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Carboxymethyl Hyaluronan-Stabilized Nanoparticles for Anticancer Drug Delivery.

Woodman JL, Suh MS, Zhang J, Kondaveeti Y, Burgess DJ, White BA, Prestwich GD, Kuhn LT - Int J Cell Biol (2015)

Bottom Line: Carboxymethyl hyaluronic acid (CMHA) is a semisynthetic derivative of HA that is recognized by HA binding proteins but contains an additional carboxylic acid on some of the 6-hydroxyl groups of the N-acetyl glucosamine sugar units.Subcutaneous BT-474EMT tumors were more reproducibly inhibited by a near tumor dose of 2.8 mg/kg CDDP than a 7 mg/kg dose nCaP(CMHA)CDDP.This was likely due to a lack of distribution of nCaP(CMHA)CDDP throughout the dense tumor tissue that limited drug diffusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Materials Science and Engineering, University of Connecticut, Storrs, CT 06269, USA ; Department of Reconstructive Sciences, UConn Health, Farmington, CT 06030, USA.

ABSTRACT
Carboxymethyl hyaluronic acid (CMHA) is a semisynthetic derivative of HA that is recognized by HA binding proteins but contains an additional carboxylic acid on some of the 6-hydroxyl groups of the N-acetyl glucosamine sugar units. These studies tested the ability of CMHA to stabilize the formation of calcium phosphate nanoparticles and evaluated their potential to target therapy resistant, CD44(+)/CD24(-/low) human breast cancer cells (BT-474EMT). CMHA stabilized particles (nCaP(CMHA)) were loaded with the chemotherapy drug cis-diamminedichloroplatinum(II) (CDDP) to form nCaP(CMHA)CDDP. nCaP(CMHA)CDDP was determined to be poorly crystalline hydroxyapatite, 200 nm in diameter with a -43 mV zeta potential. nCaP(CMHA)CDDP exhibited a two-day burst release of CDDP that tapered resulting in 86% release by 7 days. Surface plasmon resonance showed that nCaP(CMHA)CDDP binds to CD44, but less effectively than CMHA or hyaluronan. nCaP(CMHA-AF488) was taken up by CD44(+)/CD24(-) BT-474EMT breast cancer cells within 18 hours. nCaP(CMHA)CDDP was as cytotoxic as free CDDP against the BT-474EMT cells. Subcutaneous BT-474EMT tumors were more reproducibly inhibited by a near tumor dose of 2.8 mg/kg CDDP than a 7 mg/kg dose nCaP(CMHA)CDDP. This was likely due to a lack of distribution of nCaP(CMHA)CDDP throughout the dense tumor tissue that limited drug diffusion.

No MeSH data available.


Related in: MedlinePlus

Cellular uptake study using BT-474EMT cells. Cells were plated and allowed to adhere for 24 hours, after which nCaPCMHA-AF488 was added at a concentration of 2 mg/mL. After 18 hours, nCaPCMHA-AF488 can clearly be seen within cells as confirmed by Z-stack confocal images. (a) Differential interference contrast (DIC) image, (b) cells stained with DAPI, (c) cells imaged containing nCaPCMHA-AF488, and (d) overlay of DAPI and AF488 images, showing nCaPCMHA-AF488 uptake.
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fig6: Cellular uptake study using BT-474EMT cells. Cells were plated and allowed to adhere for 24 hours, after which nCaPCMHA-AF488 was added at a concentration of 2 mg/mL. After 18 hours, nCaPCMHA-AF488 can clearly be seen within cells as confirmed by Z-stack confocal images. (a) Differential interference contrast (DIC) image, (b) cells stained with DAPI, (c) cells imaged containing nCaPCMHA-AF488, and (d) overlay of DAPI and AF488 images, showing nCaPCMHA-AF488 uptake.

Mentions: Flow cytometry analysis confirmed BT-474 are 1.71% positive for both CD44 and CD24 and the majority of cells stained positively for CD24 alone, 98.3% (Figures 5(a)–5(c)). These cells were thus a good negative control for CD44 targeting. BT-474EMT cells stained 99.7% positive for CD44 and negative for CD24, which corresponds well to the phenotype of therapy resistant breast cancer cells in the literature, CD44+/CD24−/low (Figures 5(d)–5(f)) [11, 44]. BT-474EMT cells were therefore used as the CD44+ test groups of cells to examine CD44 specificity with cellular uptake and cytotoxicity of nCaPCMHA-AF488. In the cell uptake studies no significant uptake was determined for the 200 μg/mL or 1 mg/mL concentrations at any time tested (images not shown). At the 2 mg/mL dose, significant cellular uptake was determined at 18 hours posttreatment (Figures 6(a)–6(d)). Z-stack images were obtained, confirming the nCaPCMHA-AF488 was within the cell with nuclei counterstained with DAPI. This was a preliminary test of cellular uptake. These were insufficient to prove that nCaPCMHA-AF488 cellular uptake was mediated by CD44. To do so, at least two controls are necessary, a CD44− cell type and a CD44+ cell type pretreated with HA to saturate the CD44 receptors [45] and should be included in future studies. SPR showed nCaPCMHACDDP had lower binding than CMHA, which is likely due to two factors. Only 30% of the 4 mg/mL CMHA in the precipitation is incorporated into nCaPCMHA. Additionally, nCaPCMHACDDP is stored as a suspension which allows the CaP to undergo Ostwald ripening incorporating much of the CMHA within the nCaP core [46, 47]. The exposure time required for cellular uptake of nCaPCMHA-AF488 was more than four times that of mesoporous silica nanoparticles targeted to CD44 via HA, where a 200 kDa HA was used [15].


