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Phospholipid-modified PEI-based nanocarriers for in vivo siRNA therapeutics against multidrug-resistant tumors.

Essex S, Navarro G, Sabhachandani P, Chordia A, Trivedi M, Movassaghian S, Torchilin VP - Gene Ther. (2014)

Bottom Line: First, we studied the biodistribution of DOPE-PEI nanocarriers and the effect of PEG coating in a subcutaneous breast tumor model.Four hours postinjection, PEGylated carriers showed an 8% injected dose (ID) accumulation in solid tumor via the enhanced permeability and retention effect and 22% ID in serum due to a prolonged, PEG-mediated circulation.Second, we established the therapeutic efficacy and safety of DOPE-PEI/siRNA-mediated P-gp downregulation in combination with doxorubicin (Dox) chemotherapy in MCF-7/MDR xenografts.

View Article: PubMed Central - PubMed

Affiliation: Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, MA 02115, USA.

ABSTRACT
Multidrug resistance (MDR) mediated by P-glycoprotein overexpression in solid tumors is a major factor in the failure of many forms of chemotherapy. Here we evaluated phospholipid-modified, low-molecular-weight polyethylenimine (DOPE-PEI) nanocarriers for intravenous delivery of anti-P-pg siRNA to tumors with the final goal of modulating MDR in breast cancer. First, we studied the biodistribution of DOPE-PEI nanocarriers and the effect of PEG coating in a subcutaneous breast tumor model. Four hours postinjection, PEGylated carriers showed an 8% injected dose (ID) accumulation in solid tumor via the enhanced permeability and retention effect and 22% ID in serum due to a prolonged, PEG-mediated circulation. Second, we established the therapeutic efficacy and safety of DOPE-PEI/siRNA-mediated P-gp downregulation in combination with doxorubicin (Dox) chemotherapy in MCF-7/MDR xenografts. Weekly injection of siRNA nanopreparations and Dox for up to 5 weeks sensitized the tumors to otherwise non-effective doses of Dox and decreased the tumor volume by threefold vs controls. This therapeutic improvement in response to Dox was attributed to the significant, sequence-specific P-gp downregulation in excised tumors mediated by the DOPE-PEI formulations.

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General scheme of MDR phenomena (A) and anti-P-gp siRNA/drug combination treatment (B). It is well known that there is an active efflux of chemotherapeutic agents like Dox as a result of overexpression of P-gp (encoded by the MDR1 gene) on the tumor cell surface. This leads to a low intracellular drug accumulation (below minimum effective concentration or MEC) and hence, results in failure of chemotherapy. One of the solutions is treatment with therapeutic siRNA that targets the MDR1 gene and thus, brings about the down-regulation of the surface P-gp expression. With conventional chemotherapeutic treatment, drug concentration can reach the therapeutic index window, exert its cytotoxic effects and lead to a successful chemotherapeutic treatment.
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Figure 1: General scheme of MDR phenomena (A) and anti-P-gp siRNA/drug combination treatment (B). It is well known that there is an active efflux of chemotherapeutic agents like Dox as a result of overexpression of P-gp (encoded by the MDR1 gene) on the tumor cell surface. This leads to a low intracellular drug accumulation (below minimum effective concentration or MEC) and hence, results in failure of chemotherapy. One of the solutions is treatment with therapeutic siRNA that targets the MDR1 gene and thus, brings about the down-regulation of the surface P-gp expression. With conventional chemotherapeutic treatment, drug concentration can reach the therapeutic index window, exert its cytotoxic effects and lead to a successful chemotherapeutic treatment.

