Anthracyclines induce cardiotoxicity through a shared gene expression response signature.

TOP2 inhibitors (TOP2i) are effective drugs for breast cancer treatment. However, they can cause cardiotoxicity in some women. The most widely used TOP2i include anthracyclines (AC) Doxorubicin (DOX), Daunorubicin (DNR), Epirubicin (EPI), and the anthraquinone Mitoxantrone (MTX). It is unclear whether women would experience the same adverse effects from all drugs in this class, or if specific drugs would be preferable for certain individuals based on their cardiotoxicity risk profile. To investigate this, we studied the effects of treatment of DOX, DNR, EPI, MTX, and an unrelated monoclonal antibody Trastuzumab (TRZ) on iPSC-derived cardiomyocytes (iPSC-CMs) from six healthy females. All TOP2i induce cell death at concentrations observed in cancer patient serum, while TRZ does not. A sub-lethal dose of all TOP2i induces limited cellular stress but affects calcium handling, a function critical for cardiomyocyte contraction. TOP2i induce thousands of gene expression changes over time, giving rise to four distinct gene expression response signatures, denoted as TOP2i early-acute, early-sustained, and late response genes, and non-response genes. There is no drug- or AC-specific signature. TOP2i early response genes are enriched in chromatin regulators, which mediate AC sensitivity across breast cancer patients. However, there is increased transcriptional variability between individuals following AC treatments. To investigate potential genetic effects on response variability, we first identified a reported set of expression quantitative trait loci (eQTLs) uncovered following DOX treatment in iPSC-CMs. Indeed, DOX response eQTLs are enriched in genes that respond to all TOP2i. Next, we identified 38 genes in loci associated with AC toxicity by GWAS or TWAS. Two thirds of the genes that respond to at least one TOP2i, respond to all ACs with the same direction of effect. Our data demonstrate that TOP2i induce thousands of shared gene expression changes in cardiomyocytes, including genes near SNPs associated with inter-individual variation in response to DOX treatment and AC-induced cardiotoxicity.

Amino acid signaling in TOR activation.

The target of rapamycin (TOR) is a central cell growth regulator conserved from yeast to mammals. Uncontrolled TOR activation is commonly observed in human cancers. TOR forms two distinct structural and functional complexes, TORC1 and TORC2. TORC1 promotes cell growth and cell size by stimulating protein synthesis. A wide range of signals, including nutrients, energy levels, and growth factors, are known to control TORC1 activity. Among them, amino acids (AA) not only potently activate TORC1 but are also required for TORC1 activation by other stimuli, such as growth factors. The mechanisms of growth factors and cellular energy status in activating TORC1 have been well elucidated, whereas the molecular basis of AA signaling is just emerging. Recent advances in the role of AA signaling on TORC1 activation have revealed key components, including the Rag GTPases, protein kinases, nutrient transporters, and the intracellular trafficking machinery, in relaying AA signals to TORC1 activation.

Targeting autophagy with SAR405 alleviates doxorubicin-induced cardiotoxicity.

Anthracycline antitumor agents, such as doxorubicin (DOX), are effective in the treatment of solid tumors and hematological malignancies, but anthracycline-induced cardiotoxicity (AIC) limits their application as chemotherapeutics. Dexrazoxane (DEX) has been adopted to prevent AIC. Using a chronic AIC mouse model, we demonstrated that DEX is insufficient to reverse DOX-induced cardiotoxicity. Although therapies targeting autophagy have been explored to prevent AIC, but whether novel autophagy inhibitors could alleviate or prevent AIC in clinically relevant models needs further investigation. Here, we show that genetic ablation of Atg7, a key regulator in the early phase of autophagy, protected mice against AIC. We further demonstrated that SAR405, a novel autophagy inhibitor, attenuated DOX-induced cytotoxicity. Intriguingly, the combination of DEX and SAR405 protected cells against DOX-induced cardiotoxicity in vivo. Using the cardiomyocyte cell lines AC16 and H9c2, we determined that autophagy was initiated during AIC. Our results suggest that inhibition of autophagy at its early phase with SAR405 combined with DEX represents an effective therapeutic strategy to prevent AIC.

Metabolic and non-metabolic pathways that control cancer resistance to anthracyclines.

