Doxorubicin Fluorescence Intensity Research Articles

Sub-50 nm core-shell nanoparticles with the pH-responsive squeezing release effect for targeting therapy of hepatocellular carcinoma.

The development of drug delivery systems with high drug loading capacity, low leakage at physiological pH, and rapid release at the lesion sites remains an ongoing challenge. In this work, core-shell poly(6-O-methacryloyl-D-galactose)@poly(tert-butyl methacrylate) (PMADGal@PtBMA) nanoparticles (NPs) of sub-50 nm are facilely synthesized by reversible addition-fragmentation chain transfer (RAFT) soap-free emulsion polymerization with the assistance of 12-crown-4. A hydrophilic poly(methacrylic acid) (PMAA) core can then be revealed after deprotection of the tert-butyl groups, which is negatively charged and can adsorb nearly 100% of incubated doxorubicin (DOX) from a solution at pH 7.4. The physical shrinkage of PMAA chains below pH 6.0 endows the core with the squeezing effect, therefore realizing rapid drug release. It is demonstrated that the DOX release rate of PMADGal@PMAA NPs at pH 5 was 4 times that at pH 7.4. Cellular uptake experiments confirm the high targeting ability of the galactose modified PMADGal shell to human hepatocellular carcinoma (HepG2) cells. The fluorescence intensity of DOX in HepG2 cells is 4.86 times that of HeLa cells after 3 h incubation. Moreover, 20% cross-linked NPs show the highest uptake efficiency by HepG2 cells due to their moderate surface charge, size and hardness. In summary, both the core and the shell of PMADGal@PMAA NPs promise the rapid site-specific release of DOX in HepG2 cells. This work provides a facile and an effective strategy to synthesize core-shell NPs for hepatocellular carcinoma targeting therapy.

Enhancing Doxorubicin Delivery in Solid Tumor by Superhydrophobic Amorphous Calcium Carbonate-Doxorubicin Silica Nanoparticles with Focused Ultrasound.

The current approach of delivering chemotherapy via pH-sensitive amorphous calcium carbonate-doxorubicin silica nanoparticles (ADS NPs) faces the challenge of insufficient drug dose due to drug instability within the bloodstream and poor tumor penetration. To overcome these long-standing obstacles, we proposed a superhydrophobic coating on the surface of the ADS NPs that could be easily modified via fluorination (ADSF NPs). The surface of fluorinated ADS NPs was further modified with a phospholipid layer to reduce aggregation and improve biocompatibility (ADSFL NPs). The contact angle and mean size of ADSFL NPs were 30.2 ± 4.4° and 353.1 ± 54.2 nm, respectively. The superhydrophobic layer generated interfacial nanobubbles on the outer shell of the NPs that reduced water-induced leakage of doxorubicin (DOX) sevenfold compared with the uncoated group and induced a cavitation effect upon ultrasound (US) sonication. Moreover, release of DOX from the ADSFL NPs could be triggered by US, and this release was further improved 1.6-fold in acidic aqueous conditions, indicating that the ADSFL NPs retained pH responsiveness. Enhanced sonography contrast and histological examination demonstrated that US could trigger cavitation activities from ADSFL NPs in vivo to induce vessel disruption and enhance the fluorescence intensity of DOX within the tumor region threefold under US imaging guidance compared with the ADSFL NPs-only group.

Cetuximab-modified doxorubicin bovine serum albumin nanoparticles for the treatment of epidermal growth factor receptor-overexpressing colon cancer cells

