The association of statins plus LDL receptor‐targeted liposome‐encapsulated doxorubicin increases in vitro drug delivery across blood–brain barrier cells

Statins are reported to exert cytotoxic effects against CNS tumour cells.

“Moreover statins are reported to exert cytotoxic effects against CNS tumour cells (Bababeygy et al., 2009;Yanae et al., 2011).”

• cites Bababeygy et al., 2009Not checked — no full text of the source
• cites Yanae et al., 2011 sourceSupported

By lowering de novo synthesis of cholesterol, statins force mammalian cells to expose the LDL receptor on their surface.

“By lowering the de novo synthesis of cholesterol, statins force mammalian cells to expose the LDL receptor on their surface (Liao and Laufs, 2005).”

• cites Liao and Laufs, 2005 sourcePartly supported

  The Introduction says statins inhibit endogenous cholesterol biosynthesis and increase hepatic LDL receptor activity, which supports the claim’s basic mechanism. It does not say that statins force mammalian cells generally to display LDL receptors on their surface; its receptor finding is specific to the liver, and it describes activity rather than surface exposure. (LLM, conf=0.86)

  Experimental — accuracy not yet measured

Increased production of NO elicited by statins reduces activity of Pgp/ABCB1 and BCRP/ABCG2 in hCMEC/D3 cells.

“the increased production of NO elicited by the statins reduced the activity of at least two proteins -Pgp/ABCB1 and BCRP/ABCG2 -that are involved in the apical extrusion of drugs in hCMEC/D3 cells (Tai et al., 2009) and in vivo (Declèves et al., 2006;Mercer et al., 2009;Robey et al., 2010).”

• cites Tai et al., 2009 sourceNot checked — no full text of the source
• cites Declèves et al., 2006 sourceNot checked — no full text of the source
• cites Mercer et al., 2009 sourcePartly supported

  Section 7 (page unavailable) says P-glycoprotein and other efflux pumps can prevent drugs from crossing the blood–brain barrier, supporting the general point that these proteins limit drug entry. It does not say that statin-induced nitric oxide reduces P-glycoprotein or BCRP activity in vivo; the citing sentence attributes the hCMEC/D3-cell finding to a different source. (LLM, conf=0.80)

  Experimental — accuracy not yet measured

hCMEC/D3 cells constitutively express Pgp/ABCB1, MRP1/ABCC1, and BCRP/ABCG2.

“hCMEC/D3 cells constitutively express several ABC transporters (Carl et al., 2010), like Pgp/ABCB1, MRP1/ABCC1, BCRP/ ABCG2 (Figure 2A).”

• cites Carl et al., 2010 sourceSupported

The brain microvascular endothelium has high levels of drug efflux pumps of the ABC transporter family.

“In addition, having no fenestrations and many tight junctions, the brain microvascular endothelium has high levels of drug efflux pumps of the ABC transporter family (Declèves et al., 2006;Mercer et al., 2009;Robey et al., 2010).”

• cites Declèves et al., 2006 sourceNot checked — no full text of the source
• cites Mercer et al., 2009 sourcePartly supported

  Section 7, “Challenges Associated with Targeted Therapies” (page unavailable), says that P-glycoprotein and other efflux pumps impede some drugs from crossing the blood–brain barrier. It does not specify that brain microvascular endothelial cells have high levels of these pumps, or identify the pumps collectively as ABC transporters. (LLM, conf=0.84)

  Experimental — accuracy not yet measured
• cites Robey et al., 2010 sourceSupported

The effects of pegylated liposomal doxorubicin (Doxil or Caelyx) in glioblastoma are unconvincing.

“The effects of a pegylated liposomal doxorubicin (Doxil or Caelyx) have recently been investigated in glioblastoma, but the results obtained are unconvincing (Glas et al., 2007;Beier et al., 2009;Ananda et al., 2011).”

• cites Glas et al., 2007 sourceNot checked — no full text of the source
• cites Beier et al., 2009 sourceSupported
• cites Ananda et al., 2011 sourceNot checked — no full text of the source

In the experimental conditions used, mevastatin and simvastatin did not affect the permeability to inulin.

“In the experimental conditions used in this study, mevastatin and simvastatin reduced the amount of cholesterol in the cell membrane by a lesser extent than b-methyl-cyclodextrin (Supporting information Figure S1A) and did not affect the permeability to inulin (Supporting information Figure S1B).”

The NO scavenger PTIO prevents the statin-induced increase in permeability to doxorubicin in hCMEC/D3 cells.

