- Email: [email protected]
Polymer inhibitors of ABC transporter overcoming multidrug resistance: Synthesis, characterization and in vitro evaluation
Abstracts / Journal of Controlled Release 213 (2015) e8–e152
[2] U. Lächelt, P. Kos, F.M. Mickler, A. Herrmann, E.E. Salcher, W. Rödl, N. Badgujar, C. Bräuchle, E. Wagner, Fine-tuning of proton sponges by precise diaminoethanes and histidines in pDNA polyplexes, Nanomed. NBM 10 (2014) 35–44. doi:10.1016/j.jconrel.2015.05.178
Synthesis of PLGA-gemcitabine conjugate and its anti-proliferative properties Vaibhav Kharea,*, Ravindra Dubeya, Noor Alama, Ankit Sanejaa, Prem N. Guptaa, Shashank K. Singhb a Formulation and Drug Delivery Division, Indian Institute of Integrative Medicine, Jammu 180001, India b Cancer Pharmacology Division, Indian Institute of Integrative Medicine, Jammu 180001, India ⁎Corresponding author. E-mail address: [email protected] (V. Khare). The use of gemcitabine is limited, owing to its rapid metabolism by cytidine-deaminase and fast kidney excretion [1]. A polymeric conjugate of gemcitabine was prepared by covalent coupling with poly (lactic-co-glycolic) acid (PLGA) using DCC/NHS chemistry, in order to improve the anticancer properties of the drug. The synthesized conjugate was characterized by various analytical techniques including FTIR, NMR and mass spectroscopic analysis. The stability study indicated that the polymeric conjugate was more stable in plasma as compared to native gemcitabine. Further, the in-vitro cytotoxicity in a panel of cell lines including pancreatic cancer (MIAPaCa-2), breast cancer (MCF-7) and colon cancer (HCT-116), demonstrated that the cytotoxic activity of gemcitabine was retained following conjugation with the polymeric carrier. The nucleoside transportation inhibition assay showed that the prepared conjugate was not dependent on nucleoside transporter for entering into the cells and this, in turn, reflecting potential implication of this conjugate in the therapy of transporter-deficient resistance cancer. Further, the cell cycle analysis (Fig. 1) showed that the sub-G1 (G0) apoptotic population was 46.6% and 60.6% for gemcitabine and PLGA-gemcitabine conjugate, respectively. The conjugate produced remarkable decrease in mitochondrial membrane potential, a marker of apoptosis [2]. In addition, there was a marked increase in PARP cleavage and P-H2AX expression (Fig. 1) with PLGA-gemcitabine conjugate as compared to native gemcitabine indicative of improved apoptotic activity. The findings demonstrated the potential of PLGA-gemcitabine conjugate to improve clinical outcome of gemcitabine based chemotherapy of cancer.
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References [1] A.M. Storniolo, S.R. Allerheiligen, H.L. Pearce, Preclinical, pharmacologic, and phase I studies of gemcitabine, Semin. Oncol. 24 (1997) (S7-2-S7-7). [2] C.D. Bortner, J.A. Cidlowski, Caspase independent/dependent regulation of K+, cell shrinkage, and mitochondrial membrane potential during lymphocyte apoptosis, J. Biol. Chem. 274 (1999) 21953–21962. doi:10.1016/j.jconrel.2015.05.179