In this study, we developed an active targeting nano-immunoplatform of doxorubicin (DOX)-loaded bovine serum albumin (BSA) modified with the humanized antibody cetuximab, which can target the epidermal growth factor receptor (EGFR) of EGFR-overexpressing colon cancer, and subjected it to a series ofin vitroevaluation. The obtained cetuximab-modified nanoparticles (cetuximab-DOX-immuno-NPs) had an average particle size of 218.2±2.2 nm, a polydispersity index of 0.162±0.023, a zeta potential of −23.28±0.75 mV, and a cetuximabloading efficiency of 33.6%±3.5%. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity experiments showed that the cytotoxic of cetuximab-DOX-immuno-NPs in EGFR-overexpressing RKO colon cancer cells was significantly higher than it was in EGFR-under-expressing LS174 T cells. The results of flow cytometry showed that when DOX-immuno-NPs were used, DOX was eliminated soon after entering the cells, whereas when cetuximab-DOX-immuno-NPs were used, DOX efflux was reduced. Confocal laser-scanning microscopy was used to visualize the endocytosis of nanoparticles, and revealed that the fluorescence intensity of cetuximab-DOX-immuno-NPs was significantly higher than that of non-specific IgG-DOX-immuno-NPs in RKO cells. Moreover, in EGFR-under-expressing LS174 T cells, both cetuximab-DOXimmuno-NPs and Immunoglobulin G-DOX-immuno-NPs showed weak DOX fluorescence intensity, reflecting the specificity and selectivity of cetuximab-modified nanoparticles toward EGFR-overexpressing cancer cells. This study demonstrated that cetuximab modification of DOX-loaded BSA nanoparticles can increase their selectivity and reduce their toxicity. As this would decrease the side effects of these treatments in patients, this study shows that cetuximab-modified doxorubicin-loaded bovine serum albumin nanoparticles are promising candidates for targeted colon cancer therapy.

In vitro Anti-cancer Efficacy and Cellular Interaction of Cubic Phases Containing Cinnamic Acid, Poly(ethyleneimine), and Doxorubicin

Folate-decorated monoolein (MO) cubosomes containing poly(ethyleneimine) (PEI), cinnamic acid (CA), and doxorubicin (DOX) were prepared to deliver the anticancer drug specifically to cancer cells (KB cells) and boost the anti-cancer efficacy without causing an acute toxicity. Hydrophobically modified (Hm) gelatin (Gel)-folate (FA) conjugate (HmGel-FA) was prepared through amidation reaction among Gel, decanoyl chloride, and FA. HmGel-FA and Pluronic F127 were used as stabilizers for the cubosomal particles. According to the measurement of air/water interfacial tension, the surface activity was greater in the order of HmGel > HmGel-FA > Gel. Since it was reported that PEI and CA formed a self-assembly in the water channel of cubosomes and the dissolving property of the self-assembly was dependent on the pH value of medium, they were included in the water channel as a controller for DOX release. On the TEM micrographs of the cubosomes, water channels were surrounded by MO bilayers, and the payload (i.e. PEI, CA, DOX) had little effect on the structure. The in vitro anti-cancer efficacy of folate-decorated cubosomes containing PEI, CA, and DOX was higher than that of folate-free ones. According to flow cytometric analysis and confocal laser scanning microscopy, DOX fluorescence intensity was greater in the order of KB cells treated with folate-decorated cubosomes containing PEI, CA, and DOX > folate-free ones > free DOX. The folate-decorated cubosomes would be able to target the cancer cells and subsequently the receptor-mediated endocytosis would take place.

Synergistic Treatment of Tumor by Targeted Biotherapy and Chemotherapy via Site-Specific Anchoring of Aptamers on DNA Nanotubes.

