Development of a routine analysis method for liposome encapsulated recombinant interleukin-2

Koppenhagen, Frank J.; Storm, Gert; Underberg, W.J.M.

doi:10.1016/s0378-4347(98)00271-0

“…Prior to analysis, lipids were extracted following stardard protocols [28] in order to release the entrapped drug. Briefly, 100 μl of liposome sample was mixed with 225 μl of cold methanol and 125 μl of chloroform.…”

Section: Methodsmentioning

confidence: 99%

Study of the efficacy of antimalarial drugs delivered inside targeted immunoliposomal nanovectors

Urbán

¹

,

Estelrich

²

,

Adeva

³

et al. 2011

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Paul Ehrlich's dream of a 'magic bullet' that would specifically destroy invading microbes is now a major aspect of clinical medicine. However, a century later, the implementation of this medical holy grail continues being a challenge in three main fronts: identifying the right molecular or cellular targets for a particular disease, having a drug that is effective against it, and finding a strategy for the efficient delivery of sufficient amounts of the drug in an active state exclusively to the selected targets. In a previous work, we engineered an immunoliposomal nanovector for the targeted delivery of its contents exclusively to Plasmodium falciparum-infected red blood cells [pRBCs]. In preliminary assays, the antimalarial drug chloroquine showed improved efficacy when delivered inside immunoliposomes targeted with the pRBC-specific monoclonal antibody BM1234. Because difficulties in determining the exact concentration of the drug due to its low amounts prevented an accurate estimation of the nanovector performance, here, we have developed an HPLC-based method for the precise determination of the concentrations in the liposomal preparations of chloroquine and of a second antimalarial drug, fosmidomycin. The results obtained indicate that immunoliposome encapsulation of chloroquine and fosmidomycin improves by tenfold the efficacy of antimalarial drugs. The targeting antibody used binds preferentially to pRBCs containing late maturation stages of the parasite. In accordance with this observation, the best performing immunoliposomes are those added to Plasmodium cultures having a larger number of late form-containing pRBCs. An average of five antibody molecules per liposome significantly improves in cell cultures the performance of immunoliposomes over non-functionalized liposomes as drug delivery vessels. Increasing the number of antibodies on the liposome surface correspondingly increases performance, with a reduction of 50% parasitemia achieved with immunoliposomes encapsulating 4 nM chloroquine and bearing an estimated 250 BM1234 units. The nanovector prototype described here can be a valuable platform amenable to modification and improvement with the objective of designing a nanostructure adequate to enter the preclinical pipeline as a new antimalarial therapy.

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“…In contrast to interleukin-2 and interferon-gamma, which associate to liposomes by simple electrostatic, nontransforming interactions, rh-Epo binds with defined positively charged amino acids, as demonstrated by experiments with carbamylated rh-Epo (Koppenhagen et al, 1998a(Koppenhagen et al, , 1998bKuboi et al, 2000;Van Slooten et al, 2001;Zschörnig et al, 2005). Within these examinations, the positively charged lysines and arginines were neutralized by carbamylation, which results in the deprivation of the membrane-modulating activity of rh-Epo (Arcasoy, 2008;Brines et al, 2004;Leist et al, 2004;Tilbrook and Klinken, 1999;Wang et al, 2007).…”

Section: Resultsmentioning

confidence: 99%

Topological transformation of liposomes by a membrane-affecting domain of recombinant human erythropoietin

