Sang Mun Bae

Hydrophobically modified glycol chitosan nanoparticles-encapsulated camptothecin enhance the drug stability and tumor targeting in cancer therapy

To prepare a water-insoluble camptothecin (CPT) delivery carrier, hydrophobically modified glycol chitosan (HGC) nanoparticles were constructed by chemical conjugation of hydrophobic 5beta-cholanic acid moieties to the hydrophilic glycol chitosan backbone. Insoluble anticancer drug, CPT, was easily encapsulated into HGC nanoparticles by a dialysis method and the drug loading efficiency was above 80%. CPT-encapsulated HGC (CPT-HGC) nanoparticles formed nano-sized self-aggregates in aqueous media (280-330 nm in diameter) and showed sustained release of CPT for 1 week. Also, HGC nanoparticles effectively protected the active lactone ring of CPT from the hydrolysis under physiological condition, due to the encapsulation of CPT into the hydrophobic cores in the HGC nanoparticles. The CPT-HGC nanoparticles exhibited significant antitumor effects and high tumor targeting ability towards MDA-MB231 human breast cancer xenografts subcutaneously implanted in nude mice. Tumor growth was significantly inhibited after i.v. injection of CPT-HGC nanoparticles at doses of 10 mg/kg and 30 mg/kg, compared to free CPT at dose of 30 mg/kg. The significant antitumor efficacy of CPT-HGC nanoparticles was attributed to the ability of the nanoparticles to show both prolonged blood circulation and high accumulation in tumors, as confirmed by near infrared (NIR) fluorescence imaging systems. Thus, the delivery of CPT to tumor tissues at a high concentration, with the assistance of HGC nanoparticles, exerted a potent therapeutic effect. These results reveal the promising potential of HGC nanoparticles-encapsulated CPT as a stable and effective drug delivery system in cancer therapy.

Smart Nanocarrier Based on PEGylated Hyaluronic Acid for Cancer Therapy

Tumor targetability and site-specific drug release of therapeutic nanoparticles are key factors for effective cancer therapy. In this study, poly(ethylene glycol) (PEG)-conjugated hyaluronic acid nanoparticles (P-HA-NPs) were investigated as carriers for anticancer drugs including doxorubicin and camptothecin (CPT). P-HA-NPs were internalized into cancer cells (SCC7 and MDA-MB-231) via receptor-mediated endocytosis, but were rarely taken up by normal fibroblasts (NIH-3T3). During in vitro drug release tests, P-HA-NPs rapidly released drugs when incubated with cancer cells, extracts of tumor tissues, or the enzyme Hyal-1, which is abundant in the intracellular compartments of cancer cells. CPT-loaded P-HA-NPs (CPT-P-HA-NPs) showed dose-dependent cytotoxicity to cancer cells (MDA-MB-231, SCC7, and HCT 116) and significantly lower cytotoxicity against normal fibroblasts (NIH-3T3) than free CPT. Unexpectedly, high concentrations of CPT-P-HA-NPs demonstrated greater cytotoxicity to cancer cells than free CPT. An in vivo biodistribution study indicated that P-HA-NPs selectively accumulated into tumor sites after systemic administration into tumor-bearing mice, primarily due to prolonged circulation in the blood and binding to a receptor (CD44) that was overexpressed on the cancer cells. In addition, when CPT-P-HA-NPs were systemically administrated into tumor-bearing mice, we saw no significant increases in tumor size for at least 35 days, implying high antitumor activity. Overall, P-HA-NPs showed promising potential as a drug carrier for cancer therapy.

