Cellular Uptake Of Doxorubicin Research Articles

PEGylated magnetic Prussian blue nanoparticles as a multifunctional therapeutic agent for combined targeted photothermal ablation and pH-triggered chemotherapy of tumour cells

Multifunctional nanoagents have become popular and valuable pharmaceuticals for effective cancer treatment. Moreover, there is an increasing tendency to develop therapeutic agents with excellent biocompatibility, high efficiency, specific targeting and combinatorial treatment effects. In this study, we proposed a facile technique to synthesize PEGylated (polyethylene glycol modified) magnetic Prussian blue (PB) nanoparticles with encapsulated doxorubicin (DOX), abbreviated as Fe3O4@PB/PEG/DOX NPs, for combined targeted photothermal ablation and pH-triggered chemotherapy of tumour cells. The PEGylation of Fe3O4@PB core-shell structure was achieved through a thin-film hydration process; DOX was loaded into the nanocapsule via hydrophobic interactions. An in vitro study indicated increased drug release under acidic conditions, mimicking mild acidic tumour microenvironments. Additionally, the nanocomposites exhibited superparamagnetism, contributing to an improved therapeutic effect guided by a localized magnetic field. Cytotoxicity studies demonstrated outstanding photothermal-chemotherapy combinatorial effects on HeLa cells, attributed to the targeted photothermic effect mediated by the pH-triggered cellular uptake of DOX. Specifically, the viability of HeLa cells decreased to 8.5% after treatment with the nanoagent (DOX=10μgmL−1) and near infrared irradiation, indicating an evident tumour inhibition effect in vitro. This study presented a nanoplatform for efficient and targeted cancer treatment, which may lead to the development of multifunctional nanodrug vehicles for cancer therapy.

Rational design of multifunctional micelles against doxorubicin-sensitive and doxorubicin-resistant MCF-7 human breast cancer cells.

Even though a tremendous number of multifunctional nanocarriers have been developed to tackle heterogeneous cancer cells, little attention has been paid to elucidate how to rationally design a multifunctional nanocarrier. In this study, three individual functions (active targeting, stimuli-triggered release and endo-lysosomal escape) were evaluated in doxorubicin (DOX)-sensitive MCF-7 cells and DOX-resistant MCF-7/ADR cells by constructing four kinds of micelles with active-targeting (AT-M), passive targeting, pH-triggered release (pHT-M) and endo-lysosomal escape (endoE-M) function, respectively. AT-M demonstrated the strongest cytotoxicity against MCF-7 cells and the highest cellular uptake of DOX due to the folate-mediated endocytosis. However, AT-M failed to exhibit the best efficacy against MCF-7/ADR cells, while endoE-M exhibited the strongest cytotoxicity against MCF-7/ADR cells and the highest cellular uptake of DOX due to the lowest elimination of DOX from the cells. This was attributed to the carrier-facilitated endo-lysosomal escape of DOX, which avoided exocytosis by lysosome secretion, resulting in an effective accumulation of DOX in the cytoplasm. The enhanced elimination of DOX from the MCF-7/ADR cells also accounted for the remarkable decrease in cytotoxicity against the cells of AT-M. Three micelles were further evaluated with MCF-7 cells and MCF-7/ADR-resistant cells xenografted mice model. In accordance with the in vitro results, AT-M and endoE-M demonstrated the strongest inhibition on the MCF-7 and MCF-7/ADR xenografted tumor, respectively. Active targeting and active targeting in combination with endo-lysosomal escape have been demonstrated to be the primary function for a nanocarrier against doxorubicin-sensitive and doxorubicin-resistant MCF-7 cells, respectively. These results indicate that the rational design of multifunctional nanocarriers for cancer therapy needs to consider the heterogeneous cancer cells and the primary function needs to be integrated to achieve effective payload delivery.

An MSN-PEG-IP drug delivery system and IL13Rα2 as targeted therapy for glioma.