Polymer inhibitors of ABC transporter overcoming multidrug resistance: Synthesis, characterization and in vitro evaluation Vladimír Šubra, Eva Koziolováa, Ladislav Sivákb, Blanka Říhováb, Marek Kovářb, Karel Ulbricha a Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, v.v.i., Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic b Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20 Prague 4, Czech Republic E-mail address: [email protected] (V. Šubr). One of the most significant problems of chemotherapy is that even originally very sensitive tumors become often resistant to cytostatic after several cycles of treatment. This so-called multidrug resistance (MDR) phenomenon is caused by the overexpression of the MDR gene-encoded proteins, e.g. P-glycoprotein (P-gp), an ATP-dependent exporter which increases the efflux of anticancer drugs from cancer cells [1]. Two P-gp inhibitors of ABC transporters, reversin 121(Boc-Asp(OBzl)-Lys(Z)-OtBu) and ritonavir which are able to restore the cytotoxicity of anticancer drugs in MDR cells, were used in this study. The aim of this work was to synthesize water-soluble polymer conjugates based on N-(2hydroxypropyl)methacrylamide copolymers (pHPMA) containing either inhibitor of ABC transporters, anticancer drug doxorubicin (DOX) or both. These conjugates due to their prolonged blood circulation and passive accumulation in tumor tissue (EPR effect) can improve the efficacy of anticancer drugs and overcome nonspecific side effects on healthy tissue [2]. The 5-methyl-4-oxohexanoyl reversin 121 (MeOHe–R121) and 5-methyl-4-oxohexanoyl ritonavir ester (MeOHe–RIT), DOX or both were conjugated to pHPMA via pH sensitive hydrazone bond and the activity of conjugates was tested in murine leukemic P388/MDR and parental P388 cells. P-Ahx-NHN = MeOHe–R121 and P-Ahx-NH-N = MeOHe–RIT, showed very good P-gp inhibitory activity. At 24 μM, the P-Ahx-NHN = MeOHe–R121 conjugate increases the sensitivity of the P388/
Fig. 1. (a) Effect of PLGA-gemcitabine conjugate and gemcitabine on cell cycle distribution in MiaPaCa-2 cells. (b) Expression of proteins (PARP and P-H2AX) detected by western blotting after treatment with PLGA-gemcitabine conjugate and gemcitabine in MiaPaCa-2 cells.
Keywords: polymer-drug conjugate, drug delivery, gemcitabine, PLGA
Fig. 1. Proliferation of P388/MDR cells in vitro incubated with polymer conjugates P-Ahx-NH-N = Dox ( ), P-Ahx-NH-N = MeOHe–R121(DOX) ( ) and combination of conjugates P-Ahx-NH-N = DOX and P-Ahx-NH-N = MeOHe–R121 (●). The equimolar ratio of drug/inhibitor was used.
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Abstracts / Journal of Controlled Release 213 (2015) e8–e152
MDR cells to DOX by approximately 50-fold. The IC50 for the P-Ahx-NHN = MeOHe–R121(DOX) conjugate, which contains both DOX and the P-gp inhibitor MeOHe–R121 bound to the same carrier via hydrazone bonds, was almost 30 times lower than that for the P-Ahx-NH-N = DOX conjugate (Fig. 1). This IC50 was less than two times lower than the IC50 for the comparable mixture of the two conjugates, P-Ahx-NHN = MeOHe–R121 and P-Ahx-NH-N = DOX. Similarly, conjugate P-Ahx-NH-N = MeOHe–RIT(DOX) was almost 10 times more cytostatic than P-Ahx-NH-N = DOX.
nanogels can efficiently improve the DOX encapsulation due to the electrostatic interaction. More interestingly, the pH-sensitive terminator enables nanogels to reverse their surface charge from negative to positive at tumor extracellular pH (6.5–6.8) to facilitate cell internalization. Subsequently, the decrease of electrostatic interaction between COOH and DOX at endosomal pH, and the cleavage of the intervening disulfide bonds in response to a high GSH concentration in cytosol would lead to fast and complete release of DOX in the cells. These novel tumor-pH activated charge-conversional and reducible PVA nanogels are highly promising for targeted intracellular anticancer drug release.
