Doxorubicin Fluorescence Research Articles

Remote loading of doxorubicin into liposomes by transmembrane pH gradient to reduce toxicity toward H9c2 cells

The use of doxorubicin (DOX) is limited by its dose-dependent cardiotoxicity. Entrapped DOX in liposome has been shown to reduce cardiotoxicity. Results showed that about 92% of the total drug was encapsulated in liposome. The release experiments showed a weak DOX leakage in both culture medium and in PBS, more than 98% and 90% of the encapsulated DOX respectively was still retained in liposomes after 24h of incubation. When the release experiments were carried out in phosphate buffer pH5.3, the leakage of DOX from liposomes reached 37% after 24h of incubation. Evaluation of cellular uptake of the liposomal DOX indicated the possible endocytosis of liposomes because the majority of visible fluorescence of DOX was mainly in the cytoplasm, whereas the nuclear compartment showed a weak intensity. When using unloaded fluorescent-liposomes, the fluorescence was absent in nuclei suggests that liposomes cannot cross the nuclear membrane. MTT assay and measurement of LDH release suggest that necrosis is the form of cellular death predominates in H9c2 cells exposed to high doses of DOX, while for weak doses apoptosis could be the predominate form. Entrapped DOX reduced significantly DOX toxicity after 3 and 6h of incubation, but after 20h entrapped DOX is more toxic than free one.

Cholesterol-Enhanced Polylactide-Based Stereocomplex Micelle for Effective Delivery of Doxorubicin.

Nanoscale micelles as an effective drug delivery system have attracted increasing interest in malignancy therapy. The present study reported the construction of the cholesterol-enhanced doxorubicin (DOX)-loaded poly(D-lactide)-based micelle (CDM/DOX), poly(L-lactide)-based micelle (CLM/DOX), and stereocomplex micelle (CSCM/DOX) from the equimolar enantiomeric 4-armed poly(ethylene glycol)–polylactide copolymers in aqueous condition. Compared with CDM/DOX and CLM/DOX, CSCM/DOX showed the smallest hydrodynamic size of 96 ± 4.8 nm and the slowest DOX release. The DOX-loaded micelles exhibited a weaker DOX fluorescence inside mouse renal carcinoma cells (i.e., RenCa cells) compared to free DOX·HCl, probably because of a slower DOX release. More importantly, all the DOX-loaded micelles, especially CSCM/DOX, exhibited the excellent antiproliferative efficacy that was equal to or even better than free DOX·HCl toward RenCa cells attributed to their successful internalization. Furthermore, all of the DOX-loaded micelles exhibited the satisfactory hemocompatibility compared to free DOX·HCl, indicating the great potential for systemic chemotherapy through intravenous injection.

Copper Prussian blue analogue: investigation into multifunctional activities for biomedical applications.

We have designed and developed stable and biocompatible copper analogues of a Prussian blue (PB) nanoparticle based drug delivery system containing doxorubicin that show selective quenching of fluorescence of doxorubicin compared to PB analogues with other metals and inhibition of cancer cell proliferation, suggesting future potential multifunctional applications in biomedical sciences.

Interaction of doxorubicin with a regulatory element of hmga1 and its in vitro anti-cancer activity associated with decreased HMGA1 expression

High mobility group A1 (HMGA1) non-histone chromatin protein is known as an architectural transcription factor that regulates transcription of various genes. HMGA1 is highly expressed in almost all human cancers and considered as a potent tumor marker. Because of its association with cancers, hmga1 is considered as a critical target for anti-cancer drugs. In the present study, we report interaction of doxorubicin (DOX) with a short deoxyoligonucleotide (−1917 to −1940) within a regulatory element of hmga1 and its subsequent effect on expression of HMGA1 in breast cancer MCF7 cells. Binding of DOX to DNA was found to be strong (Ka, 5.2×105M−1) and thermodynamically favorable by both negative enthalpy (ΔH, −8.1±0.25kcalM−1) and positive entropy changes (TΔS, 21.1±5.2kcalM−1) at 20°C. A significant increase in melting temperature of DNA in presence of DOX by +10°C was accompanied by substantial quenching of fluorescence of DOX (∼85%) at 595nm and hypochromic change (∼40%) at 500nm absorption spectra of DOX along with a bathochromic shift of ∼5nm. Reduced expression of HMGA1 by ∼60% both at mRNA and protein level and associated cell death in presence of DOX was observed in breast cancer cells. Therefore, hmga1 is a promising chemotherapeutic target in treating human malignancies.