Anthracyclines Doxorubicin, Epirubicin, Daunorubicin and Idarubicin are used to treat a variety of tumor types in the clinics, either alone or, most often, in combination therapies. While their cardiotoxicity is well known, the emergence of chemoresistance is also a major issue accounting for treatment discontinuation. Resistance to anthracyclines is associated to the acquisition of multidrug resistance conferred by overexpression of permeability glycoprotein-1 or other efflux pumps, by altered DNA repair, changes in topoisomerase II activity, cancer stemness and metabolic adaptations. This review further details the metabolic aspects of resistance to anthracyclines, emphasizing the contributions of glycolysis, the pentose phosphate pathway and nucleotide biosynthesis, glutathione, lipid metabolism and autophagy to the chemoresistant phenotype.

Evaluation of the anti-cervical cancer effect of a prodrug ：CBZ-AAN-DOX with hypoxic cell culture and tumor-bearing zebrafish models.

BACKGROUND: Prodrugs are medications or compounds that, after administration, can be converted into pharmacologically active drugs through metabolism. Unlike conventional drugs, prodrugs have reduced adverse or unintended effects, which could become critical limitations in treatments such as chemotherapy. Previously through computer-aided drug design and chemical synthesis, we have obtained and examined a prodrug N-benzyloxycarbonyl-Ala-Asn-Doxorubicin (CBZ-AAN-DOX). CBZ-AAN-DOX is essentially Doxorubicin that is chemically-modified with tripeptides to target Legumain, a highly expressed protein in cancer cells and is involved in tumor metastasis and tumor microvessel formation. The difficulty to test the safety and efficacy of the prodrug (including the pharmacodynamic parameters of CBZ-AAN-DOX on metastasis and invasion of tumors, as well as cardiac and vascular toxicity) primarily comes from the lack of appropriate experimental models. METHODS: Human cervical cancer cell lines CaSki under hypoxic conditions were used to evaluate the cell viability by CCK-8 assay after the prodrug treatment. Western blotting method was performed for Legumain protein determination in the cell culture. Wound healing and transwell invasion assays were performed to determine the effects of the prodrug on tumor metastasis and invasion, respectively. Zebrafish models were constructed for toxicity and angiogenesis visual analysis after in vivo treatment with the prodrug. RESULTS: The CCK-8 results showed that CBZ-AAN-DOX exhibits an IC50 of 28.7 µM in 48 h on CaSki cells that had a lower cell inhibition rate than DOX 80.3 µM for 24 h. Legumain expression was significantly increased in a time-dependent manner in 48 h under hypoxia conditions. The results also showed that 13.9 µM of the prodrug significantly inhibited the migration and invasion of cells and the effects were significantly stronger than that of 41.8 µM of DOX under hypoxia conditions after 48 h. The effects of 160 µM of the prodrug on the survival rate of zebrafish after 72 h and heart-toxicity showed no obvious abnormalities. Cell metastasis and angiogenesis were also inhibited in tumor-bearing zebrafish model. CONCLUSION: The findings in this study demonstrated that CBZ-AAN-DOX is a promising chemotherapy candidate with low toxicity and high efficiency for cervical cancer. Remarkably, the hypoxic culture model together with the zebrafish model serve as a good system for the evaluation of the toxicity, targeting and impact of the prodrug on tumor invasion and metastasis.

Polymorphic G:G mismatches act as hotspots for inducing right-handed Z DNA by DNA intercalation.

DNA mismatches are highly polymorphic and dynamic in nature, albeit poorly characterized structurally. We utilized the antitumour antibiotic CoII(Chro)2 (Chro = chromomycin A3) to stabilize the palindromic duplex d(TTGGCGAA) DNA with two G:G mismatches, allowing X-ray crystallography-based monitoring of mismatch polymorphism. For the first time, the unusual geometry of several G:G mismatches including syn-syn, water mediated anti-syn and syn-syn-like conformations can be simultaneously observed in the crystal structure. The G:G mismatch sites of the d(TTGGCGAA) duplex can also act as a hotspot for the formation of alternative DNA structures with a GC/GA-5' intercalation site for binding by the GC-selective intercalator actinomycin D (ActiD). Direct intercalation of two ActiD molecules to G:G mismatch sites causes DNA rearrangements, resulting in backbone distortion to form right-handed Z-DNA structures with a single-step sharp kink. Our study provides insights on intercalators-mismatch DNA interactions and a rationale for mismatch interrogation and detection via DNA intercalation.

