A Nanoparticle Cocktail: Temporal Release of Predefined Drug Combinations

A single magic bullet is not enough for treatment of metastatic cancers. However, administration of a combination of free drugs can be extremely challenging because of the inability to control the correct choice of dosages and definitive delivery of the effective drug ratio at the target tissue due to the differences in pharmacokinetics and biodistribution of individual drugs. Here we report an engineered biodegradable polymer containing combination therapeutics that can be self-assembled into a controlled release nanoparticle with abilities to deliver multiple therapeutics in a predefined ratio following temporal release patterns. This platform technology can lead to a rationally designed combination therapy

<i>Ex Vivo</i> Programming of Dendritic Cells by Mitochondria-Targeted Nanoparticles to Produce Interferon-Gamma for Cancer Immunotherapy

One of the limitations for clinical applications of dendritic cell (DC)-based cancer immunotherapy is the low potency in generating tumor antigen specific T cell responses. We examined the immunotherapeutic potential of a mitochondria-targeted nanoparticle (NP) based on a biodegradable polymer and zinc phthalocyanine (ZnPc) photosensitizer (T-ZnPc-NPs). Here, we report that tumor antigens generated from treatment of breast cancer cells with T-ZnPc-NPs upon light stimulation activate DCs to produce high levels of interferon-gamma, an important cytokine considered as a product of T and natural killer cells. The remarkable <i>ex vivo</i> DC stimulation ability of this tumor cell supernatant is a result of an interleukin (IL)-12/IL-18 autocrine effect. These findings contribute to the understanding of how <i>in situ</i> light activation amplifies the host immune responses when NPs deliver the photosensitizer to the mitochondria and open up the possibility of using mitochondria-targeted-NP-treated, light-activated cancer cell supernatants as possible vaccines

Mito-DCA: A Mitochondria Targeted Molecular Scaffold for Efficacious Delivery of Metabolic Modulator Dichloroacetate

Tumor growth is fueled by the use of glycolysis, which normal cells use only in the scarcity of oxygen. Glycolysis makes tumor cells resistant to normal death processes. Targeting this unique tumor metabolism can provide an alternative strategy to selectively destroy the tumor, leaving normal tissue unharmed. The orphan drug dichloroacetate (DCA) is a mitochondrial kinase inhibitor that has the ability to show such characteristics. However, its molecular form shows poor uptake and bioavailability and limited ability to reach its target mitochondria. Here, we describe a targeted molecular scaffold for construction of a multiple DCA loaded compound, Mito-DCA, with three orders of magnitude enhanced potency and cancer cell specificity compared to DCA. Incorporation of a lipophilic triphenylphosphonium cation through a biodegradable linker in Mito-DCA allowed for mitochondria targeting. Mito-DCA did not show any significant metabolic effects toward normal cells but tumor cells with dysfunctional mitochondria were affected by Mito-DCA, which caused a switch from glycolysis to glucose oxidation and subsequent cell death <i>via</i> apoptosis. Effective delivery of DCA to the mitochondria resulted in significant reduction in lactate levels and played important roles in modulating dendritic cell (DC) phenotype evidenced by secretion of interleukin-12 from DCs upon activation with tumor antigens from Mito-DCA treated cancer cells. Targeting mitochondrial metabolic inhibitors to the mitochondria could lead to induction of an efficient antitumor immune response, thus introducing the concept of combining glycolysis inhibition with immune system to destroy tumor

DZNep alters histone lysine methylation and methyltransferase and demethylase expressions in pancreatic cancer.

<p>A. Changes in methylation levels of H3K4, H3K9, H3K27, and H4K20 in HPDE and MIA PaCa-2 treated with DZNep (0–100 µM). Cells were treated with DZNep for 24 h, and whole cell lysates (50 µg) were subjected to Western blotting analysis. β-actin, the internal loading control, is shown with a representative blot. B. Western blotting analysis of histone lysine methyltransferases and demethylases in MIA PaCa-2 treated with DZNep (1 µM) for up to 48 h. C. Histone methylation dynamics in MIA PaCa-2 treated with DZNep (1 µM) for up to 72 h.</p

Acyl modifications of DZNep further enhance cytotoxicity.

<p>A. The chemical structures of DZNep and its two acyl prodrugs (Prodrug 1: C₂₀H₂₉ClN₄O₄, and Prodrug 2: C₁₈H₂₄N₄O₄). B. Cytotoxicity of DZNep versus its prodrugs in HPDE and MIA PaCa-2. IC₅₀ values are designated in each legend. Significance between each prodrug and DZNep was identified using the Student’s t test. C. Average IC₅₀ values of the various drug combinations in HPDE, Capan-1, and MIA PaCa-2. Twenty-four hours after 3×10³ cells/well were seeded in a 96-well plate, cells were treated for 72 h. Cellular viabilities were measured using MTT assays. IC₅₀ values are plotted. Significance of each prodrug combination was compared with Gem+DZNep using one-way ANOVA followed by Tukey’s post-hoc test. <i>Bars</i>, SD. <i>n</i> = 3. *<i>p</i><0.05, **<i>p</i><0.01.</p

DZNep and gemcitabine sensitivity, singly or in combination, and interactions within a panel of pancreatic cell lines.

