CoChat
Sign Up
Cited by 72Nanoparticle-Based Drug DeliveryAdvanced Drug Delivery SystemsAdvancements in Transdermal Drug Delivery

Fabrication Principles and Their Contribution to the Superior In Vivo Therapeutic Efficacy of Nano-Liposomes Remote Loaded with Glucocorticoids

Open access
Avnir, Yuval, Turjeman, Keren, Tulchinsky, Deborah, Sigal, Alex, Kizelsztein, Pablo, Tzemach, Dina, Gabizon, Alberto, Barenholz, Yechezkel·2011·PLoS ONE
DOI 10.1371/journal.pone.0025721PMC PMC3188566
Open this paper in CoChat — free accountOpen PDF

Abstract

We report here the design, development and performance of a novel formulation of liposome- encapsulated glucocorticoids (GCs). A highly efficient (>90%) and stable GC encapsulation was obtained based on a transmembrane calcium acetate gradient driving the active accumulation of an amphipathic weak acid GC pro-drug into the intraliposome aqueous compartment, where it forms a GC-calcium precipitate. We demonstrate fabrication principles that derive from the physicochemical properties of the GC and the liposomal lipids, which play a crucial role in GC release rate and kinetics. These principles allow fabrication of formulations that exhibit either a fast, second-order (t(1/2) ~1 h), or a slow, zero-order release rate (t(1/2) ~ 50 h) kinetics. A high therapeutic efficacy was found in murine models of experimental autoimmune encephalomyelitis (EAE) and hematological malignancies.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper reports a nano-liposomal glucocorticoid formulation (nSSL-MPS) remote loaded via a transmembrane calcium acetate gradient, achieving >90% stable encapsulation and lipid-composition-dependent release kinetics. In murine EAE and hematological malignancy models, nSSL-MPS showed superior pharmacokinetics, biodistribution, and therapeutic efficacy versus free methylprednisolone hemisuccinate.

Key findings

  • Remote loading via calcium acetate gradient was highly efficient for GC-succinates: MPS 96%, HCS 100%, BMS 80%, while the GC-phosphate BMP showed minimal encapsulation. Remote loading gave 40-fold higher encapsulation than passive loading (96% vs 2%) and 3.2-fold higher intraliposome MPS concentration (38 vs 12 [GC]/[PL]); intraliposome MPS reached 358 mM, replacing 90% of nSSL acetate.
  • Release kinetics depended on liposome lipid composition: high-Tm HSPC- and DSPC-based nSSL showed slow zero-order release with t1/2 of 50 h and 58 h, respectively, and ~1% of original MPS load released per hour; low-Tm POPC-based nSSL showed fast second-order release with t1/2 of 0.87 h. Release was similar in human plasma and PBS (t1/2 50 h vs ~64 h), with activation energy 37.8 kcal/mole.
  • Pharmacokinetics improved markedly: free MPS cleared with t1/2 ~9 min, whereas encapsulated MPS cleared with t1/2 ~12 h; in vivo bloodstream release t1/2 was 17 h. In acute EAE mice, brain/liver MPS ratio was 0.075 at 1 h for nSSL-MPS and 0.180 at 24 h, versus 0.003 at 0.3 h for free MPS.
  • Therapeutic efficacy was superior to free MPS and comparator MS drugs: in acute EAE, nSSL-MPS caused no mortality in 10 mice versus 3/10 deaths in control and free-MPS groups, with day-22 clinical score ~0.0 versus 2.5 for free MPS and 3.0 for control, and mean maximal score 1.9 versus 4.3 for free MPS. In BCL-1 leukemia, median survival was 35 days (5 mg/kg) and 47 days (50 mg/kg) for nSSL-MPS versus 19 days for free MPS; in J6456 lymphoma, median survival was 23 days versus 16.5 days for free MPS.