Redox-dependent ferric oxide nanoparticles loaded with doxorubicin and their influence on the functions of mitochondria

The spectral, fluorescent and functional properties of ferric oxide and ferric hydroxide nanoparticles loaded with doxorubicin and stabilized with citric acid or lysine were studied in comparison with free doxorubicin. Their effect on the opening of calcium-induced mitochondrial pore and the possibility of the controlled release of doxorubicin under the influence of redox stimuli were investigated. The data show that the effect of nanoparticles on mitochondria depends on the type of a stabilizer. The spectral and fluorescence methods used allow us to estimate the presence or absence of free doxorubicin in solution of nanoparticles and the:concentration of bound doxorubicin. It is shown that the dithiotreitol and glutathione increase the amplitude of absorption and fluorescence of doxorubicin during incubation with nanoparticles. It is assumed that this effect may be associated with the reduction of the oxidized iron by thiols with subsequent release of doxorubicin.

Doxorubicin Encapsulation Investigated by Capillary Electrophoresis with Laser-Induced Fluorescence Detection

Doxorubicin (DOX) belongs to the group of anthracycline antibiotics with very effective anticancer properties. On the other hand, the cardiotoxic effects limit its application over the maximum cumulative dose. To overcome this obstacle, encapsulation of this drug into the protective nanotransporter such as apoferritin is beneficial. In this study, fluorescent behavior of DOX in various solvents was determined by fluorescence spectrometry, demonstrating the fluorescence quenching effect of water, which is often used as a solvent. It was found that by increasing the amount of the organic phase in the DOX solvent the dynamic quenching is significantly suppressed. Ethanol, acetonitrile and dimethyl sulfoxide were tested and the best linearity of the calibration curve was obtained when above 50 % of the solvent was present in the binary mixture with water. Moreover, pH influence on the DOX fluorescence was also observed within the range of 4–10. Two times higher fluorescence intensity was observed at pH 4 compared to pH 10. Further, the DOX behavior in capillary electrophoresis (CE) was investigated. Electrophoretic mobilities (CE) in various pH of the background electrolyte were determined within the range from 16.3 to −13.3 × 10 −9 m−2 V−1 s−1. Finally, CE was also used to monitor the encapsulation of DOX into the cavity of apoferritin as well as the pH-triggered release.

Responsive delivery of drug cocktail via mesoporous silica nanolamps

After a substantial advancement in single drug nanocarrier, nanomedicine now demands an integration of nanotechnology with combination therapy to achieve synergistic therapeutic effects. In this respect, a smart and multiple drug shuttling nanotheranostic system is developed which transport diverse kinds of anticancer drugs to cancer cells in a controlled and responsive manner respectively. Synthetically, a significantly high dose of hydrophobic camptothecin (CPT) is first loaded into the porous structure of quantum dots (CdS) coupled mesoporous silica nanocomposite. Subsequently, fluorescent doxorubicin (DOX) molecules are exclusively anchored onto the surface of CdS; as a result, the fluorescence of both CdS and DOX is quenched. Upon exposing to mildly acidic conditions, the fluorescence of both species is recovered, such fluorescent “on–off” states provides an added opportunity to real time sense drug release. In-vitro cell experiment reveals an excellent anticancer efficacy of drug cocktail, merely 3μg/ml concentration of multiple drugs loaded nanocarrier reduces the cell viability to 30%. Furthermore, confocal imaging indicates a successful release of both therapeutic entities. We visualize that our newly fabricated multifunctional double drug-carrying nanoparticles can be a valuable addition to next generation of materials that simultaneously deliver cocktail of drugs with imaging functionality.