A combination of gene therapy and chemotherapy has recently received interest as a targeted therapy for glioma. A mesoporous silica nanoparticle (MSN)-based vehicle coated with IL13Rα2-targeted peptide (IP) using polyethylene glycol (PEG), MSN-PEG-IP (MPI), was constructed and confirmed as a potential glioma-targeted drug delivery system in vitro. In this work, tissue microarray (TMA) results revealed that IL13Rα2 was over-expressed in human glioma tissues and that high expression of IL13Rα2 in patients was associated with poor survival. Doxorubicin (DOX)-loaded MPI (MPI/D) crossed the blood-brain barrier, specifically targeting glioma cells and significantly enhancing the cellular uptake of DOX in glioma cells compared with MSN/DOX (M/D) and MSN-PEG/DOX (MP/D), whereas the normal brain was not affected. Magnetic Resonance Imaging (MRI) examinations showed that the tumour size of glioma-bearing rats in the MPI/D-treated group was much smaller than those in the M/D and MP/D treated groups. Immunofluorescence results demonstrated that MPI/D treatment induced more apoptosis and much less proliferation than the other two treatments. However, the therapeutic effect was weak when IL13Rα2 was knocked down. Furthermore, U87 cells treated with IL-13 and MPI together could increase both STAT6 and P63 expression, which attenuated glioma cell proliferation, invasion and migration compared with cells treated with IL-13 alone. The results of the subcutaneous tumour model also revealed that IL13Rα2 knockdown could hinder cell proliferation and induce more apoptosis. The promising results suggested that MPI can not only deliver DOX to glioma in a targeted manner but also occupy IL13Rα2, which can promote IL-13 binding to IL13Rα1 and activation of the JAK-STAT pathway to induce an anti-glioma effect.

Selenadiazole derivatives antagonize hyperglycemia-induced drug resistance in breast cancer cells by activation of AMPK pathways.

Hyperglycemia is an important factor for chemoresistance of breast cancer patients with diabetes. In the present study, a novel selenadiazole derivative has been evaluated and found to be able to antagonize the doxorubicin (DOX) resistance of MCF-7 cells under simulated diabetes conditions. Hyperglycemia promotes the proliferation, invasion and migration of MCF-7 cells through activation of ERK and AKT pathways, which could be inhibited by the synthetic selenadiazole derivative. The antitumor effects of the selenadiazole derivative were attributed to its ability to activate AMPK pathways. Furthermore, the high lipophilicity (log P = 1.9) of the synthetic selenadiazole derivative facilitated its uptake by cancer cells and subsequently potentiated the cellular uptake of DOX, leading to a strong enhancment of the antiproliferative activity of DOX on MCF-7 cells by induction of apoptosis. The apoptosis was initiated by the ROS overproduction induced by the cooperation of the selenadiazole derivative and DOX. The excessive ROS then caused damage to DNA, which upregulated the expression of proapoptosis Bcl-2 family proteins and led to fragmentation of mitochondria, which finally caused apoptosis of the cancer cells. Taken together, this study provides a rational strategy for using selenadiazole derivatives to overcome hyperglycemia-induced drug resistance in breast cancer by activation of AMPK-mediated pathways.

Co-assembly of doxorubicin and curcumin targeted micelles for synergistic delivery and improving anti-tumor efficacy

Chemotherapeutic drugs have a series of limitations in anti-tumor treatment, mainly including multidrug resistance (MDR) and serious adverse reactions. Co-delivery system with two or more synergistic therapeutic drugs is an effective strategy to settle these limitations. In this study, active tumor-targeted co-delivery micelles (DOX+Cur)-PMs, with two synergistic drugs of a therapeutic drug of doxorubicin (DOX) and a chemosensitizer of curcumin (Cur) co-encapsulated into hyaluronic acid-vitamin E succinate (HA-VES) graft copolymer, were prepared and delivered simultaneously into tumor cells for improving therapeutic effects of DOX. (DOX+Cur)-PMs had uniform particle size, high encapsulation efficacy, sustained release profile and good colloidal stability. In vitro cytotoxicity study, (DOX+Cur)-PMs exerted the strongest cytotoxicity and highest cell apoptosis-inducing activities against DOX-resistant MCF-7/Adr cells. Moreover, (DOX+Cur)-PMs more efficiently internalized into cancer cells and enhanced the cellular uptake of DOX via energy-dependent and caveolae-mediated endocytosis, and significantly reversed MDR effects via CD44 targeting delivery and the synergic effect of released Cur. More importantly, in vivo results illustrated that (DOX+Cur)-PMs not only displayed better tumor accumulation and tumor targeting, and more efficiently inhibited the growth of tumor in 4T1 tumor-bearing mice, but also induced significantly less pathological damage to the cardiac tissue in comparison with free DOX, even DOX-PMs and DOX-PMs+Cur. In summary, this targeted combinational micellar delivery system with DOX and Cur could be a promising vehicle in tumor therapy.