“The presence of the NO scavenger 2-phenyl-4,4,5,5,tetramethylimidazoline-1-oxyl 3-oxide (PTIO) prevented the increase in permeability to doxorubicin (Figure 2E)”

The synthesis of NO, measured as the amount of the stable NO-derivative nitrite in culture supernatant, was significantly increased in hCMEC/D3 cells treated with mevastatin or simvastatin.

“As a consequence of these changes, the global enzymatic activity of NOS in cell lysates and the synthesis of NO, measured as the amount of the stable NO-derivative nitrite in culture supernatant, were significantly increased in hCMEC/D3 cells treated with mevastatin or simvastatin (Figure 1F).”

• Also stated in: § Abstract · plain

  “Statins reduced the efflux activity of Pgp/ABCB1 and BCRP/ABCG2 in hCMEC/D3 cells by increasing the synthesis of NO, which elicits the nitration of critical tyrosine residues on these transporters.”

Addition of PTIO dramatically decreases the permeability of apo-Lipodox in hCMEC/D3 cells.

“Again, the addition of PTIO dramatically decreased the permeability of apo-Lipodox”

All cell lines (U87-MG, SJKNP, MDA-MB-231, A549) express at various levels Pgp/ABCB1, MRP1/ABCC1, and BCRP/ABCG2.

“A preliminary characterization of these cell lines showed that they all expressed at various levels Pgp/ABCB1, MRP1/ ABCC1 and BCRP/ABCG2 (Figure 4A)”

The association of statins plus drug-loaded nanoparticles engineered as LDLs was effective as a vehicle for non-permeant drugs like doxorubicin to cross the BBB.

“We showed that the association of statins plus drug-loaded nanoparticles engineered as LDLs was effective as a vehicle for non-permeant drugs like doxorubicin to cross the BBB, allowing its delivery into primary and metastatic brain tumour cells and to achieve significant anti-tumour cytotoxicity.”

• AssumptionRests on an assumption: Permeability measured across hCMEC/D3 cell monolayers reflects drug permeability across the blood-brain barrier in vivo.

The increase in phospho-IKKα/β was accompanied by a decrease in the inhibitory protein IκB-α in hCMEC/D3 cells.

“Such an increase was accompanied by a decrease in the inhibitory protein Ik-Ba (Figure 1C) and by the activation of NF-kB, measured as the ability of the transcription factor translocated into the nucleus to bind its specific target sequence (Figure 1D).”

Drug delivery into tumour cells is improved by pretreating BBB cells with simvastatin for 48 h followed by doxorubicin or apo-Lipodox in the last 24 h.

“the drug delivery into tumour cells was improved by pretreating BBB cells with simvastatin for 48 h followed by doxorubicin or apo-Lipodox in the last 24 h.”

The lowest concentration that significantly decreased cholesterol and GGPP level after 24 h was 0.1 mmol•L -1.

“The lowest concentration that significantly decreased cholesterol and GGPP level after 24 h was 0.1 mmol•L -1 and this was chosen for all the subsequent experiments.”

Apo-Lipodox exhibits a mean concentration of LDL receptor-targeted peptide of 20 ± 1.6 mg/mL, corresponding to about 3 × 10^12 peptides/mL.

“Apo-Lipodox exhibited a mean concentration of LDL receptor-targeted peptide of 20 Ϯ 1.6 mg•mL -1 , corresponding to about 3 ¥ 10 12 peptides•mL -1 (n = 3).”

Statins induce a persistent inhibition of Pgp/ABCB1 activity after 48 h.

“The reduction was even more pronounced after 48 h, indicating that statins induce a persistent inhibition of Pgp/ ABCB1 activity after a longer time.”

Permeability of pegylated and non-pegylated apo-Lipodox is superimposable in both untreated and simvastatin-treated hCMEC/D3 cells.

“The permeability of pegylated and nonpegylated apo-Lipodox was superimposable in both untreated and simvastatin-treated hCMEC/D3 cells (Supporting information Figure S2)”

The activity of the geranylgeranylated protein RhoA, measured as the amount of active GTP-bound protein, was reduced by both statins in hCMEC/D3 cells.

“In keeping with the decrease in GGPP, the activity of the geranylgeranylated protein RhoA, measured as the amount of active GTP-bound protein, was reduced by both statins, resulting in a lower activation of the downstream effector RhoA kinase (Figure 1B).”

In untreated hCMEC/D3 cells, permeability for apo-Lipodox is significantly greater than that of free doxorubicin.