Host-guest interactions in Fe(III)-trimesate MOF nanoparticles loaded with doxorubicin.

Doxorubicin (DOX) entrapment in porous Fe(III)-trimesate metal organic frameworks (MIL-100(Fe)) nanoparticles was investigated in neutral Tris buffer via UV-vis absorption, circular dichroism (CD), and fluorescence. The binding constants and the absolute spectra of the DOX-MIL-100(Fe) complexes were determined via absorption and fluorescence titrations. A binding model where DOX associates as monomer to the dehydrated Fe3O (OH)(H2O)2 [(C6H3)(CO2)3]2 structural unit in 1:1 stoichiometry, with apparent association constant of (1.1 to 1.8) × 10(4) M(-1), was found to reasonably fit the experimental data. Spectroscopic data indicate that DOX binding occurs via the formation of highly stable coordination bonds between one or both deprotonated hydroxyl groups of the aglycone moiety and coordinatively unsaturated Fe(III) centers. Complete quenching of the DOX fluorescence and remarkable thermal and photochemical stability were observed for DOX incorporated in the MIL-100(Fe) framework.

HIGH INTERSTITIAL FLUID PRESSURE REGULATES TUMOR GROWTH AND DRUG UPTAKE IN HUMAN GLIOBLASTOMA

BACKGROUND: (blind field). METHODS: We developed methodology to measure IFP in human GBM xenografts and mechanical compression in 3D-cultures. We used a cell counter and calcein-AM-loaded cells to measure cell volume following treatment with temozolomide and/or blockade of NKCC1 activity. Fluorescent MQAE-loaded cells were recorded to measure intracellular chloride levels. To study uptake of chemotherapy following IFP-reduction in GBM xenografts following AF-induction or treatment with bumetanide, we injected doxorubicin and measured fluorescent doxorubicin levels in tissue sections. RESULTS: Elevated compression increased proliferation in GBM cultures and IFP levels rapidly increased during the exponential growth phase in GBM xenografts. In contrast to Avastin that targets the vasculature, SPC diet induced AF expression only in tumor cells. In addition to reducing IFP levels, AF-induction also inhibited proliferation, induced apoptosis, and increased survival in mice xenografted with human GBM cells. SPC diet and intranasal injection of AF increased uptake of doxorubicin in GBM xenografts. In vitro, AF augmented TMZ-induced apoptosis and reduced proliferation at both baseline and increased hydrostatic compression. CONCLUSIONS: In contrast to IFP-reducing therapies targeting the vasculature, treatments that reduced osmotic pressure in GBM cells effectively reduced tumor growth and invasion in vivo. AF and bumetanide increased uptake and cytotoxic response from chemotherapies by inhibiting NKCC1 activity in GBM cells. Our studies suggest that elevated IFP promotes tumor growth, reduces drug uptake, and limits therapy-response in GBM. AF-induction represents an attractive strategy to reduce invasion, inhibit tumor growth, increase drug uptake, and ultimately improve the survival of GBM patients. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

Targeted cytotoxic analog of luteinizing hormone-releasing hormone (LHRH), AEZS-108 (AN-152), inhibits the growth of DU-145 human castration-resistant prostate cancer in vivo and in vitro through elevating p21 and ROS levels.