Methods

  • nSSL were fabricated by lyophilization, hydration in calcium acetate, extrusion above lipid Tm, and dialysis against 5% dextrose to create a >800 transmembrane calcium acetate gradient; final nSSL-MPS were 83±15 nm, zeta potential -5 mV, and remote loading was done at 60–65°C for 20 min.
  • In vitro characterization included surface tension/CAC, octanol-buffer logD, XRD, cryo-TEM, HPLC, atomic absorption spectrometry, and release studies at 37°C in human plasma or PBS using gel permeation chromatography.
  • In vivo studies used SJL/J, C57Bl/6, and BALB/c mice: acute PLP-induced EAE, chronic MOG 35–55-induced EAE, BCL-1 B-cell leukemia, and J6456 T-cell lymphoma; pharmacokinetics/biodistribution used 3H-cholesteryl hexadecyl ether-labeled nSSL-MPS, with clinical scoring and survival analysis.

Limitations

  • The paper states that the reason for the discrepancy between in vitro release t1/2 (50 h) and in vivo release t1/2 (17 h) is not yet clear; it suggests static in vitro conditions versus in vivo clearance to interstitial fluids may enhance release, and notes this assumption is under evaluation.
  • Evident from the text: all therapeutic efficacy, pharmacokinetic, and biodistribution data are from murine models (with prior rat arthritis data cited), and no human clinical trial data are reported.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1371/journal.pone.0025721

Cites

69 of 69 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 38 cited sources checked · 0 supported · 0 flagged

  • Findingreasoning

    The accumulated GC forms a GC-calcium precipitate in the intraliposome aqueous compartment.

    “A highly efficient (>90%) and stable GC encapsulation was obtained based on a transmembrane calcium acetate gradient driving the active accumulation of an amphipathic weak acid GC pro-drug into the intraliposome aqueous compartment, where it forms a GC-calcium precipitate.”
    Quote in the paperSubstantial claim
  • Findingown data

    A highly efficient (>90%) and stable GC encapsulation was obtained based on a transmembrane calcium acetate gradient.

    “A highly efficient (>90%) and stable GC encapsulation was obtained based on a transmembrane calcium acetate gradient driving the active accumulation of an amphipathic weak acid GC pro-drug into the intraliposome aqueous compartment, where it forms a GC-calcium precipitate.”
    Quote in the paperSubstantial claim
  • Findingown data

    XRD analysis shows the precipitate formed by calcium and MPS is crystalline Ca(MPS)2.

    “Fig. 2a demonstrates that calcium ions (but not sodium ions) cause MPS aggregation, whereby characterization of the precipitate by XRD analysis shows a clear crystalline nature of Ca(MPS)2 ( Fig. 2b ).”
    Quote in the paperSubstantial claim
  • Findingown data

    Calcium as counterion improves MPS loading more than sodium as counterion.

    “This stems from the findings that nSSL hydrated in either calcium acetate or sodium acetate show a much better loading efficiency than nSSL hydrated in calcium chloride or sodium chloride, signifying the necessity of the acetate anion, while calcium as counterion improves MPS loading more than sodium as counterion as is proven from comparing nSSL prepared from lipids hydrated in calcium acetate”
    Quote in the paperSubstantial claim
  • Findingnone given

    High therapeutic efficacy was found in murine models of experimental autoimmune encephalomyelitis (EAE) and hematological malignancies.

    “A high therapeutic efficacy was found in murine models of experimental autoimmune encephalomyelitis (EAE) and hematological malignancies.”
    Quote in the paperSubstantial claim
  • May combine two claims. 42
  • Classified as probably not a substantial claim. 6

  • May combine two claims.

    Findingreasoning

    The trapped volume of nSSL was 1.2 μl/μmol PL; encapsulation of 14.96 μmol MPS for 34.72 μmol PL gave an intraliposome MPS concentration of 358 mM, meaning 90% of nSSL acetate was replaced with MPS.

    “A value of 1.2 µl/µmol PL was obtained. Therefore, encapsulation of 14.96 µmol MPS for 34.72 µmol PL calculates into MPS intraliposome concentration of 358 mM, which means that 90% of the nSSL acetate was replaced with MPS.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    Both GC-succinate and GC-phosphate sodium salts meet the condition of sufficient water solubility.

