Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 23, 2015
Lipid nanoparticle (LNP) formulations facilitate tumor uptake and intracellular processing throug... more Lipid nanoparticle (LNP) formulations facilitate tumor uptake and intracellular processing through an enhanced permeation and retention effect (EPR), and currently multiple products are undergoing clinical evaluation. Clusterin (CLU) is a cytoprotective chaperone induced by androgen receptor (AR) pathway inhibition to facilitate adaptive survival pathway signalling and treatment resistance. In our study, we investigated the efficacy of siRNA tumor delivery using LNP systems in an enzalutamide-resistant (ENZ-R) castration-resistant prostate cancer (CRPC) model. Gene silencing of a luciferase reporter gene in the PC3-M-luc stable cell line was first assessed in subcutaneous and metastatic PC-3 xenograft tumors. Upon validation, the effect of LNP siRNA targeting CLU in combination with AR antisense oligonucleotides (ASO) was assessed in ENZ-R CRPC LNCaP in vitro and in vivo models. LNP LUC-siRNA silenced luciferase expression in PC-3M-luc subcutaneous xenograft and metastatic models. L...
The ability of a systemically administered gene therapy vector to exhibit extended circulation li... more The ability of a systemically administered gene therapy vector to exhibit extended circulation lifetimes, accumulate at a distal tumor site, and enable transgene expression is unique to SPLP. The flexibility and low toxicity of SPLP as a platform technology for systemic gene therapy allows for further optimization of tumor transfection properties following systemic administration. For example, the PEG coating of SPLP is necessary to engender the long circulation lifetimes required to achieve tumor delivery. However, PEG coatings have also been shown to inhibit cell association and uptake required for transfection. The dissociation rate of the PEG coating from SPLP can be modulated by varying the acyl chain length of the ceramide anchor, suggesting the possibility of developing PEG-Cer molecules that remain associated with SPLP long enough to promote tumor delivery, but which dissociate quickly enough to allow transfection. Alternatively, improvements may be expected from inclusion of cell-specific targeting ligands in SPLP to promote cell association and uptake. Finally, the nontoxic properties of SPLP allow the possibility of higher doses. A dose of 100 micrograms plasmid DNA per mouse corresponds to a dose of approximately 5 mg plasmid DNA per kg body weight. This compares well to small molecules used for cancer therapy, which typically are used at dose levels of 10 to 50 mg per kg body weight. In summary, SPLP consist of plasmid encapsulated in a lipid vesicle that, in contrast to naked plasmid or complexes, exhibit extended circulation lifetimes following intravenous injection, resulting in accumulation and transgene expression at a distal tumor site in a murine model. The pharmacokinetics, biodistribution, and tumor transfection properties of SPLP are highly sensitive to the nature of the ceramide anchor employed to attach the PEG to the SPLP surface. The SPLP-CerC20 system in which the PEG-Cer does not readily dissociate exhibits good serum stability, long circulation lifetimes, and high levels of tumor accumulation and mediates marker gene expression at the tumor site. The flexibility of the SPLP system offers the potential of further optimization to achieve therapeutically effective levels of gene transfer and clearly has considerable potential as a nontoxic systemic gene therapy vehicle with general applicability. These features of SPLP contrast favorably with previous plasmid encapsulation procedures. Plasmid DNA has been encapsulated by a variety of methods, including reverse phase evaporation, ether injection, detergent dialysis in the absence of PEG stabilization, lipid hydration and dehydration-rehydration techniques, and sonication, among others. The characteristics of these protocols are summarized in Table I. None of these procedures yields small, serum-stable particles at high plasmid concentrations and plasmid-to-lipid ratios in combination with high plasmid-encapsulation efficiencies. Trapping efficiencies comparable with the SPLP procedure can be achieved employing methods relying on sonication. However, sonication is a harsh technique that can shear nucleic acids. Size ranges of 100 mm diameter or less can be achieved by reverse-phase techniques; however, this requires an extrusion step through filters with 100 nm or smaller pore size which can often lead to significant loss of plasmid. Finally, it may be noted that the plasmid DNA-to-lipid ratios that can be achieved for SPLP are significantly higher than those achievable by any other encapsulation procedure.