Management of castration-resistant prostate cancer (CRPC) is challenging due to lack of efficacious therapy. Luteinizing hormone-releasing hormone analogs appear to act directly on cells based on the LHRH receptors on human prostate adenocarcinoma cells. We explored anticancer activity of a cytotoxic analog of LHRH, AEZS-108 consisting of LHRH agonist linked to doxorubicin. Nude mice bearing DU-145 tumors were used to compare antitumor effects of AEZS-108 with its individual constituents or their unconjugated combination. The tumor growth inhibition of conjugate was greatest among treatment groups (90.5% inhibition vs. 41% by [D-Lys(6)]LHRH+DOX). The presence of LHRH receptors on DU-145 cells was confirmed by immunocytochemistry. In vitro, AEZS-108 significantly inhibited cell proliferation (61.2% inhibition) and elevated apoptosis rates (by 46%). By the detection of the inherent doxorubicin fluorescence, unconjugated doxorubicin was seen in the nucleus; the conjugate was perinuclear and at cell membrane. Autophagy, visualized by GFP-tagged p62 reporter, was increased by AEZS-108 (7.9-fold vs. 5.3-fold by DOX+[D-Lys(6)]LHRH. AEZS-108 more effectively increased reactive oxygen species (ROS, 2-fold vs. 1.4-fold by DOX+[D-Lys(6)]LHRH) and levels of the apoptotic regulator p21 in vivo and in vitro. We demonstrate robust inhibitory effects of the targeted cytotoxic LHRH analog AEZS-108 on LHRHR positive castration-resistant prostate cancer cells.

Co-delivery of doxorubicin and paclitaxel by PEG-polypeptide nanovehicle for the treatment of non-small cell lung cancer

Despite progress, combination therapy of different functional drugs to increase the efficiency of anticancer treatment still remains challenges. An amphiphilic methoxy poly(ethylene glycol)-b-poly(l-glutamic acid)-b-poly(l-lysine) triblock copolymer decorated with deoxycholate (mPEsG-b-PLG-b-PLL/DOCA) was synthesized and developed as a nanovehicle for the co-delivery of anticancer drugs: doxorubicin (DOX) and paclitaxel (PTX). The amphiphilic copolymer spontaneously self-assembled into micellar-type nanoparticles in aqueous solutions and the blank nanoparticles possessed excellent stability. Three different domains of the copolymer performed distinct functions: PEG outer corona provided prolonged circulation, middle biodegradable and hydrophilic PLG shell was designed for DOX loading through electrostatic interactions, and hydrophobic deoxycholate modified PLL served as the container for PTX. In vitro cytotoxicity assays against A549 human lung adenocarcinoma cell line demonstrated that the DOX + PTX co-delivered nanoparticles (Co-NPs) exhibited synergistic effect in inducing cancer cell apoptosis. Ex vivo DOX fluorescence imaging revealed that Co-NPs had highly efficient targeting and accumulation at the implanted site of A549 xenograft tumor in vivo. Co-NPs exhibited significantly higher antitumor efficiency in reducing tumor size compared to free drug combination or single drug-loaded nanoparticles, while no obvious side effects were observed during the treatment, indicating this co-delivery system with different functional antitumor drugs provides the clinical potential in cancer therapy.

Mechanism of preventing doxorubicin‐induced mitochondrial toxicity with cardiolipin‐targeted peptide, SS‐31 (966.1)

Doxorubicin (DOX) is an effective chemotherapeutic that intercalates DNA and inhibits topoisomerase II in cancer cells. However, clinical use of DOX is limited by its cardiac toxicity, resulting from its ability to bind to cardiolipin (CL) and inhibit mitochondrial function. SS‐31, a mitochondria‐targeted tetrapeptide was shown to prevent DOX‐induced toxicity in cardiac and skeletal muscle, and cultured myocytes. However, the mechanism of protection against DOX by SS‐31 is not well understood. Here, by taking advantage of the intrinsic fluorescence of DOX, we developed assays to measure DOX binding to free CL, CL‐containing liposomes, mitoplasts and mitochondria. We demonstrated that SS‐31 prevents DOX binding in different CL‐containing model systems. Furthermore, we showed that SS‐31 could inhibit DOX binding to endogenous CL and uptake in mitoplasts and mitochondria in a dose dependent manner. Therefore, SS‐31 protects against mitochondrial DOX toxicity by preventing its binding to CL. Targeting CL with SS‐31 has clinical implications in increasing the therapeutic window of DOX and potentially addressing other therapeutics with off‐target side effects associated with CL and mitochondrial toxicity.