    “Both GC acid prodrug families, the succinate and the phosphate sodium salts, meet the conditions of sufficient water solubility.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In silico database screening of commercially available GC candidates for remote loading yielded only water-soluble weak acid GCs (GC-succinates and GC-phosphates) and no amphipathic weak base GCs.

    “An in silico database screening of suitable commercially available GC candidates for remote loading yielded only water-soluble weak acid GCs: the GC-succinates (GC-S) and the GC-Phosphates (GC-P). No GCs that are amphipathic weak bases were found.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    There is no appreciable effect of plasma components on MPS release rates; at 37°C t1/2 is similar in human plasma (50 h) and PBS (~64 h).

    “Fig. 4a and its inserted Table also demonstrate that there is no appreciable effect of plasma components on the release rates, which at 37°C are similar in human plasma (t1/2 of 50 h) and in PBS (t1/2 of ∼64 h).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Remote loading achieved 40-fold higher encapsulation efficiency (96% vs 2%) and 3.2-fold higher intraliposome MPS concentration (38 [GC]/[PL] vs 12 [GC]/[PL]) than passive loading.

    “This comparison showed a clear advantage of the remote loading method, which achieved 40-fold higher encapsulation efficiency (96% versus 2%) and a 3.2-fold higher intraliposome MPS concentration for the remote loaded nSSL-MPS (38 [GC]/[PL] versus 12[GC]/[PL]).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Calcium ions, but not sodium ions, cause MPS aggregation.

    “Fig. 2a demonstrates that calcium ions (but not sodium ions) cause MPS aggregation, whereby characterization of the precipitate by XRD analysis shows a clear crystalline nature of Ca(MPS)2 ( Fig. 2b ).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    MPS release from HSPC/CHOL/2000-PEG-DSPE nSSL in human plasma was not accompanied by loss of liposome calcium ions or change in liposome size distribution, indicating controlled release not membrane destruction.

    “Moreover, as we have reported in our previous study [27], the release of MPS from nSSL based on HSPC/CHOL/2000-PEG-DSPE when incubated in human plasma was neither accompanied by loss of liposome calcium ions nor by change in liposome size distribution (data not shown), indicating that MPS release is not a result of a destructive effect on membrane barrier properties, but rather a controlled”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [27] Y Avnir, 2008, Amphipathic weak acid glucocorticoid prodrugs remote-loaded into sterically stabilized nanoliposomes evaluated in arthritic rats and in a Beagle dog: a novel approach to treating autoimmune arthritis., Arthritis Rheum source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    nSSL based on high-Tm PCs (HSPC or DSPC) show similar zero-order MPS release profiles with t1/2 of 50 h for HSPC and 58 h for DSPC, and a constant release rate of ~1% of original MPS load per hour.

    “nSSL based on high-Tm PCs (either HSPC or DSPC) are characterized by similar zero-order release profiles, which translate to similar long t1/2 (50 h for HSPC and 58 h for DSPC), and to a constant release rate of ∼1% of the original MPS load per hour, while the low-Tm “fluid” POPC-based nSSL showed a much faster release rate that is best described by second-order kinetics, having a t1/2 of 0.87 h (”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    GC-S HCS, BMS, and MPS were remote loaded into HSPC/CHOL/2000-PEG-DSPE nSSL with high encapsulation efficiencies (HCS 100%, BMS 80%, MPS 96%), while GC-P BMP showed minimal encapsulation.

    “As expected, from the logD values, all of the GC-S tried were successfully remote loaded into HSPC/CHOL/2000-PEG-DSPE nSSL with high encapsulation efficiencies: HCS 100%, BMS 80%, and MPS 96%, while the representative of GC-P, BMP, showed minimal encapsulation as explained by its lack of amphipathicity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Only GC-S (represented by MPS), but not GC-P (represented by DEXP), are surface-active, as shown by reduction of surface tension.

    “However, only the GC-S, represented by MPS, but not the GC-P ,represented by dexamethasone phosphate (DEXP), were shown to be surface-active, as was demonstrated by the reduction of surface tension of MPS, but not DEXP ( Fig. 1a ).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    All calcium ions remain entrapped in the intraliposome aqueous phase during acetate release, MPS loading, and upon storage, indicating liposome membrane integrity.

