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Design and Evaluation of Tumor-Specific Dendrimer Epigenetic Therapeutics.

Zong H, Shah D, Selwa K, Tsuchida RE, Rattan R, Mohan J, Stein AB, Otis JB, Goonewardena SN - ChemistryOpen (2015)

Bottom Line: Here we report the design and evaluation of tumor-specific dendrimer-HDACi conjugates.The HDACi was conjugated to the dendrimer using an ester linkage through its hydroxamic acid group, inactivating the HDACi until it is released from the dendrimer.Furthermore, we demonstrate that unlike traditional HDACi, dendrimer-HDACi conjugates do not affect tumor-associated macrophages, a recently recognized mechanism through which drug resistance emerges.

View Article: PubMed Central - PubMed

Affiliation: Michigan Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan, Room 9220C MSRBIII 1150 W. Medical Center Drive, Ann Arbor, MI, 48109, USA).

ABSTRACT
Histone deacetylase inhibitors (HDACi) are promising therapeutics for cancer. HDACi alter the epigenetic state of tumors and provide a unique approach to treat cancer. Although studies with HDACi have shown promise in some cancers, variable efficacy and off-target effects have limited their use. To overcome some of the challenges of traditional HDACi, we sought to use a tumor-specific dendrimer scaffold to deliver HDACi directly to cancer cells. Here we report the design and evaluation of tumor-specific dendrimer-HDACi conjugates. The HDACi was conjugated to the dendrimer using an ester linkage through its hydroxamic acid group, inactivating the HDACi until it is released from the dendrimer. Using a cancer cell model, we demonstrate the functionality of the tumor-specific dendrimer-HDACi conjugates. Furthermore, we demonstrate that unlike traditional HDACi, dendrimer-HDACi conjugates do not affect tumor-associated macrophages, a recently recognized mechanism through which drug resistance emerges. We anticipate that this new class of cell-specific epigenetic therapeutics will have tremendous potential in the treatment of cancer.

No MeSH data available.


Related in: MedlinePlus

Biological evaluation of the dendrimer SAHA conjugates. a)  Mean fluorescence intensity (MFI) demonstrates concentration-dependent uptake of the G5-FA-eSAHA compared to other conjugates. Pretreatment of KB cells with free FA blocked this uptake confirming the FR specificity of the targeted dendrimer conjugates. KB cells were incubated with dendrimer SAHA conjugates for 1 h and analyzed by flow cytometry for cell-specific uptake. Values represent the mean ± S.E.M. for n=3 independent experiments. b) Confocal images using the in situ click detection methodology confirm the cell specificity of the FA-targeted dendrimer conjugates (punctate, green cytoplasmic signal; left panel) compared with the nontargeted controls (no cytoplasmic signal; right panel). KB cells were incubated with 100 nm of the dendrimer SAHA conjugates for 1 h and subsequently analyzed using confocal microscopy. c) KB cells treated with the targeted G5-FA-eSAHA showed significantly greater levels of apoptosis than KB cells treated with the nontargeted G5-eSAHA. KB cells were treated with dendrimer–SAHA conjugates for 5 d and assessed for apoptosis using the annexin/7AAD assay. Values represent the mean ± S.E.M. for n=3 independent experiments.
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fig02: Biological evaluation of the dendrimer SAHA conjugates. a)  Mean fluorescence intensity (MFI) demonstrates concentration-dependent uptake of the G5-FA-eSAHA compared to other conjugates. Pretreatment of KB cells with free FA blocked this uptake confirming the FR specificity of the targeted dendrimer conjugates. KB cells were incubated with dendrimer SAHA conjugates for 1 h and analyzed by flow cytometry for cell-specific uptake. Values represent the mean ± S.E.M. for n=3 independent experiments. b) Confocal images using the in situ click detection methodology confirm the cell specificity of the FA-targeted dendrimer conjugates (punctate, green cytoplasmic signal; left panel) compared with the nontargeted controls (no cytoplasmic signal; right panel). KB cells were incubated with 100 nm of the dendrimer SAHA conjugates for 1 h and subsequently analyzed using confocal microscopy. c) KB cells treated with the targeted G5-FA-eSAHA showed significantly greater levels of apoptosis than KB cells treated with the nontargeted G5-eSAHA. KB cells were treated with dendrimer–SAHA conjugates for 5 d and assessed for apoptosis using the annexin/7AAD assay. Values represent the mean ± S.E.M. for n=3 independent experiments.