BackgroundAptamers have been widely used as targeted therapeutic agents due to its relatively small physical size, flexible structure, high specificity, and selectivity. Aptamers functionalized nanomaterials, not only enhance the targeting of nanomaterials, but can also improve the stability of the aptamers. We developed aptamer C2NP (Apt) conjugated straight DNA nanotubes (S-DNT-Apt) and twisted DNA nanotubes (T-DNT-Apt) as nanocarriers for doxorubicin (DOX).MethodsThe twisted DNA nanotubes (T-DNT) and straight DNA nanotubes (S-DNT) were assembled with a scaffold and hundreds of staples. Apt was site-specifically anchored on DNA nanotubes with either different spatial distribution (3 or 6 nm) or varied stoichiometry (15Apt or 30Apt). The developed nanocarriers were characterized with agarose gel electrophoresis and transmission electron microscopy. The drug loading and release in vitro were evaluated by measuring the fluorescence intensity of DOX using a microplate reader. The stability of DNT in cell culture medium plus 10% of FBS was evaluated by agarose gel electrophoresis. The cytotoxicity of DNA nanostructures against K299 cells was tested with a standard CCK8 method. Cellular uptake, cell apoptosis, cell cycle and reactive oxygen species level were investigated by flow cytometry. The expression of p53 was examined by Western Blot.ResultsT-DNT-30Apt-6 exhibited the highest cytotoxicity when the concentration of Apt was 120 nM. After intercalation of DOX, the cytotoxicity of DOX@T-DNT-30Apt-6 was further enhanced due to the combination of chemotherapy of DOX and biotherapy of Apt. The enhanced cytotoxicity of DOX@T-DNT-30Apt-6 can be explained by the increase in the cellular uptake, cell apoptosis and intracellular ROS levels. Additionally, the interaction between Apt and its receptor CD30 could upregulate the expression of p53.ConclusionThese results demonstrate that both stoichiometry and spatial arrangement of Apt on T-DNT-Apt influence the anticancer activity. The developed twisted DNA nanotubes may be a solution for the synergistic treatment of cancer.

Effect of oxaliplatin induced autophagy on drug resistance of SGC7901 gastric cancer cells

Objective To evaluate the effect of oxaliplatin induced autophagy on drug resistance of gastric cancer cells. Methods Gastric cancer SGC7901 cells were cultured in vitro and treated with different concentrations of oxaliplatin. Control group (oxaliplatin 0 μmol/L), oxaliplatin treatment groups (oxaliplatin 1, 2, 4 μmol/L) and oxaliplatin combined with doxorubicin groups (oxaliplatin 0, 1, 2, 4 μmol/L + doxorubicin 4 μmol/L) were set up. Cells were treated with different conditions for 24 hours. Western blotting and flow cytometry were used to detect the expression of autophagy-related molecule Beclin-1. Methyl thiazolyl tetrazolium (MTT) assay was used to detect the cell viability. Flow cytometry was used to analyze the uptake of doxorubicin in SGC7901 cells. Results The expression of Beclin-1 increased in gastric cancer cells treated with oxaliplatin. The percentages of positive cells treated with 1, 2 and 4 μmol/L oxaliplatin were respectively (9.51±0.27)%, (13.73±0.80)% and (20.17±1.03)%, control group was (2.17±0.15)%, and the difference was statistically significant (F=111.10, P<0.001). Compared with the control group, Beclin-1 expression significantly increased in each treatment group (P<0.05; P<0.001; P<0.001). Different concentrations of 1, 2, 4 μmol/L oxaliplatin combined with doxorubicin were used to treat gastric cancer SGC7901 cells, fluorescence intensities of doxorubicin were 11 567±802, 13 433±808, 15 967±472, control group was 10 257±367, and the difference was statistically significant (F=79.81, P<0.001). Compared with the control group, the fluorescence intensity of doxorubicin significantly increased in each treatment group (P<0.05; P<0.001; P<0.001). After treatment with oxaliplatin (1, 2, 4 μmol/L) and doxorubicin (4 μmol/L) for 24 hours, the cell viability levels were (68.27±1.64)%, (51.72±1.93)%, (39.60±1.80)% respectively. The cell viability levels of oxaliplatin treated with 1, 2 and 4 μmol/L were respectively (93.70±1.15)%, (76.53±1.10)%, (74.00±1.65)%. Compared with oxaliplatin alone, oxaliplatin combined with doxorubicin decreased cell viability more obviously, and the differences were statistically significant (t=8.91, P<0.001; t=9.21, P<0.001; t=10.34, P<0.001). The doxorubicin fluorescence intensity of oxaliplatin (2 μmol/L) combined with doxorubicin (4 μmol/L) group with pretreatment of autophagy inhibitor was 16 898±105, oxaliplatin combined with doxorubicin group was 22 245±168, and doxorubicin alone group was 17 562±67, and the difference was statistically significant (F=92.16, P<0.001). Compared with the pretreatment group, the doxorubicin fluorescence intensity of oxaliplatin combined with doxorubicin group was significantly increased (P<0.001). On the other hand, the cell viability levels of the above three groups were (81.33±3.54)%, (65.00±2.61)% and (101.02±3.58)%, and the difference was statistically significant (F=90.66, P<0.001). Compared with the pretreatment group, the cell viability level of oxaliplatin combined with doxorubicin group was significantly lower (P<0.001). Conclusion Oxaliplatin can reduce the resistance of gastric cancer cells to chemotherapeutics by increasing the level of autophagy in gastric cancer cells, and improve the therapeutic effect of chemotherapy drugs. Key words: Antineoplastic agents; Autophagy; Stomach neoplasms; Antineoplastic combined chemotherapy protocols