    “The fact that all calcium ions remain entrapped in the intraliposome aqueous phase during acetate release, MPS loading, and upon storage points to the integrity of the liposome membrane under the conditions that the uncharged acetic acid can diffuse via this membrane.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MPS logD is linearly pH-dependent from pH 5 to 8.5, with logD 2 at pH 5 and -0.75 at pH 8.5.

    “The amphipathic weak-acid characteristic is further supported by the linear pH-dependency of logD for the pH range of 5–8.5 ( Fig. 1b ). At pH 5 the logD is 2 and at pH 8.5 the logD is -0.75.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the acute EAE model, nSSL-MPS are highly efficacious in reducing disease severity compared with the untreated control group or the group treated with a high dose of free drug, with a mean maximal score of 1.9 versus 4.3 for free drug.

    “Thirdly, from the statistical evaluation of the data collected from this experiment, it is obvious that nSSL-MPS are highly efficacious in reducing disease severity compared with the control untreated group, or the group treated with a high dose of free drug [compare the mean maximal score of 1.9 in the nSSL-MPS group (limp tail with righting reflex) with the score of 4.3 (early paralysis) for the”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Chronic MOG 35-55 peptide-induced EAE is characterized by milder clinical features than PLP-induced EAE, as seen by low mean clinical score and lack of mortality for all groups.

    “the chronic EAE induced by the MOG protein, which is characterized by milder clinical features than the PLP-induced EAE (as seen by the low mean clinical score and lack of mortality for all groups)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    At 1 hour post injection, the brain/liver distribution ratio for nSSL-MPS is 0.076 vs 0.006 for free MPS; at 24h only MPS derived from nSSL is found in the brain at a brain/liver ratio of 0.18.

    “At 1 hour post injection the MPS ratio of brain to liver distribution is 12 times higher than at 0.3 hour for free MPS (compare 0.006 for free with 0.076 for nSSL-MPS). At 24h post injection only MPS derived from nSSL is found in the brain at a brain to liver ratio of 0.18.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The in vivo release t1/2 in bloodstream is 17 h, 3 times faster than the in vitro t1/2 of 50 h.

    “The t1/2 of 17 h determined for the release in vivo in the bloodstream ( Fig. 5 ) although slow, is 3 time faster than the t1/2 of 50 h found in vitro ( Fig. 4a and inserted Table).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the EAE mouse model, free-MPS has almost no significant effect, while nSSL-MPS lowers the mean burden of disease by 4-fold.

    “Overall, when we summarize the efficacy of the treatment in the EAE mouse model used by the parameter of “mean burden of disease”, free-MPS has almost no significant effect, while nSSL-MPS was able to lower the mean burden by 4-fold ( Fig. 6a ).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the acute EAE model, nSSL-MPS lower the mean burden of disease to 0.8±0.17 and are clearly superior to Copaxone and Betaferon.

    “while nSSL-MPS, which lower the mean burden to 0.8±0.17, was clearly superior.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Pharmacokinetics and biodistribution of nSSL-MPS in healthy and EAE mice show lack of major differences, except brain MPS concentration at 1 and 6 h post IV injection was about twice in diseased EAE mice.

    “Comparison of pharmacokinetics and biodistribution of nSSL-MPS in healthy and EAE mice show lack of major differences (data not shown), with the brain being an exception: the concentration of MPS at 1 and 6 h post IV injection in the diseased EAE mice brains was about twice that in the healthy mice Fig. S1a (Supporting information).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    During the first hour post IV injection, the liver gets much higher levels of free MPS than of MPS derived from nSSL-MPS, suggesting much less GC first-pass metabolism.

    “During the first hour post intravenous (IV) injection, the liver gets much higher levels of non-encapsulated (free) MPS than of MPS derived from nSSL-MPS. This suggests much less GC first-pass metabolism, which is relevant to GC side effects [2].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [2] RP Schleimer, 2003, Glucocorticoids.

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Encapsulated MPS is cleared in hours (t1/2 ~12 h), while non-encapsulated free MPS is rapidly cleared in minutes (t1/2 ~9 min).