Synthesis and in vitro evaluation of pH-sensitive PEG-I-dC16 block polymer micelles for anticancer drug delivery.

To develop an acid trigger release of antitumour drug delivery carriers, pH-sensitive amphiphilic poly (ethyleneglycol)-imine-benzoic-dipalmitate (PEG-I-dC16 ) polymers were designed and synthesized and the drug-loaded micelles were evaluated in vitro. PEG-I-dC16 synthesized by Schiff base synthetic method and characterized by (1) H-NMR. To determine the drug-loading capacity, doxorubicin (DOX) was encapsulated in the micelles using membrane dialysis method. Zeta potential, particle size, drug-loading capacity, in vitro drug release in different pH conditions and cytotoxicity evaluation of micelles were carried out comparing with non-acid liable PEG-amide-benzoic-dipalmitate (PEG-A-dC16) polymers micelles. The cellular uptake and intracellular distribution of DOX were detected by flow cytometry and confocal laser scanning microscope. Drug-loading capacity and encapsulation efficiency of micelle (PEG molecular weight 2k) were 12.7 ± 1.1% and 49.8 ± 2.2%, respectively. The average particle size was 72.3 ± 2.5 nm. The DOX release rate of PEG-I-dC16 micelles is much higher at pH 6.5 than at pH 7.4. DOX cellular uptake and nuclear accumulation of PEG-I-dC16 micelles were more efficiency than that of PEG-A-dC16 micelles. The pH-sensitive PEG-I-dC16 micelles could be a promising drug delivery system for anticancer drugs.

Iodinated hyaluronic acid oligomer-based nanoassemblies for tumor-targeted drug delivery and cancer imaging

Nano-sized self-assemblies based on amphiphilic iodinated hyaluronic acid (HA) were developed for use in cancer diagnosis and therapy. 2,3,5-Triiodobenzoic acid (TIBA) was conjugated to an HA oligomer as a computed tomography (CT) imaging modality and a hydrophobic residue. Nanoassembly based on HA-TIBA was fabricated for tumor-targeted delivery of doxorubicin (DOX). Cellular uptake of DOX from nanoassembly, compared to a DOX solution group, was enhanced via an HA-CD44 receptor interaction, and subsequently, the in vitro antitumor efficacy of DOX-loaded nanoassembly was improved in SCC7 (CD44 receptor positive squamous cell carcinoma) cells. Cy5.5, a near-infrared fluorescence (NIRF) dye, was attached to the HA-TIBA conjugate and the in vivo tumor targetability of HA-TIBA nanoassembly, which is based on the interaction between HA and CD44 receptor, was demonstrated in a NIRF imaging study using an SCC7 tumor-xenografted mouse model. Tumor targeting and cancer diagnosis with HA-TIBA nanoassembly were verified in a CT imaging study using the SCC7 tumor-xenografted mouse model. In addition to efficient cancer diagnosis using NIRF and CT imaging modalities, improved antitumor efficacies were shown. HA and TIBA can be used to produce HA-TIBA nanoassembly that may be a promising theranostic nanosystem for cancers that express the CD44 receptor.

Multifunctional PLGA Nanobubbles as Theranostic Agents: Combining Doxorubicin and P-gp siRNA Co-Delivery Into Human Breast Cancer Cells and Ultrasound Cellular Imaging.

Multidrug resistance (MDR) is a major impediment to the success of cancer chemotherapy. One of the effective approaches to overcome MDR is to use nanoparticle-mediated the gene silence of chemotherapeutic export proteins by RNA interference to increase drug accumulation in drug resistant cancer cells. In this work, a new co-delivery system, DOX-PLGA/PEI/P-gp shRNA nanobubbles (NBs) around 327 nm, to overcome doxorubicin (DOX) resistance in MCF-7 human breast cancer was designed and developed. Positively charged polyethylenimine (PEI) were modified onto the surface of DOX-PLGA NBs through DCC/NHS crosslinking, and could efficiently condense P-gp shRNA into DOX-PLGA/PEI NBs at vector/shRNA weight ratios of 70:1 and above. An in vitro release profile demonstrated an efficient DOX release (more than 80%) from DOX-PLGA/PEI NBs at pH 4.4, suggesting a pH-responsive drug release for the multifunctionalized NBs. Cellular experimental results further showed that DOX-PLGA/PEI/P-gp shRNA NBs could facilitate cellular uptake of DOX into cells and increase the cell proliferation suppression effect of DOX against MCF-7/ADR cells (a DOX-resistant and P-glycoprotein (P-gp) over-expression cancer cell line). The IC50 of DOX-PLGA NBs against MCF-7/ADR cells was 2-fold lower than that of free DOX. The increased cellular uptake and nuclear accumulation of DOX delivered by DOX-PLGA/PEI/P-gp shRNA NBs in MCF-7/ADR cells was confirmed by fluorescence microscopy and fluorescence spectrophotometry, and might be owning to the down-regulation of P-gp and reduced the efflux of DOX. The cellular uptake mechanism of DOX-PLGA/PEI/P-gp shRNA NBs indicated that the macropinocytosis was one of the pathways for the uptake of NBs by MCF-7/ADR cells, which was also an energy-dependent process. Furthermore, the in vitro cellular ultrasound imaging suggested that the employment of the DOX-PLGA/PEI/P-gp shRNA NBs could efficiently enhance ultrasound imaging of cancer cells. These results demonstrated that the developed DOX-PLGA/PEI/P-gp shRNA NBs is a potential, safe and efficient theranotic agent for cancer therapy and diagnostics.