Keywords: HPMA copolymers, multidrug resistance, P-glycoprotein, reversin 121, ritonavir Acknowledgments This work was supported by the Grant Agency of the Czech Republic (Grant No. P301/12/1254). References [1] M.M. Gottesman, How cancer-cells evade chemotherapy-16th Richard-And-Hinda-Rosenthal-Foundation Award Lecture, Cancer Res. 53 (1993) 747–754. [2] H. Maeda, Y. Matsumura, EPR effect based drug design and clinical outlook for enhanced cancer chemotherapy, Adv. Drug Deliv. Rev. 63 (2011) 129–130. doi:10.1016/j.jconrel.2015.05.180
Tumor-pH activated charge-conversional and reducible poly(vinyl alcohol) nanogels for enhanced cell uptake and intracellular DOX release Wei Chen, Katharina Achazi, Rainer Haag* Freie Universität Berlin, Institut für Chemie und Biochemie, Takustraße 3, 14195 Berlin, Germany ⁎Corresponding author. E-mail addresses: [email protected] (W. Chen), [email protected] (R. Haag). Drug delivery systems (DDS) have appeared as a promising and reliable approach to delivering potent drugs to the site of action precisely and timely, in which polymeric nanosystems including micelles, nanoparticles, and nanogels, have been mostly investigated, due to their prolonged circulation time, enhanced accumulation in the tumor sites via the enhanced permeability and retention (EPR) effect, decreased adverse effects, and improved drug tolerance [1]. As compared to other nanosystems, nanogels with internally cross-linked 3D structures are highly interesting for controlling drug delivery at the target site in fast response to external stimuli, as well as improving the drug bioavailability. Taking the advantage of the high redox potential in cytoplasms and nuclei of cancer cells which contain a 100 to 1000-fold higher concentration of reducing glutathione (GSH) tripeptide than body fluids and extracellular milieu (0.5–10 mM versus 2–20 μM GSH), reduction-sensitive nanogels have recently been designed and exploited for active intracellular release of various potent chemotherapeutics. Poly(vinyl alcohol) (PVA) has a good history of biomedical applications, specifically in the form of micro/hydrogel materials used for enzyme immobilization, cell encapsulation, and clinical applications as embolic bodies. However, its physical gels failed to meet the requirements of nanomedicine, such as drug loading capability, controlled drug release, and degradation. Herein, we developed tumor-pH activated charge-conversional and reducible PVA nanogels for enhanced cell uptake and intracellular DOX release. These PVA nanogels are facilely prepared by inverse nanoprecipitation via “click” reaction (Scheme 1) [2]. The introduction of COOH into the PVA
Scheme 1. Formation of tumor-pH activated charge-conversional and reducible PVA nanogels for enhanced cell uptake and intracellular DOX release.
Keywords: poly(vinyl alcohol), nanogel, charge-conversion, reductionsensitive, intracellular release References [1] R. Haag, F. Kratz, Polymer therapeutics: concepts and applications, Angew. Chem. Int. Ed. 45 (2006) 1198–1215. [2] D. Steinhilber, M. Witting, X. Zhang, M. Staegemann, F. Paulus, W. Friess, S. Kuechler, R. Haag, Surfactant free preparation of biodegradable dendritic polyglycerol nanogels by inverse nanoprecipitation for encapsulation and release of pharmaceutical biomacromolecules, J. Control. Release 169 (2013) 289–295. doi:10.1016/j.jconrel.2015.05.181