    “While non-encapsulated MPS is rapidly cleared in minutes (t1/2 of ∼9 min), encapsulated MPS is cleared in hours (t1/2 of ∼12 h).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    nSSL-MPS show in vivo zero-order slow release kinetics in blood and tissues, similar to in vitro.

    “The pharmacokinetic and biodistribution studies done with the nSSL-MPS clearly demonstrate in vivo zero- order, slow release kinetics in blood ( Fig. 5 ) and tissues (Fig. S1c Supporting information), which is similar to what was found for these nSSL in vitro.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    nSSL-MPS treated group had lower disease severity and faster recovery; on day 22 clinical score ~0.0 vs 2.5 for free MPS and 3.0 for control.

    “Secondly, looking at the progress of the clinical score, not only was the severity of disease of the nSSL-MPS-treated group much lower, but also recovery from the acute disease attack was much faster. On day 22 mice of the nSSL-MPS treated group reached a clinical score of ∼0.0 (no clinical symptoms) compared with a clinical score of 2.5 for the free-MPS-treated mice, and 3.0 for the control group”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    MPS is released from nSSL-MPS due to its amphipathicity and not due to a "dumping" effect related to damaged or permeable liposome membrane.

    “MPS is released from the nSSL-MPS due to its amphipathicity and not due to a “dumping” effect related to damaged or permeable liposome membrane.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    A high (37.8 kcal/mole) activation energy for nSSL based on high-Tm PCs (HSPC and DSPC) supports slow, but sufficient for activity, release at 37°C, concomitantly with minimal release at 4°C.

    “A high (37.8 kcal/mole) activation energy for nSSL based on high-Tm PCs (HSPC and DSPC) supports slow, but sufficient for activity, release at 37°C, concomitantly with minimal release at 4°C (important in order to achieve an acceptable product shelf life).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingcitation

    The final nSSL-MPS are small (mean size 83±15 nm SD) unilamellar vesicles in 5% dextrose, with a unimodal narrow size distribution and low negative charge (zeta potential of -5 mV).

    “Our final nSSL-MPS are small (mean size of 83±15 nm SD) unilamellar vesicles in 5% dextrose, having a unimodal narrow size distribution and low negative charge (zeta potential of -5mV), which is related to the hindered charge of pegylated liposomes [58].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [58] O Garbuzenko, 2005, Electrostatics of PEGylated micelles and liposomes containing charged and neutral lipopolymers., Langmuir source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    In the J6456 mouse T-cell lymphoma model, nSSL-MPS had a significant therapeutic effect, increasing survival median from 17.5 to 23 days, while free-MPS had no effect (survival median 16.5 days).

    “The survival experiment done ( Fig. 6e ) shows that, indeed, the nSSL-MPS had a significant therapeutic effect, where survival median of the J6456 mouse T-cell lymphoma-bearing mice increased from 17.5 to 23 days, while free-MPS treatment had no effect at all (survival median of 16.5 days).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingreasoning

    The optimized nSSL-MPS features result in a high drug-to-lipid ratio, high encapsulation efficiency, good stability during 4°C storage, superior pharmacokinetic and biodistribution, and slow zero-order drug release in vivo.

    “Overall, the features of the optimized nSSL-MPS described here result in a high drug to lipid ratio (high MPS level per liposome), high efficiency of encapsulation, good stability during 4°C storage, superior pharmacokinetic and biodistribution, as well as a slow zero-order drug release in vivo.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationnone given

    Precipitation of loaded GC is related to the steroid ring structure and to the nano-intraliposomal aqueous volume of the nSSL.

    “This precipitation is related to the steroid ring structure and to the nano-intraliposomal aqueous volume of the nSSL.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    A high activation energy (slope of the Arrhenius plot) enables sufficient GC release at 37°C and minimal release at 2-8°C.

    “Such a high slope enables a sufficient release at 37°C (needed to achieve efficacy), concomitant with a minimal release at 2–8°C (to ensure long-term storage shelf life), both of which were demonstrated for Doxil [17], [19].”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [17] G Haran, 1993, Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases., Biochim Biophys Acta source

      Not checked in the free run.

