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Effect of PEG pairing on the efficiency of cancer-targeting liposomes.

Saw PE, Park J, Lee E, Ahn S, Lee J, Kim H, Kim J, Choi M, Farokhzad OC, Jon S - Theranostics (2015)

Bottom Line: A class of high-affinity peptides (aptides) specific to extra domain B of fibronectin (APT(EDB)) was used as a representative model for a cancer-targeting ligand.Furthermore, in a U87MG xenograft model, APT(EDB)‑PEG(2000))/PEG(2000)) liposomes retarded tumor growth to the greatest extent, followed closely by APT(EDB)‑PEG(2000))/PEG(2000)) liposomes.Among the PEGylated liposomes tested, pairs in which the methoxy-capped PEG length was about half that of the targeting ligand-displaying PEG exhibited the best performance, suggesting that PEG pairing is a key consideration in the design of drug-delivery vehicles.

View Article: PubMed Central - PubMed

Affiliation: 1. KAIST Institute for the BioCentury, Department of Biological Sciences, Korea Advanced Institute of Science and technology (KAIST), 291 Daehak-ro, Daejeon 305-701, Republic of Korea. ; 2. Laboratory of Nanomedicine and Biomaterials, Department of Anesthesiology, Brigham and Women's Hospital Harvard Medical School, Boston, MA 02115, U.S.A.

ABSTRACT
Standardized poly(ethylene glycol)-modified (PEGylated) liposomes, which have been widely used in research as well as in pre-clinical and clinical studies, are typically constructed using PEG with a molecular weight of 2000 Da (PEG(2000)). Targeting ligands are also generally conjugated using various functionalized PEG(2000)). However, although standardized protocols have routinely used PEG(2000), it is not because this molecular weight PEG has been optimized to enhance tumor uptake of nanoparticles. Herein, we investigated the effect of various PEG lipid pairings--that is, PEG lipids for targeting-ligand conjugation and PEG lipids for achieving 'stealth' function--on in vitro cancer cell- and in vivo tumor-targeting efficacy. A class of high-affinity peptides (aptides) specific to extra domain B of fibronectin (APT(EDB)) was used as a representative model for a cancer-targeting ligand. We synthesized a set of aptide-conjugated PEGylated phospholipids (APT(EDB)‑PEG(2000))‑DSPE and APT(EDB)‑PEG(2000))‑DSPE) and then paired them with methoxy-capped PEGylated phospholipids with diverse molecular weights (PEG(2000)), PEG(2000)), PEG(2000)), and PEG(2000))) to construct various aptide-conjugated PEGylated liposomes. The liposomes with APT(EDB)‑PEG(2000))/PEG(2000)) and APT(EDB)‑PEG(2000))/PEG(2000)) pairings had the highest uptake in EDB-positive cancer cells. Furthermore, in a U87MG xenograft model, APT(EDB)‑PEG(2000))/PEG(2000)) liposomes retarded tumor growth to the greatest extent, followed closely by APT(EDB)‑PEG(2000))/PEG(2000)) liposomes. Among the PEGylated liposomes tested, pairs in which the methoxy-capped PEG length was about half that of the targeting ligand-displaying PEG exhibited the best performance, suggesting that PEG pairing is a key consideration in the design of drug-delivery vehicles.

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In vitro cellular uptake of all liposomal systems in U87MG cells. (a) Uptake of each liposomal formulation in U87MG cells in vitro as determined by confocal microscopy. U87MG cells were co-incubated with a respective liposomal system for 1 h in serum containing medium, washed and fixed prior to confocal imaging. APTEDB-PEG2000/PEG1000 LS showed the highest cellular uptake among formulations in the APTEDB-PEG2000-DSPE set (left column), and APTEDB-PEG1000/PEG550 LS showed the highest cellular uptake among formulations in the APTEDB-PEG1000-DSPE set (right column). (b) Image J quantitative analysis on uptake efficiency of each liposomal system. Five confocal images were taken from each liposomal formulation and the mean value of rhodamine intensity was calculated for the respective liposome system. Statistical significance was calculated using Student's T test; with standard deviation (S.D.) being used in calculation.
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Figure 2: In vitro cellular uptake of all liposomal systems in U87MG cells. (a) Uptake of each liposomal formulation in U87MG cells in vitro as determined by confocal microscopy. U87MG cells were co-incubated with a respective liposomal system for 1 h in serum containing medium, washed and fixed prior to confocal imaging. APTEDB-PEG2000/PEG1000 LS showed the highest cellular uptake among formulations in the APTEDB-PEG2000-DSPE set (left column), and APTEDB-PEG1000/PEG550 LS showed the highest cellular uptake among formulations in the APTEDB-PEG1000-DSPE set (right column). (b) Image J quantitative analysis on uptake efficiency of each liposomal system. Five confocal images were taken from each liposomal formulation and the mean value of rhodamine intensity was calculated for the respective liposome system. Statistical significance was calculated using Student's T test; with standard deviation (S.D.) being used in calculation.