Morphologically homogeneous, pH-responsive gold nanoparticles for non-invasive imaging of HeLa cancer.

Gold nanoparticles (AuNPs) have been widely used as nanocarriers in drug delivery to improve the efficiency of chemotherapy treatment and enhance early disease detection. The advantages of AuNPs include their excellent biocompatibility, easy modification and functionalization, facile synthesis, low toxicity, and controllable particle size. This study aimed to synthesize a conjugated citraconic anhydride link between morphologically homogeneous AuNPs and doxorubicin (DOX) (DOX-AuNP). The carrier was radiolabeled for tumor diagnosis using positron emission tomography (PET). The systemically designed DOX-AuNP was cleaved at the citraconic anhydride linker site under the mild acidic conditions of a cancer cell, thereby releasing DOX. Subsequently, the AuNPs aggregated via electrostatic attraction. HeLa cancer cells exhibited a high uptake of the radiolabeled DOX-AuNP. Moreover, PET tumor images were obtained using radiolabeled DOX-AuNP in cancer xenograft mouse models. Therefore, DOX-AuNP is expected to provide a valuable insight into the use of radioligands to detect tumors using PET.

Sungeidines from a Non-canonical Enediyne Biosynthetic Pathway.

We report the genome-guided discovery of sungeidines, a class of microbial secondary metabolites with unique structural features. Despite evolutionary relationships with dynemicin-type enediynes, the sungeidines are produced by a biosynthetic gene cluster (BGC) that exhibits distinct differences from known enediyne BGCs. Our studies suggest that the sungeidines are assembled from two octaketide chains that are processed differently than those of the dynemicin-type enediynes. The biosynthesis also involves a unique activating sulfotransferase that promotes a dehydration reaction. The loss of genes, including a putative epoxidase gene, is likely to be the main cause of the divergence of the sungeidine pathway from other canonical enediyne pathways. The findings disclose the surprising evolvability of enediyne pathways and set the stage for characterizing the intriguing enzymatic steps in sungeidine biosynthesis.

Fluorometer for Screening of Doxorubicin in Perfusate Solution and Tissue with Solid-Phase Microextraction Chemical Biopsy Sampling.

The recent development of an in vivo solid-phase microextraction (SPME) method capable of analyzing drugs and metabolic products in biofluids and living tissues holds great promise. The standard in vivo SPME protocol based on mass spectrometry is a very powerful analytical approach, but it is not practical for on-site analysis in many cases. In this paper, we present a fluorescence-based SPME method and a prototype of a portable fluorometer that is capable of quickly quantifying concentrations of the anticancer drug, doxorubicin (DOX). The instrument uses thin coated, biocompatible SPME fibers, which we have previously presented as a chemical biopsy tool for use during in vivo lung perfusion (IVLP) procedures within a hospital setting. In this research, we test SPME fibers with C8-SCX, C18, and HLB coatings with our fluorometer. The mixed-mode C8-SCX fibers showed the best sensitivity of the three and were therefore used to examine DOX extraction from perfusate solution and a homogenized lamb lung tissue. The maximum concentration of free active sites in the C8-SCX fiber and the adsorption equilibrium constant were determined to be (9.1 ± 0.3) × 10-7 mol m-2 and 420 ± 30 m3 mol-1, respectively. Finally, the detection limits for DOX extracted from buffer, perfusate, and lung tissue were 40, 100, and 3700 µg L-1, respectively.

Deletion of the Trichoderma virens NRPS, Tex7, induces accumulation of the anti-cancer compound heptelidic acid.

The anticancer antibiotic heptelidic acid is a sesquiterpene lactone produced by the beneficial plant fungus Trichoderma virens. This species has been separated into two strains, referred to as P and Q, based on its biosynthesis of secondary metabolites; notably, only P-strains were reported to produce heptelidic acid. While characterizing a Q-strain of T. virens containing a directed mutation in the non-ribosomal peptide synthetase encoding gene Tex7, the appearance of an unknown compound in anomalously large quantities was visualized by TLC. Using a combination of HPLC, LC-MS/MS, and NMR spectroscopy, this compound was identified as heptelidic acid. This discovery alters the strain classification structure of T. virens. Additionally, the Tex7 mutants inhibited growth of maize seedlings, while retaining the ability to induce systemic resistance against the foliar fungal pathogen, Cochliobolus heterostrophus.