Identification of dual DNA-PK MDR1 inhibitors for the potentiation of cytotoxic drug activity

Inhibition of DNA repair is an attractive therapeutic approach to enhance the activity of DNA-damaging anticancer chemotherapeutic agents. Similarly, blockade of the multidrug-resistance protein 1 (MDR1) can overcome efflux-mediated resistance. DNA-dependent protein kinase (DNA-PK) is essential for the non-homologous end-joining DNA repair pathway. NU7441 is a potent DNA-PK inhibitor (IC50=14nM) that is used widely to study the effects of DNA-PK inhibition in vitro. In growth inhibition studies, 1μM NU7441 sensitised vincristine-resistant CCRF-CEM VCR/R leukaemia cells (1200-fold resistant) to a range of MDR1 substrates, including doxorubicin (8-fold, p=0.03), vincristine (14-fold, p=0.01) and etoposide (63-fold, p=0.02), compared with 1.4-fold (p=0.02), 2.2-fold (p=0.04) and 3.6-fold (p=0.01) sensitisation, respectively, in parental CCRF-CEM cells. This difference in NU7441 sensitivity was confirmed in another two parental and MDR1-overexpressing cell line pairs. A doxorubicin fluorescence assay showed that in MDR1-overexpressing canine kidney MDCKII-MDR1 cells, 1μM NU7441 increased doxorubicin nuclear fluorescence 16-fold. NU7441 and 3 structurally related compounds (NU7742 (an NU7441 analogue that does not inhibit DNA-PK – IC50>10μM), DRN1 (DNA-PK-inhibitory atropisomeric NU7441 derivative – IC50=2nM) and DRN2 (DNA-PK non-inhibitory atropisomeric NU7441 derivative - IC50=7μM)) all increased intracellular vincristine accumulation in the CCRF-CEM VCR/R cells to a level similar to verapamil, as measured by LC–MS. This paper demonstrates that NU7441 is a dual DNA-PK and MDR1 inhibitor, and this extends the therapeutic potential of the compound when used in combination with MDR substrates.

Mitochondrial Delivery of Doxorubicin by Triphenylphosphonium-Functionalized Hyperbranched Nanocarriers Results in Rapid and Severe Cytotoxicity

To develop a novel hyperbranched polymer-based nanocarrier for efficient drug delivery to cell mitochondria. Also to study for the first time the cytotoxic effect of doxorubicin via mitochondria-specific delivery system. We introduced alkyltriphenylphosphonium groups (TPP) to a poly(ethylene imine) hyperbranched polymer (PEI). We harnessed the hydrophobic assembly of these alkylTPP functionalized PEI molecules into ~100nm diameter nanoparticles (PEI-TPP) and further encapsulated the chemotherapy agent doxorubicin (DOX), to produce the mitotropic nanoparticles PEI-TPP-DOX. By administering PEI-TPP-DOX to human prostate carcinoma cells DU145, we found that: (i) PEI-TPP-DOX specifically localized at cell mitochondria as revealed by the inherent DOX fluorescence; (ii) in contrast to the slow apoptotic cell death incurred by DOX over the period of days at micromolar concentrations, PEI-TPP-DOX triggered rapid and severe cytotoxicity within few hours of incubation and at submicromolar incubation concentrations. This cytotoxicity was mainly found to be of a necrotic nature, not precluding autophagy related death pathways to a smaller extent. We have elaborated a versatile mitotropic nanocarrier; furthermore, using this platform, we have developed a mitochondrial-doxorubicin formulation with exceptional cytocidal properties, even in nanomolar concentrations.

Abstract 3329: The DNA-PK inhibitor, NU7441, sensitises cancer cells to microtubule-targeting agents and modulates vincristine efflux in multidrug resistant cells.