A drug delivery system based on novel hollow mesoporous silica nanospheres Wei Feng, Liang Chen, Xiaojun Zhou, Zhiqi Yin, Wei Nie, Kexin Qiu, Chuanglong He* College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China ⁎Corresponding author. E-mail address: [email protected] (C. He). In the last decade, mesoporous silica nanoparticles (MSNs) have received considerable attention for drug delivery due to their nontoxic nature, adjustable pore size and high specific surface area. To date, extensive studies have witnessed rapid advances in the elaborate synthesis of the versatile MSNs [1,2]. Compared with the conventional MSNs (such as MCM-41 and SBA-15), hollow mesoporous silica nanospheres (HMSNs) are of substantial significance for drug delivery applications because of their high pore volume, exceptional drug-loading capacity and excellent biocompatibility. In this work, novel HMSNs that possess a definite huge hollow cavity in the core part and a number of smaller silica nanospheres in the shell have been successfully synthesized by utilizing the cationic polystyrene (PS) nanospheres as the core template, 3-aminopropyltriethoxysilane (APTES) as an additive, and cetyltrimethyl ammonium bromide (CTAB) as the structure-directing surfactant. The shell thickness and the size of cavity could be easily adjusted by altering the amounts of TEOS and the size of PS nanospheres during synthesis process, respectively. Both PS and CTAB can be simultaneously removed via calcination at 550 °C for 6 h. Fig. 1a presents a typical transmission electron microscopy (TEM)
[2] U. Lächelt, P. Kos, F.M. Mickler, A. Herrmann, E.E. Salcher, W. Rödl, N. Badgujar, C. Bräuchle, E. Wagner, Fine-tuning of proton sponges by precise diaminoethanes and histidines in pDNA polyplexes, Nanomed. NBM 10 (2014) 35–44. doi:10.1016/j.jconrel.2015.05.178
Synthesis of PLGA-gemcitabine conjugate and its anti-proliferative properties Vaibhav Kharea,*, Ravindra Dubeya, Noor Alama, Ankit Sanejaa, Prem N. Guptaa, Shashank K. Singhb a Formulation and Drug Delivery Division, Indian Institute of Integrative Medicine, Jammu 180001, India b Cancer Pharmacology Division, Indian Institute of Integrative Medicine, Jammu 180001, India ⁎Corresponding author. E-mail address: [email protected] (V. Khare). The use of gemcitabine is limited, owing to its rapid metabolism by cytidine-deaminase and fast kidney excretion [1]. A polymeric conjugate of gemcitabine was prepared by covalent coupling with poly (lactic-co-glycolic) acid (PLGA) using DCC/NHS chemistry, in order to improve the anticancer properties of the drug. The synthesized conjugate was characterized by various analytical techniques including FTIR, NMR and mass spectroscopic analysis. The stability study indicated that the polymeric conjugate was more stable in plasma as compared to native gemcitabine. Further, the in-vitro cytotoxicity in a panel of cell lines including pancreatic cancer (MIAPaCa-2), breast cancer (MCF-7) and colon cancer (HCT-116), demonstrated that the cytotoxic activity of gemcitabine was retained following conjugation with the polymeric carrier. The nucleoside transportation inhibition assay showed that the prepared conjugate was not dependent on nucleoside transporter for entering into the cells and this, in turn, reflecting potential implication of this conjugate in the therapy of transporter-deficient resistance cancer. Further, the cell cycle analysis (Fig. 1) showed that the sub-G1 (G0) apoptotic population was 46.6% and 60.6% for gemcitabine and PLGA-gemcitabine conjugate, respectively. The conjugate produced remarkable decrease in mitochondrial membrane potential, a marker of apoptosis [2]. In addition, there was a marked increase in PARP cleavage and P-H2AX expression (Fig. 1) with PLGA-gemcitabine conjugate as compared to native gemcitabine indicative of improved apoptotic activity. The findings demonstrated the potential of PLGA-gemcitabine conjugate to improve clinical outcome of gemcitabine based chemotherapy of cancer.
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References [1] A.M. Storniolo, S.R. Allerheiligen, H.L. Pearce, Preclinical, pharmacologic, and phase I studies of gemcitabine, Semin. Oncol. 24 (1997) (S7-2-S7-7). [2] C.D. Bortner, J.A. Cidlowski, Caspase independent/dependent regulation of K+, cell shrinkage, and mitochondrial membrane potential during lymphocyte apoptosis, J. Biol. Chem. 274 (1999) 21953–21962. doi:10.1016/j.jconrel.2015.05.179