Dual-pH Sensitive Charge-Reversal Drug Delivery System for Highly Precise and Penetrative Chemotherapy.

PURPOSE: The complex physiological barriers impose extremely conflicting demands on systemic drug delivery, so both particle size and surface charge of the nanoplatforms become vital factors. As a carbon-based nanomaterial with excellent optical properties, carbon dots are not suitable for direct systemic transport in vivo, which limits their application in the field of biomedical imaging, especially in the areas of diagnosis and cancer treatment. Liposomes have been developed as universal nanocarriers for various drugs. In this study, we aimed to build a highly precise and penetrative drug delivery system (DDS) using carbon dots encapsulated by liposomes. METHODS: Carbon dots (CDs) were synthesized by the hydrothermal method using citric acid and ethylenediamine. Furthermore, simian virus 40 large T-antigen derived the nuclear targeting sequence (NLS) was bonded on the surface of CDs to obtain CDs-NLS. The antitumor drug doxorubicin was loaded onto the CDs-NLS through an acid-labile hydrazine bond to obtain DOX@CDs. Finally, DOX@CDs were encapsulated in aqueous centers of folate-coated and pH-sensitive liposomes, named pHSL-FA. RESULTS: In this paper, a nucleus-targeted nanocomposite (DOX@CDs), which bonds with the nuclear targeting sequence (NLS) and the anticancer drug doxorubicin (DOX), has physicochemical properties of particle size of about 3.8 nm, zeta potential of +31.8 mV and high quantum yield of 64.53%. The negatively charged folate-coated and pH-sensitive liposomes (pHSL-FA) are used as a carrier to reverse the surface charge of DOX@CDs. Compared to free DOX@CDs, pHSL-FA show higher tumor accumulation in 4 T1 tumor-bearing mice and further improve cytotoxicity to tumor cells. CONCLUSIONS: This work proposes a unique nanomedical approach that enables the precise delivery of chemotherapy drugs and significantly reduces side effects, which is promising for clinical translation.

Cooperative recognition of T:T mismatch by echinomycin causes structural distortions in DNA duplex.

Small-molecule compounds that target mismatched base pairs in DNA offer a novel prospective for cancer diagnosis and therapy. The potent anticancer antibiotic echinomycin functions by intercalating into DNA at CpG sites. Surprisingly, we found that the drug strongly prefers to bind to consecutive CpG steps separated by a single T:T mismatch. The preference appears to result from enhanced cooperativity associated with the binding of the second echinomycin molecule. Crystallographic studies reveal that this preference originates from the staggered quinoxaline rings of the two neighboring antibiotic molecules that surround the T:T mismatch forming continuous stacking interactions within the duplex. These and other associated changes in DNA conformation allow the formation of a minor groove pocket for tight binding of the second echinomycin molecule. We also show that echinomycin displays enhanced cytotoxicity against mismatch repair-deficient cell lines, raising the possibility of repurposing the drug for detection and treatment of mismatch repair-deficient cancers.

Liquid Chromatography-Tandem Mass Spectrometry for the Simultaneous Determination of Doxorubicin and its Metabolites Doxorubicinol, Doxorubicinone, Doxorubicinolone, and 7-Deoxydoxorubicinone in Mouse Plasma.

Doxorubicin, an anthracycline antitumor antibiotic, acts as a cancer treatment by interfering with the function of DNA. Herein, liquid chromatography-tandem mass spectrometry was for the first time developed and validated for the simultaneous determination of doxorubicin and its major metabolites doxorubicinol, doxorubicinone, doxorubicinolone, and 7-deoxydoxorubicinone in mouse plasma. The liquid-liquid extraction of a 10 µL mouse plasma sample with chloroform:methanol (4:1, v/v) and use of the selected reaction monitoring mode led to less matrix effect and better sensitivity. The lower limits of quantification levels were 0.5 ng/mL for doxorubicin, 0.1 ng/mL for doxorubicinol, and 0.01 ng/mL for doxorubicinone, doxorubicinolone, and 7-deoxydoxorubicinone. The standard curves were linear over the range of 0.5-200 ng/mL for doxorubicin; 0.1-200 ng/mL for doxorubicinol; and 0.01-50 ng/mL for doxorubicinone, doxorubicinolone, and 7-deoxydoxorubicinone in mouse plasma. The intra and inter-day relative standard deviation and relative errors for doxorubicin and its four metabolites at four quality control concentrations were 0.9-13.6% and -13.0% to 14.9%, respectively. This method was successfully applied to the pharmacokinetic study of doxorubicin and its metabolites after intravenous administration of doxorubicin at a dose of 1.3 mg/kg to female BALB/c nude mice.