The pH-dependent fusion properties of large unilamellar vesicles (LUVs) composed of binary mixtur... more The pH-dependent fusion properties of large unilamellar vesicles (LUVs) composed of binary mixtures of anionic and cationic lipids have been investigated. It is shown that stable LUVs can be prepared from the ionizable anionic lipid cholesteryl hemisuccinate (CHEMS) and the permanently charged cationic lipid N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC) at neutral pH values and that these LUVs undergo fusion as the pH
Lipid nanoparticles (LNPs) encapsulating short interfering RNAs that target hepatic genes are adv... more Lipid nanoparticles (LNPs) encapsulating short interfering RNAs that target hepatic genes are advancing through clinical trials, and early results indicate the excellent gene silencing observed in rodents and nonhuman primates also translates to humans. This success has motivated research to identify ways to further advance this delivery platform. Here, we characterize the polyethylene glycol lipid (PEG-lipid) components, which are required to control the self-assembly process during formation of lipid particles, but can negatively affect delivery to hepatocytes and hepatic gene silencing in vivo. The rate of transfer from LNPs to plasma lipoproteins in vivo is measured for three PEG-lipids with dialkyl chains 14, 16, and 18 carbons long. We show that 1.5 mol % PEG-lipid represents a threshold concentration at which the chain length exerts a minimal effect on hepatic gene silencing but can still modify LNPs pharmacokinetics and biodistribution. Increasing the concentration to 2.5 and 3.5 mol % substantially compromises hepatocyte gene knockdown for PEG-lipids with distearyl (C18) chains but has little impact for shorter dimyristyl (C14) chains. These data are discussed with respect to RNA delivery and the different rates at which the steric barrier disassociates from LNPs in vivo.Molecular Therapy-Nucleic Acids (2013) 2, e139; doi:10.1038/mtna.2013.66; published online 17 December 2013.
The discovery of RNA interference (RNAi) in mammalian cells has created a new class of therapeuti... more The discovery of RNA interference (RNAi) in mammalian cells has created a new class of therapeutics based on the reversible silencing of specific disease-causing genes. This therapeutic potential depends on the ability to deliver inducers of RNAi, such as short-interfering RNA (siRNA) and micro-RNA (miRNA), to cells of target tissues. This chapter reviews various challenges and delivery strategies for siRNA, with a particular focus on the development of lipid nanoparticle (LNP) delivery technologies. Currently, LNP delivery systems are the most advanced technology for systemic delivery of siRNA, with numerous formulations under various stages of clinical trials. We also discuss methods to improve gene silencing potency of LNP-siRNA, as well as application of LNP technologies beyond siRNA to the encapsulation of other nucleic acids such as mRNA and clustered regularly interspaced short palindromic repeats (CRISPR).
Doxorubicin is the best known and most widely used member of the anthracycline antibiotic group o... more Doxorubicin is the best known and most widely used member of the anthracycline antibiotic group of anticancer agents. It was first introduced in the 1970s, and since that time has become one of the most commonly used drugs for the treatment of both hematological and solid tumors. The therapy-limiting toxicity for this drug is cardiomyopathy, which may lead to congestive heart failure and death. Approximately 2% of patients who have received a cumulative (lifetime) doxorubicin dose of 450-500 mg?m(2) will experience this condition. An approach to ameliorating doxorubicin-related toxicity is to use drug carriers, which engender a change in the pharmacological distribution of the drug, resulting in reduced drug levels in the heart. Examples of these carrier systems include lipid-based (liposome) formulations that effect a beneficial change in doxorubicin biodistribution, with two formulations approved for clinical use. Drug approval was based, in part, on data suggesting that beneficial changes in doxorubicin occurred in the absence of decreased therapeutic activity. Preclinical (animal) and clinical (human) studies showing that liposomes can preferentially accumulate in tumors have provided a rationale for improved activity. Liposomes represent ideal drug delivery systems, as the microvasculature in tumors is typically discontinuous, having pore sizes (100-780 nm) large enough for liposomes to move from the blood compartment into the extravascular space surrounding the tumor cells. Liposomes, in the size range of 100-200 nm readily extravasate within the site of tumor growth to provide locally concentrated drug delivery, a primary role of liposomal formulation. Although other liposomal drugs have been prepared and characterized due to the potential for liposomes to improve antitumor potency of the encapsulated drug, the studies on liposomal doxorubicin have been developed primarily to address issues of acute and chronic toxicity that occur as a consequence of using this drug. It is important to recognize that research programs directed toward the development of liposomal doxorubicin occurred concurrently with synthetic chemistry programs attempting to introduce safer and more effective anthracycline analogues. Although many of these drugs are approved for use, and preliminary liposomal formulations of these analogues have been prepared, doxorubicin continues to be a mainstay of drug cocktails used in the management of most solid tumors. It will be of great interest to observe how the approved formulations of liposomal doxorubicin are integrated into combination regimes for treatment of cancer. In the meantime, we have learned a great deal about liposomes as drug carriers from over 20 years of research on different liposomal doxorubicin formulations, the very first of which were identified in the late 1970s. This chapter will discuss the various methods for encapsulation of doxorubicin into liposomes, as well as some of the important interactions between the formulation components of the drug and how this may impact the biological activity of the associated drug. This review of methodology, in turn, will highlight research activities that are being pursued to achieve better performance parameters for liposomal formulations of doxorubicin, as well as other anticancer agents being considered for use with lipid-based carriers.