“In untreated hCMEC/D3 cells, the permeability for apo-Lipodox was significantly greater than that of free doxorubicin”

Mevastatin and simvastatin reduced the endogenous synthesis of cholesterol and GGPP in hCMEC/D3 cells in a dose- and time-dependent manner.

“The human brain microvascular endothelial hCMEC/D3 cells were sensitive to both mevastatin and simvastatin, two lipophilic statins that reduced the endogenous synthesis of cholesterol and GGPP in a dose-and time-dependent manner (Figure 1A).”

NOS II was undetectable in untreated hCMEC/D3 cells.

“Statins increased the expression of NOS II; this was undetectable in untreated hCMEC/D3 cells (Figure 1E).”

The association of statins plus drug-loaded nanoparticles engineered as LDLs achieved significant anti-tumour cytotoxicity.

“We showed that the association of statins plus drug-loaded nanoparticles engineered as LDLs was effective as a vehicle for non-permeant drugs like doxorubicin to cross the BBB, allowing its delivery into primary and metastatic brain tumour cells and to achieve significant anti-tumour cytotoxicity.”

• AssumptionRests on an assumption: The co-culture of hCMEC/D3 cells with tumour cells in Transwell devices reflects drug delivery to brain tumours in vivo.

Statins did not induce NOS I in hCMEC/D3 cells.

“The statins did not induce NOS I and did not change the expression of NOS III, but a slight increase in the amount of the active phospho-(Ser1177)-NOS III was detected following statins treatment (Figure 1E).”

Intracellular retention of rhodamine 123 and Hoechst 33342 is increased by statins in hCMEC/D3 cells.

“the intracellular retention of rhodamine 123 (Figure 2C) that is inversely related to the activity of Pgp/ABCB1, and the intracellular retention of Hoechst 33342 (Figure 2D) that is inversely related to the activity of BCRP/ABCG2, were both increased by the statins.”

Effects of statins on Pgp/ABCB1 and BCRP/ABCG2 activity are stronger after 48 h than after 24 h.

“the effects of these changes on the activity of Pgp/ABCB1 and BCRP/ABCG2 were stronger after 48 h (Figure 2C and D).”

The transport of apo-Lipodox across the BBB monolayer is higher than that of free doxorubicin in hCMEC/D3 cells exposed to statins.

“The transport of apo-Lipodox across the BBB monolayer was even higher than that of free doxorubicin in cells exposed to statins (Figures 2E and 3D; significance of apo-Lipodox vs. doxorubicin in statin-treated cells: P < 0.001).”

Statins increased the expression of NOS II in hCMEC/D3 cells.

“Statins increased the expression of NOS II; this was undetectable in untreated hCMEC/D3 cells (Figure 1E).”

In hCMEC/D3 cells, the permeability coefficient of doxorubicin is low.

“also in hCMEC/D3 cells, the permeability coefficient was low (Figure 2E).”

• AssumptionRests on an assumption: Doxorubicin is transported by Pgp/ABCB1, MRP1/ABCC1 and BCRP/ABCG2.

The final result of treating BBB cells with statins is a dramatic increase in permeability of doxorubicin, a substrate of both Pgp/ABCB1 and BCRP/ABCG2.

“Whatever the pleiotropic effects of statins on ABC transporters are, the final result of treating BBB cells with statins was a dramatic increase in the permeability of doxorubicin, a substrate of both Pgp/ABCB1 and BCRP/ABCG2.”

Statins increased the number of LDL receptors exposed on the surface of tumour cells like human glioblastoma.

“Statins also increased the number of low-density lipoprotein (LDL) receptors exposed on the surface of BBB cells, as well as on tumour cells like human glioblastoma.”

The amount of LDL receptor protein on the cell surface is further increased by statins after 48 h in hCMEC/D3 cells.

“The amount of LDL receptor protein on the cell surface was further increased by statins after 48 h (Figure 3B).”

Mevastatin and simvastatin did not significantly change the expression of IKKα/β protein in hCMEC/D3 cells.

“In parallel, the amount of IKKa/b complex phosphorylated on serine 176/180, which was nearly undetectable in untreated hCMEC/D3 cells, was increased by mevastatin and simvastatin, without significant changes in the expression of IKKa/b protein (Figure 1C).”

For all these cell lines, the maximal increase in drug retention and LDH release is achieved by the combination of simvastatin plus apo-Lipodox.

“For all the cell lines, the maximal increase in these parameters was achieved by the combination of simvastatin plus apo-Lipodox (Figure 4C).”