Cathepsin B-Cleavable Cyclopeptidic Chemotherapeutic Prodrugs.

Cyclopeptidic chemotherapeutic prodrugs (cPCPs) are macromolecular protease-sensitive doxorubicin (DOX) prodrugs synthesized from a cyclodecapeptidic scaffold, termed Regioselectively Addressable Functionalized Template (RAFT). In order to increase the chemotherapeutic potential of DOX and limit its toxicity, we used a Cathepsin B (Cat B)-sensitive prodrug concept for its targeted release since this enzyme is frequently overexpressed in cancer cells. Copper-free "click" chemistry was used to synthesize cPCPs containing up to four DOX moieties tethered to the upper face of the scaffold through a Cat B-cleavable peptidic linker (GAGRRAAG). On the lower part, PEG 5, 10 and 20 kDa and a fifth peptidyl DOX moiety were grafted in order to improve the solubility, bioavailability and pharmacokinetic profiles of the compound. In vitro results on HT1080 human fibrosarcoma cells showed that cPCPs display a delayed action that consists of a cell cycle arrest in the G2 phase comparable to DOX alone, and increased cell membrane permeability.

Chemometric Approaches for Developing Infrared Nanosensors To Image Anthracyclines.

Generation, identification, and validation of optical probes to image molecular targets in a biological milieu remain a challenge. Synthetic molecular recognition approaches leveraging the intrinsic near-infrared fluorescence of single-walled carbon nanotubes are promising for long-term biochemical imaging in tissues. However, generation of nanosensors for selective imaging of molecular targets requires a heuristic approach. Here, we present a chemometric platform for rapidly screening libraries of candidate single-walled carbon nanotube nanosensors against biochemical analytes to quantify the fluorescence response to small molecules, including vitamins, neurotransmitters, and chemotherapeutics. We further show this method can be applied to identify biochemical analytes that selectively modulate the intrinsic near-infrared fluorescence of candidate nanosensors. Chemometric analysis thus enables identification of nanosensor-analyte "hits" and also nanosensor fluorescence signaling modalities such as wavelength shifts that are optimal for translation to biological imaging. Through this approach, we identify and characterize a nanosensor for the chemotherapeutic anthracycline doxorubicin (DOX), which provides a ≤17 nm fluorescence red-shift and exhibits an 8 µM limit of detection, compatible with peak circulatory concentrations of doxorubicin common in therapeutic administration. We demonstrate the selectivity of this nanosensor over dacarbazine, a chemotherapeutic commonly co-injected with doxorubicin. Lastly, we establish nanosensor tissue compatibility for imaging of doxorubicin in muscle tissue by incorporating nanosensors into the mouse hindlimb and measuring the nanosensor response to exogenous DOX administration. Our results motivate chemometric approaches to nanosensor discovery for chronic imaging of drug partitioning into tissues and toward real-time monitoring of drug accumulation.

Preparation of superparamagnetic iron oxide/doxorubicin loaded chitosan nanoparticles as a promising glioblastoma theranostic tool.

Theranostic nanoparticles (NPs) are promising for opening new windows toward personalized disease management. Using a single particle capable of both diagnosis and drug delivery, is the major benefit of such particles. In the present study, chitosan NPs were used as a dual action carrier for doxorubicin (DOX; chemotherapeutic agent) and superparamagnetic iron oxide nanoparticles (SPIONs; imaging agent). SPIONs and DOX were loaded at different concentrations within poly-l-arginine-chitosan-triphosphate matrix (ACSD) using the ionic gelation method. NPs' size were in the range of 184.33 ± 4.4 nm. Drug release analysis of DOX loaded NPs (NP-DOX) showed burst release at pH 5.5 (as in tumor environment) and slow release at pH 7.4 (physiological condition), demonstrating pH-sensitive drug release profile. NP-DOX internalization was confirmed by flowcytometry and fluorescent microscopy. Uptake process results were corroborated by accumulation of drug in the intracellular space. Iron content was evaluated by inductively coupled plasma and prussian blue staining. In vitro magnetic resonance imaging (MRI) showed a decline in T 2 relaxation times by increasing iron concentration. MRI analysis also confirmed uptake of NPs at the optimum concentration in C6 glioma cells. In conclusion, ACSD NPs could be utilized as a promising theranostic formulation for both diagnosis and treatment of glioblastoma.