Activity of the dietary flavonoid, apigenin, against multidrug-resistant tumor cells as determined by pharmacogenomics and molecular docking

Natural products have been extensively studied and involved in cancer therapy field [1], apigenin has considerable cytotoxic activity in vitro and in vivo. Despite many mechanistic studies, less is known about resistance factors hampering apigenin’s activity. We investigated the ATP-binding cassette (ABC) transporters BCRP/ABCG2, P-glycoprotein/ABCB1 and its close relative ABCB5. Apigenin inhibited not only P-glycoprotein, but also BCRP by increasing cellular uptake of doxorubicin and showed synergistic inhibitory effect in combination with doxorubicin or docetaxel against multidrug-resistant cells. To perform in silico studies, we first generated homology models for human P-glycoprotein and ABCB5 based on the crystal structure of murine P-glycoprotein. Their nucleotide binding domains (NBDs) revealed the highest degrees of sequence homologies (89%-100%), indicating that ATP binding and cleavage is of crucial importance for ABC transporters. In silico studies showed a pigenin bound to the NBDs of P-glycoprotein and ABCB5. Hence, apigenin may compete with ATP for NBD-binding leading to energy depletion to fuel the transport of ABC transporter substrates. Furthermore, we performed COMPARE and hierarchical cluster analyses of transcriptome-wide mRNA expression profiles of the National Cancer Institute tumor cell line panel. Microarray-based mRNA expressions of genes of diverse biological functions significantly predicted responsiveness of tumor cells to apigenin [2].

Bile acid-conjugated chondroitin sulfate A-based nanoparticles for tumor-targeted anticancer drug delivery

Chondroitin sulfate A-deoxycholic acid (CSA-DOCA)-based nanoparticles (NPs) were produced for tumor-targeted delivery of doxorubicin (DOX). The hydrophobic deoxycholic acid (DOCA) derivative was conjugated to the hydrophilic chondroitin sulfate A (CSA) backbone via amide bond formation, and the structure was confirmed by 1H-nuclear magnetic resonance (NMR) analysis. Loading the DOX to the CSA-DOCA NPs resulted in NPs with an approximately 230nm mean diameter, narrow size distribution, negative zeta potential, and relatively high drug encapsulation efficiency (up to 85%). The release of DOX from the NPs exhibited sustained and pH-dependent release profiles. The cellular uptake of DOX from the CSA-DOCA NPs in CD44 receptor-positive human breast adenocarcinoma MDA-MB-231 cells was reduced when co-treated with free CSA, indicating the interaction between CSA and the CD44 receptor. The lower IC50 value of DOX from the CSA-DOCA NPs compared to the DOX solution was also probably due to this interaction. Moreover, the ability of the developed NPs to target tumors could be inferred from the in vivo and ex vivo near-infrared fluorescence (NIRF) imaging results in the MDA-MB-231 tumor-xenografted mouse model. Both passive and active strategies appear to have contributed to the in vivo tumor targetability of the CSA-DOCA NPs. Therefore, these CSA-DOCA NPs could further be developed into a theranostic nanoplatform for CD44 receptor-positive cancers.