Stimuli‐responsive Supra‐biomolecular Nanoassemblies of Cucurbit[7]uril with Bovine Serum Albumin: Drug Delivery and Sensor Applications

AbstractConstruction and characterization of stimuli‐responsive supra‐biomolecular nanoassembly between cucurbit[7]uril (CB7) and bovine serum albumin (BSA), uptake and release of doxorubicin (DOX) in live cells, the enhanced sensitivity of brilliant green (BG) and the metal ion‐induced relocation of neutral red (NR) dye to BSA have been discussed in this review. The fluorescence intensity of DOX is largely quenched in the presence of nanoassembly which recovers with adamantylamine or by changing the pH of the solution, indicating the significant uptake and release of DOX. Whereas, the interaction of BG with CB7‐BSA assembly leads to a huge fluorescence enhancement ∼350‐fold through ternary complex formation. In another study, the supramolecular pKa tuning of nanoassembly encapsulated NR dye with metal ion and the consequent relocation of NR from CB7 cavity to the hydrophobic pocket of BSA have been demonstrated. All these studies show promising applications in drug delivery and on‐off sensor.

An eco-friendly stability-indicating spectrofluorimetric method for the determination of two anticancer stereoisomer drugs in their pharmaceutical preparations following micellar enhancement: Application to kinetic degradation studies

A new rapid and highly sensitive stability-indicating spectrofluorimetric method was developed for the determination of two stereoisomers anticancer drugs, doxorubicin (DOX) and epirubicin (EPI) in pure form and in pharmaceutical preparations. The fluorescence spectral behavior of DOX and EPI in a sodium dodecyl sulfate (SDS) micellar system was investigated. It was found that the fluorescence intensity of DOX and EPI in an aqueous solution of phosphate buffer pH4.0 and in the presence of SDS was greatly (about two fold) enhanced and the mechanism of fluorescence enhancement effect of SDS on DOX was also investigated. The fluorescence intensity of DOX or EPI was measured at 553nm after excitation at 497nm. The plots of fluorescence intensity versus concentration were rectilinear over a range of 0.03–2μg/mL for both DOX and EPI with good correlation coefficient (r>0.999). High sensitivity to DOX and EPI was attained using the proposed method with limits of detection of 10 and 9ng/mL and limits of quantitation of 29 and 28ng/mL, for DOX and EPI, respectively. The method was successfully applied for the determination of DOX and EPI in biological fluids and in their commercial pharmaceutical preparations and the results were concordant with those obtained using a previously reported method. The application of the proposed method was extended to stability studies of DOX following different forced degradation conditions (acidic, alkaline, oxidative and photolytic) according to ICH guidelines. Moreover, the kinetics of the alkaline and oxidative degradation of DOX was investigated and the apparent first-order rate constants and half-life times were calculated.