Polymer inhibitors of ABC transporter overcoming multidrug resistance: Synthesis, characterization and in vitro evaluation Vladimír Šubra, Eva Koziolováa, Ladislav Sivákb, Blanka Říhováb, Marek Kovářb, Karel Ulbricha a Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, v.v.i., Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republic b Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i., Vídeňská 1083, 142 20 Prague 4, Czech Republic E-mail address: [email protected] (V. Šubr). One of the most significant problems of chemotherapy is that even originally very sensitive tumors become often resistant to cytostatic after several cycles of treatment. This so-called multidrug resistance (MDR) phenomenon is caused by the overexpression of the MDR gene-encoded proteins, e.g. P-glycoprotein (P-gp), an ATP-dependent exporter which increases the efflux of anticancer drugs from cancer cells [1]. Two P-gp inhibitors of ABC transporters, reversin 121(Boc-Asp(OBzl)-Lys(Z)-OtBu) and ritonavir which are able to restore the cytotoxicity of anticancer drugs in MDR cells, were used in this study. The aim of this work was to synthesize water-soluble polymer conjugates based on N-(2hydroxypropyl)methacrylamide copolymers (pHPMA) containing either inhibitor of ABC transporters, anticancer drug doxorubicin (DOX) or both. These conjugates due to their prolonged blood circulation and passive accumulation in tumor tissue (EPR effect) can improve the efficacy of anticancer drugs and overcome nonspecific side effects on healthy tissue [2]. The 5-methyl-4-oxohexanoyl reversin 121 (MeOHe–R121) and 5-methyl-4-oxohexanoyl ritonavir ester (MeOHe–RIT), DOX or both were conjugated to pHPMA via pH sensitive hydrazone bond and the activity of conjugates was tested in murine leukemic P388/MDR and parental P388 cells. P-Ahx-NHN = MeOHe–R121 and P-Ahx-NH-N = MeOHe–RIT, showed very good P-gp inhibitory activity. At 24 μM, the P-Ahx-NHN = MeOHe–R121 conjugate increases the sensitivity of the P388/
Fig. 1. (a) Effect of PLGA-gemcitabine conjugate and gemcitabine on cell cycle distribution in MiaPaCa-2 cells. (b) Expression of proteins (PARP and P-H2AX) detected by western blotting after treatment with PLGA-gemcitabine conjugate and gemcitabine in MiaPaCa-2 cells.
Keywords: polymer-drug conjugate, drug delivery, gemcitabine, PLGA
Fig. 1. Proliferation of P388/MDR cells in vitro incubated with polymer conjugates P-Ahx-NH-N = Dox ( ), P-Ahx-NH-N = MeOHe–R121(DOX) ( ) and combination of conjugates P-Ahx-NH-N = DOX and P-Ahx-NH-N = MeOHe–R121 (●). The equimolar ratio of drug/inhibitor was used.
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Abstracts / Journal of Controlled Release 213 (2015) e8–e152
MDR cells to DOX by approximately 50-fold. The IC50 for the P-Ahx-NHN = MeOHe–R121(DOX) conjugate, which contains both DOX and the P-gp inhibitor MeOHe–R121 bound to the same carrier via hydrazone bonds, was almost 30 times lower than that for the P-Ahx-NH-N = DOX conjugate (Fig. 1). This IC50 was less than two times lower than the IC50 for the comparable mixture of the two conjugates, P-Ahx-NHN = MeOHe–R121 and P-Ahx-NH-N = DOX. Similarly, conjugate P-Ahx-NH-N = MeOHe–RIT(DOX) was almost 10 times more cytostatic than P-Ahx-NH-N = DOX.
nanogels can efficiently improve the DOX encapsulation due to the electrostatic interaction. More interestingly, the pH-sensitive terminator enables nanogels to reverse their surface charge from negative to positive at tumor extracellular pH (6.5–6.8) to facilitate cell internalization. Subsequently, the decrease of electrostatic interaction between COOH and DOX at endosomal pH, and the cleavage of the intervening disulfide bonds in response to a high GSH concentration in cytosol would lead to fast and complete release of DOX in the cells. These novel tumor-pH activated charge-conversional and reducible PVA nanogels are highly promising for targeted intracellular anticancer drug release.