A comparative study of the loading and retention properties of three structurally very closely re... more A comparative study of the loading and retention properties of three structurally very closely related vinca alkaloids (vincristine, vinorelbine and vinblastine) in liposomal formulations has been performed. All three vinca alkaloids showed high levels of encapsulation when accumulated into egg sphingomyelin/cholesterol vesicles in response to a transmembrane pH gradient generated by the use of the ionophore A23187 and encapsulated MgSO4. However, despite the close similarities of their structures the different vinca drugs exhibited very different release behavior, with vinblastine and vinorelbine being released faster than vincristine both in vitro and in vivo. The differences in loading and retention can be related to the lipophilicity of the drugs tested, where the more hydrophobic drugs are released more rapidly. It was also found that increasing the drug-to-lipid ratio significantly enhanced the retention of vinca alkaloids when the ionophore-based method was used for drug loading. In contrast, drug retention was not dependent on the initial drug-to-lipid ratio for vinca drugs loaded into liposomes containing an acidic citrate buffer. The differences in retention can be explained on the basis of differences in the physical state of the drug inside the liposomes. The drug-to-lipid ratio dependence of retention observed for liposomes loaded with the ionophore technique may provide a way to improve the retention characteristics of liposomal formulations of vinca drugs.
Clinical cancer research : an official journal of the American Association for Cancer Research, Jan 23, 2015
Lipid nanoparticle (LNP) formulations facilitate tumor uptake and intracellular processing throug... more Lipid nanoparticle (LNP) formulations facilitate tumor uptake and intracellular processing through an enhanced permeation and retention effect (EPR), and currently multiple products are undergoing clinical evaluation. Clusterin (CLU) is a cytoprotective chaperone induced by androgen receptor (AR) pathway inhibition to facilitate adaptive survival pathway signalling and treatment resistance. In our study, we investigated the efficacy of siRNA tumor delivery using LNP systems in an enzalutamide-resistant (ENZ-R) castration-resistant prostate cancer (CRPC) model. Gene silencing of a luciferase reporter gene in the PC3-M-luc stable cell line was first assessed in subcutaneous and metastatic PC-3 xenograft tumors. Upon validation, the effect of LNP siRNA targeting CLU in combination with AR antisense oligonucleotides (ASO) was assessed in ENZ-R CRPC LNCaP in vitro and in vivo models. LNP LUC-siRNA silenced luciferase expression in PC-3M-luc subcutaneous xenograft and metastatic models. L...