Mentions: P-gp-mediated MDR can be reversed using small molecules acting as P-gp substrates or inhibitors. However, their clinical use is associated with intolerable side-effects and toxicity due to off-target distribution and pharmacokinetic interactions with anticancer drugs 5-11. An alternative approach to inhibition of P-gp activity is to down-regulate its expression at the transcriptional level by using short double-stranded RNAs, so-called siRNA, that trigger the catalytic degradation of complementary mRNAs 12-14. Clear advantages of the siRNA approach compared to chemical inhibitors include its reduced toxicity towards non-specific tissues (since siRNA cannot diffuse passively through cellular membranes) and its high specificity. However, the targeted delivery of siRNA into tumor cells following a systemic application is difficult due to its poor in vivo stability (rapid degradation in plasma and cellular cytoplasm) and poor cellular uptake 15. The development of parenteral siRNA formulations that are stable in the circulation, promote tumor accumulation and that lack carrier-related toxicities is essential for the clinical use of siRNA-based drugs. One of the most investigated carriers is polyethylenimine (PEI), which functions as a transfection reagent based on its ability to compact siRNA into nano-scale complexes. This feature offers protection from harsh enzymatic degradation and improves both intracellular delivery of siRNA and trafficking uptake through endocytosis and escape from lysosomes 16, 17. The transfection efficiency/cytotoxicity profile of PEIs is largely influenced by their molecular weight. With the increase in their molecular weight, branched PEIs exhibit increased transfection that also correlates with increased non-specific toxicity due to excessive interactions with cell membranes by the cationic charges of PEI. Low molecular weight (LMW) PEIs have low toxicity and are hemocompatible but display poor transfection 18-20. We previously reported that chemical conjugation of DOPE phospholipid to low molecular weight PEI (1.8 kDa) significantly improved the intracellular siRNA delivery and the gene silencing of non-modified PEI while keeping cytotoxicity levels low 21, 22. The present study reports on the in vivo utility of these DOPE-PEI nanocarriers. In particular, we evaluated their tissue distribution upon intravenous injection in tumor-bearing mice and the effect of PEG coating as a strategy to improve their stability in circulation and enhance permeability and retention by the tumor 23-25. In a second step, we evaluated the anti-cancer activity of DOPE-PEI/anti-P-gp siRNA nanopreparations and Dox combinations in resistant breast cancer MCF-7/ADR xenografts. The scheme of the proposed experiment is depicted in Fig. 1. We used different dosing regimens, i.e. simultaneous vs. sequential administration of siRNA and Dox to optimize their anti-cancer activity. To the best of our knowledge, this is the first study that demonstrates the utility of LMW PEI (1.8 kDa) for i.v. siRNA delivery to tumors for the reversal of P-gp resistance to Dox and investigates the impact of different delivery modes on the therapeutic outcome of siRNA/drug combinations.


Phospholipid-modified PEI-based nanocarriers for in vivo siRNA therapeutics against multidrug-resistant tumors.

Essex S, Navarro G, Sabhachandani P, Chordia A, Trivedi M, Movassaghian S, Torchilin VP - Gene Ther. (2014)

General scheme of MDR phenomena (A) and anti-P-gp siRNA/drug combination treatment (B). It is well known that there is an active efflux of chemotherapeutic agents like Dox as a result of overexpression of P-gp (encoded by the MDR1 gene) on the tumor cell surface. This leads to a low intracellular drug accumulation (below minimum effective concentration or MEC) and hence, results in failure of chemotherapy. One of the solutions is treatment with therapeutic siRNA that targets the MDR1 gene and thus, brings about the down-regulation of the surface P-gp expression. With conventional chemotherapeutic treatment, drug concentration can reach the therapeutic index window, exert its cytotoxic effects and lead to a successful chemotherapeutic treatment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4352110&req=5

