Low generation PAMAM dendrimer and CpG free plasmids allow targeted and extended transgene expression in tumors after systemic delivery

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Abstract

Polyplexes consisting of a standard CMV promoter driven luciferase plasmid condensed with PAMAM starburst dendrimers (generation 4 and 5) efficiently transfected tumor cells in vitro. Tail vein injection of PAMAM polyplexes into immune competent mice bearing subcutaneous, well vascularized murine neuroblastoma tumors (Neuro2A) led to predominant luciferase reporter gene expression in the tumor, and negligible transgene expression levels in other organs. Repeated PAMAM polyplex applications were well tolerated and prolonged transgene expression in the tumor. In vivo imaging studies using polyplexes fluorescently labeled with near infrared emitting semiconductor quantum dots (quantoplexes) revealed lung accumulation for both PAMAM and linear PEI (LPEI) based polyplexes, but only LPEI polyplexes induced high luciferase expression in lung, demonstrating that biodistribution and transgene expression of polyplexes does not necessarily correlate. With a luciferase plasmid devoid of immune modulatory CpG sequences and a combination of human CMV enhancer and human elongation factor 1 alpha promoter elements, Neuro2A tumor transgene expression after a single intravenous injection of generation 5 PAMAM polyplexes was observed for up to 1 week as measured by luciferase bioluminescence imaging. Utilizing a human xenograft model (HUH7) in immune compromised nude mice, a low level of luciferase activity in the tumor area was observed after systemic PAMAM polyplex application.

Introduction

PAMAM dendrimers are well known carriers for different drugs including nucleic acids delivering them efficiently into tumor cells [1]. Due to the synthesis procedure dendrimers have, in contrast to other polymers, a defined molecular weight. