Magnetofluoro-Immunosensing Platform Based on Binary Nanoparticle-Decorated Graphene for Detection of Cancer Cell-Derived Exosomes.

Multi-functionalized carbon nanomaterials have attracted interest owing to their excellent synergic properties, such as plasmon resonance energy transfer and surface-enhanced Raman scattering. Particularly, nanoparticle (NP)-decorated graphene (GRP) has been applied in various fields. In this study, silver NP (AgNP)- and magnetic iron oxide NP (IONP)-decorated GRP were prepared and utilized as biosensing platforms. In this case, AgNPs and GRP exhibit plasmonic properties, whereas IONPs exhibit magnetic properties; therefore, this hybrid nanomaterial could function as a magnetoplasmonic substrate for the magnetofluoro-immunosensing (MFI) system. Conversely, exosomes were recently considered high-potential biomarkers for the diagnosis of diseases. However, exosome diagnostic use requires complex isolation and purification methods. Nevertheless, we successfully detected a prostate-cancer-cell-derived exosome (PC-exosome) from non-purified exosomes in a culture media sample using Ag/IO-GRP and dye-tetraspanin antibodies (Ab). First, the anti-prostate-specific antigen was immobilized on the Ag/IO-GRP and it could isolate the PC-exosome from the sample via an external magnetic force. Dye-tetraspanin Ab was added to the sample to induce the sandwich structure. Based on the number of exosomes, the fluorescence intensity from the dye varied and the system exhibited highly sensitive and selective performance. Consequently, these hybrid materials exhibited excellent potential for biosensing platforms.

Pharmaceutical Aspects of Nanocarriers for Smart Anticancer Therapy.

Drug delivery to tumor sites using nanotechnology has been demonstrated to overcome the drawbacks of conventional anticancer drugs. Altering the surface shape and geometry of nanocomposites alters their chemical properties, which can confer multiple attributes to nanocarriers for the treatment of cancer and their use as imaging agents for cancer diagnosis. However, heterogeneity and blood flow in human cancer limit the distribution of nanoparticles at the site of tumor tisues. For targeted delivery and controlled release of drug molecules in harsh tumor microenvironments, smart nanocarriers combined with various stimuli-responsive materials have been developed. In this review, we describe nanomaterials for smart anticancer therapy as well as their pharmaceutical aspects including pharmaceutical process, formulation, controlled drug release, drug targetability, and pharmacokinetic or pharmacodynamic profiles of smart nanocarriers. Inorganic or organic-inorganic hybrid nanoplatforms and the electrospinning process have also been briefly described here.

Exosomes and Cancer Stem Cells in Cancer Immunity: Current Reports and Future Directions.

Cancer stem cells (CSCs), which have the capacity to self-renew and differentiate into various types of cells, are notorious for their roles in tumor initiation, metastasis, and therapy resistance. Thus, underlying mechanisms for their survival provide key insights into developing effective therapeutic strategies. A more recent focus has been on exosomes that play a role in transmitting information between CSCs and non-CSCs, resulting in activating CSCs for cancer progression and modulating their surrounding microenvironment. The field of CSC-derived exosomes (CSCEXs) for different types of cancer is still under exploration. A deeper understanding and further investigation into CSCEXs' roles in tumorigenicity and the identification of novel exosomal components are necessary for engineering exosomes for the treatment of cancer. Here, we review the features of CSCEXs, including surface markers, cargo, and biological or physiological functions. Further, reports on the immunomodulatory effects of CSCEXs are summarized, and exosome engineering for CSC-targeting is also discussed.

Current Limitations and Recent Progress in Nanomedicine for Clinically Available Photodynamic Therapy.

Photodynamic therapy (PDT) using oxygen, light, and photosensitizers has been receiving great attention, because it has potential for making up for the weakness of the existing therapies such as surgery, radiation therapy, and chemotherapy. It has been mainly used to treat cancer, and clinical tests for second-generation photosensitizers with improved physicochemical properties, pharmacokinetic profiles, or singlet oxygen quantum yield have been conducted. Progress is also being made in cancer theranostics by using fluorescent signals generated by photosensitizers. In order to obtain the effective cytotoxic effects on the target cells and prevent off-target side effects, photosensitizers need to be localized to the target tissue. The use of nanocarriers combined with photosensitizers can enhance accumulation of photosensitizers in the tumor site, owing to preferential extravasation of nanoparticles into the tumor vasculature by the enhanced permeability and retention effect. Self-assembly of amphiphilic polymers provide good loading efficiency and sustained release of hydrophobic photosensitizers. In addition, prodrug nanomedicines for PDT can be activated by stimuli in the tumor site. In this review, we introduce current limitations and recent progress in nanomedicine for PDT and discuss the expected future direction of research.

The Biological Function and Therapeutic Potential of Exosomes in Cancer: Exosomes as Efficient Nanocommunicators for Cancer Therapy.

Cancer therapeutics must be delivered to their targets for improving efficacy and reducing toxicity, though they encounter physiological barriers in the tumor microenvironment. They also face limitations associated with genetic instability and dynamic changes of surface proteins in cancer cells. Nanosized exosomes generated from the endosomal compartment, however, transfer their cargo to the recipient cells and mediate the intercellular communication, which affects malignancy progression, tumor immunity, and chemoresistance. In this review, we give an overview of exosomes' biological aspects and therapeutic potential as diagnostic biomarkers and drug delivery vehicles for oncotherapy. Furthermore, we discuss whether exosomes could contribute to personalized cancer immunotherapy drug design as efficient nanocommunicators.

Crosstalk between Stress Granules, Exosomes, Tumour Antigens, and Immune Cells: Significance for Cancer Immunity.

RNA granules and exosomes produced by tumour cells under various stresses in the microenvironment act as critical determinants of cell survival by promoting angiogenesis, cancer metastasis, chemoresistance, and immunosuppression. Meanwhile, developmental cancer/testis (CT) antigens that are normally sequestered in male germ cells of the testes, but which are overexpressed in malignant tumour cells, can function as tumour antigens triggering immune responses. As CT antigens are potential vaccine candidates for use in cancer immunotherapy, they could be targeted together with crosstalk between stress granules, exosomes, and immune cells for a synergistic effect. In this review, we describe the effects of exosomes and exosomal components presented to the recipient cells under different types of stresses on immune cells and cancer progression. Furthermore, we discuss their significance for cancer immunity, as well as the outlook for their future application.

Properties of Heparinoids Premixed with Tumor-Derived Extracellular Vesicles.

Tumor-derived exosomes are bound and internalized to organ-specific cells, affecting metastasis. Heparan sulfate proteoglycans mediate the interaction between cells and exosomes. Exosome transfer to the recipient cell can be competitively blocked by heparinoids, because heparin is structurally similar to heparan sulfate. It is hypothesized that there may be structural requirements of heparinoids to attenuate the cellular uptake and metastatic activity of tumor-derived exosomes. Here, we compared the properties of unfractionated heparin (UFH), glycol-split UFH, low-molecular-weight heparin (LMWH), glycol-split LMWH, and ultra-LMWH premixed with A549-derived exosomes. Uptake of A549-derived exosomes (0.1 mg/mL) into BEAS-2B cells was significantly blocked by 0.4 mg/mL of heparinoids. Heparinoids attenuated migration of BEAS-2B cells stimulated by A549-derived exosomes. Glycol-split LMWH with no antifactor Xa activity exhibited the strongest antimigratory effects than other heparinoids. Thus, heparinoids with proper molecular weight and structure can inhibit tumor-derived exosomes, not proportionally to the anticoagulant activity.

Self-assembled nanocomplex of PEGylated protamine and heparin-suramin conjugate for accumulation at the tumor site.

Heparin-like sulfated polysaccharides are potential drug candidates owing to their ability to interact with angiogenic factors and inhibit angiogenesis, tumor growth, and metastasis. This study aimed to improve the delivery of heparin-like anticancer polysaccharides for accumulation at the tumor site. We designed a nanocarrier system using protamine attached to polyethylene glycol (PEG) and evaluated the stability, tumor targeting, and tumor growth inhibition of the nanocarrier loaded with heparin derivatives. When mixed with various polyanionic heparin derivatives, the polycationic PEG-protamine formed stable self-assembled nanocomplexes via ionic interactions, with flexible PEG chains located on the outside. Among the complexes, a nanocomplex loaded with a low-molecular-weight heparin-suramin conjugate (LHsura) had the most suitable average size (101.9nm) for the enhanced permeability and retention effect and allowed accumulation of LHsura at the tumor site for up to 48h. In a tumor-bearing mouse model, the PEG-protamine and LHsura nanocomplex (10mg/kg/3days, intravenously), which could be extravasated through the tumor vasculature, significantly inhibited tumor growth, more than LHsura alone did. Overall, the self-assembled nanocomplexation of PEG-protamine and LHsura helped control the release and extravasation of LHsura, which resulted in an antitumor effect on the target tumor cells.

Multi-stage inhibition in breast cancer metastasis by orally active triple conjugate, LHTD4 (low molecular weight heparin-taurocholate-tetrameric deoxycholate).

Targeting multiple stages in metastatic breast cancer is one of the effective ways to inhibit metastatic progression. To target human metastatic breast cancer as well as improving patient compliance, we developed an orally active low molecular weight heparin (LMWH)-taurocholate conjugated with tetrameric deoxycholic acid, namely LHTD4, which followed by physical complexation with a synthetic bile acid enhancer, DCK. In breast cancer, both transforming growth factor-ß1 (TGF-ß1) and CXCL12 exhibit enhanced metastatic activity during the initiation and progression stages of breast cancer, thus we direct the focus on investigating the antimetastatic effect of LHTD4/DCK complex by targeting TGF-ß1 and CXCL12. Computer simulation study and SPR analysis were performed for the binding confirmation of LHTD4 with TGF-ß1 and CXCL12. We carried out in vitro phosphorylation assays of the consecutive receptors of TGF-ß1 and CXCL12 (TGF-ß1R1 and CXCR4, respectively). Effects of LHTD4 on in vitro cell migration (induced by TGF-ß1) and chemotaxis (mediated by CXCL12) were investigated. The in vivo anti-metastatic effect of LHTD4 was evaluated in an accelerated metastasis model and an orthotopic MDA-MB-231 breast cancer model. The obtained KD values of TGF-ß1 and CXCL12 with LHTD4 were 0.85 and 0.019 µM respectively. The simulation study showed that binding affinities of LHTD4 fragment with either TGF-ß1 or CXCL12 through additional electrostatic interaction was more stable than that of LMWH fragment. In vitro phosphorylation assays of TGF-ß1R1 and CXCR4 showed that the effective inhibition of receptor phosphorylation was observed with the treatment of LHTD4. The expressions of epithelial to mesenchymal transition (EMT) marker proteins such as vimentin and Snail were prevented by LTHD4 treatment in in vitro studies with TGF-ß1 treated MDA-MB-231 cells. Moreover, we observed that LHTD4 negatively regulated the functions of TGF-ß1 and CXCL12 on migration and invasion of breast cancer cell. In several advanced orthotopic and experimental breast cancer metastasis murine models, the treatment with LHTD4 (5 mg/kg daily, p.o.) significantly inhibited metastasis compared to the control. Overall, LHTD4 exhibited anti-metastatic effects by inhibiting TGF-ß1 and CXCL12, and the clinically relevant dose of orally active LHTD4 was found to be effective in preclinical studies without any apparent toxicity.

Safety studies on intravenous infusion of a potent angiogenesis inhibitor: taurocholate-conjugated low molecular weight heparin derivative LHT7 in preclinical models.

CONTEXT: As a class of angiogenesis inhibitors, heparin conjugates have shown significant effectiveness in several studies. OBJECTIVES: The purpose of our current study is to evaluate the effectiveness and safety of infusing the conjugate of low molecular weight heparin and taurocholate (LHT7), which has been developed as a potent angiogenesis inhibitor. METHODS: To evaluate its safety, the method of intravenous infusion was compared with its i.v. bolus administration. Intravenous infusion was administered at a rate of 400 µl/min/kg of body weight for 30 min. Pharmacokinetic (PK) analysis, organ accumulation, and plasma concentration profiles of LHT7 were measured. The anticancer effect of LHT7 was evaluated in murine and human xenograft models, and preclinical studies were performed in SD rats and beagle dogs. RESULTS: The results of the PK studies showed reduced organ accumulation in mice and the AUC(0-96 h) (area under the curve) was increased up to 1485 ± 125 h × µg/ml. The efficacy, at dose 1 mg/kg/2 d was higher for i.v. infusion than for i.v. bolus administration in both murine and human cancer models. The preclinical studies showed the safety dose of LHT7 is less than 20 mg/kg in SD rats and in the next safety analysis in beagle dogs showed that there were no organ-specific adverse effects in higher doses, such as, 12 mg/kg. LHT7 showed sustained effects with minimized adverse events when administered through i.v. infusion. CONCLUSIONS: LHT7 (i.v. infusion) could be safely used for further clinical development as a multi-targeting anti-angiogenic agent.

Chemical Conjugate of Low Molecular Weight Heparin and Suramin Fragment Inhibits Tumor Growth Possibly by Blocking VEGF165.

Low molecular weight heparin (LMWH) and its derivatives have been reported to possess antiangiogenic effect via electrostatic interaction with various angiogenic growth factors such as VEGF165. However, clinical applications of LMWH for anticancer therapy have been restricted due to its anticoagulant effect and insufficient therapeutic efficacy. To overcome these limitations and enhance the antiangiogenic efficacy, LMWH was conjugated with suramin fragments that have a binding affinity to the heparin-binding domain (HBD) of proteins. The conjugation of suramin fragments to LMWH enhanced the antiangiogenic effect of LMWH by increasing the binding affinity to VEGF165, while decreasing its anticoagulant activity. The chemical conjugate of LMWH and suramin fragments (LHsura) showed a substantial inhibitory effect on VEGF165-mediated cell proliferation, migration, and tube formation of HUVECs without significant cytotoxicity in vitro. Finally, we confirmed the anticancer effect of LHsura (61.4% vs control) in a SCC7-bearing mouse model.

Formulations of deoxycholic for therapy: a patent review (2011 - 2014).

INTRODUCTION: Deoxycholic acid (DOCA) is involved in many physiological functions and has been used in various fields of pharmaceutical formulations as a natural active solubilizing and permeation-enhancing agent. Although DOCA has been suggested to be a promoter of colon cancer, it has also been used extensively as a starting material to obtain new derivatives for potential therapeutic applications. AREA COVERED: In this review, we focus on patents and research reports from 2011 to 2014 related to pharmaceutical formulations and therapeutic applications using DOCA and its derivatives as surfactants or absorption enhancers, drug delivery carriers, and anti-cancer agents. EXPERT OPINION: In recent few years, DOCA and its derivatives have been used mostly as pharmaceutical excipients for solubilizing lipophilic compounds to improve their bioavailability. Other studies have expanded its applications to include enhanced drug permeability and have designed more effective drug carriers by conjugation with polymeric materials. Recently, a synthetic DOCA injection, ATX-101, has shown long-term efficacy in the non-surgical treatment of unwanted submental fat and acceptable tolerability in humans. Thus, it may be used for reducing specific localized fat accumulations. Additionally, DOCA has been a starting material for anti-cancer drugs, and some derivatives showed strong inhibitory activities against several carcinoma cells.

Preliminary safety evaluation of a taurocholate-conjugated low-molecular-weight heparin derivative (LHT7): a potent angiogenesis inhibitor.

In our previous studies, taurocholic acid (TA)-conjugated low-molecular-weight heparin derivative (LHT7) has been proven to be a potent anti-angiogenic agent by demonstrated successful blockage capability of vascular endothelial growth factors (VEGF). Preliminary safety evaluations were conducted based on its mechanism of action and chemical behavior. For this purpose, acute toxicity study, and hematological and serological evaluations were carried out. Additionally, in order to evaluate mechanism-related side effects, both blood pressure and the occurrence of proteinuria were measured using a treatment regime of multiple high doses of LHT7 in a biodistribution study. LD50 values for LHT7 in female and male mice were 56.9 and 64.7 mg kg(-1) doses, respectively. There were no vital fluctuations in the serological and hematological parameters, except for the elevated levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) at 100 and 200 mg kg(-1) doses of LHT7, representing vital changes in the liver function. Moreover, the results of mechanism-related studies showed that blood pressure at 50 mg kg(-1) did not change but showed elevated levels of protein in urine. In the biodistribution study, a slight accumulation of LHT7 in the kidney and the liver were observed at the 50 mg kg(-1) repeated dose owing to the presence of bile acid. No fatal damage was observed in this study; most observations were related to the chemical composition or the mechanism of action of the material.