Tumor-Targeting Peptide Conjugated pH-Responsive Micelles as a Potential Drug Carrier for Cancer Therapy

Herein, we prepared tumor-targeting peptide (AP peptide; CRKRLDRN) conjugated pH-responsive polymeric micelles (pH-PMs) in cancer therapy by active and pH-responsive tumor targeting delivery systems, simultaneously. The active tumor targeting and tumoral pH-responsive polymeric micelles were prepared by mixing AP peptide conjugated PEG-poly(d,l-lactic acid) block copolymer (AP-PEG-PLA) into the pH-responsive micelles of methyl ether poly(ethylene glycol) (MPEG)-poly(beta-amino ester) (PAE) block copolymer (MPEG-PAE). These mixed amphiphilic block copolymers were self-assembled to form stable AP peptide-conjugated and pH-responsive AP-PEG-PLA/MPEG-PAE micelles (AP-pH-PMs) with an average size of 150 nm. The AP-pH-PMs containing 10 wt % of AP-PEG-PLA showed a sharp pH-dependent micellization/demicellization transition at the tumoral acid pH. Also, they presented the pH-dependent drug release profile at the acidic pH of 6.4. The fluorescence dye, TRITC, encapsulated AP-pH-PMs (TRITC-AP-pH-PMs) presented the higher tumor-specific targeting ability in vitro cancer cell culture system and in vivo tumor-bearing mice, compared to control pH-responsive micelles of MPEG-PAE. For the cancer therapy, the anticancer drug, doxorubicin (DOX), was efficiently encapsulated into the AP-pH-PMs (DOX-AP-pH-PMs) with a higher loading efficiency. DOX-AP-pH-PMs efficiently deliver anticancer drugs in MDA-MB231 human breast tumor-bearing mice, resulted in excellent anticancer therapeutic efficacy, compared to free DOX and DOX encapsulated MEG-PAE micelles, indicating the excellent tumor targeting ability of AP-pH-PMs. Therefore, these tumor-targeting peptide-conjugated and pH-responsive polymeric micelles have great potential application in cancer therapy.

Tumoral acidic pH-responsive MPEG-poly(β-amino ester) polymeric micelles for cancer targeting therapy

Herein, we evaluated the tumoral low pH targeting characteristics of pH-responsive polymer micelles in cancer targeting therapy. To design the pH-responsive polymeric micelles, hydrophilic methyl ether poly(ethylene glycol) (MPEG) and pH-responsive/biodegradable poly(beta-amino ester) (PAE) were copolymerized using a Michael-type step polymerization, resulting in an MEPG-PAE block copolymer. The amphiphilic MPEG-PAE block copolymer formed polymeric micelles with nano-sized diameter by self-assembly, which showed a sharp pH-dependant micellization/demicellization transition at the tumoral acidic pH value (pH 6.4). For the cancer image and therapy, fluorescence dye, tetramethylrhodamine isothiocyanate (TRITC), or anticancer drug, camptothecin (CPT), was efficiently encapsulated into the pH-responsive polymeric micelles (pH-PMs) by a simple solvent casting method. The TRITC or CPT encapsulated pH-PMs (TRITC-pH-PMs or CPT-pH-PMs) showed rapid release of TRITC or CPT in weakly acidic aqueous (pH 6.4) because they still presented a sharp tumoral acid pH-responsive micellization/demicellization transition. The pH-PMs with 10wt.% of TRITC could deliver substantially more fluorescence dyes to the target tumor tissue in MDA-MB231 human breast tumor-bearing mice, compared to the control polymeric micelles of PEG-poly(l-lactic acid) (PEG-PLLA). Importantly, CPT-pH-PMs exhibited significantly increased therapeutic efficacy with minimum side effects by other tissues in breast tumor-bearing mice, compared to free CPT and CPT encapsulated PEG-PLLA micelles. The tumoral acidic pH-responsive polymeric micelles are highly useful for cancer targeting therapy.

Polyproline‐type helical‐structured low‐molecular weight heparin (LMWH)‐taurocholate conjugate as a new angiogenesis inhibitor