In BBB cells treated with statins that inhibited RhoA kinase, phosphorylation of eNOS is increased.

“In accord with these observations, we detected an increased phosphorylation of eNOS in BBB cells treated with statins that inhibited RhoA kinase.”

In the experimental conditions used, mevastatin and simvastatin reduced the amount of cholesterol in the cell membrane by a lesser extent than β-methyl-cyclodextrin.

“In the experimental conditions used in this study, mevastatin and simvastatin reduced the amount of cholesterol in the cell membrane by a lesser extent than b-methyl-cyclodextrin (Supporting information Figure S1A) and did not affect the permeability to inulin (Supporting information Figure S1B).”

The amount of IKKα/β complex phosphorylated on serine 176/180 was increased by mevastatin and simvastatin in hCMEC/D3 cells.

“In parallel, the amount of IKKa/b complex phosphorylated on serine 176/180, which was nearly undetectable in untreated hCMEC/D3 cells, was increased by mevastatin and simvastatin, without significant changes in the expression of IKKa/b protein (Figure 1C).”

The association of statins plus drug-loaded nanoparticles engineered as LDLs allowed delivery of doxorubicin into primary and metastatic brain tumour cells.

“We showed that the association of statins plus drug-loaded nanoparticles engineered as LDLs was effective as a vehicle for non-permeant drugs like doxorubicin to cross the BBB, allowing its delivery into primary and metastatic brain tumour cells and to achieve significant anti-tumour cytotoxicity.”

• AssumptionRests on an assumption: The co-culture of hCMEC/D3 cells with tumour cells in Transwell devices reflects drug delivery to brain tumours in vivo.

Statins increased the number of LDL receptors exposed on the surface of BBB cells.

“Statins also increased the number of low-density lipoprotein (LDL) receptors exposed on the surface of BBB cells, as well as on tumour cells like human glioblastoma.”

• AssumptionRests on an assumption: The hCMEC/D3 cell line is a suitable in vitro model of the human blood-brain barrier.

The strongest fluorescent signal is achieved by the association of simvastatin (for 48 h) plus apo-Lipodox (in the last 24 h).

“The strongest fluorescent signal was achieved by the association of simvastatin (for 48 h) plus apo-Lipodox (in the last 24 h; Figure 5A).”

Co-incubation of free doxorubicin with empty liposomes, with or without LDL receptor-targeted peptide, in hCMEC/D3 cells pretreated or not with simvastatin does not change permeability compared with free doxorubicin alone.

“Furthermore, when we co-incubated free doxorubicin with empty liposomes, with or without the LDL receptor-targeted peptide, in hCMEC/D3 cells pretreated or not with simvastatin (0.1 mmol•L -1 for 48 h), we did not detect a different permeability compared with free doxorubicin alone (Supporting information Figure S3D).”

Doxorubicin alone does not exert any relevant cytotoxic effect in tumour cells, in terms of LDH release and caspase-3 activation.

“In keeping with these results, doxorubicin alone did not exert any relevant cytotoxic effect in tumour cells, in terms of LDH release (Figure 5B) and caspase-3 activation (Figure 5C).”

The up-regulation of iNOS isoform together with increased eNOS phosphorylation leads to a significant increase in NO levels.

“This event, together with the up-regulation of iNOS isoform, led to a significant increase in NO levels”

A slight increase in the amount of the active phospho-(Ser1177)-NOS III was detected following statins treatment in hCMEC/D3 cells.

“The statins did not induce NOS I and did not change the expression of NOS III, but a slight increase in the amount of the active phospho-(Ser1177)-NOS III was detected following statins treatment (Figure 1E).”

The global enzymatic activity of NOS in cell lysates was significantly increased in hCMEC/D3 cells treated with mevastatin or simvastatin.

“As a consequence of these changes, the global enzymatic activity of NOS in cell lysates and the synthesis of NO, measured as the amount of the stable NO-derivative nitrite in culture supernatant, were significantly increased in hCMEC/D3 cells treated with mevastatin or simvastatin (Figure 1F).”

Free doxorubicin co-incubated with empty liposomes, with or without LDL receptor-targeted peptide, does not accumulate within U87-MG cells more than doxorubicin alone, either in absence or presence of simvastatin.

“Free doxorubicin co-incubated with empty liposomes, with or without the LDL receptor-targeted peptide, did not accumulate within U87-MG cells more than doxorubicin alone, either in the absence or in the presence of simvastatin (Supporting information Figure S4A).”