Self-assembled cationic biodegradable nanoparticles from a new family of pH-responsive amino acid-based poly(ester urea urethane) and their application as a doxorubicin delivery vehicle

Event Abstract Back to Event Self-assembled cationic biodegradable nanoparticles from a new family of pH-responsive amino acid-based poly(ester urea urethane) and their application as a doxorubicin delivery vehicle Mingyu He1 and Chih-Chang Chu1 1 Cornell University, Department of Fiber Science and Apparel Design, United States Introduction: The objective of this study is to develop a new family of Arg-based biodegradable and biologically active copolymers (Arg-Leu PEUU) and their subsequent self-assembled cationic nanoparticles as a better delivery vehicle for anticancer drugs like doxorubicin (DOX) to achieve the synergism of cytotoxicity effect between the loaded drugs and macrophage inflammatory response of the delivery vehicle. This new strategy is expected to not only minimize severe cytotoxicity side effect of DOX upon conventional infusion administration but also enhance the cytotoxicity toward cancer cells via the synergism between the loaded DOX and the macrophage inflammatory response. Materials and Methods: Arginine-Leucine (Arg-Leu) based poly(ester urea urethane) (Arg-Leu PEUU) synthesized using a two-step solution polymerization[1]. Arg-Leu PEUU nanoparticles (NPs) and their DOX loading were performed by a facile dialysis method in water. Results and Discussion: The Arg-Leu PEUU copolymers have weight-average molecular weight from 13.4 to 16.8 KDa and glass-transition temperature from -3.4 to -4.6 oC. The self-assembled cationic nanoparticles (Arg-Leu PEUU NPs) were prepared by a facile dialysis method. The zeta potential of the Arg-Leu PEUU NPs ranged from +31.2 to +40.9 mV. The TEM images of the self-assembled 6-Arg-4-Leu-4 A/L-1/4 NPs and their DOX-loaded self-assemblies showed spherical morphology with a diameter ranging from 100 nm to 300 nm (Fig. 1). Arg-Leu PEUU NPs showed good biocompatibility toward 3T3 fibroblast and good bovine aortic endothelial cell (BAEC) proliferation and adhesion. DOX of 13.2~14.2 wt% could be physically incorporated into the Arg-Leu PEUU NPs. The merged fluorescence images provide a clear visual evidence (green fluorescent dots) of the blank Arg-Leu PEUU NPs engulfed by HeLa cells (Fig. 2A). The red fluorescence of the DOX-loaded Arg-Leu PEUU NP (Fig. 2B) was similarly distributed in the HeLa cells. While the free DOX treated HeLa cell (Fig. 2C) has red fluorescence concentrated around the cell nuclei. The quantified DOX fluorescence intensity data inside HeLa cells (Fig. 2D) show that the DOX-loaded Arg-Leu PEUU NPs have higher DOX fluorescence intensity than the free DOX. Arg-Leu PEUU NPs also enhanced the production of tumor necrosis factor-α (TNF-α) and nitric oxide (NO) of RAW 264.7 macrophages, but relatively low interleukin-10 (IL-10) production, thereof anti-tumour activity of macrophages might be enhanced by the nanocarrier. The in vitro hemolysis assay showed the cationic Arg-Leu PEUU NPs increased their chance of endosomal escape at a more acidic pH. The DOX-loaded Arg-Leu PEUU NPs exhibited a pH-dependent drug release profile with accelerated release kinetics in a mild acidic condition. Free DOX showed the highest cytotoxicity potency at the shortest duration of treatment (i.e., 1 hr, Fig. 3 A), but the DOX-loaded Arg-Leu PEUU NPs caught up with the free DOX potency at the longer duration of treatment (4 and 24 hrs, Fig. 3 B & C). 6-Arg-4-Leu-4 A/L-2/1 NPs with encapsulated DOX showed a higher potency toward HeLa cell toxicity than the free DOX at the same concentration after 24 hours treatment (Fig 3. C). Conclusion: In this study, a new family of cationic Arg-Leu PEUU biomaterials and their self-assembled nanoparticles (NPs) were developed as an advanced nano-carrier for doxorubicin. The Arg-Leu PEUU biomaterials showed high macrophage-induced inflammatory cytokine production of TNF-α and NO but low IL-10, indicating the leaning toward the M-1 macrophages inflammatory response. By adjusting the chemical composition of the Arg to Leu feed ratio in the Arg-Leu PEUU NP system, the DOX release profile could be altered, hence an either enhanced or reduced DOX cytotoxicity against cancer cells could be achieved.