Although heparin can regulate angiogenesis, tumor growth and metastasis, its clinical application, as well as that of low‐molecular heparin (LMWH), for treating cancer are limited because of heparins anticoagulant activity and risk of hemorrhages. LMWH‐taurocholate conjugates (LHT7), which have low anticoagulant activity, were synthesized. The structural property of LHT was evaluated by circular dichroism and the binding affinity of LHT7 to vascular endothelial growth factor 165 (VEGF165) was measured by isothermal titration calorimetry. The inhibitory effect of LHT7 on VEGF‐mediated KDR (VEGF‐receptor 2) phosphorylation in Human umbilical vein endothelial cells was evaluated. The VEGF165 dependent Matrigel plug assay was performed to verify the antiangiogenic potential of LHT7 on a VEGF165 inhibitor. Finally, tumor growth inhibition effects of LHT7 on SCC7 and the survival rate of animal models were investigated. Moreover, MDA‐MB231 xenograft mouse model was additionally used to confirm the therapeutic effect of LHT7 on human breast cancer cell line. As a result, LHT7 which has 12.7% of anticoagulant activity of the original LMWH showed a peculiar polyproline‐type helical structure. LHT7 binds to VEGF strongly and inhibits VEGF dependent KDR phosphorylation. The results of Matrigel plug assay proved LHT7 as a strong antiangiogenic agent inhibiting VEGF165. Remarkably, LHT7 showed a significant tumor growth inhibition potential on SCC7 with an increased survival rate. LHT7 also delayed tumor growth in MDA‐MB231 human breast cancer cell lines.

Facilitated intracellular delivery of peptide-guided nanoparticles in tumor tissues

Macromolecular nanoparticles can extravasate and accumulate within tumor tissues via the passive targeting system, reflecting enhanced permeability and the retention effect. However, the unsatisfactory tumor therapeutic efficacy of the passive-targeting system, attributable to the retention of extravasated nanoparticles in the vicinity of tumor vessels, argues that a new system that facilitates intracellular delivery of nanoparticles within tumors is needed. Here, we developed hydrophobically modified glycol chitosan (HGC) nanoparticles conjugated with interleukin-4 receptor (IL-4R) binding peptides, termed I4R, and tested them in mice bearing IL-4R-positive tumors. These HGC-I4R nanoparticles exhibited enhanced IL-4R-dependent cellular uptake in tumors compared to nonconjugated nanoparticles, leading to better therapeutic and imaging efficacy. We conclude that I4R facilitates and enhances cellular uptake of nanoparticles in tumor tissues. This study suggests that the intracelluar uptake of nanoparticles in tumors is an essential factor to consider in designing nanoparticles for tumor-targeted drug delivery and imaging.

Transforming growth factor-β-induced protein (TGFBIp/β ig-h3) activates platelets and promotes thrombogenesis

Transforming growth factor-beta-induced protein (TGFBIp)/beta ig-h3 is a 68-kDa extracellular matrix protein that is functionally associated with the adhesion, migration, proliferation, and differentiation of various cells. The presence of TGFBIp in platelets led us to study the role of this protein in the regulation of platelet functions. Upon activation, platelet TGFBIp was released and associated with the platelets. TGFBIp mediates not only the adhesion and spread of platelets but also activates them, resulting in phosphatidylserine exposure, alpha-granule secretion, and increased integrin affinity. The fasciclin 1 domains of TGFBIp are mainly responsible for the activation of platelets. TGFBIp promotes thrombus formation on type I fibrillar collagen under flow conditions in vitro and induces pulmonary embolism in mice. Moreover, transgenic mice, which have approximately a 1.7-fold greater blood TGFBIp concentration, are significantly more susceptible to collagen- and epinephrine-induced pulmonary embolism than wild-type mice. These results suggest that TGFBIp, a human platelet protein, plays important roles in platelet activation and thrombus formation. Our findings will increase our understanding of the novel mechanism of platelet activation, contributing to a better understanding of thrombotic pathways and the development of new antithrombotic therapies.

Construction and application of elastin like polypeptide containing IL-4 receptor targeting peptide.