    • cites [19] DD Lasic, 1995, Transmembrane gradient driven phase transitions within vesicles: lessons for drug delivery., Biochim Biophys Acta source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    nSSL-GC based on high (>37°C) Tm liposome-forming phosphatidylcholines exhibit zero-order slow drug release in vitro in human plasma and inflamed synovial fluid, and in vivo in rat systemic circulation and inflamed paw.

    “nSSL-GC based on high (>37°C) Tm “liposome-forming phosphatidylcholines” are characterized by the unique property of being a zero-order, slow drug release DDS, as shown in vitro in human plasma and inflamed synovial fluid, and in vivo in the rat's systemic circulation and inflamed paw [27].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [27] Y Avnir, 2008, Amphipathic weak acid glucocorticoid prodrugs remote-loaded into sterically stabilized nanoliposomes evaluated in arthritic rats and in a Beagle dog: a novel approach to treating autoimmune arthritis., Arthritis Rheum source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    GC-S are highly favorable candidates for remote loading via the calcium acetate transmembrane gradient, while GC-P are not.

    “Thus, we concluded that GC-S are highly favorable candidates for remote loading via the calcium acetate transmembrane gradient, while the GC-P are not.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Efficient remote loading of GCs requires the GC to be fairly water-soluble and to have sufficient hydrophobicity in its uncharged form to diffuse across the liposome lipid bilayer, while inside the intraliposome aqueous phase the hydrophobicity/hydrophilicity ratio should be reduced to slow release.

    “Efficient remote loading of GCs requires the GC candidate to have a specific set of physicochemical features. Firstly, the GC should be fairly water-soluble, and secondly, while present in the extraliposomal medium, a part of the GC molecule has to be hydrophobic enough to allow sufficient GC diffusion across the liposome lipid bilayer in its uncharged form (see Scheme S1b, Supporting information”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    When GCs are given intravenously or orally, their pharmacokinetics obey first-order release kinetics with an initial high plasma concentration followed by rapid exponential clearance.

    “When GCs are given intravenously or orally, their pharmacokinetics obey first-order release kinetics [4] with an initial high plasma concentration followed by rapid exponential clearance.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [4] D Czock, 2005, Pharmacokinetics and pharmacodynamics of systemically administered glucocorticoids., Clin Pharmacokinet source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Size-dependent passive targeting allows nano-particles but not micro-particles to extravasate into inflamed and cancerous tissues.

    “This size-dependent “passive targeting” to the inflamed and cancerous tissues [10]–[12] is the Achilles heel of the disease permitting better treatment of inflammation and cancer.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [10]–[12]

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    MPS remote loading by nSSL with a calcium acetate gradient is an exchange process between MPS and acetate anions followed by precipitation of a calcium MPS salt in the intraliposome aqueous phase.

    “Thus, overall ( Figs. 1 – 3 ) the “heart” of MPS remote loading by nSSL exhibiting a calcium acetate transmembrane gradient is basically an exchange process between MPS and acetate anions, which is followed by precipitation of a calcium MPS salt in the intraliposome aqueous phase.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    High-Tm PC nSSL have low free volume defects that become saturated at low MPS concentration, leading to slow zero-order release.

    “Thus, in nSSL based on high-Tm PCs the small number of free volume defects becomes saturated already at low MPS concentration and consequently the system operates under conditions of Vmax, leading to a slow, zero-order release.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    PLP-induced acute mouse EAE is a routinely used model for multiple sclerosis.

    “We also studied therapeutic efficacy in the proteolipid protein (PLP)-induced acute mouse EAE, a routinely used model for the autoimmune inflammatory neurodegenerative disease multiple sclerosis [39], [40], in which the involvement of inflammation in the brain enhance the permeability of this difficult to reach organ.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [39] L Steinman, 2006, How to successfully apply animal studies in experimental allergic encephalomyelitis to research on multiple sclerosis., Ann Neurol source

      Not checked in the free run.