Mentions: Because only the SAHA-eAzide 1 demonstrated therapeutic efficacy, further evaluations were conducted using only the ester-modified SAHA compound. Next, we wanted to compare the cell specificity of the tumor-specific dendrimer–SAHA conjugates. We and others have shown that FA-functionalized dendrimers internalize through the FR in vitro and in vivo.14 Using our recently described in situ click reporter strategy, we monitored the uptake of the FA-targeted and nontargeted control dendrimer–SAHA conjugates by flow cytometry.14b This strategy allows us to track dendrimer therapeutics after they have been delivered to cells by using CuAAC to conjugate fluorescent reporters in situ. We incubated G5-FA-eSAHA-Alkyne 6 and the nontargeted control G5-eSAHA-Alkyne 5 with KB cells for one hour at 37 °C. After incubation, the cells were washed and processed, and Alexa Fluor 647-azide (AF647) was conjugated to the internalized dendrimer conjugates using the CuAAC reaction as we have previously described.14b The G5-FA-eSAHA-Alkyne 6 demonstrated a dose-dependent uptake into KB cells while the nontargeted G5-eSAHA-Alkyne 5 did not demonstrate any uptake (Figure 2). Importantly, the uptake of G5-FA-eSAHA-Alkyne 6 could be blocked by pretreatment with 100 μm FA, confirming the FR-specificity of the tumor-targeted dendrimer–HDACi (Figure 2). To further confirm the cell specificity of the dendrimer conjugates, we used our in situ click reporter strategy and confocal microscopy to demonstrate the FR-dependent uptake of the targeted-dendrimer SAHA conjugates. KB cells were treated as described above for one hour. After incubation, the KB cells were processed, and Alexa Fluor 555-azide (AF555) was conjugated to the internalized dendrimer conjugates using the CuAAC reaction. The G5-FA-eSAHA-Alkyne 6 had significantly more uptake at one hour compared with the nontargeted G5-eSAHA-Alkyne 5 (Figure 2).


Design and Evaluation of Tumor-Specific Dendrimer Epigenetic Therapeutics.

Zong H, Shah D, Selwa K, Tsuchida RE, Rattan R, Mohan J, Stein AB, Otis JB, Goonewardena SN - ChemistryOpen (2015)