Stereocomplex micelle from nonlinear enantiomeric copolymers efficiently transports antineoplastic drug.

Nanoscale polymeric micelles have attracted more and more attention as a promising nanocarrier for controlled delivery of antineoplastic drugs. Herein, the doxorubicin (DOX)-loaded poly(D-lactide)-based micelle (PDM/DOX), poly(L-lactide)-based micelle (PLM/DOX), and stereocomplex micelle (SCM/DOX) from the equimolar mixture of the enantiomeric four-armed poly(ethylene glycol)-polylactide (PEG-PLA) copolymers were successfully fabricated. In phosphate-buffered saline (PBS) at pH 7.4, SCM/DOX exhibited the smallest hydrodynamic diameter (Dh) of 90 ± 4.2 nm and the slowest DOX release compared with PDM/DOX and PLM/DOX. Moreover, PDM/DOX, PLM/DOX, and SCM/DOX exhibited almost stable Dhs of around 115, 105, and 90 nm at above normal physiological condition, respectively, which endowed them with great potential in controlled drug delivery. The intracellular DOX fluorescence intensity after the incubation with the laden micelles was different degrees weaker than that incubated with free DOX · HCl within 12 h, probably due to the slow DOX release from micelles. As the incubation time reached to 24 h, all the cells incubated with the laden micelles, especially SCM/DOX, demonstrated a stronger intracellular DOX fluorescence intensity than free DOX · HCl-cultured ones. More importantly, all the DOX-loaded micelles, especially SCM/DOX, exhibited potent antineoplastic efficacy in vitro, excellent serum albumin-tolerance stability, and satisfactory hemocompatibility. These encouraging data indicated that the loading micelles from nonlinear enantiomeric copolymers, especially SCM/DOX, might be promising in clinical systemic chemotherapy through intravenous injection.

Reversal of multidrug resistance by 5,5′-dimethoxylariciresinol-4-O-β-D-glucoside in doxorubicin-resistant human leukemia K562/DOX

Objective:The objective of this study was to investigate the reversal effects of 5,5’-dimethoxylariciresinol-4’-O-β-D-glucoside (DMAG) extracted from traditional Chinese medicines Mahonia on multidrug resistance (MDR) of human leukemia cells to chemotherapeutic agents.Materials and Methods:MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was performed to determine the effect of DMAG on doxorubicin sensitivity to K562/DOX cells. Propidium iodide /Hoechst 33342 double staining assay was used to investigate the effect of DMAG on doxorubicin-induced cellular apoptosis. Intracellular accumulation of doxorubicin and rhodamine 123 assay were performed to evaluate the effect of DMAG on drugs efflux activity of P-glycoprotein.Results:DMAG significantly enhanced the doxorubicin cytotoxicity to K562/DOX cells. In the presence of 1.0 μM of DMAG, the IC50 of doxorubicin decreased from 34.93 ± 1.37 μM to 12.51 ± 1.28 μM. DMAG of 1.0 μM significantly enhanced doxorubicin-induced cell apoptosis in K562/DOX cells and the enhancement was time-dependent. A significant increase in accumulation of doxorubicin in the presence of DMAG was observed. After treatment of the K562/DOX cells for 1 h with 15.0 μM doxorubicin alone, the fluorescence intensity was 33093.12. With the addition of 1.0 μM of DMAG, the fluorescence intensity of doxorubicin was 2.3-fold higher. A significant increase of accumulation of rhodamine 123 in the presence of DMAG was also observed. With the addition of 1.0 μM of DMAG, the fluorescence intensity was increased by 49.11% compared with rhodamine 123 alone.Conclusion:DMAG was shown to effectively enhance chemosensitivity of resistant cells, which makes it might be a suitable candidate for potential MDR-reversing agents.