Amphiphilic copolymers with pendent carboxyl groups for high-efficiency loading and controlled release of doxorubicin

In this paper, biodegradable amphiphilic block copolymer based on methoxy poly(ethylene glycol)-b-poly(5-allyloxy-1,3-dioxan-2-one) (mPEG-b-PATMC) was successfully synthesized in bulk using immobilized porcine pancreas lipase (IPPL) as the catalyst. After thiol-ene “click” reactions occur between thiol group of thioglycolic acid and carbon–carbon double bonds of PATMC segments, the pendent carboxyl-modified copolymer mPEG-b-PATMC-g-SCH2COOH was obtained for high-efficiency loading and controlled release of doxorubicin (DOX) to cancer cells. Both the carboxyl-modified and unmodified copolymers could self-assemble to form nano-sized micelles in aqueous solution, while transmission electron microscopy (TEM) observation showed that the micelles dispersed in spherical shape with nano-size before and after DOX loading. Compared with the unmodified copolymer, the pendent carboxyl-modified structure in mPEG-b-PATMC-g-SCH2COOH could markedly enhance the drug-loading capacity and entrapment efficiency via the electrostatic interaction. The in vitro release studies showed more sustained drug release behavior of mPEG-b-PATMC-g-SCH2COOH without an initial burst, which could be further adjusted by the conditions of ionic strength and pH. Confocal laser scanning microscopy (CLSM) indicated efficient cellular uptake of DOX delivered by mPEG-b-PATMC-g-SCH2COOH, while MTT assays also demonstrated potent cytotoxic activity against HeLa cells.

Synergistic effect of pH-responsive folate-functionalized poloxamer 407-TPGS-mixed micelles on targeted delivery of anticancer drugs.

BackgroundDoxorubicin (DOX), an anthracycline anticancer antibiotic, is used for treating various types of cancers. However, its use is associated with toxicity to normal cells and development of resistance due to overexpression of drug efflux pumps. Poloxamer 407 (P407) and vitamin E TPGS (D-α-tocopheryl polyethylene glycol succinate, TPGS) are widely used polymers as drug delivery carriers and excipients for enhancing the drug retention times and stability. TPGS reduces multidrug resistance, induces apoptosis, and shows selective anticancer activity against tumor cells. Keeping in view the problems, we designed a mixed micelle system encapsulating DOX comprising TPGS for its selective anticancer activity and P407 conjugated with folic acid (FA) for folate-mediated receptor targeting to cancer cells.MethodsFA-functionalized P407 was prepared by carbodiimide crosslinker chemistry. P407-TPGS/FA-P407-TPGS-mixed micelles were prepared by thin-film hydration method. Cytotoxicity of blank micelles, DOX, and DOX-loaded micelles was determined by alamarBlue® assay.ResultsThe size of micelles was less than 200 nm with encapsulation efficiency of 85% and 73% for P407-TPGS and FA-P407-TPGS micelles, respectively. Intracellular trafficking study using nile red-loaded micelles indicated improved drug uptake and perinuclear drug localization. The micelles show minimal toxicity to normal human cell line WRL-68, enhanced cellular uptake of DOX, reduced drug efflux, increased DOX–DNA binding in SKOV3 and DOX-resistant SKOV3 human ovarian carcinoma cell lines, and enhanced in vitro cytotoxicity as compared to free DOX.ConclusionFA-P407-TPGS-DOX micelles show potential as a targeted nano-drug delivery system for DOX due to their multiple synergistic factors of selective anticancer activity, inhibition of multidrug resistance, and folate-mediated selective uptake.

Rational design of selenadiazole derivatives to antagonize hyperglycemia-induced drug resistance in cancer cells.

Hyperglycemia is an important factor for chemoresistance of hepatocellular carcinoma patients with diabetes to therapeutics. In the present study, a series of selenadiazole derivatives have been rationally designed, synthesized, and found be able to antagonize drug resistance in HepG2 cells to doxorubicin (DOX) under simulated diabetes conditions. Hyperglycemia could promote the cell proliferation through upregulation of ERK and AKT phosphorylation. However, the synthetic selenadiazole derivatives effectively potentiated the cellular uptake of DOX and enhanced the antiproliferative activity of DOX on HepG2 cells by induction of apoptosis, via regulation of ROS-mediated AMPK activation, inhibition of mTORC1, and an increase in DNA damage. The selenadiazole derivatives that possess an increased lipophilicity could enhance the cellular uptake and anticancer efficacy of DOX. Taken together, this study provides a rational design strategy of selenadiazole derivatives to overcome hyperglycemia-induced drug resistance.