The ability of a systemically administered gene therapy vector to exhibit extended circulation li... more The ability of a systemically administered gene therapy vector to exhibit extended circulation lifetimes, accumulate at a distal tumor site, and enable transgene expression is unique to SPLP. The flexibility and low toxicity of SPLP as a platform technology for systemic gene therapy allows for further optimization of tumor transfection properties following systemic administration. For example, the PEG coating of SPLP is necessary to engender the long circulation lifetimes required to achieve tumor delivery. However, PEG coatings have also been shown to inhibit cell association and uptake required for transfection. The dissociation rate of the PEG coating from SPLP can be modulated by varying the acyl chain length of the ceramide anchor, suggesting the possibility of developing PEG-Cer molecules that remain associated with SPLP long enough to promote tumor delivery, but which dissociate quickly enough to allow transfection. Alternatively, improvements may be expected from inclusion of cell-specific targeting ligands in SPLP to promote cell association and uptake. Finally, the nontoxic properties of SPLP allow the possibility of higher doses. A dose of 100 micrograms plasmid DNA per mouse corresponds to a dose of approximately 5 mg plasmid DNA per kg body weight. This compares well to small molecules used for cancer therapy, which typically are used at dose levels of 10 to 50 mg per kg body weight. In summary, SPLP consist of plasmid encapsulated in a lipid vesicle that, in contrast to naked plasmid or complexes, exhibit extended circulation lifetimes following intravenous injection, resulting in accumulation and transgene expression at a distal tumor site in a murine model. The pharmacokinetics, biodistribution, and tumor transfection properties of SPLP are highly sensitive to the nature of the ceramide anchor employed to attach the PEG to the SPLP surface. The SPLP-CerC20 system in which the PEG-Cer does not readily dissociate exhibits good serum stability, long circulation lifetimes, and high levels of tumor accumulation and mediates marker gene expression at the tumor site. The flexibility of the SPLP system offers the potential of further optimization to achieve therapeutically effective levels of gene transfer and clearly has considerable potential as a nontoxic systemic gene therapy vehicle with general applicability. These features of SPLP contrast favorably with previous plasmid encapsulation procedures. Plasmid DNA has been encapsulated by a variety of methods, including reverse phase evaporation, ether injection, detergent dialysis in the absence of PEG stabilization, lipid hydration and dehydration-rehydration techniques, and sonication, among others. The characteristics of these protocols are summarized in Table I. None of these procedures yields small, serum-stable particles at high plasmid concentrations and plasmid-to-lipid ratios in combination with high plasmid-encapsulation efficiencies. Trapping efficiencies comparable with the SPLP procedure can be achieved employing methods relying on sonication. However, sonication is a harsh technique that can shear nucleic acids. Size ranges of 100 mm diameter or less can be achieved by reverse-phase techniques; however, this requires an extrusion step through filters with 100 nm or smaller pore size which can often lead to significant loss of plasmid. Finally, it may be noted that the plasmid DNA-to-lipid ratios that can be achieved for SPLP are significantly higher than those achievable by any other encapsulation procedure.
The pH-dependent fusion properties of large unilamellar vesicles (LUVs) composed of binary mixtur... more The pH-dependent fusion properties of large unilamellar vesicles (LUVs) composed of binary mixtures of anionic and cationic lipids have been investigated. It is shown that stable LUVs can be prepared from the ionizable anionic lipid cholesteryl hemisuccinate (CHEMS) and the permanently charged cationic lipid N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC) at neutral pH values and that these LUVs undergo fusion as the pH
Lipid nanoparticles (LNPs) encapsulating short interfering RNAs that target hepatic genes are adv... more Lipid nanoparticles (LNPs) encapsulating short interfering RNAs that target hepatic genes are advancing through clinical trials, and early results indicate the excellent gene silencing observed in rodents and nonhuman primates also translates to humans. This success has motivated research to identify ways to further advance this delivery platform. Here, we characterize the polyethylene glycol lipid (PEG-lipid) components, which are required to control the self-assembly process during formation of lipid particles, but can negatively affect delivery to hepatocytes and hepatic gene silencing in vivo. The rate of transfer from LNPs to plasma lipoproteins in vivo is measured for three PEG-lipids with dialkyl chains 14, 16, and 18 carbons long. We show that 1.5 mol % PEG-lipid represents a threshold concentration at which the chain length exerts a minimal effect on hepatic gene silencing but can still modify LNPs pharmacokinetics and biodistribution. Increasing the concentration to 2.5 and 3.5 mol % substantially compromises hepatocyte gene knockdown for PEG-lipids with distearyl (C18) chains but has little impact for shorter dimyristyl (C14) chains. These data are discussed with respect to RNA delivery and the different rates at which the steric barrier disassociates from LNPs in vivo.Molecular Therapy-Nucleic Acids (2013) 2, e139; doi:10.1038/mtna.2013.66; published online 17 December 2013.
The discovery of RNA interference (RNAi) in mammalian cells has created a new class of therapeuti... more The discovery of RNA interference (RNAi) in mammalian cells has created a new class of therapeutics based on the reversible silencing of specific disease-causing genes. This therapeutic potential depends on the ability to deliver inducers of RNAi, such as short-interfering RNA (siRNA) and micro-RNA (miRNA), to cells of target tissues. This chapter reviews various challenges and delivery strategies for siRNA, with a particular focus on the development of lipid nanoparticle (LNP) delivery technologies. Currently, LNP delivery systems are the most advanced technology for systemic delivery of siRNA, with numerous formulations under various stages of clinical trials. We also discuss methods to improve gene silencing potency of LNP-siRNA, as well as application of LNP technologies beyond siRNA to the encapsulation of other nucleic acids such as mRNA and clustered regularly interspaced short palindromic repeats (CRISPR).