    • cites [40] RH Whitham, 1991, Lymphocytes from SJL/J mice immunized with spinal cord respond selectively to a peptide of proteolipid protein and transfer relapsing demyelinating experimental autoimmune encephalomyelitis., J Immunol source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The high activation energy ensures nSSL-MPS loading stability upon long-term storage at 4°C without compromising slow zero-order release at 37°C.

    “This is an important advantage of this formulation, as it ensures nSSL-MPS loading stability upon long term storage at 4°C without compromising the needed slow zero-order release at 37°C.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Only nSSL-MPS, but not standard (non-liposomal) MPS, acts as a disease-modifying drug in the EAE mouse model.

    “These results suggest that only nSSL-MPS, but not standard (non-liposomal) MPS acts as a disease-modifying drug.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Storm and coworkers described successful treatment of B6F10 murine melanoma by nano-liposomes passively loaded with prednisolone phosphate, with the underlying mechanism described as inhibition of angiogenesis.

    “Recently, Storm and coworkers [15], [48] described a successful treatment of B6F10 murine melanoma by nano-liposomes passively loaded with the GC water-soluble prodrug prednisolone phosphate, where the underlying mechanism was described as inhibition of angiogenesis [15].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [15] M Banciu, 2008, Liposomal glucocorticoids as tumor-targeted anti-angiogenic nanomedicine in B16 melanoma-bearing mice., J Steroid Biochem Mol Biol source

      Not checked in the free run.

    • cites [48] E Kluza, 2011, Anti-tumor activity of liposomal glucocorticoids: The relevance of liposome-mediated drug delivery, intratumoral localization and systemic activity., J Control Release source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In multiple sclerosis patients, Copaxone causes a decrease in relapses and lesions.

    “Though the exact therapeutic mechanism of Copaxone is not clear [45], in multiple sclerosis patients it causes decrease in relapses and lesions [46].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [46] T Moreau, 2009, Link of the mechanisms of action of glatiramer acetate to its long-term clinical data., J Neurol Sci source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    By increasing the therapeutic index of glucocorticoids, it might be possible to lower the dosage of other drugs in treatment protocols, lowering overall toxic effect and perhaps increasing overall therapeutic success.

    “Namely, by increasing the therapeutic index of GCs we might lower the dosage of other drugs in the treatment protocol and by that lower the overall toxic effect and perhaps increase the overall therapeutic success of the treatment regime.”
    Quote in the paperNot a substantial claim
    • The authors state this as an assumption: The same pharmacokinetic and biodistribution advantages demonstrated for nSSL-GC in the EAE mouse model apply to cancers.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Betaferon (interferon beta-1b) is a bacterial-derived recombinant cytokine having anti-inflammatory activity.

    “Betaferon (interferon beta-1b) [43], is a bacterial-derived recombinant cytokine having anti-inflammatory activity.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [43] M Buttmann, 2007, Interferon-beta1b in multiple sclerosis., Expert Rev Neurother source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    For human cancer therapy, nSSL-GC may increase the therapeutic index of glucocorticoids in both MPS-sensitive and MPS-insensitive tumors, with a larger direct impact for MPS-sensitive tumors.

    “The implication of treatment of MPS-sensitive and MPS-insensitive mice tumors for human cancer therapy is that the nSSL-GC may increase the therapeutic index of GCs in both tumor types, with larger direct impact for MPS-sensitive tumors.”
    Quote in the paperSubstantial claim
    • The authors state this as an assumption: The same pharmacokinetic and biodistribution advantages demonstrated for nSSL-GC in the EAE mouse model apply to cancers.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Experimental use of animals was authorized by the Ethics Committee of the Hebrew University Medical School.

    “Experimental use of animals was authorized by the Ethics Committee of the Hebrew University Medical School.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Copaxone (glatiramer acetate) is a polypeptide composed of amino acids that mimic amino acids found in the main myelin-sheath protein.

    “Copaxone [44] (glatiramer acetate) is a polypeptide composed of amino acids that mimic amino acids found in the main myelin-sheath protein.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [44] T Ziemssen, 2007, Glatiramer acetate: mechanisms of action in multiple sclerosis., Int Rev Neurobiol source

      Not checked in the free run.