Advances in oral macromolecular drug delivery.

INTRODUCTION: Various macromolecules including polypeptides, proteins, genes and polysaccharides have been drawing attention for their therapeutic potential. The passage through intestinal epithelium is the major barrier for the oral delivery of macromolecules, by either paracellular or transcellular pathways. However, most macromolecules are poorly absorbed in oral route due to their high molecular weight and low stability in the gastrointestinal (GI) tract. Nonetheless, advancing in oral macromolecular drug delivery will be significant in expanding the clinical use of therapeutic macromolecules. AREAS COVERED: Technologies using chemical conjugation, absorption enhancers and nano-/micro-particulate systems have been developed to improve oral bioavailability of macromolecules, and some of them are in the process of clinical trials. In this review, they are discussed in the context of their progression states, hurdles and modes of action. EXPERT OPINION: According to the better understanding of receptor or transporter structure and transport mechanisms in the GI tract, the progress ineffective oral delivery systems for therapeutic macromolecules is anticipated over the next decades. In addition, the advent of numerous particulate systems will also speed up the development of novel drug delivery technologies. This offers an optimistic perspective on the potential clinical usage of oral macromolecular drugs.

TNF-α gene silencing using polymerized siRNA/thiolated glycol chitosan nanoparticles for rheumatoid arthritis.

Among various proinflammatory cytokines involved in the pathogenesis of rheumatoid arthritis (RA), tumor necrosis factor (TNF)-α plays a pivotal role in the release of other cytokines and induction of chronic inflammation. Even though siRNA has the therapeutic potential, they have a challenge to be delivered into the target cells because of their poor stability in physiological fluids. Herein, we design a nanocomplex of polymerized siRNA (poly-siRNA) targeting TNF-α with thiolated glycol chitosan (tGC) polymers for the treatment of RA. Poly-siRNA is prepared through self-polymerization of thiol groups at the 5' end of sense and antisense strand of siRNA and encapsulated into tGC polymers, resulting in poly-siRNA-tGC nanoparticles (psi-tGC-NPs) with an average diameter of 370 nm. In the macrophage culture system, psi-tGC-NPs exhibit rapid cellular uptake and excellent in vitro TNF-α gene silencing efficacy. Importantly, psi-tGC-NPs show the high accumulation at the arthritic joint sites in collagen-induced arthritis (CIA) mice. Treatment monitoring data obtained by the matrix metalloproteinase 3-specific nanoprobe and microcomputed tomography show that intravenous injection of psi-tGC-NPs significantly inhibits inflammation and bone erosion in CIA mice, comparable to methotrexate (5 mg/kg). Therefore, the availability of psi-tGC-NP therapy that target specific cytokines may herald new era in the treatment of RA.

Enhanced oral absorption of ibandronate via complex formation with bile acid derivative.

Bisphosphonates are recommended for the treatment of postmenopausal osteoporosis, Paget's disease, bone metastasis, and multiple myeloma. However, the efficacy of oral preparations is limited because of their low bioavailabilities and adverse effects from the gastrointestinal tract. This study was conducted to investigate whether N(α)-deoxycholyl-L-lysyl-methylester (DCK), an absorption enhancer derived from deoxycholic acid, can increase the oral bioavailability of ibandronate. We prepared a physical complex of ibandronate with DCK, and evaluated its permeability across a parallel artificial membrane. Furthermore, pharmacokinetic profile and oral absorption of the optimized formulation were also studied in rats. DCK enhanced the apparent membrane permeability of ibandronate by 14.4-fold in a parallel artificial membrane permeability assay model, compared with when ibandronate was applied alone. When ibandronate-DCK complex was intrajejunally administered to rats, it resulted in a 2.8- and 4.3-fold increase in maximum plasma concentration and area under the concentration-time curve from time zero to the last measurable time point, respectively. These results demonstrate that the ibandronate-DCK formulation can improve the oral absorption of ibandronate, allowing less frequent dosing to avoid side effects as well to enhance patient compliance.