Targeting of a Thermosensitive Nanogel Copolymerized With Macromonomer for Cell Uptake and Drug Controlled Release

In this study, N-isopropylacrylamide (NIPAAm), propyl acrylic acid (PAAC), and poly(N-isopropylacrylamide-co-undecylenic acid) acrylamide were introduced to synthesize PNIPAAm-based nanogels (NG). The morphology of NG particles was characterized via transmission electron microscopy (TEM). The nanogels exhibited a lower critical solution temperature (LCST) of 34°C and a temperature-induced drug release in vitro. After conjugation of arginine-glycine-aspartic acid (RGD)–containing peptide (GRGDS), the cellular uptake of doxorubicin (Dox)-loaded nanogel by HeLa cells had been greatly enhanced. The Dox molecules in endocytosed nanogels could be released efficiently at 37°C and subsequently kill tumor cells, suggesting that the nanogel has great potential for targeting delivery.

Self-assembled nanocomplexes of anionic pullulan and polyallylamine for DNA and pH-sensitive intracellular drug delivery

The amalgamation of chemotherapy and gene therapy is promising treatment option for cancer. In this study, novel biocompatible self-assembled nanocomplexes (NCs) between carboxylmethylated pullulan t335 (CMP) with polyallylamine (CMP–PAA NCs) were developed for plasmid DNA (pDNA) and pH-sensitive doxorubicin (DOX) delivery. DOX was conjugated to CMP (DOX–CMP) via hydrazone and confirmed by FTIR and 1H-NMR. In vitro release studies of pH-sensitive DOX–CMP conjugate showed 23 and 85 % release after 48 h at pH 7.4 (physiological pH) and pH 5 (intracellular/tumoral pH), respectively. The CMP–PAA NCs or DOX–CMP–PAA NCs self-assembled into a nanosized (<250 nm) spherical shape as confirmed by DLS and TEM. The hemolysis and cytotoxicity study indicated that the CMP–PAA NCs did not show cytotoxicity in comparison with plain polyallylamine. Gel retardation assay showed complete binding of pDNA with CMP–PAA NCs at 1:2 weight ratio. CMP–PAA NCs/pDNA showed significantly higher transfection in HEK293 cells compared to PAA/pDNA complexes. Confocal imaging demonstrated successful cellular uptake of DOX–CMP–PAA NCs in HEK293 cells. Thus, NCs hold great potential for targeted pDNA and pH-sensitive intratumoral drug delivery.

Abstract 5382: A breakthrough in application of a drug delivery nanoparticle system for therapy and diagnosis of solid tumor

Abstract Introduction: Nanoparticle systems are usually designed to deliver the incorporated molecular to tumors. However, we found an interesting breakthrough in conducting the super carbonate apatite (sCA) nanoparticles itself. So far we have introduced the sCA as an in vivo pH sensitive delivery system of doxorubicin (PLoS ONE 8(4): e60428. 2013) or siRNA, which simply consist of inorganic ions and quickly accumulate specifically in tumors yet yield no serious adverse events in mice and monkeys. Intravenously administered sCA-siRNA markedly accumulated in the tumor cells at 90 min and survivin-siRNA delivered by sCA significantly inhibited in vivo tumor growth, compared with the two other in vivo commercially available delivery reagents, Invivofectamine 2.0 and Atelocollagen. In this study, we confirmed a unique characteristic of sCA itself, not as a drug delivery system. Empty sCA alone markedly enhanced the uptake of low molecular chemicals into tumor cells in vitro and in vivo. Methods: Doxorubicin (DXR), fluorouracil (5-FU), oxaliplatin (L-OHP) and indocyanine green (ICG) are used as low molecular chemicals. Cellular uptake of doxorubicin was analyzed by flow cytometry using a BD FACS Aria II instrument (BD Biosciences). Cell viability was examined by WST-8 assay (Dojindo). As for the in vivo tumor imaging assay, sCA and ICG were simultaneously administered via the separate routes to the mice bearing colon cancer HT29; sCA was administered i.v. (intravenously) and ICG was administered i.p. (intraperitoneally). Mice were imaged under anesthesia using IVIS (PerkinElmer) for ex vivo and in vivo imaging. Results and Discussions: Empty sCA itself enhanced the uptake and cytotoxic effect of anti-cancer agents in vitro, although sCA itself was not toxic. The flow cytometric analysis showed the mean florescence index (MFI: 2186) of the cells, first treated with sCA for 24 h and then treated with DXR for another 24 h, was higher than the MFI (1684) of the cells directly treated with DXR for 24 h. The IC50 (μM; mean ± SD) of 5-FU and L-OHP in colon cancer HCT116 were 47.6 ± 10.0 and 31.3 ± 2.2, whereas the IC50 of both drugs in the sCA pre-treated cells for 24 h were 11.7 ± 2.0 and 3.70 ± 1.1, respectively. Furthermore, the empty sCA itself also improved the efficacy of in vivo tumor imaging by ICG, even when ICG and sCA were administered through the separate routes; sCA was administered i.v. and ICG was administered i.p.. We should mention that we had not been able to incorporate ICG into sCA nanoparticles before this experiment. Time course studies showed that ICG levels in tumors with treatment of sCA plus ICG were significantly higher throughout the examined time points than those treated with ICG alone (P = 0.0142 for 4 h, P = 0.0139 for 8 h, P = 0.0433 for 12 h, P = 0.0304 for 24 and 48 h). Conclusion: Our data suggest that sCA itself, while designed as an in vivo delivery device, can facilitate entrance of low molecular chemicals into tumor cells in vitro and in vivo. Citation Format: Xin Wu, Hirofumi Yamamoto, Mamoru Uemura, Taishi Hata, Junichi Nishimura, Ichiro Takemasa, Tsunekazu Mizushima, Yuichiro Doki, Masaki Mori. A breakthrough in application of a drug delivery nanoparticle system for therapy and diagnosis of solid tumor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5382. doi:10.1158/1538-7445.AM2014-5382