Hyperthermia improves therapeutic efficacy of doxorubicin carried by mesoporous silica nanocontainers in human lung cancer cells

Purpose: We investigated the use of hyperthermia to improve the anti-cancer efficacy of doxorubicin (DOX)-loaded mesoporous silica nanocontainer Si-SS-CD-PEG. The hypothesis was that heat stimulates glutathione-mediated degradation of cyclodextrin gatekeeper, thereby causing the release of DOX from the carrier and DOX-induced cell death.Materials and methods: The release of DOX from DOX-loaded Si-SS-CD-PEG suspended in PBS containing glutathione (GSH) was studied by assessing the changes in DOX fluorescence intensity. The effect of heating at 42°C on the release of DOX from the intracellular carriers was determined with confocal microscopy. The extents of clonogenic and apoptotic cell death caused by DOX-loaded Si-SS-CD-PEG were determined.Results: The release of DOX from DOX-loaded Si-SS-CD-PEG in PBS occurred only when GSH presented in the suspension, and heating at 42°C slightly increased the release of DOX from the carriers. Heating significantly elevated the GSH content in A549 cells and increased the release of DOX from the internalised carriers. Heating the cancer cells treated with the carriers at 42°C markedly increased the clonogenic death and apoptosis. The GSH content in A549 cells was greater than that in L-132 cells, and A549 cells were far more sensitive than L-132 cells to DOX-loaded Si-SS-CD-PEG at both 37°C and 42°C.Conclusions: Hyperthermia increased the GSH-mediated release of DOX from DOX-loaded Si-SS-CD-PEG. Furthermore, hyperthermia markedly elevated the GSH content in cancer cells, thereby increasing the release of DOX from the internalised carriers and potentiating the DOX-induced clonogenic and apoptotic cell death.

Doxorubicin as a Molecular Nanotheranostic Agent: Effect of Doxorubicin Encapsulation in Micelles or Nanoemulsions on the Ultrasound-Mediated Intracellular Delivery and Nuclear Trafficking

Doxorubicin (DOX) is one of the most commonly used chemotherapeutic drugs and is a popular research tool due to the inherent fluorescence of the DOX molecule. After DOX injection, fluorescence imaging of organs or cells can provide information on drug biodistribution. Therapeutic and imaging capabilities combined in a DOX molecule make it an excellent theranostic agent. However, DOX fluorescence depends on a number of factors that should be taken into consideration when interpreting results of DOX fluorescence measurements. Discussing these problems is the main thrust of the current paper. The sensitivity of DOX fluorescence intensity to DOX concentration, local microenvironment, and interaction with model cellular components is illustrated by fluorescence spectra of paired DOX/phospholipid, DOX/histone, DOX/DNA, and triple DOX/histone/DNA and DOX/phospholipid/DNA systems. DOX fluorescence is dramatically quenched upon intercalation into the DNA; DOX fluorescence is also self-quenched at high concentrations of molecularly dissolved DOX; in contrast, DOX fluorescence is increased after binding to the histone or partitioning into the phospholipid phase of PEG-phospholipid micelles or hydrophobic cores of polymeric micelles. While flow cytometry is commonly used for characterization of DOX intracellular uptake, the above aspects of DOX fluorescence may significantly complicate interpretation of flow cytometry results. High cell fluorescence measured by flow cytometry may provide deceptive information on the actual intracellular DOX concentration and may not correlate with the therapeutic efficacy if DOX does not penetrate into the site of action in cell nuclei. These problems are illustrated in the experiments on the intracellular trafficking of DOX encapsulated in poly(ethylene glycol)-co-polycaprolactone (PEG-PCL) micelles or PEG-PCL stabilized perfluorocarbon nanodroplets, with and without the application of ultrasound used as an external trigger. For efficient encapsulation in micelle cores, DOX is usually deprotonated, which removes the positive charge and enhances hydrophobicity of DOX molecule. It was found that the deprotonated DOX accumulated in the cell cytoplasm but did not penetrate into the cell nuclei. The same was true for the DOX encapsulated in micelles or nanodroplets, which may explain their low therapeutic efficacy in the absence of ultrasound. Ultrasound triggers DOX trafficking into the cell nuclei, which is especially pronounced in the presence of nanoemulsions that convert into microbubbles under the ultrasound action. Microbubble cavitation results in the transient permeabilization of both plasma and nuclear membranes, thus allowing DOX penetration into the cell nuclei, which dramatically enhances therapeutic efficacy of DOX-loaded nanodroplet systems.