Tumor-Acidity-Cleavable Maleic Acid Amide (TACMAA): A Powerful Tool for Designing Smart Nanoparticles To Overcome Delivery Barriers in Cancer Nanomedicine.

Over the past few decades, cancer nanomedicine has been under intensive development for applications in drug delivery, cancer therapy, and molecular imaging. However, there exist a series of complex biological barriers in the path of a nanomedicine from the site of administration to the site of action. These barriers considerably prevent a nanomedicine from reaching its targets in a sufficient concentration and thus severely limit its therapeutic benefits. According to the delivery process, these biological delivery barriers can be briefly summarized in the following order: blood circulation, tumor accumulation, tumor penetration, cellular internalization, and intracellular drug release. The therapeutic effect of a nanomedicine is strongly determined by its ability to overcome these barriers. However, advances in cancer biology have revealed that each barrier has its own distinct microenvironment, which imposes different requirements on the optimal design of nanocarriers, thus further complicating the delivery process. For example, the pH of blood is neutral, while the tumor extracellular environment features an acidic pH (pHe ≈ 6.5-7.0) and the endosome and lysosome are more acidic (pH 5.5-4.5). The nanoparticles (NPs) should be able to change their properties to adapt to each individual environment for robust and effective delivery. This demand promotes the design and development of smart delivery carriers that can respond to endogenous and exogenous stimuli. It is well-documented that tumors develop acidic extracellular microenvironments with pH ≈ 6.5-7.0 due to their abnormal metabolism in comparison with normal tissues. This provides a unique tool for designing smart NP drug delivery systems. Our studies have revealed that the NPs' physiochemical properties, such as particle size and surface charge, have profound effects on their systemic transport in the body. In different delivery stages, the NPs should possess different sizes or surface charges for optimal performance. We developed a class of stimuli-responsive NPs by incorporating tumor-acidity-cleavable maleic acid amide (TACMAA) as a design feature. TACMAA is produced by the facile reaction of an amino group with 2,3-dimethylmaleic anhydride (DMMA) and its derivatives and can be cleaved under tumor acidity. By virtue of such characteristics, NPs containing TACMAA enable size or surface charge switching at tumor sites so that they can overcome those delivery barriers for improved drug delivery and cancer therapy. In this Account, we systemically review the development and evolution of TACMAA-based delivery systems and elaborate how TACMAA helps the innovation and design of intelligent nanocarriers for overcoming the delivery barriers. In particular, our Account focuses on five parts: TACMAA chemistry, tumor-acidity-triggered charge reversal, tumor-acidity-triggered shell detachment, tumor-acidity-triggered size transition, and tumor-acidity-triggered ligand reactivation. We provide detailed information on how tumor-acidity-triggered property changes correlate with the ability of NPs to overcome delivery barriers.

Transarterial Infusion of iRGD-Modified ZrO2 Nanoparticles with Lipiodol Improves the Tissue Distribution of Doxorubicin and Its Antitumor Efficacy.