Biological evaluation of the dendrimer SAHA conjugates. a)  Mean fluorescence intensity (MFI) demonstrates concentration-dependent uptake of the G5-FA-eSAHA compared to other conjugates. Pretreatment of KB cells with free FA blocked this uptake confirming the FR specificity of the targeted dendrimer conjugates. KB cells were incubated with dendrimer SAHA conjugates for 1 h and analyzed by flow cytometry for cell-specific uptake. Values represent the mean ± S.E.M. for n=3 independent experiments. b) Confocal images using the in situ click detection methodology confirm the cell specificity of the FA-targeted dendrimer conjugates (punctate, green cytoplasmic signal; left panel) compared with the nontargeted controls (no cytoplasmic signal; right panel). KB cells were incubated with 100 nm of the dendrimer SAHA conjugates for 1 h and subsequently analyzed using confocal microscopy. c) KB cells treated with the targeted G5-FA-eSAHA showed significantly greater levels of apoptosis than KB cells treated with the nontargeted G5-eSAHA. KB cells were treated with dendrimer–SAHA conjugates for 5 d and assessed for apoptosis using the annexin/7AAD assay. Values represent the mean ± S.E.M. for n=3 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig02: Biological evaluation of the dendrimer SAHA conjugates. a)  Mean fluorescence intensity (MFI) demonstrates concentration-dependent uptake of the G5-FA-eSAHA compared to other conjugates. Pretreatment of KB cells with free FA blocked this uptake confirming the FR specificity of the targeted dendrimer conjugates. KB cells were incubated with dendrimer SAHA conjugates for 1 h and analyzed by flow cytometry for cell-specific uptake. Values represent the mean ± S.E.M. for n=3 independent experiments. b) Confocal images using the in situ click detection methodology confirm the cell specificity of the FA-targeted dendrimer conjugates (punctate, green cytoplasmic signal; left panel) compared with the nontargeted controls (no cytoplasmic signal; right panel). KB cells were incubated with 100 nm of the dendrimer SAHA conjugates for 1 h and subsequently analyzed using confocal microscopy. c) KB cells treated with the targeted G5-FA-eSAHA showed significantly greater levels of apoptosis than KB cells treated with the nontargeted G5-eSAHA. KB cells were treated with dendrimer–SAHA conjugates for 5 d and assessed for apoptosis using the annexin/7AAD assay. Values represent the mean ± S.E.M. for n=3 independent experiments.
Mentions: Because only the SAHA-eAzide 1 demonstrated therapeutic efficacy, further evaluations were conducted using only the ester-modified SAHA compound. Next, we wanted to compare the cell specificity of the tumor-specific dendrimer–SAHA conjugates. We and others have shown that FA-functionalized dendrimers internalize through the FR in vitro and in vivo.14 Using our recently described in situ click reporter strategy, we monitored the uptake of the FA-targeted and nontargeted control dendrimer–SAHA conjugates by flow cytometry.14b This strategy allows us to track dendrimer therapeutics after they have been delivered to cells by using CuAAC to conjugate fluorescent reporters in situ. We incubated G5-FA-eSAHA-Alkyne 6 and the nontargeted control G5-eSAHA-Alkyne 5 with KB cells for one hour at 37 °C. After incubation, the cells were washed and processed, and Alexa Fluor 647-azide (AF647) was conjugated to the internalized dendrimer conjugates using the CuAAC reaction as we have previously described.14b The G5-FA-eSAHA-Alkyne 6 demonstrated a dose-dependent uptake into KB cells while the nontargeted G5-eSAHA-Alkyne 5 did not demonstrate any uptake (Figure 2). Importantly, the uptake of G5-FA-eSAHA-Alkyne 6 could be blocked by pretreatment with 100 μm FA, confirming the FR-specificity of the tumor-targeted dendrimer–HDACi (Figure 2). To further confirm the cell specificity of the dendrimer conjugates, we used our in situ click reporter strategy and confocal microscopy to demonstrate the FR-dependent uptake of the targeted-dendrimer SAHA conjugates. KB cells were treated as described above for one hour. After incubation, the KB cells were processed, and Alexa Fluor 555-azide (AF555) was conjugated to the internalized dendrimer conjugates using the CuAAC reaction. The G5-FA-eSAHA-Alkyne 6 had significantly more uptake at one hour compared with the nontargeted G5-eSAHA-Alkyne 5 (Figure 2).

Bottom Line: Here we report the design and evaluation of tumor-specific dendrimer-HDACi conjugates.The HDACi was conjugated to the dendrimer using an ester linkage through its hydroxamic acid group, inactivating the HDACi until it is released from the dendrimer.Furthermore, we demonstrate that unlike traditional HDACi, dendrimer-HDACi conjugates do not affect tumor-associated macrophages, a recently recognized mechanism through which drug resistance emerges.

View Article: PubMed Central - PubMed

Affiliation: Michigan Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan, Room 9220C MSRBIII 1150 W. Medical Center Drive, Ann Arbor, MI, 48109, USA).

ABSTRACT
Histone deacetylase inhibitors (HDACi) are promising therapeutics for cancer. HDACi alter the epigenetic state of tumors and provide a unique approach to treat cancer. Although studies with HDACi have shown promise in some cancers, variable efficacy and off-target effects have limited their use. To overcome some of the challenges of traditional HDACi, we sought to use a tumor-specific dendrimer scaffold to deliver HDACi directly to cancer cells. Here we report the design and evaluation of tumor-specific dendrimer-HDACi conjugates. The HDACi was conjugated to the dendrimer using an ester linkage through its hydroxamic acid group, inactivating the HDACi until it is released from the dendrimer. Using a cancer cell model, we demonstrate the functionality of the tumor-specific dendrimer-HDACi conjugates. Furthermore, we demonstrate that unlike traditional HDACi, dendrimer-HDACi conjugates do not affect tumor-associated macrophages, a recently recognized mechanism through which drug resistance emerges. We anticipate that this new class of cell-specific epigenetic therapeutics will have tremendous potential in the treatment of cancer.

No MeSH data available.


Related in: MedlinePlus