency in antiproliferation against U87MG cells was in the order of MA–TX–ETP-SLNs > TX–ETP-SLNs > ETP-SLNs > SLNs. The capability of MA–TX–ETP-SLNs to target HBMECs and U87MG cells during internalization was verified by immunochemical staining of expressed melanotransferrin. MA–TX–ETP-SLNs can be a potent pharmacotherapy to deliver ETP across the BBB to GBM.     

Dexamethasone reduced invasiveness of human malignant glioblastoma cells through a MAPK phosphatase-1 (MKP-1) dependent mechanism

Dexamethasone has been shown to inhibit tumor invasiveness. In the present study, the effects of dexamethasone on matrix metalloproteinases-2 (MMP-2) secretion, cell invasiveness, and intravasation in human U87MG glioma cells were examined. Dexamethasone decreased MMP-2 secretion and cell invasiveness in human glioma cells. Incubation of cells with dexamethasone increased mitogen activated protein kinase phosphatase-1 (MKP-1) expression. Ectopic expression of MKP-1 decreased cell invasiveness in vitro and intravasation in vivo. Because expression of inducible nitric oxide synthase (iNOS) has been implicated in the progression of malignant gliomas, we next investigated the possible roles of NO(-) in MMP-2 secretion and cell invasiveness in human U87MG glioma cells. Treatment of glioma cells with nitric oxide donor, sodium nitroprusside (SNP), increased MMP-2 secretion and the capacity of cell invasion in U87MG cells. Addition of dexamethasone or ectopic expression of wild-type MKP-1 suppressed the SNP-stimulated MMP-2 activation and glioma cell invasiveness in U87MG cells. Taken together, these results suggest that dexamethasone may suppress MMP-2 secretion and cell invasion through MKP-1 induction in human glioma cells.     

Targeting malignant glioma cells in vitro using platelet-derived growth factor AA-based conjugates

Glioblastoma multiforme (GBM) is an unusually aggressive brain tumor; it is also highly heterogeneous. Poor prognosis and a median survival of less than 1 year, using conventional treatment, calls for development of new treatment strategies. Overexpression and/or amplification of platelet-derived growth factor alpha receptors (PDGFαRs) in GBM might act as potential targets for a novel therapeutic approach. In this study, conjugates based on PDGFAA-ligand and dextran, of different sizes (10 and 40?kDa dextran), were prepared and investigated regarding targeting properties in vitro. Three human malignant glioma cell lines, U343MGa31L, U343MGaCl2:6, and U563MG, were used because of their previously reported differences in receptor expression and behavior. PDGFAA-based 10?kDa dextran iodine-125 radiolabeled conjugates showed the most favorable properties according to results achieved in accumulation, retention, and localization of cell-associated radioactivity. In comparison with dextran-¹²⁵I-tyrosine delivered radioactivity, the PDGFAA-based dextran conjugates confirm the potential of receptor targeting.     

Cisplatin-incorporated hyaluronic acid nanoparticles based on ion-complex formation

The aim of this study is to prepare cisplatin-incorporated nanoparticles based on ion complex formation between hyaluronic acid (HA) and cisplatin for antitumor drug delivery. To prepare nanoparticles using HA, bulk HA was degraded by hyaluronidases (HAses). Cisplatin-incorporated HA nanoparticles were prepared by mixing cisplatin with an aqueous solution of HA and then the nanoparticle solution was dialyzed to remove trace elements. Since glioma tumor cell lines are able to secrete HAse, extracts from U343MG and U87MG cell lines were used to test the release of cisplatin from the nanoparticles. The morphological observation of the cisplatin-incorporated nanoparticles showed that they had spherical shapes with a particle size around 100-200 nm. The loading efficiency of cisplatin in the nanoparticles was about 67-81% (w/w) and cisplatin was continuously released from the nanoparticles for 4 days. Especially, the release rate of cisplatin from the nanoparticles increased when HAse was added to the release medium. In the results of the HA zymography, the U343MG cell line secreted HAse, while the U87MG cell line did not. When the extracts from U343MG were added to the release medium, the release rate of cisplatin was slightly increased, while the extracts from U87MG did not significantly affect the release rate of cisplatin. In conclusion, cisplatin-incorporated nanoparticles have sufficiently small particle sizes to use as a drug targeting system. The release of cisplatin from the nanoparticles was responsive to the secretion of HAse. These nanoparticles are suitable vehicles for an antitumor drug targeting system.     