Mentions: Cellular uptake for all the prepared liposomal formulations was assessed in U87MG cells, a human glioblastoma cell line known to overexpress EDB.24 The expression levels of EDB in these cell lines studied were confirmed by conventional reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), and immunocytohistochemistry (Fig. S5). To qualitatively evaluate the uptake efficiency, we fluorescently labeled all the liposomes by incorporating 0.5% rhodamine-modified phospholipid during the liposome preparation. As shown in Fig. 2a, the uptake of APTEDB-PEG2000/PEG1000 LS was higher than that of APTEDB-PEG2000/PEG2000 LS as well as APTEDB-PEG2000/PEG550 LS and APTEDB-PEG2000/PEG350 LS. As for the right column, the APTEDB-PEG1000/PEG550 LS had the highest uptake compared with both APTEDB-PEG1000/PEG1000 LS and APTEDB-PEG1000/PEG350 LS. Quantification of these images with Image J further confirmed that the increase in uptake of APTEDB-PEG2000/PEG1000 LS and APTEDB-PEG1000/PEG550 LS were statistically significant compared to the other groups (Fig. 2b). These results indicate that liposomes containing a methoxy-capped PEG lipid shorter than the targeting ligand-displaying PEG lipid have a higher cellular uptake than PEG lipid pairs of equal length. Interestingly, both APTEDB-PEG2000/PEG1000 LS and APTEDB-PEG1000/PEG550 LS in which the length of the methoxy-capped PEG lipids was nearly half that of the ligand-displaying PEG lipid had a much higher cellular uptake than that of the other combinations.


Effect of PEG pairing on the efficiency of cancer-targeting liposomes.

Saw PE, Park J, Lee E, Ahn S, Lee J, Kim H, Kim J, Choi M, Farokhzad OC, Jon S - Theranostics (2015)

In vitro cellular uptake of all liposomal systems in U87MG cells. (a) Uptake of each liposomal formulation in U87MG cells in vitro as determined by confocal microscopy. U87MG cells were co-incubated with a respective liposomal system for 1 h in serum containing medium, washed and fixed prior to confocal imaging. APTEDB-PEG2000/PEG1000 LS showed the highest cellular uptake among formulations in the APTEDB-PEG2000-DSPE set (left column), and APTEDB-PEG1000/PEG550 LS showed the highest cellular uptake among formulations in the APTEDB-PEG1000-DSPE set (right column). (b) Image J quantitative analysis on uptake efficiency of each liposomal system. Five confocal images were taken from each liposomal formulation and the mean value of rhodamine intensity was calculated for the respective liposome system. Statistical significance was calculated using Student's T test; with standard deviation (S.D.) being used in calculation.
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Related In: Results  -  Collection