Abstract Microtubules are involved in the formation of the mitotic spindle, and correct separation of the sister chromatids at mitosis is essential for maintaining genomic stability. Microtubule-targeting drugs arrest rapidly-dividing cells and are important chemotherapeutic agents, but the development of multidrug-resistance (MDR) is a serious clinical problem. Along with its role in DNA double-strand break repair, DNA-dependent protein kinase (DNA-PK) has also been shown to play an important role in stabilising spindle formation and preventing mitotic catastrophe (Shang et al, Cancer Res. 2010). In light of this novel finding, we hypothesised that inhibition of DNA-PK would sensitise cells to microtubule-targeting agents. Pilot studies showed that inhibition of DNA-PK by the selective inhibitor, NU7441, caused greater sensitisation to vincristine in multidrug-resistant cells compared to parental cells. Here, we further investigate the role of DNA-PK in response to microtubule-targeting drugs using a novel series of NU7441 analogues, and investigate whether these inhibitors interact with the efflux transporter, P-glycoprotein. In growth inhibition studies, CCRF-CEM leukaemia cells were sensitised 1.4-fold to vincristine by NU7441 (1μM) whereas their vincristine-resistant derivative CCRF-CEM VCR/R cells were sensitised 8-fold to vincristine. DNA-PK-deficient M059J glioblastoma cells were 2-fold more sensitive to docetaxel than DNA-PK-proficient cells, and NU7441 sensitised only DNA-PK-proficient M059J-Fus1 cells to docetaxel. CCRF-CEM VCR/R cells have a higher basal level of autophosphorylation of DNA-PKcs (Western blotting) than the CCRF-CEM cells and autophosphorylation in response to vincristine was abrogated by NU7441. To investigate the effects of NU7441 on drug transport, a doxorubicin fluorescence assay showed that in P-glycoprotein-overexpressing canine kidney MDCKII-MDR1 cells, 1μM NU7441 increased doxorubicin cellular fluorescence 16-fold by blocking drug efflux via P-glycoprotein. NU7441 and 3 structurally-related compounds (NU7742 (inactive NU7441 analogue), DRN1 (DNA-PK-inhibitory atropisomeric NU7441 derivative) or DRN2 (DNA-PK non-inhibitory atropisomeric NU7441 derivative)) all increased intracellular vincristine accumulation in the CCRF-CEM VCR/R cells to a level similar to verapamil, as measured by LC-MS. However, the non-inhibitory compound NU7742 was 5-fold less effective at sensitising CCRF-CEM VCR/R cells to vincristine than NU7441 (p=0.008). The novel finding that DNA-PK inhibition sensitises cells to microtubule-targeting agents suggests that along with its function in DNA repair, this enzyme plays a role in the mitotic spindle checkpoint. Additionally, in multidrug-resistant cells, NU7441 is able to block drug efflux via interaction with P-glycoprotein. Citation Format: Emily Mould, Philip Berry, David Jamieson, Christopher Hill, Niu Tan, Sarah Elliott, Barbara Durkacz, David Newell, Elaine Willmore. The DNA-PK inhibitor, NU7441, sensitises cancer cells to microtubule-targeting agents and modulates vincristine efflux in multidrug resistant cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3329. doi:10.1158/1538-7445.AM2013-3329

Use of Molecular Biomarkers to Quantify the Spatial Distribution of Effects of Anticancer Drugs in Solid Tumors