Block copolymer micelles conjugated with anti‐EGFR antibody for targeted delivery of anticancer drug

AbstractAntiepidermal growth factor receptor antibody (anti‐EGFR antibody) was conjugated with the block copolymer micelle based on poly(ethylene glycol) (PEG) and poly(ε‐caprolactone) (PCL) for active targeting to EGFR overexpressing cancer cells. Doxorubicin (DOX) was encapsulated in the core of the block copolymer (MePEG‐b‐PCL) micelle (DOX‐micelle). The mean diameters of the DOX‐micelle and the anti‐EGFR‐PEG‐b‐PCL copolymer micelles loaded with DOX (DOX‐anti‐EGFR‐micelle) were about 25 and 31 nm, respectively. The RKO human colorectal cancer cells expressing moderate degree of EGFR were incubated with free DOX, DOX‐micelle, or DOX‐anti‐EGFR‐micelle to study the distribution of DOX in the cells. When cells were incubated with free DOX, moderate degree of DOX fluorescence was observed in the nuclei. In the cells treated with DOX‐micelle, the DOX fluorescence intensity in the cytoplasm was much greater than that in the nuclei. On the other hand, the nuclei of the cells treated with DOX‐anti‐EGFR‐micelle exhibited DOX fluorescence intensity similar to that in the cytoplasm. The cytotoxicity of DOX‐anti‐EGFR‐micelle to induce apoptosis in RKO cells was significantly greater than that of free DOX or DOX‐micelle. These results demonstrated that the presence of anti‐EGFR antibody on the DOX‐micelle surface (DOX‐anti‐EGFR‐micelle) increased the internalization of the DOX‐micelle and nuclear accumulation of DOX, and enhanced the DOX‐induced cell death. © 2008 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 46: 7321–7331, 2008

Selective Determination of the Doxorubicin Content of Individual Acidic Organelles in Impure Subcellular Fractions

Since organelle preparations often contain more than one organelle type (e.g., acidic organelles and mitochondria), techniques that measure the properties of a given organelle type while avoiding biases caused by ancillary subcellular compartments are highly desirable. We report here the use of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) dual-channel detection to identify acidic organelles containing doxorubicin (DOX) in crude subcellular fractions from CCRF-CEM and CEM/C2 cell lines. As confirmed by confocal microscopy, acidic organelles are identified by their accumulation of fluorescently labeled nanospheres. Using CE-LIF analysis, individually detected organelles are classified into three kinds: acidic organelles containing only nanospheres, acidic organelles containing nanospheres and DOX, and other organelles containing DOX (e.g., mitochondria) with no detectable nanospheres. Electrophoretic mobility, DOX fluorescence intensity, and nanosphere fluorescence intensity distributions of individual acidic organelles and other organelles containing DOX are determined in the same CE-LIF run. The acidic organelle mobilities range from (-0.7 to -2.0) x 10(-4) cm(2) V(-1) s(-1) while those of the other organelles spread from (-0.6 to -3.5) x 10(-4) cm(2) V(-1) s(-1). In addition, by calibrating the detector response, DOX content in individual acidic organelles and other organelles can be estimated. The average amounts of DOX per acidic organelle in CEM/C2 and CCRF-CEM cells are 11.1 +/- 0.5 and 10.6 +/- 0.4 zmol, respectively. This first report of an analysis of the accumulation of DOX in individual acidic organelles presents a procedure for analyzing the accumulation of fluorescent compounds in acidic organelles that could be useful for investigating acidic organelle maturation and the role of these organelles in drug resistance.