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Figure 2: In vitro cellular uptake of all liposomal systems in U87MG cells. (a) Uptake of each liposomal formulation in U87MG cells in vitro as determined by confocal microscopy. U87MG cells were co-incubated with a respective liposomal system for 1 h in serum containing medium, washed and fixed prior to confocal imaging. APTEDB-PEG2000/PEG1000 LS showed the highest cellular uptake among formulations in the APTEDB-PEG2000-DSPE set (left column), and APTEDB-PEG1000/PEG550 LS showed the highest cellular uptake among formulations in the APTEDB-PEG1000-DSPE set (right column). (b) Image J quantitative analysis on uptake efficiency of each liposomal system. Five confocal images were taken from each liposomal formulation and the mean value of rhodamine intensity was calculated for the respective liposome system. Statistical significance was calculated using Student's T test; with standard deviation (S.D.) being used in calculation.
Mentions: Cellular uptake for all the prepared liposomal formulations was assessed in U87MG cells, a human glioblastoma cell line known to overexpress EDB.24 The expression levels of EDB in these cell lines studied were confirmed by conventional reverse transcription-polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), and immunocytohistochemistry (Fig. S5). To qualitatively evaluate the uptake efficiency, we fluorescently labeled all the liposomes by incorporating 0.5% rhodamine-modified phospholipid during the liposome preparation. As shown in Fig. 2a, the uptake of APTEDB-PEG2000/PEG1000 LS was higher than that of APTEDB-PEG2000/PEG2000 LS as well as APTEDB-PEG2000/PEG550 LS and APTEDB-PEG2000/PEG350 LS. As for the right column, the APTEDB-PEG1000/PEG550 LS had the highest uptake compared with both APTEDB-PEG1000/PEG1000 LS and APTEDB-PEG1000/PEG350 LS. Quantification of these images with Image J further confirmed that the increase in uptake of APTEDB-PEG2000/PEG1000 LS and APTEDB-PEG1000/PEG550 LS were statistically significant compared to the other groups (Fig. 2b). These results indicate that liposomes containing a methoxy-capped PEG lipid shorter than the targeting ligand-displaying PEG lipid have a higher cellular uptake than PEG lipid pairs of equal length. Interestingly, both APTEDB-PEG2000/PEG1000 LS and APTEDB-PEG1000/PEG550 LS in which the length of the methoxy-capped PEG lipids was nearly half that of the ligand-displaying PEG lipid had a much higher cellular uptake than that of the other combinations.

Bottom Line: A class of high-affinity peptides (aptides) specific to extra domain B of fibronectin (APT(EDB)) was used as a representative model for a cancer-targeting ligand.Furthermore, in a U87MG xenograft model, APT(EDB)‑PEG(2000))/PEG(2000)) liposomes retarded tumor growth to the greatest extent, followed closely by APT(EDB)‑PEG(2000))/PEG(2000)) liposomes.Among the PEGylated liposomes tested, pairs in which the methoxy-capped PEG length was about half that of the targeting ligand-displaying PEG exhibited the best performance, suggesting that PEG pairing is a key consideration in the design of drug-delivery vehicles.

View Article: PubMed Central - PubMed

Affiliation: 1. KAIST Institute for the BioCentury, Department of Biological Sciences, Korea Advanced Institute of Science and technology (KAIST), 291 Daehak-ro, Daejeon 305-701, Republic of Korea. ; 2. Laboratory of Nanomedicine and Biomaterials, Department of Anesthesiology, Brigham and Women's Hospital Harvard Medical School, Boston, MA 02115, U.S.A.

ABSTRACT
Standardized poly(ethylene glycol)-modified (PEGylated) liposomes, which have been widely used in research as well as in pre-clinical and clinical studies, are typically constructed using PEG with a molecular weight of 2000 Da (PEG(2000)). Targeting ligands are also generally conjugated using various functionalized PEG(2000)). However, although standardized protocols have routinely used PEG(2000), it is not because this molecular weight PEG has been optimized to enhance tumor uptake of nanoparticles. Herein, we investigated the effect of various PEG lipid pairings--that is, PEG lipids for targeting-ligand conjugation and PEG lipids for achieving 'stealth' function--on in vitro cancer cell- and in vivo tumor-targeting efficacy. A class of high-affinity peptides (aptides) specific to extra domain B of fibronectin (APT(EDB)) was used as a representative model for a cancer-targeting ligand. We synthesized a set of aptide-conjugated PEGylated phospholipids (APT(EDB)‑PEG(2000))‑DSPE and APT(EDB)‑PEG(2000))‑DSPE) and then paired them with methoxy-capped PEGylated phospholipids with diverse molecular weights (PEG(2000)), PEG(2000)), PEG(2000)), and PEG(2000))) to construct various aptide-conjugated PEGylated liposomes. The liposomes with APT(EDB)‑PEG(2000))/PEG(2000)) and APT(EDB)‑PEG(2000))/PEG(2000)) pairings had the highest uptake in EDB-positive cancer cells. Furthermore, in a U87MG xenograft model, APT(EDB)‑PEG(2000))/PEG(2000)) liposomes retarded tumor growth to the greatest extent, followed closely by APT(EDB)‑PEG(2000))/PEG(2000)) liposomes. Among the PEGylated liposomes tested, pairs in which the methoxy-capped PEG length was about half that of the targeting ligand-displaying PEG exhibited the best performance, suggesting that PEG pairing is a key consideration in the design of drug-delivery vehicles.

Show MeSH
Related in: MedlinePlus