Low levels of endogenous NO induced by statins are sufficient to elicit detectable nitration of Pgp/ABCB1, which decreases its activity.

“Such low levels of endogenous NO, however, were sufficient to elicit a detectable nitration of the transporter, which decreased its activity.”

β-methyl-cyclodextrin significantly depleted the cell membrane of cholesterol in hCMEC/D3 cells.

“The cholesterol chelator b-methyl-cyclodextrin significantly depleted the cell membrane of cholesterol (Supporting information Figure S1A) and in parallel disrupted the integrity of the tight junctions in hCMEC/D3 cells, as demonstrated by the increased permeability coefficient of inulin (Supporting information Figure S1B).”

Lipodox permeability is the same in the presence of simvastatin or of the competing peptide.

“In contrast, Lipodox permeability was the same in the presence of simvastatin or of the competing peptide (Figure 3E)”

The human brain microvascular endothelial hCMEC/D3 cells were sensitive to both mevastatin and simvastatin.

“The human brain microvascular endothelial hCMEC/D3 cells were sensitive to both mevastatin and simvastatin, two lipophilic statins that reduced the endogenous synthesis of cholesterol and GGPP in a dose-and time-dependent manner (Figure 1A).”

Statins activated NF-kB in hCMEC/D3 cells, measured as the ability of the transcription factor translocated into the nucleus to bind its specific target sequence.

“Such an increase was accompanied by a decrease in the inhibitory protein Ik-Ba (Figure 1C) and by the activation of NF-kB, measured as the ability of the transcription factor translocated into the nucleus to bind its specific target sequence (Figure 1D).”

Treatment with statins for 48 h significantly increases the permeability of doxorubicin across hCMEC/D3 cells.

“However, the treatment with statins for 48 h significantly increased the permeability of the drug.”

β-methyl-cyclodextrin disrupted the integrity of the tight junctions in hCMEC/D3 cells, as demonstrated by the increased permeability coefficient of inulin.

“The cholesterol chelator b-methyl-cyclodextrin significantly depleted the cell membrane of cholesterol (Supporting information Figure S1A) and in parallel disrupted the integrity of the tight junctions in hCMEC/D3 cells, as demonstrated by the increased permeability coefficient of inulin (Supporting information Figure S1B).”

Statins reduced the efflux activity of Pgp/ABCB1 and BCRP/ABCG2 in hCMEC/D3 cells.

“Statins reduced the efflux activity of Pgp/ABCB1 and BCRP/ABCG2 in hCMEC/D3 cells by increasing the synthesis of NO, which elicits the nitration of critical tyrosine residues on these transporters.”

• AssumptionRests on an assumption: The hCMEC/D3 cell line is a suitable in vitro model of the human blood-brain barrier.

The reduction in RhoA activity by statins resulted in a lower activation of the downstream effector RhoA kinase in hCMEC/D3 cells.

“In keeping with the decrease in GGPP, the activity of the geranylgeranylated protein RhoA, measured as the amount of active GTP-bound protein, was reduced by both statins, resulting in a lower activation of the downstream effector RhoA kinase (Figure 1B).”

Quantitative fluorimetric analysis confirms that intratumour doxorubicin content is significantly higher when simvastatin is combined with apo-Lipodox rather than with doxorubicin in all cell lines co-cultured with hCMEC/D3 cells.

“Quantitative fluorimetric analysis of intratumour doxorubicin confirmed that the drug content was significantly higher when simvastatin was combined with apo-Lipodox rather than with doxorubicin in all the cell lines co-cultured with hCMEC/D3 cells (Figure 5B).”

In hCMEC/D3 cells treated with mevastatin and simvastatin, mRNA level for LDL receptors is significantly increased after 24 h.

“In hCMEC/D3 cells treated with mevastatin and simvastatin, the mRNA level for LDL receptors was significantly increased after 24 h (Figure 3A)”

Mevastatin and simvastatin promote tyrosine nitration of Pgp/ABCB1 and BCRP/ABCG2 in hCMEC/D3 cells.

“mevastatin and simvastatin promoted a nitration on tyrosine of Pgp/ABCB1 and BCRP/ABCG2, similar to the one produced by the NO donor sodium nitroprusside (Figure 2A).”

Death of glioblastoma cells, measured as release of LDH and activation of caspase-3, is not increased by addition of free doxorubicin to empty liposomes.