47 of 47

  • The paper relies on this without support.

    The challenge of making a stable and efficacious GC-loaded nanoliposome formulation having slow zero-order release kinetics was not met prior to this study.

    “Despite the progress achieved, the challenge of making a stable and efficacious GC-loaded nanoliposome formulation having slow zero-order release kinetics was not met.”

  • The authors state this as an assumption.

    The same pharmacokinetic and biodistribution advantages demonstrated for nSSL-GC in the EAE mouse model apply to cancers.

    “Treatment based on use of nSSL-GC may overcome these major deficiencies, as the same PK and BD advantages that were demonstrated in the EAE mouse model should also apply to the cancers.”

  • The paper relies on this without support.

    The unfavorable pharmacokinetics and biodistribution of glucocorticoids are major obstacles to achieving efficacious and nontoxic treatment.

    “However, the unfavorable pharmacokinetics and biodistribution of GCs are major obstacles to achieving efficacious and nontoxic treatment.”

  • The paper relies on this without support.

    The EAE mouse models used are valid models for multiple sclerosis.

    “The chronic MOG 35–55 peptide-induced EAE mouse model serves as another common disease model for multiple sclerosis.”
4 of 4

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Brain-to-liver distribution ratios imply a 25-fold difference

    Major

    Table 1 reports brain/liver ratios of 0.075 for nSSL-MPS at 1 hour and 0.003 for free MPS at 0.3 hour. Their ratio is 25, not the stated 12; the prose also gives ratios that differ from the table.

    Reported 12 · recomputed 25

  • Hydrocortisone succinate has conflicting abbreviations

    Minor

    The same prodrug is abbreviated HCS in Materials but HDS in the remote-loading method.

  • Low-Tm release formulation is identified as both DOPC and POPC

    Minor

    The Figure 4 caption attributes the fast, second-order release result to DOPC, whereas the results text identifies the tested low-Tm formulation as POPC.

3 of 3

Cites

Flagged first, then by citations

1 of the 70 references couldn’t be matched to a known paper.

Research works printed but not linked: 1

  1. NJ Zuidam, 1997, Electrostatic parameters of cationic liposomes commonly used for gene delivery as determined by 4-heptadecyl-7-hydroxycoumarin., Biochim Biophys Acta, 1329, 211

Cited by

Sorted by citations

Related

Authors
Avnir, Y., Turjeman, K., Tulchinsky, D., Sigal, A., Kizelsztein, P., Tzemach, D., Gabizon, A., & Barenholz, Y.
Affiliations
  • Hebrew University of Jerusalem (IL)
  • Shaare Zedek Medical Center (IL)
Corresponding author
Barenholz, Yechezkel
ORCID
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2011-10-06
Volume
6
Issue
10
Pages
e25721
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0025721
PMC
PMC3188566
OpenAlex
W1987358985
Cited by
72
References
70
Citations by year
2025: 3 · 2024: 4 · 2023: 6 · 2022: 8 · 2021: 5 · 2020: 7 · 2019: 6 · 2018: 5 · 2017: 6 · 2016: 7 · 2015: 4 · 2014: 4 · 2013: 2 · 2012: 2
FWCI
1.44
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Figshare (submittedVersion, cc-by)
Funders
Hebrew University of Jerusalem
Topics
  • Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmaceutical Science › Advanced Drug Delivery Systems
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmaceutical Science › Advancements in Transdermal Drug Delivery
Keywords
nanoliposomes, experimental autoimmune encephalomyelitis, hematologic malignancies
MeSH terms
Animals, Capsules, Chemistry, Pharmaceutical, Encephalomyelitis, Autoimmune, Experimental, Female, Glucocorticoids, Humans, Leukemia, Liposomes, Methylprednisolone Hemisuccinate, Multiple Sclerosis, Solubility, Water, Lymphoma, T-Cell, Myelin Proteolipid Protein, Nanostructures, Mice, Chemical Phenomena, Hydrophobic and Hydrophilic Interactions

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-10.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-10.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review