Crosslinked triblock copolymeric micelle for redox-responsive drug delivery

In this paper, novel biodegradable amphiphilic triblock copolymer based on methoxy poly(ethylene glycol)-b-poly(ɛ-caprolactone)-b-poly(2-(2-oxo-1,3,2-dioxaphospholoyloxy)ethyl methacrylate) (mPEG-b-PCL-b-PPEMA) was successfully synthesized. After Michael-addition reactions between amine groups of cystamine and carbon–carbon double bonds of PPEMA segments, the crosslinked reduction-sensitive copolymer mPEG-b-PCL-b-PPEMA-SS- was obtained for efficient delivery and controlled release of doxorubicin (DOX) to cancer cells. Both the uncrosslinked and crosslinked copolymers could self-assemble to form nano-sized micelles in aqueous solution, while transmission electron microscopy (TEM) observation showed that the micelles dispersed in spherical shape with nano-size before and after DOX loading. Meanwhile, 1H NMR spectra in D2O indicated the formation of a lower mobility core by crosslinking method and a solid-like rigid core via further DOX-loaded. As compared to the uncrosslinked copolymer, the core crosslinking structure in mPEG-b-PCL-b-PPEMA-SS- could improve the micellar stability and enhance the drug loading capacity and entrapment efficiency. The in vitro release studies showed more sustained drug release behavior of crosslinked mPEG-b-PCL-b-PPEMA-SS- which could be accelerated quantitatively in response to the reductive condition similar to intracellular environment. Furthermore, confocal laser scanning microscopy (CLSM) indicated much more efficient cellular uptake of DOX delivered by mPEG-b-PCL-b-PPEMA-SS-, while MTT assays also demonstrated potent cytotoxic activity against HeLa cells.

Tea nanoparticles for immunostimulation and chemo-drug delivery in cancer treatment.

Many health benefits have been associated with tea consumption. In an effort to elucidate the source of these health benefits, numerous phytochemicals have been extracted from tea infusions, some of which have demonstrated promise as clinical therapeutics for cancer therapy. Considering the advantageous properties of organic nanoparticles, the purpose of this study is to develop a method for isolating nanoparticles from tea leaves, and explore potential biomedical applications for these nanoparticles. First, an infusion-dialysis procedure for isolating tea nanoparticles (TNPs) from green tea infusions is developed. Second, atomic force microscopy and scanning electron microscopy reveal that the TNPs are spherical with diameters of 100-300 nm. Third, electrophoretic light scattering is used to determine that the TNPs have a zeta potential of -26.52 mV at pH 7.0. Finally, chemical analysis demonstrates that (-) Epigallocatechin gallate, caffeine, and theobromine are not found in the TNPs. Interestingly, the TNPs do enhance the in vitro secretion of cytokines IL-6, TNF-alpha, and G-CSF, as well as the chemokines RANTES, IP-10, MDC from mouse macrophages RAW264.7, indicating an immunostimulatory effect. As a nanocarrier, the TNPs are able to form complexes with doxorubicin (DOX) and have the potential for applications in drug delivery. Further the DOX-loaded TNPs increase the cellular DOX uptake, compared to free DOX, leading to higher cytotoxicity in the A549 human lung cancer and MCF-7 breast cancer cells. More importantly, the DOX-loaded TNPs significantly increase the DOX uptake and cytotoxicity in MCF-7/ADR multidrug resistant breast cancer cells. In this work, an infusion-dialysis procedure is developed for isolation of the TNPs from green tea, and the potential of these nanoparticles as a multifunctional nanocarrier for cancer therapy in vitro is explored.