Keywords: HPMA copolymers, multidrug resistance, P-glycoprotein, reversin 121, ritonavir Acknowledgments This work was supported by the Grant Agency of the Czech Republic (Grant No. P301/12/1254). References [1] M.M. Gottesman, How cancer-cells evade chemotherapy-16th Richard-And-Hinda-Rosenthal-Foundation Award Lecture, Cancer Res. 53 (1993) 747–754. [2] H. Maeda, Y. Matsumura, EPR effect based drug design and clinical outlook for enhanced cancer chemotherapy, Adv. Drug Deliv. Rev. 63 (2011) 129–130. doi:10.1016/j.jconrel.2015.05.180
Tumor-pH activated charge-conversional and reducible poly(vinyl alcohol) nanogels for enhanced cell uptake and intracellular DOX release Wei Chen, Katharina Achazi, Rainer Haag* Freie Universität Berlin, Institut für Chemie und Biochemie, Takustraße 3, 14195 Berlin, Germany ⁎Corresponding author. E-mail addresses: [email protected] (W. Chen), [email protected] (R. Haag). Drug delivery systems (DDS) have appeared as a promising and reliable approach to delivering potent drugs to the site of action precisely and timely, in which polymeric nanosystems including micelles, nanoparticles, and nanogels, have been mostly investigated, due to their prolonged circulation time, enhanced accumulation in the tumor sites via the enhanced permeability and retention (EPR) effect, decreased adverse effects, and improved drug tolerance [1]. As compared to other nanosystems, nanogels with internally cross-linked 3D structures are highly interesting for controlling drug delivery at the target site in fast response to external stimuli, as well as improving the drug bioavailability. Taking the advantage of the high redox potential in cytoplasms and nuclei of cancer cells which contain a 100 to 1000-fold higher concentration of reducing glutathione (GSH) tripeptide than body fluids and extracellular milieu (0.5–10 mM versus 2–20 μM GSH), reduction-sensitive nanogels have recently been designed and exploited for active intracellular release of various potent chemotherapeutics. Poly(vinyl alcohol) (PVA) has a good history of biomedical applications, specifically in the form of micro/hydrogel materials used for enzyme immobilization, cell encapsulation, and clinical applications as embolic bodies. However, its physical gels failed to meet the requirements of nanomedicine, such as drug loading capability, controlled drug release, and degradation. Herein, we developed tumor-pH activated charge-conversional and reducible PVA nanogels for enhanced cell uptake and intracellular DOX release. These PVA nanogels are facilely prepared by inverse nanoprecipitation via “click” reaction (Scheme 1) [2]. The introduction of COOH into the PVA
Scheme 1. Formation of tumor-pH activated charge-conversional and reducible PVA nanogels for enhanced cell uptake and intracellular DOX release.
Keywords: poly(vinyl alcohol), nanogel, charge-conversion, reductionsensitive, intracellular release References [1] R. Haag, F. Kratz, Polymer therapeutics: concepts and applications, Angew. Chem. Int. Ed. 45 (2006) 1198–1215. [2] D. Steinhilber, M. Witting, X. Zhang, M. Staegemann, F. Paulus, W. Friess, S. Kuechler, R. Haag, Surfactant free preparation of biodegradable dendritic polyglycerol nanogels by inverse nanoprecipitation for encapsulation and release of pharmaceutical biomacromolecules, J. Control. Release 169 (2013) 289–295. doi:10.1016/j.jconrel.2015.05.181
A drug delivery system based on novel hollow mesoporous silica nanospheres Wei Feng, Liang Chen, Xiaojun Zhou, Zhiqi Yin, Wei Nie, Kexin Qiu, Chuanglong He* College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, Shanghai 201620, China ⁎Corresponding author. E-mail address: [email protected] (C. He). In the last decade, mesoporous silica nanoparticles (MSNs) have received considerable attention for drug delivery due to their nontoxic nature, adjustable pore size and high specific surface area. To date, extensive studies have witnessed rapid advances in the elaborate synthesis of the versatile MSNs [1,2]. Compared with the conventional MSNs (such as MCM-41 and SBA-15), hollow mesoporous silica nanospheres (HMSNs) are of substantial significance for drug delivery applications because of their high pore volume, exceptional drug-loading capacity and excellent biocompatibility. In this work, novel HMSNs that possess a definite huge hollow cavity in the core part and a number of smaller silica nanospheres in the shell have been successfully synthesized by utilizing the cationic polystyrene (PS) nanospheres as the core template, 3-aminopropyltriethoxysilane (APTES) as an additive, and cetyltrimethyl ammonium bromide (CTAB) as the structure-directing surfactant. The shell thickness and the size of cavity could be easily adjusted by altering the amounts of TEOS and the size of PS nanospheres during synthesis process, respectively. Both PS and CTAB can be simultaneously removed via calcination at 550 °C for 6 h. Fig. 1a presents a typical transmission electron microscopy (TEM)