“Similarly, the death of glioblastoma cells, measured as release of LDH and activation of caspase-3, was not increased by the addition of free doxorubicin to empty liposomes (Supporting information Figure S4B).”

When added to the upper chamber of the Transwell containing confluent hCMEC/D3 cells on the insert and U87-MG in the lower chamber, doxorubicin poorly enters glioblastoma cells.

“When added to the upper chamber of the Transwell containing confluent hCMEC/D3 cells on the insert and U87-MG in the lower chamber, doxorubicin poorly entered glioblastoma cells, as shown by the low red fluorescence in U87-MG cells recovered by the lower chamber (Figure 5A)”

Statins did not change the expression of NOS III in hCMEC/D3 cells.

“The statins did not induce NOS I and did not change the expression of NOS III, but a slight increase in the amount of the active phospho-(Ser1177)-NOS III was detected following statins treatment (Figure 1E).”

ATPase activity of Pgp/ABCB1 and BCRP/ABCG2, but not MRP1/ABCC1, is decreased in hCMEC/D3 cells in the presence of statins.

“the ATPase activity of Pgp/ABCB1 and BCRP/ABCG2, but not that MRP1/ABCC1, was decreased in the presence of the statins (Figure 2B)”

Statins affect NF-kB pathway in BBB cells.

“Effects of statins on cholesterol and GGPP synthesis, RhoA/RhoA kinase activity, NF-kB pathway and NO synthesis in BBB cells.”

Statins, at concentrations found in blood of patients receiving anti-cholesterolemic therapy, enhance efficacy of doxorubicin against glioblastoma cells.

“It is noteworthy, however, that statins, at concentrations that are found in the blood of patients receiving anti-cholesterolemic therapy, enhanced the efficacy of a chemotherapeutic drug like doxorubicin against glioblastoma cells.”

Statins affect doxorubicin permeability across BBB cells.

“Effects of statins on ABC transporters activity and doxorubicin permeability across BBB cells.”

In co-culture models, application of free doxorubicin on luminal side of hCMEC/D3 monolayer does not yield any strong drug delivery into tumour cells growing under BBB.

“In co-culture models, the application of free doxorubicin on the luminal side of hCMEC/D3 monolayer did not yield any strong drug delivery into the tumour cells growing under the BBB.”

Statins affect tight junctions integrity in hCMEC/D3 cells.

“Effects of statins on membrane cholesterol and tight junctions integrity in hCMEC/D3 cells.”

Statins affect GGPP synthesis in BBB cells.

“Effects of statins on cholesterol and GGPP synthesis, RhoA/RhoA kinase activity, NF-kB pathway and NO synthesis in BBB cells.”

Empty liposomal shells do not exert any permeabilizing activity on delivery of doxorubicin across BBB.

“Empty liposomal shells, which inhibited the activity of Pgp/ABCB1 in chemoresistant tumours (Riganti et al., 2011), did not exert any permeabilizing activity on the delivery of doxorubicin across the BBB”

• cites Riganti et al., 2011 sourceNot checked — no full text of the source

Statins affect NO synthesis in BBB cells.

“Effects of statins on cholesterol and GGPP synthesis, RhoA/RhoA kinase activity, NF-kB pathway and NO synthesis in BBB cells.”

Statins affect the permeability of LDL receptor-targeted liposomal doxorubicin across the BBB.

“Effects of statins on the expression of LDL receptors and on the permeability of LDL receptor-targeted liposomal doxorubicin across the BBB.”

Statins affect membrane cholesterol in hCMEC/D3 cells.

“Effects of statins on membrane cholesterol and tight junctions integrity in hCMEC/D3 cells.”

Statins affect cholesterol synthesis in BBB cells.

“Effects of statins on cholesterol and GGPP synthesis, RhoA/RhoA kinase activity, NF-kB pathway and NO synthesis in BBB cells.”

Statins affect RhoA/RhoA kinase activity in BBB cells.

“Effects of statins on cholesterol and GGPP synthesis, RhoA/RhoA kinase activity, NF-kB pathway and NO synthesis in BBB cells.”

Statins affect the expression of LDL receptors in BBB cells.

“Effects of statins on the expression of LDL receptors and on the permeability of LDL receptor-targeted liposomal doxorubicin across the BBB.”

Statins affect ABC transporters activity in BBB cells.

“Effects of statins on ABC transporters activity and doxorubicin permeability across BBB cells.”

We did not detect direct cytotoxic effects of statins in co-cultured glioblastoma cells.

“We did not detect direct cytotoxic effects of statins in co-cultured glioblastoma cells, perhaps because we used simvastatin at lower dose.”