PURPOSE: To evaluate the effect of transarterial infusion of iRGD-modified and doxorubicin-loaded zirconia-composite nanoparticles (R-DZCNs) with lipiodol in the improvement of the distribution of doxorubicin (DOX) in liver tumors and its antitumor efficacy. MATERIALS AND METHODS: The effect of R-DZCNs was evaluated in vitro by tumor cellular uptake and cytotoxicity assays. For the in vivo study, DOX distribution and antitumor efficiency were assessed. In the DOX distribution study, VX2 tumor-bearing rabbits received transarterial infusion of lipiodol with DOX, doxorubicin-loaded zirconia-composite nanoparticles (DZCNs), or R-DZCNs, respectively. DOX distribution was assessed by immunofluorescence. In the antitumor study, tumor-bearing rabbits received transarterial infusions of lipiodol with DOX, DZCNs, R-DZCNs, or saline respectively. Tumor volume was measured using magnetic resonance imaging, and the expression of apoptosis-related factors (caspase-3, Bax, Bcl-2) was analyzed by immunohistochemistry and Western blotting. RESULTS: R-DZCNs increased cellular uptake and caused stronger cytotoxicity. Compared with the DOX + lipiodol or DZCNs + lipiodol group, the R-DZCNs + lipiodol group showed more DOX fluorescence spots (2,449.15 ± 444.14 vs. 3,464.73 ± 632.75 or 5,062.25 ± 585.62, respectively; P < .001) and longer penetration distance (117.58 ± 19.36 vs 52.64 ± 8.53 or 83.37 ± 13.76 µm, respectively; P < .001). In the antitumor study, the R-DZCNs + lipiodol group showed smaller tumor volumes than the DOX + lipiodol or DZCNs + lipiodol group (1,223.87 ± 223.58 vs. 3,695.26 ± 666.25 or 2281.06 ± 457.21 mm3, respectively; P = .005).The greatest extent of tumor cell apoptosis was observed in R-DZCNs + lipiodol group immunohistochemistry and Western blotting results. CONCLUSIONS: Transarterial infusion of R-DZCNs with lipiodol improved the distribution of DOX and enhanced its antitumor efficacy.

Synthesis, in vitro stability, and antiproliferative effect of d-cysteine modified GnRH-doxorubicin conjugates.

Overexpression of gonadotropin-releasing hormone (GnRH) receptor in many tumors but not in normal tissues makes it possible to use GnRH analogs as targeting peptides for selective delivery of cytotoxic agents, which may help to enhance the uptake of anticancer drugs by cancer cells and reduce toxicity to normal cells. The GnRH analogs [d-Cys6 , desGly10 , Pro9 -NH2 ]-GnRH, [d-Cys6 , desGly10 , Pro9 -NHEt]-GnRH, and [d-Cys6 , α-aza-Gly10 -NH2 ]-GnRH were conjugated with doxorubicin (Dox), respectively, through N-succinimidyl-3-maleimidopropionate as a linker to afford three new GnRH-Dox conjugates. The metabolic stability of these conjugates in human serum was determined by RP-HPLC. The antiproliferative activity of the conjugates was examined in GnRH receptor-positive MCF-7 human breast cancer cell line by MTT assay. The three GnRH-Dox conjugates showed improved metabolic stability in human serum in comparison with AN-152. The antiproliferative effect of conjugate II ([d-Cys6 , desGly10 , Pro9 -NHEt]-GnRH-Dox) on MCF-7 cells was higher than that of conjugate I ([d-Cys6 , desGly10 , Pro9 -NH2 ]-GnRH-Dox) and conjugate III ([d-Cys6 , α-aza-Gly10 -NH2 ]-GnRH-Dox), and the cytotoxicity of conjugate II against GnRH receptor-negative 3T3 mouse embryo fibroblast cells was decreased in comparison with free Dox. GnRH receptor inhibition test suggested that the antiproliferative activity of conjugate II might be due to the cellular uptake mediated by the targeting binding of [d-Cys6 -des-Gly10 -Pro9 -NHEt]-GnRH to GnRH receptors. Our study indicates that targeting delivery of conjugate II mediated by [d-Cys6 -des-Gly10 -Pro9 -NHEt]-GnRH is a promising strategy for chemotherapy of tumors that overexpress GnRH receptors.

Quantitative evaluation of liposomal doxorubicin and its metabolites in spheroids.

Accurate measurement and understanding of therapeutic uptake and metabolism is key in the drug development process. This work examines the amount of doxorubicin that can penetrate into spheroids after being encapsulated in a liposomal configuration in comparison with free drug. Through a process known as serial trypsinization, three distinct cellular populations of a spheroid were successfully separated and a small molecule extraction was used to isolate the chemotherapeutic. Doxorubicin showed a time-dependent permeability into spheroids with the most drug accumulating in the core at 24 h of treatment. Entrapment of the chemotherapeutic delayed the permeability of the drug and resulted in reduced amounts quantified at the earlier time points. These findings validate the claim that liposomal therapeutics have the ability to alter the pharmacokinetics and pharmacodynamics profiles of a drug while also demonstrating the combined power of mass spectrometry and three-dimensional cell cultures to evaluate drug penetration and metabolism. Graphical abstract.