Doxorubicin is the best known and most widely used member of the anthracycline antibiotic group o... more Doxorubicin is the best known and most widely used member of the anthracycline antibiotic group of anticancer agents. It was first introduced in the 1970s, and since that time has become one of the most commonly used drugs for the treatment of both hematological and solid tumors. The therapy-limiting toxicity for this drug is cardiomyopathy, which may lead to congestive heart failure and death. Approximately 2% of patients who have received a cumulative (lifetime) doxorubicin dose of 450-500 mg?m(2) will experience this condition. An approach to ameliorating doxorubicin-related toxicity is to use drug carriers, which engender a change in the pharmacological distribution of the drug, resulting in reduced drug levels in the heart. Examples of these carrier systems include lipid-based (liposome) formulations that effect a beneficial change in doxorubicin biodistribution, with two formulations approved for clinical use. Drug approval was based, in part, on data suggesting that beneficial changes in doxorubicin occurred in the absence of decreased therapeutic activity. Preclinical (animal) and clinical (human) studies showing that liposomes can preferentially accumulate in tumors have provided a rationale for improved activity. Liposomes represent ideal drug delivery systems, as the microvasculature in tumors is typically discontinuous, having pore sizes (100-780 nm) large enough for liposomes to move from the blood compartment into the extravascular space surrounding the tumor cells. Liposomes, in the size range of 100-200 nm readily extravasate within the site of tumor growth to provide locally concentrated drug delivery, a primary role of liposomal formulation. Although other liposomal drugs have been prepared and characterized due to the potential for liposomes to improve antitumor potency of the encapsulated drug, the studies on liposomal doxorubicin have been developed primarily to address issues of acute and chronic toxicity that occur as a consequence of using this drug. It is important to recognize that research programs directed toward the development of liposomal doxorubicin occurred concurrently with synthetic chemistry programs attempting to introduce safer and more effective anthracycline analogues. Although many of these drugs are approved for use, and preliminary liposomal formulations of these analogues have been prepared, doxorubicin continues to be a mainstay of drug cocktails used in the management of most solid tumors. It will be of great interest to observe how the approved formulations of liposomal doxorubicin are integrated into combination regimes for treatment of cancer. In the meantime, we have learned a great deal about liposomes as drug carriers from over 20 years of research on different liposomal doxorubicin formulations, the very first of which were identified in the late 1970s. This chapter will discuss the various methods for encapsulation of doxorubicin into liposomes, as well as some of the important interactions between the formulation components of the drug and how this may impact the biological activity of the associated drug. This review of methodology, in turn, will highlight research activities that are being pursued to achieve better performance parameters for liposomal formulations of doxorubicin, as well as other anticancer agents being considered for use with lipid-based carriers.
A comparative study of the loading and retention properties of three structurally very closely re... more A comparative study of the loading and retention properties of three structurally very closely related vinca alkaloids (vincristine, vinorelbine and vinblastine) in liposomal formulations has been performed. All three vinca alkaloids showed high levels of encapsulation when accumulated into egg sphingomyelin/cholesterol vesicles in response to a transmembrane pH gradient generated by the use of the ionophore A23187 and encapsulated MgSO4. However, despite the close similarities of their structures the different vinca drugs exhibited very different release behavior, with vinblastine and vinorelbine being released faster than vincristine both in vitro and in vivo. The differences in loading and retention can be related to the lipophilicity of the drugs tested, where the more hydrophobic drugs are released more rapidly. It was also found that increasing the drug-to-lipid ratio significantly enhanced the retention of vinca alkaloids when the ionophore-based method was used for drug loading. In contrast, drug retention was not dependent on the initial drug-to-lipid ratio for vinca drugs loaded into liposomes containing an acidic citrate buffer. The differences in retention can be explained on the basis of differences in the physical state of the drug inside the liposomes. The drug-to-lipid ratio dependence of retention observed for liposomes loaded with the ionophore technique may provide a way to improve the retention characteristics of liposomal formulations of vinca drugs.
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Papers by Pieter Cullis