Cyclic RGDyk-conjugated LMWH-taurocholate derivative as a targeting angiogenesis inhibitor.

LMWH-taurocholate derivative (LHT7) has been reported as a novel angiogenesis inhibitor, due to its ability to bind to several kinds of growth factors, which play critical roles in tumor angiogenesis. In this study, we have highlighted the enhanced antiangiogenic activity of LHT7, by using cyclic RGDyk (cRGD), a targeting moiety that was chemically conjugated to LHT7 via amide bond. The SPR study revealed that cRGD-LHT7 bound to α(v)ß(3) integrin as strongly as cRGD, and it bound to VEGF as strongly as LHT7. Importantly, in vitro anti-angiogenesis studies revealed that cRGD-LHT7 had a significant inhibition effect on HUVEC tubular formation. Finally, cRGD-LHT7 showed a greater inhibitory efficiency on the tumor growth in the U87MG xenograft model than the original LHT7, which was owed to its ability to target the tumor cells. All of these findings demonstrated that cRGD-LHT7 targeted α(v)ß(3) integrin-positive cancer cells and endothelial cells, resulting in a greater anti-angiogenesis effect on the solid tumors.

Orally active desulfated low molecular weight heparin and deoxycholic acid conjugate, 6ODS-LHbD, suppresses neovascularization and bone destruction in arthritis.

The regulation of angiogenesis is an interesting area to consider for novel therapeutic approaches to rheumatoid arthritis (RA). Chemically modified heparins have been developed as possible candidates for angiogenesis inhibitor; however, they have a major clinical drawback in exhibiting poor oral bioavailability. Here, orally absorbable O-desulfated low molecular weight heparin (ODS-LMWH) derivatives were newly synthesized by conjugating 2-O- or 6-O-desulfated LMWH with deoxycholic acid (DOCA) or bisDOCA (a dimer of DOCA), and their physicochemical properties, antiangiogenic potency and pharmacokinetic profiles were assessed. After selecting the best candidate among those derivatives, its therapeutic efficacy on arthritis was investigated in a murine collagen antibody-induced arthritis (CAIA) model. ODS-LMWH derivatives significantly inhibited the capillary-like tube formation of human umbilical vein endothelial cells (HUVECs) and basic fibroblast growth factor (bFGF)-induced angiogenesis in the Matrigel plug assay. Among all the compounds, 6ODS-LHbD showed the highest oral bioavailability in rats (19.3%). In the CAIA mouse model, 6ODS-LHbD (10 mg/kg, p.o., S.I.D.) significantly inhibited neovascularization in the joint, the increase of hind-paw thickness, and the structural damage in the bone. Therefore, 6ODS-LHbD would be a promising candidate for an orally active drug for the treatment of RA.

Phospholipid-based microemulsion formulation of all-trans-retinoic acid for parenteral administration.

All-trans-retinoic acid (ATRA) shows anti-cancer activities, especially in patients with acute promyelocytic leukemia. Due to the highly variable bioavailability of ATRA and induction of its own metabolism after oral treatment, development of alternative parenteral dosage form is required. The principal aim of this study was to develop a parenteral formulation of ATRA by overcoming its solubility limitation by utilizing phospholipid-based microemulsion system as a carrier. Microemulsion was prepared with pharmaceutically acceptable ingredients such as soybean oil and phospholipids. The mean particle diameter and polydispersity of ATRA microemulsion could be decreased to be applicable for parenteral administration by modulation of composition of microemulsion. The loading concentration of ATRA in microemulsion increased by increasing the oil contents and also by inclusion of distearoylphosphatidyl-ethanolamine-N-poly(ethyleneglycol) 2000 (DSPE-PEG). Furthermore, loading of ATRA in microemulsion improved the chemical stability of ATRA. The pharmacokinetic profile of ATRA after intravenous injection of microemulsion formulation to rats was similar to that of sodium ATRA. The growth inhibitory effects of ATRA on human cancer HL-60 and MCF-7 cell lines were also similar between free ATRA and microemulsion formulation of ATRA, suggesting that its anti-cancer activity was not impaired by loading in microemulsion. Our study herein demonstrates that phospholipid-based microemulsion may provide an alternative parenteral formulation of ATRA.