Mitochondrial delivery of doxorubicin via triphenylphosphine modification for overcoming drug resistance in MDA-MB-435/DOX cells.

In this study, doxorubicin (DOX) was conjugated to a lipophilic triphenylphosphonium (TPP) that is selectively taken up by the mitochondrial membrane of cells. This new derivative of DOX, i.e., TPP-DOX, was characterized by infrared spectroscopy (IR), nuclear magnetic resonance ((1)H NMR, (13)C NMR), and mass spectrometry. The effect of TPP modification on DOX cell uptake, intracellular trafficking, eventual DOX induced cytotoxicity, and the level of cleaved caspase 3 and PARP in wild type MDA-MB-435/WT and DOX resistant MDA-MB-435/DOX cells was then evaluated and compared to that for free DOX. In general, free DOX cellular uptake appeared to be significantly higher in MDA-MB-435/WT than MDA-MB-435/DOX cells. Moreover, free DOX was able to enter the nucleus of MDA-MB-435/WT cells, but in MDA-MB-435/DOX cells, it was confined within the cytoplasm. The TPP-DOX, on the other hand, was localized in the cytoplasm of both cell phenotypes and showed preferential distribution to the mitochondria. Correspondingly, in MDA-MB-435/DOX cells, an enhanced cytotoxicity was observed for TPP-DOX (IC50 of 33.6 and 21.0 μM at 48 and 72 h incubation, respectively) in comparison to free DOX (IC50 of 126.7 and 77.96 μM at 48 and 72 h incubation, respectively). This observation was accompanied by the increased level of cleaved caspase 3 and PARP indicating enhanced apoptosis in both cell lines, particularly that of MDA-MB-435/DOX, for TPP-DOX compared to free DOX following 24 h treatment. The present study highlights promising application of TPP-DOX in reversing drug resistance in tumor cells.

Temperature-triggered tumor-specific delivery of anticancer agents by cRGD-conjugated thermosensitive liposomes

One of the most effective methods to treat cancer is the specific delivery of anticancer drugs to the target site. To achieve this goal, we designed an anticancer drug with mild hyperthermia-mediated triggering and tumor-specific delivery. To enhance the thermosensitive drug release, we incorporated elastin-like polypeptide (ELP), which is known to be a thermally responsive phase transition peptide into the dipalmitoylphosphatidylcholine (DPPC)-based liposome surface. Additionally, cyclic arginine-glycine-aspartic acid (cRGD) binds to αvβ3 integrin, which is overexpressed in angiogenic vasculature and tumor cells, was introduced on the liposome. ELP-modified liposomes with the cRGD targeting moiety were prepared using a lipid film hydration method, and doxorubicin (DOX) was loaded into the liposome by the ammonium sulfate-gradient method. The cRGD-targeted and ELP-modified DOX-encapsulated liposomes (RELs) formed spherical vesicles with a mean diameter of 181nm. The RELs showed 75% and 83% DOX release at 42°C and 45°C, respectively. The stability of RELs was maintained up to 12h without the loss of their thermosensitive function for drug release. Flow cytometry results showed that the cellular uptake of DOX in RELs into αvβ3 integrin-overexpressing U87MG and HUVEC cells was 8-fold and 10-fold higher, respectively, than that of non-targeting liposomes. Confocal microscopy revealed that REL released DOX only under the mild hyperthermia condition at 42°C by showing the localization of DOX in nuclei and the liposomes in the cytosol. The cell cytotoxicity results demonstrated that REL can efficiently kill U87MG cells through cRGD targeting and thermal triggering. The in vivo tumoral accumulation measurement showed that the tumor-targeting effect of RELs was 5-fold higher than that of non-targeting liposomes. This stable, target-specific, and thermosensitive liposome shows promise to enhance therapeutic efficacy if it is applied along with a relevant external heat-generating medical system.