Apo-Lipodox has been previously validated for efficacy in solid tumours with high expression of LDL receptors.

“We have previously validated the efficacy of an engineered LDL receptor-targeted liposome-encapsulated doxorubicin ('apo-Lipodox') in solid tumours with high expression of LDL receptors (Kopecka et al., 2011).”

• cites Kopecka et al., 2011Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

It is unlikely that statins, at least at concentrations used in this study, increase paracellular transport across BBB.

“According to these data, it seems unlikely that statins -at least at the concentrations used in this study -increase the paracellular transport across BBB.”

The mechanism by which statins reduce vascular tone and increase cerebral flow is via increased NO levels.

“this suggests that this is the mechanism by which statins reduce the vascular tone and increase cerebral flow, as inhibitors of Rho kinase produce similar effects (Rikitake et al., 2005).”

• cites Rikitake et al., 2005Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Certain types of drug-containing nanoparticles adsorb apoB-100 and apo-E proteins from human low-density lipoproteins (LDLs).

“Certain types of drug-containing nanoparticles adsorb apoB-100 and apo-E proteins from human low-density lipoprotein (LDLs; Kim et al., 2007), are recognized by the LDL receptor and achieve an efficient drug delivery into brain parenchyma (Michaelis et al., 2006;Nikanjam et al., 2007) because both neurons and glial cells express LDL receptors (Ambruosi et al., 2006).”

• cites Kim et al., 2007 sourceNot checked — no full text of the source

The liposomal shell itself does not exert a permeabilizing effect on doxorubicin delivery across hCMEC/D3 cells.

“We thus excluded a permeabilizing effect exerted by the liposomal shell itself.”

Liposome-encapsulated drugs have been proposed as vehicles able to cross the BBB (through a diffusion process or a receptor-mediated endocytosis) and deliver their cargo into the CNS.

“As alternative strategy, liposome-encapsulated drugs that have shown selective intratumour drug delivery, lower side effects and particular efficacy against aggressive and chemoresistant tumours (Jabr-Milane et al., 2008;Riganti et al., 2011), have been proposed as vehicles able to cross the BBB (through a diffusion process or a receptor-mediated endocytosis) and deliver their cargo into the CNS (”

• cites Deelen and Loscher, 2007Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Nitration probably impairs the catalytic cycle of Pgp/ABCB1 and BCRP/ABCG2 pumps.

“suggesting that the nitration probably impairs the catalytic cycle of the pumps.”

NO levels play a crucial role in regulating the activity of Pgp/ABCB1 and BCRP/ABCG2 in hCMEC/D3 cells.

“suggesting that NO levels play a crucial role in regulating the activity of Pgp/ABCB1 and BCRP/ABCG2 in hCMEC/D3 cells.”

Liposome-encapsulated drugs have shown particular efficacy against aggressive and chemoresistant tumours.

“As alternative strategy, liposome-encapsulated drugs that have shown selective intratumour drug delivery, lower side effects and particular efficacy against aggressive and chemoresistant tumours (Jabr-Milane et al., 2008;Riganti et al., 2011), have been proposed as vehicles able to cross the BBB (through a diffusion process or a receptor-mediated endocytosis) and deliver their cargo into the CNS (”

• cites Jabr-Milane et al., 2008Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Riganti et al., 2011 sourceNot checked — no full text of the source

The reduction in efflux activity of Pgp/ABCB1 and BCRP/ABCG2 by statins is mediated by increased synthesis of NO.

“Statins reduced the efflux activity of Pgp/ABCB1 and BCRP/ABCG2 in hCMEC/D3 cells by increasing the synthesis of NO, which elicits the nitration of critical tyrosine residues on these transporters.”

Liposome-encapsulated drugs have shown selective intratumour drug delivery.

“As alternative strategy, liposome-encapsulated drugs that have shown selective intratumour drug delivery, lower side effects and particular efficacy against aggressive and chemoresistant tumours (Jabr-Milane et al., 2008;Riganti et al., 2011), have been proposed as vehicles able to cross the BBB (through a diffusion process or a receptor-mediated endocytosis) and deliver their cargo into the CNS (”

• cites Jabr-Milane et al., 2008Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Riganti et al., 2011 sourceNot checked — no full text of the source

ABC transporters have also been identified in CNS tumours, where they contribute to frequently observed chemoresistance.

“The latter have also been identified in CNS tumours (Nakagawa et al., 2009), where they contribute to the frequently observed chemoresistance.”