Figure 1: General scheme of MDR phenomena (A) and anti-P-gp siRNA/drug combination treatment (B). It is well known that there is an active efflux of chemotherapeutic agents like Dox as a result of overexpression of P-gp (encoded by the MDR1 gene) on the tumor cell surface. This leads to a low intracellular drug accumulation (below minimum effective concentration or MEC) and hence, results in failure of chemotherapy. One of the solutions is treatment with therapeutic siRNA that targets the MDR1 gene and thus, brings about the down-regulation of the surface P-gp expression. With conventional chemotherapeutic treatment, drug concentration can reach the therapeutic index window, exert its cytotoxic effects and lead to a successful chemotherapeutic treatment.
Mentions: P-gp-mediated MDR can be reversed using small molecules acting as P-gp substrates or inhibitors. However, their clinical use is associated with intolerable side-effects and toxicity due to off-target distribution and pharmacokinetic interactions with anticancer drugs 5-11. An alternative approach to inhibition of P-gp activity is to down-regulate its expression at the transcriptional level by using short double-stranded RNAs, so-called siRNA, that trigger the catalytic degradation of complementary mRNAs 12-14. Clear advantages of the siRNA approach compared to chemical inhibitors include its reduced toxicity towards non-specific tissues (since siRNA cannot diffuse passively through cellular membranes) and its high specificity. However, the targeted delivery of siRNA into tumor cells following a systemic application is difficult due to its poor in vivo stability (rapid degradation in plasma and cellular cytoplasm) and poor cellular uptake 15. The development of parenteral siRNA formulations that are stable in the circulation, promote tumor accumulation and that lack carrier-related toxicities is essential for the clinical use of siRNA-based drugs. One of the most investigated carriers is polyethylenimine (PEI), which functions as a transfection reagent based on its ability to compact siRNA into nano-scale complexes. This feature offers protection from harsh enzymatic degradation and improves both intracellular delivery of siRNA and trafficking uptake through endocytosis and escape from lysosomes 16, 17. The transfection efficiency/cytotoxicity profile of PEIs is largely influenced by their molecular weight. With the increase in their molecular weight, branched PEIs exhibit increased transfection that also correlates with increased non-specific toxicity due to excessive interactions with cell membranes by the cationic charges of PEI. Low molecular weight (LMW) PEIs have low toxicity and are hemocompatible but display poor transfection 18-20. We previously reported that chemical conjugation of DOPE phospholipid to low molecular weight PEI (1.8 kDa) significantly improved the intracellular siRNA delivery and the gene silencing of non-modified PEI while keeping cytotoxicity levels low 21, 22. The present study reports on the in vivo utility of these DOPE-PEI nanocarriers. In particular, we evaluated their tissue distribution upon intravenous injection in tumor-bearing mice and the effect of PEG coating as a strategy to improve their stability in circulation and enhance permeability and retention by the tumor 23-25. In a second step, we evaluated the anti-cancer activity of DOPE-PEI/anti-P-gp siRNA nanopreparations and Dox combinations in resistant breast cancer MCF-7/ADR xenografts. The scheme of the proposed experiment is depicted in Fig. 1. We used different dosing regimens, i.e. simultaneous vs. sequential administration of siRNA and Dox to optimize their anti-cancer activity. To the best of our knowledge, this is the first study that demonstrates the utility of LMW PEI (1.8 kDa) for i.v. siRNA delivery to tumors for the reversal of P-gp resistance to Dox and investigates the impact of different delivery modes on the therapeutic outcome of siRNA/drug combinations.

Bottom Line: First, we studied the biodistribution of DOPE-PEI nanocarriers and the effect of PEG coating in a subcutaneous breast tumor model.Four hours postinjection, PEGylated carriers showed an 8% injected dose (ID) accumulation in solid tumor via the enhanced permeability and retention effect and 22% ID in serum due to a prolonged, PEG-mediated circulation.Second, we established the therapeutic efficacy and safety of DOPE-PEI/siRNA-mediated P-gp downregulation in combination with doxorubicin (Dox) chemotherapy in MCF-7/MDR xenografts.

View Article: PubMed Central - PubMed

Affiliation: Center for Pharmaceutical Biotechnology and Nanomedicine, Northeastern University, Boston, MA 02115, USA.

ABSTRACT
Multidrug resistance (MDR) mediated by P-glycoprotein overexpression in solid tumors is a major factor in the failure of many forms of chemotherapy. Here we evaluated phospholipid-modified, low-molecular-weight polyethylenimine (DOPE-PEI) nanocarriers for intravenous delivery of anti-P-pg siRNA to tumors with the final goal of modulating MDR in breast cancer. First, we studied the biodistribution of DOPE-PEI nanocarriers and the effect of PEG coating in a subcutaneous breast tumor model. Four hours postinjection, PEGylated carriers showed an 8% injected dose (ID) accumulation in solid tumor via the enhanced permeability and retention effect and 22% ID in serum due to a prolonged, PEG-mediated circulation. Second, we established the therapeutic efficacy and safety of DOPE-PEI/siRNA-mediated P-gp downregulation in combination with doxorubicin (Dox) chemotherapy in MCF-7/MDR xenografts. Weekly injection of siRNA nanopreparations and Dox for up to 5 weeks sensitized the tumors to otherwise non-effective doses of Dox and decreased the tumor volume by threefold vs controls. This therapeutic improvement in response to Dox was attributed to the significant, sequence-specific P-gp downregulation in excised tumors mediated by the DOPE-PEI formulations.

Show MeSH
Related in: MedlinePlus