Transferrin-modified c[RGDfK]-paclitaxel loaded hybrid micelle for sequential blood-brain barrier penetration and glioma targeting therapy

The effective chemotherapy for glioblastoma multiform (GBM) requires a nanomedicine that can both penetrate the blood-brain barrier (BBB) and target the glioma cells subsequently. In this study, Transferrin (Tf) modified cyclo-[Arg-Gly-Asp-d-Phe-Lys] (c[RGDfK])-paclitaxel conjugate (RP) loaded micelle (TRPM) was prepared and evaluated for its targeting efficiency, antiglioma activity, and toxicity in vitro and in vivo. Tf modification significantly enhanced the cellular uptake of TRPM by primary brain microvascular endothelial cells (BMEC) to 2.4-fold of RP loaded micelle (RPM) through Tf receptor mediated endocytosis, resulting in a high drug accumulation in the brain after intravenous injection.The c[RGDfK] modified paclitaxel (PTX) was released from micelle subsequently and targeted to integrin overexpressed glioma cells in vitro, and showed significantly prolonged retention in glioma tumor and peritumoral tissue. Most importantly, TRPM exhibited the strongest antiglioma activity, as the mean survival time of mice bearing intracranial U-87 MG glioma treated with TRPM (42.8 days) was significantly longer than those treated with Tf modified PTX loaded micelle (TPM) (39.5 days), PTX loaded micelle (PM) (34.8 days), Taxol (33.6 days), and saline (34.5 days). Noteworthy, TRPM did not lead to body weight loss compared with saline and was less toxic than TPM. These results indicated that TRPM could be a promising nanomedicine for glioma chemotherapy.     

Nanostructured lipid carriers,solid lipid nanoparticles,and polymeric nanoparticles: which kind of drug delivery system is better for glioblastoma chemotherapy?

Context: Glioblastoma is a malignant brain tumor originating in the central nervous system. Successfully therapy of this disease required the efficient delivery of therapeutic agents to the tumor cells and tissues. Delivery of anticancer drugs using novel nanocarriers is promising in glioma treatment.

Objective: Polymeric nanoparticles (PNPs), solid lipid nanoparticles (SLNs), and nanostructured lipid carriers (NLCs) were constructed for the delivery of temozolomide (TMZ). The anti-tumor effects of the three kinds of nanocarriers were compared to provide the optimum choice for gliomatosis cerebri treatment.

Methods: TMZ-loaded PNPs (T-PNPs), SLNs (T-SLNs), and NLCs (T-NLCs) were formulated. Their particle size, zeta potential, drug encapsulation efficiency (EE), and drug loading (DL) capacity were evaluated. Anti-tumor efficacies of the three kinds of nanocarriers were evaluated on U87 malignant glioma cells (U87?MG cells) and mice-bearing malignant glioma model.

Results: T-NLCs displayed the best anti-tumor activity than other formulations in vivo and in vitro. The most significantly glioma inhibition was observed on NLCs formulations than PNPs and SLNs.

Conclusion: This work demonstrates that NLCs can deliver TMZ into U87MG cells more efficiently, with higher inhibition efficacy than PNPs and SLNs. T-NLCs could be an excellent drug delivery system for glioblastoma chemotherapy.     

64Cu-labeled lissamine rhodamine B: a promising PET radiotracer targeting tumor mitochondria

Enhanced mitochondrial potential in carcinoma cells is an important characteristic of cancer. It is of great current interest to develop a radiotracer that is sensitive to mitochondrial potential changes at the early stage of tumor growth. In this report, we present the synthesis and evaluation of (64)Cu-labeled Lissamine rhodamine B (LRB), (64)Cu(DOTA-LRB) (DOTA-LRB = 2-(6-(diethylamino)-3-(diethyliminio)-3H-xanthen-9-yl)-5-(N-(2-(2-(4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclo-dodecan-1-yl)acetamido)ethyl)sulfamoyl)benzenesulfonate) as a new radiotracer for imaging tumors in athymic nude mice bearing U87MG human glioma xenografts by positron emission tomography (PET). We also explored its localization mechanism using Cu(DOTA-LRB) as the fluorescent probe in both the U87MG human glioma cell line and the cultured primary U87MG glioma cells. It was found that (64)Cu(DOTA-LRB) had the highest tumor uptake (6.54 ± 1.50, 6.91 ± 1.26, 5.68 ± 1.13, 7.58 ± 1.96, and 5.14 ± 1.50%ID/g at 0.5, 1, 2, 4, and 24 h postinjection, respectively) among many (64)Cu-labeled organic cations evaluated in the same animal model. The cellular staining study indicated that Cu(DOTA-LRB) was able to localize in mitochondria of U87MG glioma cells due to the enhanced negative mitochondrial potential. This statement is completely supported by the results from decoupling experiment with carbonylcyanide-m-chlorophenylhydrazone (CCCP). MicroPET data showed that the U87MG glioma tumors were clearly visualized as early as 30 min postinjection with (64)Cu(DOTA-LRB). (64)Cu(DOTA-LRB) remained stable during renal excretion, but underwent extensive degradation during hepatobiliary excretion. On the basis of the results from this study, it was concluded that (64)Cu(DOTA-LRB) represents a new class of promising PET radiotracers for noninvasive imaging of the MDR-negative tumors.     

