Nanoparticulate formulations of mithramycin analogs for enhanced cytotoxicity

Mithramycin (MTM), a natural product of soil bacteria from the Streptomyces genus, displays potent anticancer activity but has been limited clinically by severe side effects and toxicities. Engineering of the MTM biosynthetic pathway has produced the 3-side-chain-modified analogs MTM SK (SK) and MTM SDK (SDK), which have exhibited increased anticancer activity and improved therapeutic index. However, these analogs still suffer from low bioavailability, short plasma retention time, and low tumor accumulation. In an effort to aid with these shortcomings, two nanoparticulate formulations, poly(ethylene glycol)-poly(aspartate hydrazide) self-assembled and cross-linked micelles, were investigated with regard to the ability to load and pH dependently release the drugs. Micelles were successfully formed with both nanoparticulate formulations of each drug analog, with an average size of 8.36 ± 3.21 and 12.19 ± 2.77 nm for the SK and SDK micelles and 29.56 ± 4.67 nm and 30.48 ± 7.00 nm for the SK and SDK cross-linked micelles respectively. All of the drug-loaded formulations showed a pH-dependent release of the drugs, which was accelerated as pH decreased from 7.4 to 5.0. The micelles retained biological activity of SK and SDK entrapped in the micelles, suppressing human A549 lung cancer cells effectively

Compositions and Methods for Selectively Targeting Cancer Cells Using a Thiaminase Compound

Compositions and methods of treating cancer using a thiaminase compound are described. The presently-disclosed subject matter includes a method of treating cancer by administering a thiaminase compound and a thiamine-dependent enzyme inhibitor

Combination Effects of Docetaxel and Doxorubicin in Hormone-Refractory Prostate Cancer Cells

Combination effects of docetaxel (DOC) and doxorubicin (DOX) were investigated in prostate cancer cells (PC3 and DU145). Combination indices (CIs) were determined using the unified theory in various concentrations and mixing ratios (synergy: CI \u3c 0.9, additivity: 0.9 \u3c CI \u3c 1.1, and antagonism: CI \u3e 1.1). DOC showed a biphasic cytotoxicity pattern with the half maximal inhibitory concentration (IC50) at the picomolar range for PC3 (0.598 nM) and DU145 (0.469 nM), following 72 h drug exposure. The IC50s of DOX were 908 nM and 343 nM for PC3 and DU145, respectively. Strong synergy was seen when PC3 was treated with DOC at concentrations lower than its IC50 values (0.125~0.5 nM) plus DOX (2~8 times IC50). Equipotent drug combination treatments (7 × 7) revealed that the DOC/DOX combination leads to high synergy and effective cell death only in a narrow concentration range in DU145. This study provides a convenient method to predict multiple drug combination effects by the estimated CI values as well as cell viability data. The proposed DOC/DOX mixing ratios can be used to design combination drug cocktails or delivery systems to improve chemotherapy for cancer patients

Degradable Cross-Linked Nanoassemblies as Drug Carriers for Heat Shock Protein 90 Inhibitor 17-\u3cem\u3eN\u3c/em\u3e-Allylamino-17-demethoxy-geldanamycin

Cross-linked nanoassemblies (CNAs) with a degradable core were prepared for sustained release of 17-N-allylamino-17-demethoxygeldanamycin (17-AAG), a potent inhibitor of heat shock protein 90 (HSP90). The particle size of CNAs ranged between 100 and 250 nm, which changed depending on the cross-linking yields and drug entrapment method. CNAs with a 1% cross-linking yield entrapped 17-AAG in aqueous solutions, yet degraded in 3 hrs. CNAs entrapped 5.2 weight% of 17-AAG as the cross-linking yield increased to 10%, retaining more than 80% of particles for 24 hrs. CNAs with drugs entrapped after the cross-linking reactions were 100 nm and remained stable in both pH 7.4 and 5.0, corresponding to the physiological, tumoral, and intracellular environments. Drug was completely released from CNAs in 48 hrs, which would potentially maximize drug delivery and release efficiency within tumor tissues. Drug release patterns were not negatively affected by changing the cross-linking yields of CNAs. CNAs entrapping 17-AAG suppressed the growth of human non-small cell lung cancer A549 cells as equally effective as free drugs. The results demonstrated that CNAs would be a promising formulation that can be used in aqueous solutions for controlled delivery and release of 17-AAG

Alternating Magnetic Field-Induced Hyperthermia Increases Iron Oxide Nanoparticle Cell Association/Uptake and Flux in Blood-Brain Barrier Models