Carboxymethyl Hyaluronan-Stabilized Nanoparticles for Anticancer Drug Delivery.

Woodman JL, Suh MS, Zhang J, Kondaveeti Y, Burgess DJ, White BA, Prestwich GD, Kuhn LT - Int J Cell Biol (2015)

Cellular uptake study using BT-474EMT cells. Cells were plated and allowed to adhere for 24 hours, after which nCaPCMHA-AF488 was added at a concentration of 2 mg/mL. After 18 hours, nCaPCMHA-AF488 can clearly be seen within cells as confirmed by Z-stack confocal images. (a) Differential interference contrast (DIC) image, (b) cells stained with DAPI, (c) cells imaged containing nCaPCMHA-AF488, and (d) overlay of DAPI and AF488 images, showing nCaPCMHA-AF488 uptake.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4581577&req=5

fig6: Cellular uptake study using BT-474EMT cells. Cells were plated and allowed to adhere for 24 hours, after which nCaPCMHA-AF488 was added at a concentration of 2 mg/mL. After 18 hours, nCaPCMHA-AF488 can clearly be seen within cells as confirmed by Z-stack confocal images. (a) Differential interference contrast (DIC) image, (b) cells stained with DAPI, (c) cells imaged containing nCaPCMHA-AF488, and (d) overlay of DAPI and AF488 images, showing nCaPCMHA-AF488 uptake.
Mentions: Flow cytometry analysis confirmed BT-474 are 1.71% positive for both CD44 and CD24 and the majority of cells stained positively for CD24 alone, 98.3% (Figures 5(a)–5(c)). These cells were thus a good negative control for CD44 targeting. BT-474EMT cells stained 99.7% positive for CD44 and negative for CD24, which corresponds well to the phenotype of therapy resistant breast cancer cells in the literature, CD44+/CD24−/low (Figures 5(d)–5(f)) [11, 44]. BT-474EMT cells were therefore used as the CD44+ test groups of cells to examine CD44 specificity with cellular uptake and cytotoxicity of nCaPCMHA-AF488. In the cell uptake studies no significant uptake was determined for the 200 μg/mL or 1 mg/mL concentrations at any time tested (images not shown). At the 2 mg/mL dose, significant cellular uptake was determined at 18 hours posttreatment (Figures 6(a)–6(d)). Z-stack images were obtained, confirming the nCaPCMHA-AF488 was within the cell with nuclei counterstained with DAPI. This was a preliminary test of cellular uptake. These were insufficient to prove that nCaPCMHA-AF488 cellular uptake was mediated by CD44. To do so, at least two controls are necessary, a CD44− cell type and a CD44+ cell type pretreated with HA to saturate the CD44 receptors [45] and should be included in future studies. SPR showed nCaPCMHACDDP had lower binding than CMHA, which is likely due to two factors. Only 30% of the 4 mg/mL CMHA in the precipitation is incorporated into nCaPCMHA. Additionally, nCaPCMHACDDP is stored as a suspension which allows the CaP to undergo Ostwald ripening incorporating much of the CMHA within the nCaP core [46, 47]. The exposure time required for cellular uptake of nCaPCMHA-AF488 was more than four times that of mesoporous silica nanoparticles targeted to CD44 via HA, where a 200 kDa HA was used [15].

Bottom Line: Carboxymethyl hyaluronic acid (CMHA) is a semisynthetic derivative of HA that is recognized by HA binding proteins but contains an additional carboxylic acid on some of the 6-hydroxyl groups of the N-acetyl glucosamine sugar units.Subcutaneous BT-474EMT tumors were more reproducibly inhibited by a near tumor dose of 2.8 mg/kg CDDP than a 7 mg/kg dose nCaP(CMHA)CDDP.This was likely due to a lack of distribution of nCaP(CMHA)CDDP throughout the dense tumor tissue that limited drug diffusion.

View Article: PubMed Central - PubMed

Affiliation: Department of Materials Science and Engineering, University of Connecticut, Storrs, CT 06269, USA ; Department of Reconstructive Sciences, UConn Health, Farmington, CT 06030, USA.

ABSTRACT
Carboxymethyl hyaluronic acid (CMHA) is a semisynthetic derivative of HA that is recognized by HA binding proteins but contains an additional carboxylic acid on some of the 6-hydroxyl groups of the N-acetyl glucosamine sugar units. These studies tested the ability of CMHA to stabilize the formation of calcium phosphate nanoparticles and evaluated their potential to target therapy resistant, CD44(+)/CD24(-/low) human breast cancer cells (BT-474EMT). CMHA stabilized particles (nCaP(CMHA)) were loaded with the chemotherapy drug cis-diamminedichloroplatinum(II) (CDDP) to form nCaP(CMHA)CDDP. nCaP(CMHA)CDDP was determined to be poorly crystalline hydroxyapatite, 200 nm in diameter with a -43 mV zeta potential. nCaP(CMHA)CDDP exhibited a two-day burst release of CDDP that tapered resulting in 86% release by 7 days. Surface plasmon resonance showed that nCaP(CMHA)CDDP binds to CD44, but less effectively than CMHA or hyaluronan. nCaP(CMHA-AF488) was taken up by CD44(+)/CD24(-) BT-474EMT breast cancer cells within 18 hours. nCaP(CMHA)CDDP was as cytotoxic as free CDDP against the BT-474EMT cells. Subcutaneous BT-474EMT tumors were more reproducibly inhibited by a near tumor dose of 2.8 mg/kg CDDP than a 7 mg/kg dose nCaP(CMHA)CDDP. This was likely due to a lack of distribution of nCaP(CMHA)CDDP throughout the dense tumor tissue that limited drug diffusion.

No MeSH data available.


Related in: MedlinePlus