尿石素C对胶质母细胞瘤生物学行为的调控作用及机制

目的　探讨尿石素C（UC）对胶质母细胞瘤（GBM）U251和U87 MG细胞生物学行为的影响及调控机制。方法　将U251和U87 MG细胞分为0、20、40、80、120和160 μmol/L UC处理组,通过CCK-8法分别检测各组细胞24、48和72 h时增殖率。将细胞分为0、40、80和120 μmol/L UC处理组,通过细胞克隆形成实验检测细胞克隆形成能力,划痕愈合实验检测24 h和48 h时细胞迁移能力,Transwell小室侵袭实验检测24 h时细胞侵袭能力,流式细胞术检测24 h时细胞凋亡率及细胞周期,Western blot检测AMP依赖的蛋白激酶（AMPK）、细胞外调节蛋白激酶（ERK）和丝裂原活化蛋白激酶p38（p38 MAPK）蛋白表达水平和磷酸化水平。结果　与0 μmol/L组比较,U251细胞中不同浓度UC组于48 h开始增殖率均降低,而U87 MG细胞中40 μmol/L及以上浓度组于48 h时增殖率开始降低（P＜0.05）。与0 μmol/L组比较,40、80和120 μmol/L组U251和U87 MG细胞的克隆形成率、24 h和48 h时的细胞迁移能力和24 h时的细胞侵袭能力降低,且均呈浓度依赖性（P＜0.05）。与0 μmol/L组比较,120 μmol/L组U251和U87 MG细胞凋亡率均增加,40、80和120 μmol/L组U251和U87 MG细胞周期均阻滞在S期（P＜0.05）。与0 μmol/L组比较,40、80和120 μmol/L组U251和U87 MG细胞中p-AMPK和p-p38 MAPK水平均升高,80和120 μmol/L组U251细胞中p-ERK水平升高,但仅120 μmol/L组U87 MG细胞p-ERK水平升高（P＜0.05）。结论　一定浓度的UC可抑制GBM细胞增殖、迁移和侵袭,诱导肿瘤细胞凋亡和细胞周期阻滞,其机制可能与调控AMPK/ERK/p38 MAPK信号通路有关。     

Human malignant glioma cells expressing functional formylpeptide receptor recruit endothelial progenitor cells for neovascularization

Endothelial progenitor cells (EPCs) are involved in tumor neovascularization with undefined mechanisms. In this study, we explored the role of formylpeptide receptor, a G protein-coupled receptor, expressed by human malignant glioma cells in neovascularization of malignant glioma. EPCs were isolated from human umbilical cord blood and their migratory capability and tubulogenesis induced by the supernatant of U87 glioblastoma (GBM) cell line were examined. We also assessed the recruitment and incorporation of EPCs into orthotopic intracranial tumors formed by implanted U87 GBM cells. The supernatant of control U87 cells induced high levels of migration and tubule-formation in vitro by EPCs. In contrast, the chemotactic and tubule-stimulating activities on EPCs in the supernatant of U87 cells with FPR knocking down by small interference (si) RNA were significantly attenuated. In addition, the number of EPCs recruited and incorporated into intracranial glioma xenografts was significantly higher in tumors formed by control U87 cells than tumors formed by U87 cells containing FPR-siRNA. Our results suggest that expression of functional FPR in glioma cells plays an important role in regulating vasculogenesis by EPCs, which constitute a novel target for anti-angiogenic therapy in gliomas.     

Controlled release of liposome-encapsulated temozolomide for brain tumour treatment by convection-enhanced delivery

Convection-enhanced delivery (CED) is a promising technique for the delivery of drugs directly into the central nervous system (CNS) and, more specifically, the brain. CED can increase drug concentration within a brain tumour, thereby improving the therapeutic efficacy and limiting the systemic toxicity of tumoricidal agents. In this study, we evaluated a drug-liposome construct in vitro and in vivo using U87 tumour-bearing nude mice. Dipalmitoylphosphatidylcholine (DPPC)-based liposomes were designed to deliver a lipophilic temozolomide (TMZ) formulation (LipoTMZ). The LipoTMZ displayed good release of TMZ in vitro over a suitable range of time and temperatures. Encapsulating the TMZ into liposomes enhanced its tumoricidal activity against U87MG human glioma cells. The LipoTMZ also displayed good release and distribution of TMZ when delivered intracerebrally to U87MG tumour-bearing mice by CED infusion. Histological examination revealed that CED did not damage normal brain tissue. Our data indicate that CED was an effective method to deliver LipoTMZ to U87MG tumour-bearing mice, significantly inhibiting tumour growth without evidence of systemic toxicity.     