PURPOSE: Superparamagnetic iron oxide nanoparticles (IONPs) are being investigated for brain cancer therapy because alternating magnetic field (AMF) activates them to produce hyperthermia. For central nervous system applications, brain entry of diagnostic and therapeutic agents is usually essential. We hypothesized that AMF-induced hyperthermia significantly increases IONP blood-brain barrier (BBB) association/uptake and flux. METHODS: Cross-linked nanoassemblies loaded with IONPs (CNA-IONPs) and conventional citrate-coated IONPs (citrate-IONPs) were synthesized and characterized in house. CNA-IONP and citrate-IONP BBB cell association/uptake and flux were studied using two BBB Transwell® models (bEnd.3 and MDCKII cells) after conventional and AMF-induced hyperthermia exposure. RESULTS: AMF-induced hyperthermia for 0.5 h did not alter CNA-IONP size but accelerated citrate-IONP agglomeration. AMF-induced hyperthermia for 0.5 h enhanced CNA-IONP and citrate-IONP BBB cell association/uptake. It also enhanced the flux of CNA-IONPs across the two in vitro BBB models compared to conventional hyperthermia and normothermia, in the absence of cell death. Citrate-IONP flux was not observed under these conditions. AMF-induced hyperthermia also significantly enhanced paracellular pathway flux. The mechanism appears to involve more than the increased temperature surrounding the CNA-IONPs. CONCLUSIONS: Hyperthermia induced by AMF activation of CNA-IONPs has potential to increase the BBB permeability of therapeutics for the diagnosis and therapy of various brain diseases

Polymer Nanoassemblies with Hydrophobic Pendant Groups in the Core Induce False Positive siRNA Transfection in Luciferase Reporter Assays

Poly(ethylene glycol)-conjugated polyethylenimine (PEG-PEI) is a widely studied cationic polymer used to develop non-viral vectors for siRNA therapy of genetic disorders including cancer. Cell lines stably expressing luciferase reporter protein typically evaluate the transfection efficacy of siRNA/PEG-PEI complexes, however recent findings revealed that PEG-PEI can reduce luciferase expression independent of siRNA. This study elucidates a cause of the false positive effect in luciferase assays by using polymer nanoassemblies (PNAs) made from PEG, PEI, poly-(L-lysine) (PLL), palmitate (PAL), and deoxycholate (DOC): PEG-PEI (2P), PEG-PEI-PAL (3P), PEG-PLL (2P′), PEG-PLL-PAL (3P′), and PEG-PEI-DOC (2PD). In vitro transfection and western blot assays of luciferase using a colorectal cancer cell line expressing luciferase (HT29/LUC) concluded that 2P and 2P′ caused no luciferase expression reduction while hydrophobically modified PNAs induced a 35-50% reduction (3P′ \u3c 2PD \u3c 3P). Although cell viability remained stagnant, 3P triggered cellular stress responses including increased membrane porosity and decreased ATP and cellular protein concentrations. Raman spectroscopy suggested that hydrophobic groups influence PNA conformation changes, which may have caused over-ubiquitination and degradation of luciferase in the cells. These results indicate that hydrophobically modified PEG-PEI induces cellular distress causing over-ubiquitination of the luciferase protein, producing false positive siRNA transfection in the luciferase assay

Block Copolymer Self-Assembled and Cross-linked Nanoassemblies for Combination Delivery of Iron Oxide and Doxorubicin

We describe the development of nanoscale polymer drug carriers for the combinational delivery of an anticancer drug (doxorubicin: DOX) along with super paramagnetic iron oxide nanoparticles (IONPs). The drug molecules were electrostatically loaded into both block copolymer self-assembled nanoassemblies (SNAs) and cross-linked nanoassemblies (CNAs). Both nanoassemblies entrapped DOX and IONPs either individually or in tandem, maintaining sub-100 nm diameter. The IONP-loaded nanoassemblies generated heat in the presence of an alternating magnetic field (AMF). Incorporation of the drug payload, DOX, showed no adverse effects on the heating profile. Drug release from the SNAs and CNAs was accelerated as temperature increased from the normal body temperature (37°C) to a mild hyperthermic condition (40 ~ 42°C). CNAs released DOX faster than SNAs regardless of an incubation temperature. CNAs co-entrapped IONPs and DOX were more stable than SNAs in aqueous solutions for five days. These results suggest that block copolymer cross-linked nanoassemblies provide viable delivery platforms for combination delivery of inorganic molecules, anticancer drugs, and potentially other various biologically active substances

Development of Halofluorochromic Polymer Nanoassemblies for the Potential Detection of Liver Metastatic Colorectal Cancer Tumors Using Experimental and Computational Approaches

