37 research outputs found
3-D cultures as intermediary between 2-D cultures and animal models.
<p>Intermediate in complexity, 3-D cultures permit the systematic, high-throughput assessment of formulation properties in a controlled environment that approximates important properties of <i>in vivo</i> tumors in the absence of complex parameters which may confound data interpretation.</p
Codelivery of Paclitaxel and Everolimus at the Optimal Synergistic Ratio: A Promising Solution for the Treatment of Breast Cancer
Clinical
studies examining the combination of paclitaxel (PTX)
and everolimus (EVER), an mTOR inhibitor, have failed to result in
significant improvements in efficacy and toxicity in patients with
breast cancer (BC), relative to treatment with PTX alone. These disappointing
clinical trial results have been attributed to poorly designed preclinical
studies using the combination of PTX and EVER as well as the significantly
different pharmacokinetic profiles of the two drugs. In the current
work, the potential synergy between PTX and EVER was examined in a
panel of six BC cell lines that differ in terms of their molecular
subtype and drug sensitivity. Polymeric nanoparticles (NPs) were used
to encapsulate PTX and EVER at an optimal synergistic ratio to achieve
specific, colocalized delivery of the combination therapy in BC cell
lines. Combinations of PTX and EVER (especially at relatively high
doses of EVER) resulted in pronounced synergy in all BC cell lines
evaluated. The optimal molar ratio of PTX:EVER was determined to be
1:0.5. The combination was delivered to BC cells at the synergistic
ratio via encapsulation within polymeric NPs formed from the polyÂ(ethylene
glycol)-<i>b</i>-polyÂ(lactide-<i>co</i>-glycolide)
(PEG-<i>b</i>-PLGA) copolymer. The NPs had an average diameter
of less than 100 nm and were capable of in vitro retention of the
encapsulated PTX and EVER at the optimal synergistic molar ratio for
over 7 days. Cytotoxicity data demonstrated that PTX+EVER-loaded NPs
were significantly less cytotoxic than the free drug combination in
MCF-7 and SKBR3 BC cell lines following 72 h, suggesting that PTX+EVER-loaded
NPs remain stable and retain the drug combination loaded within the
core after 72 h. The uptake of FITC-labeled NPs in SKBR3 cells was
evaluated by flow cytometry, with approximately 41% of cells demonstrating
detectable fluorescence after 24 h of exposure. The thorough and systematic
approach used in this study to determine and evaluate a synergistic
PTX:EVER ratio in conjunction with a potentially promising delivery
vector for the drug combination could offer a future clinical benefit
for patients with BC
Spheroid packing density and growth.
<p>a) Cells per HeLa and HT29 spheroid of given volume, n = 12. b) Growth of HeLa and HT29 spheroids, n = 6. Data was fit using the Gompertz equation for tumor growth. The dashed lines indicate spheroid properties used in the studies.</p
Clonogenic potential of cells following treatment.
<p>Clonogenic survival of HeLa and HT29 cells following 24 h treatment with 20 ng/mL of BCM+DTX or Taxotere® as a) monolayers, b) disaggregated spheroids and c) intact spheroids.</p
Characterization of micelle morphology and size.
<p>a) Transmission electron micrograph (Scale bar in represents 100 nm) and b) size distribution of BCM+DTX as determined by dynamic light scattering at 37°C.</p
Spatial distribution of proliferating cells in spheroids.
<p>Ki67 positive signal distribution relative to radial position in a) HeLa and b) HT29 MCTS as a percent of total positive stain, n = 6.</p
Inhibition of spheroid growth.
<p>a) Sequential images of the same HeLa and HT29 spheroids following treatment with BCM+DTX at a concentration of 20 ng/mL. Bars represent 100 µm. Growth inhibition of HeLa (b,c) and HT29 (d,e) MCTS by BCM+DTX and <b>Taxotere</b>® at concentrations of 2, 20 and 200 ng/mL. Cells were re-treated after two weeks (arrow). Box represents expanded region of plots b) and d). Data is expressed as the mean volume of six spheroids (n = 6) ± SD. “*” represents a significant difference between BCM 20 and TAX 20, p<0.05.</p
Block Copolymer Micelles Target Auger Electron Radiotherapy to the Nucleus of HER2-Positive Breast Cancer Cells
Intracellular trafficking of Auger electron emitting
radionuclides
to perinuclear and nuclear regions of cells is critical to realizing
their full therapeutic potential. In the present study, block copolymer
micelles (BCMs) were labeled with the Auger electron emitter indium-111
(<sup>111</sup>In) and loaded with the radiosensitizer methotrexate.
HER2 specific antibodies (trastuzumab fab) and nuclear localization
signal (NLS; CGYGPKKKRKVGG) peptides were conjugated to the surface
of the BCMs to direct uptake in HER2 expressing cells and subsequent
localization in the cell nucleus. Cell uptake and intracellular distribution
of the multifunctional BCMs were evaluated in a panel of breast cancer
cell lines with different levels of HER2 expression. Indeed cell uptake
was found to be HER2 density dependent, confirming receptor-mediated
internalization of the BCMs. Importantly, conjugation of NLS peptides
to the surface of BCMs was found to result in a significant increase
in nuclear uptake of the radionuclide <sup>111</sup>In. Successful
nuclear targeting was shown to improve the antipoliferative effect
of the Auger electrons as measured by clonogenic assays. In addition,
a significant radiation enhancement effect was observed by concurrent
delivery of low-dose MTX and <sup>111</sup>In in all breast cancer
cell lines evaluated
Histological assessment of spheroid microenvironment.
<p>HeLa (a–c) and HT29 (d–f) MCTS cross-sections stained with H&E (a, d), Ki67 proliferation marker (b, e) and EF5 (c, f), a marker of hypoxia. Scale bars represent 100 µm. g) Properties of the spheroid microenvironment and their spatial distribution. “++”, “+”, and “–”, indicate high, intermediate and low levels of the corresponding feature, respectively.</p
Multicellular Tumor Spheroids for Evaluation of Cytotoxicity and Tumor Growth Inhibitory Effects of Nanomedicines <i>In Vitro</i>: A Comparison of Docetaxel-Loaded Block Copolymer Micelles and Taxotere®
<div><p>While 3-D tissue models have received increasing attention over the past several decades in the development of traditional anti-cancer therapies, their potential application for the evaluation of advanced drug delivery systems such as nanomedicines has been largely overlooked. In particular, new insight into drug resistance associated with the 3-D tumor microenvironment has called into question the validity of 2-D models for prediction of <i>in vivo</i> anti-tumor activity. In this work, a series of complementary assays was established for evaluating the <i>in vitro</i> efficacy of docetaxel (DTX) -loaded block copolymer micelles (BCM+DTX) and Taxotere® in 3-D multicellular tumor spheroid (MCTS) cultures. Spheroids were found to be significantly more resistant to treatment than monolayer cultures in a cell line dependent manner. Limitations in treatment efficacy were attributed to mechanisms of resistance associated with properties of the spheroid microenvironment. DTX-loaded micelles demonstrated greater therapeutic effect in both monolayer and spheroid cultures in comparison to Taxotere®. Overall, this work demonstrates the use of spheroids as a viable platform for the evaluation of nanomedicines in conditions which more closely reflect the <i>in vivo</i> tumor microenvironment relative to traditional monolayer cultures. By adaptation of traditional cell-based assays, spheroids have the potential to serve as intermediaries between traditional <i>in vitro</i> and <i>in vivo</i> models for high-throughput assessment of therapeutic candidates.</p></div