<p>A. All cancerous cell lines excluding the normal HPDE are DZNep-responsive and reduced cellular viability. B. DZNep and gemcitabine displayed antagonistic effects in HPDE. C. DZNep and gemcitabine displayed additive or synergistic effects in many of the cancerous pancreatic cell lines. Twenty-four hours after 3×10³ cells/well were seeded in a 96-well plate, cells were treated with either DZNep, gemcitabine, or a combination of both at an equimolar ratio for 72 h. Cellular viability was measured using an MTT assay. Cytotoxic IC₅₀ values are indicated. Significances between gemcitabine and DZNep as well as DZNep+Gemcitabine and DZNep were identified using one-way ANOVA followed by Tukey’s post-hoc test. Combination index (CI) plots (insets) show the interactions between the two drugs. CI>1, antagonism; CI = 1, additivity; CI<1, synergism. Bar graphs to the right indicate the relative caspase-3 activity (RCA) of each treatment as measured by fluorescence intensity. Values were background-subtracted and are presented as fold-change from the control. Significance between a single drug versus the drug combination was identified via one-way ANOVA followed by Tukey’s post-hoc analysis. Cells were treated with 1 µM DZNep, 100 nM gemcitabine, or both. <i>Bars</i>, SD. <i>n</i> = 3. *<i>p</i><0.05, **<i>p</i><0.01.</p

Restriction sites and sequences of primers used for cloning.

<p>Restriction sites and sequences of primers used for cloning.</p

Physiochemical Characterization of NPs.

<p>Physiochemical Characterization of NPs.</p

Short priming of DZNep demonstrated superior cytotoxicity and synergy with gemcitabine than co-exposure of the two drugs.

<p>A. Short exposure with DZNep for 4–8 h produced maximal cytotoxic effects. Cells were exposed with DZNep at 1 µM for varying time intervals followed by increasing concentrations of gemcitabine (0–0.1 µM). Significance between 0 and 4 h is indicated. B. Superior cytotoxicity and synergism between gemcitabine and DZNep were observed when cells were primed with DZNep, as opposed to cotreatment with gemcitabine. Representative growth inhibition curves are shown. Twenty-four hours after 3×10³ cells/well were seeded in a 96-well plate, cells were exposed to gemcitabine and DZNep concentrations at a 1∶10 ratio either as a co-treatment for 72 h (C) or a primed treatment (with DZNep for 8 h followed by gemcitabine for 72 h) (P). Cellular viabilities were measured using MTT assays. Significance between co-treatment and priming is indicated. Combination index (CI) plots (insets) show the interactions between the two drugs. CI>1, antagonism; CI = 1, additivity; CI<1, synergism. <i>Bars</i>, SD. <i>n</i> = 3. *<i>p</i><0.05, **<i>p</i><0.01. C. Apoptosis levels were significantly greater in Capan-1 and MIA PaCa-2 cells with priming compared with co-treatment, while apoptosis levels in HPDE decreased with priming. Cells were either co-treated with 10 µM DZNep and 1 µM gemcitabine or primed with 10 µM DZNep for 8 h followed by 1 µM gemcitabine. Fluorescence values were background-subtracted and are indicated as fold-change from co-treatment to priming. Significant differences between co-treatment and priming were identified using the Student’s t test. <i>Bars</i>, SD. <i>n</i> = 3. *<i>p</i><0.05, **<i>p</i><0.01. D. Maximal reduction in H3K27 trimethylation was seen with priming schedules at 1∶10 DZNep:gemcitabine. MIA PaCa-2 was treated with vehicle, gemcitabine for 72 h, DZNep for 8 h, DZNep and gemcitabine for 72 h, or DZNep for 8 h followed by gemcitabine for 72 h. 100 µg of whole cell lysates were subjected to Western blotting analysis. Blots were stripped and re-probed for β-actin, the internal loading control. Densitometry ratios are indicated.</p

DZNep partially competes with the uptake of purine nucleosides by hENT1 and hCNT3.

<p>A. DZNep hindered the uptake of radiolabeled purine nucleosides in PANC-1 and MIA PaCa-2. Twenty-four hours after 5×10⁴ cells/well were seeded in a 24-well plate, cells were allowed to uptake the indicated radiolabeled nucleoside in the presence of DZNep or its respective unlabeled nucleoside. B. Inhibition of adenosine transport in <i>Xenopus</i> oocytes with DZNep. C. Pharmacological inhibition of hENT1 and excess uridine decreased the cytotoxicity of DZNep in MIA PaCa-2. Twenty-four hours after 3×10³ cells/well were seeded in a 96-well plate, cells were treated with increasing concentrations of DZNep in the presence of DMSO (control), 10 µM NBMPR, or 200 µM uridine. Cellular viability was measured using an MTT assay. IC₅₀ values are indicated. Significant differences between the control and each treatment were determined using the Student’s t test. <i>Bars</i>, SD. <i>n</i> = 3. *<i>p</i><0.05, **<i>p</i><0.01.</p