Purpose—To develop polymer nanoassemblies (PNAs) modified with halofluorochromic dyes to allow for the detection of liver metastatic colorectal cancer (CRC) to improve therapeutic outcomes. Methods—We combine experimental and computational approaches to evaluate macroscopic and microscopic PNA distributions in patient-derived xenograft primary and orthotropic liver metastatic CRC tumors. Halofluorochromic and non-halofluorochromic PNAs (hfPNAs and n-hfPNAs) were prepared from poly(ethylene glycol), fluorescent dyes (Nile blue, Alexa546, and IR820), and hydrophobic groups (palmitate), all of which were covalently tethered to a cationic polymer scaffold [poly(ethylene imine) or poly(lysine)] forming particles with an average diameter \u3c 30 nm. Results—Dye-conjugated PNAs showed no aggregation under opsonizing conditions for 24 h and displayed low tissue diffusion and cellular uptake. Both hfPNAs and n-hfPNAs accumulated in primary and liver metastatic CRC tumors within 12 h post intravenous injection. In comparison to n-hfPNAs, hfPNAs fluoresced strongly only in the acidic tumor microenvironment (pH \u3c 7.0) and distinguished small metastatic CRC tumors from healthy liver stroma. Computational simulations revealed that PNAs would steadily accumulate mainly in acidic (hypoxic) interstitium of metastatic tumors, independently of the vascularization degree of the tissue surrounding the lesions. Conclusion—The combined experimental and computational data confirms that hfPNAs detecting acidic tumor tissue can be used to identify small liver metastatic CRC tumors with improved accuracy

Block Copolymer Cross-Linked Nanoassemblies Improve Particle Stability and Biocompatibility of Superparamagnetic Iron Oxide Nanoparticles

PURPOSE: To develop cross-linked nanoassemblies (CNAs) as carriers for superparamagnetic iron oxide nanoparticles (IONPs). METHODS: Ferric and ferrous ions were co-precipitated inside core-shell type nanoparticles prepared by cross-linking poly(ethylene glycol)-poly(aspartate) block copolymers to prepare CNAs entrapping Fe(3)O(4) IONPs (CNA-IONPs). Particle stability and biocompatibility of CNA-IONPs were characterized in comparison to citrate-coated Fe(3)O(4) IONPs (Citrate-IONPs). RESULTS: CNA-IONPs, approximately 30 nm in diameter, showed no precipitation in water, PBS, or a cell culture medium after 3 or 30 h, at 22, 37, and 43°C, and 1, 2.5, and 5 mg/mL, whereas Citrate-IONPs agglomerated rapidly (\u3e 400 nm) in all aqueous media tested. No cytotoxicity was observed in a mouse brain endothelial-derived cell line (bEnd.3) exposed to CNA-IONPs up to 10 mg/mL for 30 h. Citrate-IONPs (\u3e 0.05 mg/mL) reduced cell viability after 3 h. CNA-IONPs retained the superparamagnetic properties of entrapped IONPs, enhancing T2-weighted magnetic resonance images (MRI) at 0.02 mg/mL, and generating heat at a mild hyperthermic level (40 ~ 42°C) with an alternating magnetic field (AMF). CONCLUSION: Compared to citric acid coating, CNAs with a cross-linked anionic core improved particle stability and biocompatibility of IONPs, which would be beneficial for future MRI and AMF-induced remote hyperthermia applications

Polymer Micelle Formulation for the Proteasome Inhibitor Drug Carfilzomib: Anticancer Efficacy and Pharmacokinetic Studies in Mice

Carfilzomib (CFZ) is a peptide epoxyketone proteasome inhibitor approved for the treatment of multiple myeloma (MM). Despite the remarkable efficacy of CFZ against MM, the clinical trials in patients with solid cancers yielded rather disappointing results with minimal clinical benefits. Rapid degradation of CFZ in vivo and its poor penetration to tumor sites are considered to be major factors limiting its efficacy against solid cancers. We previously reported that polymer micelles (PMs) composed of biodegradable block copolymers poly(ethylene glycol) (PEG) and poly(caprolactone) (PCL) can improve the metabolic stability of CFZ in vitro. Here, we prepared the CFZ-loaded PM, PEG-PCL-deoxycholic acid (CFZ-PM) and assessed its in vivo anticancer efficacy and pharmacokinetic profiles. Despite in vitro metabolic protection of CFZ, CFZ-PM did not display in vivo anticancer efficacy in mice bearing human lung cancer xenograft (H460) superior to that of the clinically used cyclodextrin-based CFZ (CFZ-CD) formulation. The plasma pharmacokinetic profiles of CFZ-PM were also comparable to those of CFZ-CD and the residual tumors that persisted in xenograft mice receiving CFZ-PM displayed an incomplete proteasome inhibition. In summary, our results showed that despite its favorable in vitroperformances, the current CFZ-PM formulation did not improve in vivo anticancer efficacy and accessibility of active CFZ to solid cancer tissues over CFZ-CD. Careful consideration of the current results and potential confounding factors may provide valuable insights into the future efforts to validate the potential of CFZ-based therapy for solid cancer and to develop effective CFZ delivery strategies that can be used to treat solid cancers