Poor distribution of anticancer drugs within solid tumors may limit their effectiveness. Here, we characterize the distribution within solid tumors of biomarkers of drug effect. γ-H2AX, cleaved-caspase-3 or -6, and Ki67 were quantified in tumor sections in relation to blood vessels (recognized by CD31) using monoclonal antibodies and immunohistochemistry. To validate their use, we compared their time-dependent distribution with that of (i) fluorescent doxorubicin and (ii) a monoclonal antibody that detects melphalan-induced DNA adducts. The biomarkers were then used to quantify the distribution of docetaxel in relation to tumor blood vessels. Activation of γ-H2AX was evaluated following in vitro exposure of tumor cells to multiple drugs. Distributions of doxorubicin in MDA-MB-231 and MCF-7 xenografts and of melphalan-induced DNA adducts in MCF-7 and EMT-6 tumors decreased with distance from blood vessels, similar to the distributions of (i) γ-H2AX at 10 minutes, (ii) cleaved caspase-3 or -6, and (iii) change in Ki67 at 24 hours following treatment. The distribution of these biomarkers following treatment with docetaxel also decreased with increasing distance from tumor blood vessels. Activation of γ-H2AX occurred within 1 hour after exposure to several drugs in culture. Multiple anticancer drugs show a decrease in activity with increasing distance from tumor blood vessels; poor drug distribution is an important cause of drug resistance. The above biomarkers may be used in designing strategies to overcome therapeutic resistance by modifying or complementing the limited spatial distribution of drug activity in solid tumors.

Single-Step Assembly of DOX/ICG Loaded Lipid–Polymer Nanoparticles for Highly Effective Chemo-photothermal Combination Therapy

A combination of chemotherapy and photothermal therapy has emerged as a promising strategy for cancer therapy. To ensure the chemotherapeutic drug and photothermal agent could be simultaneously delivered to a tumor region to exert their synergistic effect, a safe and efficient delivery system is highly desirable. Herein, we fabricated doxorubicin (DOX) and indocyanine green (ICG) loaded poly(lactic-co-glycolic acid) (PLGA)-lecithin-polyethylene glycol (PEG) nanoparticles (DINPs) using a single-step sonication method. The DINPs exhibited good monodispersity, excellent fluorescence/size stability, and consistent spectra characteristics compared with free ICG or DOX. Moreover, the DINPs showed higher temperature response, faster DOX release under laser irradiation, and longer retention time in tumor. In the meantime, the fluorescence of DOX and ICG in DINPs was also visualized for the process of subcellular location in vitro and metabolic distribution in vivo. In comparison with chemo or photothermal treatment alone, the combined treatment of DINPs with laser irradiation synergistically induced the apoptosis and death of DOX-sensitive MCF-7 and DOX-resistant MCF-7/ADR cells, and suppressed MCF-7 and MCF-7/ADR tumor growth in vivo. Notably, no tumor recurrence was observed after only a single dose of DINPs with laser irradiation. Hence, the well-defined DINPs exhibited great potential in targeting cancer imaging and chemo-photothermal therapy.

Enhanced drug delivery in rabbit VX2 tumours using thermosensitive liposomes and MRI-controlled focused ultrasound hyperthermia

Purpose: The efficacy of anticancer drugs in solid tumours is impaired by their inability to reach all cancer cells in sufficient concentration to cause cytotoxicity. Hyperthermia-triggered release of drugs from thermosensitive liposomes can increase tumour drug concentration, but tumour-specific drug delivery requires precise temperature control, and effects on microregional distribution of anticancer drugs in tumours are unknown. Here we evaluate thermally triggered release of doxorubicin in a rabbit tumour model by comparing free versus thermosensitive liposomal doxorubicin administered systemically during magnetic resonance imaging (MRI)-controlled focused ultrasound hyperthermia.Materials and methods: Twelve rabbits with a transplanted VX2 tumour in each thigh had a 10 mm diameter region in one tumour heated to 43°C using focused ultrasound with temperature control by MRI thermometry. Delivery of doxorubicin to tumours and normal tissues was quantified by fluorescence in tissue homogenates, and by fluorescence microscopy.Results: Using thermosensitive liposomal doxorubicin (2.5 mg/kg), doxorubicin concentrations in heated tumours were 26.7 times higher than in unheated tumours (n = 7, p = 0.017, two-sided Wilcoxon signed-rank test). There was no significant enhancement with free doxorubicin in heated versus unheated tumours (n = 3, p = 0.5). With thermosensitive liposomes (8.3 mg/kg), fluorescence microscopy demonstrated increased doxorubicin fluorescence in heated versus unheated tumours, co-localised with nuclear staining throughout the tumour.Conclusions: Localised image-guided delivery of high concentrations of doxorubicin to cancer cells was achieved non-invasively in implanted tumours with temperature-sensitive drug carriers and a preclinical MRI-controlled focused ultrasound hyperthermia system.