TRAIL基因修饰间充质干细胞对神经胶质瘤细胞杀伤作用研究

目的 研究肿瘤坏死因子相关的凋亡配体（TRAIL）基因修饰间充质干细胞（TRAIL-MSCs）对神经胶质瘤细胞的杀伤作用。方法 复苏冻存的脐带间充质干细胞（UC-MSCs）种子库细胞,通过慢病毒转染的方法制备TRAIL-MSCs。ELISA法检测UC-MSCs、TRAIL-MSCs细胞上清中TRAIL的含量;采用CCK-8试剂盒法检测重组TRAIL蛋白（rTRAIL,0、25、50、100、200、400 ng/mL）对U87MG、U251细胞的增殖抑制情况;实时荧光定量PCR（qRT-PCR）法检测U87MG、U251细胞中死亡受体DR4、DR5 mRNA表达水平,比较2种细胞对TRAIL的敏感性;将U87MG、U251细胞分别分为对照组、UC-MSCs培养上清（阴性对照）组、TRAIL-MSCs培养上清（含TRAIL约100 ng/mL）组和rTRAIL（100 ng/mL）组,CCK-8法检测细胞增殖抑制率;Annexin V-FITC/PI法检测细胞凋亡率;qRT-PCR法检测DR4、DR5 mRNA表达水平。结果 TRAIL-MSCs培养上清中TRAIL表达量显著高于UC-MSCs（P<0.01）;rTRAIL对U87MG细胞的半数抑制浓度（IC₅₀）为162 ng/mL,而对U251细胞的IC₅₀大于800 ng/mL,U87MG对TRAIL的敏感性更高,差异显著（P<0.01）;U87MG细胞DR4、DR5 mRNA相对表达量均显著高于U251细胞（P<0.01）;与对照组比较,TRAIL-MSCs培养上清、rTRAIL对U87MG、U251细胞均有显著增殖抑制及促进凋亡的作用（P<0.05、0.01） ,TRAIL-MSCs培养上清作用效果显著优于rTRAIL（P<0.01）;与U251相比,U87MG对TRAIL-MSCs培养上清的敏感性更强;TRAIL-MSCs培养上清处理的U87MG细胞DR4、DR5 mRNA表达量显著降低（P<0.01）。结论 TRAIL-MSCs对神经胶质瘤细胞有显著增殖抑制和杀伤作用,其功能可能与其受体DR4及DR5有关。     

Investigation on the effect of nanoparticle size on the blood–brain tumour barrier permeability by in situ perfusion via internal carotid artery in mice

The blood–brain barrier (BBB) is a limiting factor in nanoparticle drug delivery to the brain, and various attempts have been made to overcome it for efficient drug delivery. Nowadays, it was considered as further issue for brain–drug delivery that the nanoparticle delivered to brain through the BBB reach cancer cells in tumour tissue. In this study, we investigated the effect of nanoparticle size on blood–brain tumour barrier (BBTB) permeation of fluorescence-labelled gold nanoparticles (AuNPs) in a mouse model of orthotopic glioblastoma multiforme (GBM), established by intracranial implantation of luciferase-expressing human glioblastoma U87MG cells. AuNPs sized 10, 50, and 100?nm were perfused into the GBM mice via internal carotid artery (ICA) for 5?min. Immediately after perfusion, the brains were fixed and prepared for LSCM observation. The AuNPs distribution in the normal and tumorous brain tissues was analysed qualitatively and quantitatively. Higher distribution of AuNPs was observed in the tumorous tissue than in the normal tissue. Furthermore, the smallest nanoparticle, 10?nm AuNPs, was widely distributed in the brain tumour tissue, whereas the 50 and 100?nm AuNPs were located near the blood vessels. Therefore, nanoparticle size affected the permeation of nanoparticles from the blood into brain tumour tissue.     

Co-delivery of doxorubicin and siRNA for glioma therapy by a brain targeting system: angiopep-2-modified poly(lactic-co-glycolic acid) nanoparticles

It is very challenging to treat brain cancer because of the blood–brain barrier (BBB) restricting therapeutic drug or gene to access the brain. In this research project, angiopep-2 (ANG) was used as a brain-targeted peptide for preparing multifunctional ANG-modified poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), which encapsulated both doxorubicin (DOX) and epidermal growth factor receptor (EGFR) siRNA, designated as ANG/PLGA/DOX/siRNA. This system could efficiently deliver DOX and siRNA into U87MG cells leading to significant cell inhibition, apoptosis and EGFR silencing in vitro. It demonstrated that this drug system was capable of penetrating the BBB in vivo, resulting in more drugs accumulation in the brain. The animal study using the brain orthotopic U87MG glioma xenograft model indicated that the ANG-targeted co-delivery of DOX and EGFR siRNA resulted in not only the prolongation of the life span of the glioma-bearing mice but also an obvious cell apoptosis in glioma tissue.