Strobach¹,

Kunert²,

Stadlmann³

et al. 2009

Journal of Liposome Research

0

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Recombinant human erythropoietin (rh-Epo) is well accepted as a hematopoietic drug, but many other pleiotropic properties are currently under investigation. Rh-Epo-induced receptor-mediated signal transductions are accompanied with membrane dynamic processes, which facilitate the activation of individual pathways. However, its direct effect on membrane dynamics is still unknown. In the present study, we have proven the capability of rh-Epo to associate to and transform artificial lipid membranes. Association studies using neutral, negatively, and positively charged liposomes with the native as well as modified rh-Epo were performed and analyzed by transmission electron microscopy and differential scanning calorimetry. By these studies, we demonstrated that rh-Epo has the capability to transform negatively charged unilamellar vesicles into so-called disc-like micelles. Rh-Epo association to the negatively charged head groups via lysine and arginine initiates this transformation. At physiological temperatures, conformational changes within the rh-Epo structure expose a defined amino-acid sequence, which is able to induce the formation of discoid membrane structures. Enzymatic digestion, analysis, and isolation of related peptides by rp-HPLC and characterization by MS/MS enabled the identification of the membrane-affecting domain of rh-Epo (MAD-E) that represents the exposed helix B of rh-Epo. Finally, association studies performed with these peptides confirmed that the MAD-E is responsible for the formation of disc-like micelles. Since this helix B of rh-Epo has recently been supposed to be involved in the activation of neuroprotective pathways, we believe that the membrane-transforming capacity of rh-Epo participates in the proliferative activity of rh-Epo.

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“…Through our findings, we conclude that both the head-group charge and the temperature are prerequisites for disc formation. In contrast to interleukin-2 and interferon-gamma, which associate to liposomes by simple electrostatic, nontransforming interactions, rh-Epo binds with defined positively charged amino acids, as demonstrated by experiments with carbamylated rh-Epo (Koppenhagen et al, 1998a, 1998b; Kuboi et al, 2000; Van Slooten et al, 2001; Zschörnig et al, 2005). Within these examinations, the positively charged lysines and arginines were neutralized by carbamylation, which results in the deprivation of the membrane-modulating activity of rh-Epo (Arcasoy, 2008; Brines et al, 2004; Leist et al, 2004; Tilbrook and Klinken, 1999; Wang et al, 2007).…”

Section: Resultsmentioning

confidence: 99%

Topological transformation of liposomes by a membrane-affecting domain of recombinant human erythropoietin

Strobach¹,

Kunert²,

Stadlmann³

et al. 2010

Journal of Liposome Research

1

0

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Recombinant human erythropoietin (rh-Epo) is well accepted as a hematopoietic drug, but many other pleiotropic properties are currently under investigation. Rh-Epo-induced receptor-mediated signal transductions are accompanied with membrane dynamic processes, which facilitate the activation of individual pathways. However, its direct effect on membrane dynamics is still unknown. In the present study, we have proven the capability of rh-Epo to associate to and transform artificial lipid membranes. Association studies using neutral, negatively, and positively charged liposomes with the native as well as modified rh-Epo were performed and analyzed by transmission electron microscopy and differential scanning calorimetry. By these studies, we demonstrated that rh-Epo has the capability to transform negatively charged unilamellar vesicles into so-called disc-like micelles. Rh-Epo association to the negatively charged head groups via lysine and arginine initiates this transformation. At physiological temperatures, conformational changes within the rh-Epo structure expose a defined amino-acid sequence, which is able to induce the formation of discoid membrane structures. Enzymatic digestion, analysis, and isolation of related peptides by rp-HPLC and characterization by MS/MS enabled the identification of the membrane-affecting domain of rh-Epo (MAD-E) that represents the exposed helix B of rh-Epo. Finally, association studies performed with these peptides confirmed that the MAD-E is responsible for the formation of disc-like micelles. Since this helix B of rh-Epo has recently been supposed to be involved in the activation of neuroprotective pathways, we believe that the membrane-transforming capacity of rh-Epo participates in the proliferative activity of rh-Epo.

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Development of a routine analysis method for liposome encapsulated recombinant interleukin-2

Cited by 12 publications

References 26 publications

Study of the efficacy of antimalarial drugs delivered inside targeted immunoliposomal nanovectors

Study of the efficacy of antimalarial drugs delivered inside targeted immunoliposomal nanovectors

Topological transformation of liposomes by a membrane-affecting domain of recombinant human erythropoietin

Topological transformation of liposomes by a membrane-affecting domain of recombinant human erythropoietin

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