Water‐Dispersible Fullerene Aggregates as a Targeted Anticancer Prodrug with both Chemo‐ and Photodynamic Therapeutic Actions

Prodrug therapy is one strategy to deliver anticancer drugs in a less reactive manner to reduce nonspecific cytotoxicity. A new multifunctional anticancer prodrug system based on water-dispersible fullerene (C60) aggregates is introduced; this prodrug system demonstrates active targeting, pH-responsive chemotherapy, and photodynamic therapeutic (PDT) properties. Incorporating (via a cleavable bond) an anticancer drug, which is doxorubicin (DOX) in this study, and a targeting ligand (folic acid) onto fullerene while maintaining an overall size of approximately 135 nm produces a more specific anticancer prodrug. This prodrug can enter folate receptor (FR)-positive cancer cells and kill the cells via intracellular release of the active drug form. Moreover, the fullerene aggregate carrier exhibits PDT action; the cytotoxicity of the system towards FR-positive cancer cells is increased in response to light irradiation. As the DOX drug molecules are conjugated onto fullerene, the DOX fluorescence is significantly quenched by the strong electron-accepting capability of fullerene. The fluorescence restores upon release from fullerene, so this fluorescence quenching-restoring feature can be used to track intracellular DOX release. The combined effect of chemotherapy and PDT increases the therapeutic efficacy of the DOX-fullerene aggregate prodrug. This study provides useful insights into designing and improving the applicability of fullerene for other targeted cancer prodrug systems.

Differential Immunomodulating Effects of Pegylated Liposomal Doxorubicin Nanoparticles on Human Macrophages

The pegylated liposomal doxorubicin (PLD) has been widely accepted in treatment of various cancers. However, the composition of two currently marketed PLD nanoparticles differs in structure and composition of lipids, and their differential effects remain unknown. Macrophages of the mononuclear phagocyte system are pivotal in determining PLD clearance in vivo. The aim of this study was to compare the effect of these two PLDs on drug uptake, cell viability, morphology and immune function of human macrophages. Two PLD nanoparticles were used in this study. The major difference between PLD-D and PLD-H is that their phospholipid bilayers are composed of distearoyl phosphatidylcholine (DSPC) and hydrogenated soybean phosphatidylcholine (HSPC), respectively. Human CD14+ monocytes were isolated from peripheral blood to prepare macrophages. Comparative assays included: flow cytometry for detection of doxorubicin penetration into cells, MTT for cell viability, Trypan blue exclusion for cell membrane integrity, Liu's stain for morphologic evaluation, and inactivated yeast co-culture for phagocytosis. The uptake of PLD-H was rapidly detected at 10 min and kept increasing to 4 h followed by a decline thereafter, whereas that of PLD-D had similar profile with much less doxorubicin fluorescence detected, indicating a greater amount of doxorubicin retention of PLD-H. PLD-H, at higher concentration, decreased the viability and impaired cell membrane integrity of macrophages with an extent greater than PLD-D. The morphological observation showed a more extensive necrosis in PLD-H-treated macrophages. The phagocytosis function of macrophage was inhibited with a greater extent in PLD-H-treated macrophages. The PLD containing HSPC may cause retention of doxorubicin with greater amount and longer period in human macrophages than that containing DSPC. This effect was accompanied by greater toxicity and more profound dysfunction. The correlation of this differential effect to clinical outcome remains to be extensively investigated by performing in vivo experiments or conducting clinical trials.