Various human solid tumors highly express IL-4 receptors which amplify the expression of some of anti-apoptotic proteins, preventing drug-induced cancer cell death. Thus, IL-4 receptor targeted drug delivery can possibly increase the therapeutic efficacy in cancer treatment. Macromolecular carriers with multivalent targeting moieties offered great advantages in cancer therapy as they not only increase the plasma half-life of the drug but also allow delivery of therapeutic drugs to the cancer cells with higher specificity, minimizing the deleterious effects of the drug on normal cells. In this study we designed a library of elastin like polypeptide (ELP) polymers containing tumor targeting AP1 peptide using recursive directional ligation method. AP1 was previously discovered as an atherosclerotic plaque and breast tumor tissue homing peptide using phage display screening method, and it can selectively bind to the interleukin 4 receptor (IL-4R). The fluorescently labeled [AP1-V12]6, an ELP polymer containing six AP1 enhanced tumor-specific targeting ability and uptake efficiency in H226 and MDA-MB-231 cancer cell lines in vitro. Surface plasmon resonance analysis showed that multivalent presentation of the targeting ligand in the ELP polymer increased the binding affinity towards IL-4 receptor compared to free peptide. The binding of [AP1-V12]6 to cancer cells was remarkably reduced when IL-4 receptors were blocked by antibody against IL-4 receptor further confirmed its binding. Importantly, the Cy5.5-labeled [AP1-V12]6 demonstrated excellent homing and longer retention in tumor tissues in MDA-MB-231 xenograft mouse model. Immunohistological studies of tumor tissues further validated the targeting efficiency of [AP1-V12]6 to tumor tissue. These results indicate that designed [AP1-V12]6 can serve as a novel carrier for selective delivery of therapeutic drugs to tumors.

LHT7, a chemically modified heparin, inhibits multiple stages of angiogenesis by blocking VEGF, FGF2 and PDGF-B signaling pathways.

Despite the therapeutic benefits of the angiogenesis inhibitors shown in the clinics, they have encountered an unexpected limitation by the occurrence of acquired resistance. Although the mechanism of the resistance is not clear so far, the upregulation of alternative angiogenic pathways and stabilization of endothelium by mural cells were reported to be responsible. Therefore, blocking multiple angiogenic pathways that are crucial in tumor angiogenesis has been highlighted to overcome such limitations. To develop an angiogenesis inhibitor that could block multiple angiogenic factors, heparin is an excellent lead compound since wide array of angiogenic factors are heparin-binding proteins. In previous study, we reported a heparin-derived angiogenesis inhibitor, LHT7, as a potent angiogenesis inhibitor and showed that it blocked VEGF signaling pathway. Here we show that LHT7 could block the fibroblast growth factor 2 (FGF2) and platelet-derived growth factor B (PDGF-B) in addition to VEGF. Simultaneous blockade of these angiogenic factors resulted in inhibition of multiple stages of the angiogenic process, including initial angiogenic response to maturation of the endothelium by pericyte coverage in vitro. In addition, the treatment of LHT7 in vivo did not show any sign of vascular normalization and directly led to decreased blood perfusion throughout the tumor. Our findings show that LHT7 could effectively inhibit tumor angiogenesis by blocking multiple stages of the angiogenesis, and could potentially be used to overcome the resistance.

Potentiation of anti-angiogenic activity of heparin by blocking the ATIII-interacting pentasaccharide unit and increasing net anionic charge.

Heparin, a potent anticoagulant used for the prevention of venous thromboembolism, has been recognized as a tumor angiogenesis inhibitor. Its limitation in clinical application for cancer therapy, however, arises from its strong anticoagulant activity, which causes associated adverse effects. In this study, we show the structural correlation of LHT7, a previously developed heparin-based angiogenesis inhibitor, with its influence on VEGF blockade and its decreased anticoagulant activity. LHT7 was characterized as having average seven molecules of sodium taurocholates conjugated to one molecule of low-molecular-weight heparin (LMWH). This study showed that the conjugation of sodium taurocholates selectively blocked interaction with antithrombin III (ATIII) while enhancing the binding with VEGF. This resulted in LHT7 to have negligible anticoagulant activity but potent anti-angiogenic activity. Following up on this finding, we showed that the bidirectional effect of sodium taurocholate conjugation was due to its unique structure, that is, the sterane core hindering the ATIII-binding pentasaccharide unit of LMWH with its bulky and rigid structural characteristics while the terminal sulfate group interacts with VEGF to produce stronger binding. In addition, we showed that LHT7 was localized in the tumor, especially on the endothelial cells. One explanation for this might be that LHT7 was delivered to the tumor via platelets.