• cites Nakagawa et al., 2009Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

The persistent inhibition of Pgp/ABCB1 and BCRP/ABCG2 activity by statins is due to continuous synthesis of NO by hCMEC/D3 cells.

“The reversal of statins' effects by the NO scavenger PTIO suggests that the persistent inhibition of Pgp/ABCB1 and BCRP/ABCG2 activity was due to a continuous synthesis of NO by hCMEC/D3 cells.”

NO levels and the consequent status of nitration on ABC transporters are critical for the transport of apo-Lipodox in hCMEC/D3 cells.

“suggesting that the NO levels -and the consequent status of nitration on ABC transporters -are also critical for the transport of apo-Lipodox in hCMEC/D3 cells.”

Certain types of drug-containing nanoparticles are recognized by the LDL receptor and achieve efficient drug delivery into brain parenchyma.

“Certain types of drug-containing nanoparticles adsorb apoB-100 and apo-E proteins from human low-density lipoprotein (LDLs; Kim et al., 2007), are recognized by the LDL receptor and achieve an efficient drug delivery into brain parenchyma (Michaelis et al., 2006;Nikanjam et al., 2007) because both neurons and glial cells express LDL receptors (Ambruosi et al., 2006).”

• cites Michaelis et al., 2006 sourceNot checked — no full text of the source
• cites Nikanjam et al., 2007Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Liposome-encapsulated drugs have shown lower side effects.

“As alternative strategy, liposome-encapsulated drugs that have shown selective intratumour drug delivery, lower side effects and particular efficacy against aggressive and chemoresistant tumours (Jabr-Milane et al., 2008;Riganti et al., 2011), have been proposed as vehicles able to cross the BBB (through a diffusion process or a receptor-mediated endocytosis) and deliver their cargo into the CNS (”

• cites Jabr-Milane et al., 2008Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites Riganti et al., 2011 sourceNot checked — no full text of the source

Uptake of non-targeted liposomes is not dependent on LDL receptor-mediated endocytosis.

“suggesting that the uptake of non-targeted liposomes was not dependent on the LDL receptor-mediated endocytosis.”

In rat C6 glioma cells, 5 mmol/L mevastatin and simvastatin induce apoptosis by decreasing intracellular availability of GGPP and activation of ERK1/2 and Akt.

“for instance, in rat C6 glioma cells, 5 mmol•L -1 mevastatin and simvastatin induced apoptosis by decreasing the intracellular availability of GGPP and the activation of ERK1/2 and Akt (Yanae et al., 2011)”

• cites Yanae et al., 2011 sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Effects of lipidic envelope alone on doxorubicin delivery across BBB may be tissue-dependent.

“suggesting that the effects of the lipidic envelope alone may be tissue-dependent.”

Liposomes have a facilitated entry within BBB cells by simple endocytosis.

“Since both the non-LDL receptor-targeted Lipodox and the LDL receptor-targeted apo-Lipodox had a higher uptake than free doxorubicin in the absence of statins, it is likely that liposomes have a facilitated entry within BBB cells by simple endocytosis.”

The extent of increase in drug delivery and toxicity is variable, depending on different levels and activity of ABC transporters and LDL receptors, and on existence of other mechanisms of resistance characterizing each cell line.

“However, the extent of this increase was variable, depending on the different levels and activity of ABC transporters and LDL receptors, and on the existence of other mechanisms of resistance characterizing each cell line.”

The approach proposed in this work differs from other studies on CNS-penetrating nanoparticles because it is more 'physiological': it takes advantage of usual metabolic effects of therapeutic doses of statins and from properties of liposomes.

“The approach proposed in our work differs from the other studies on CNS-penetrating nanoparticles because it is more 'physiological': it takes advantage of the usual metabolic effects of therapeutic doses of statins -the increase in LDL receptors exposed and the synthesis of NO -and from the properties of liposomes, that induce permeability across the BBB and efficacy against drugresistant tumours”

The greater permeability of apo-Lipodox in untreated hCMEC/D3 cells is probably a consequence of decreased efflux via Pgp/ABCB1 and increased uptake.

“probably as a consequence of the decreased efflux via Pgp/ABCB1 and of the increased uptake.”

The LDL receptor becomes a helpful tool to increase endocytosis of targeted liposomes when its levels increase on the cells' surface after exposure to statins.

“In contrast, the LDL receptor became a helpful tool to increase the endocytosis of targeted liposomes when its levels increased on the cells' surface, that is, after exposure to statins.”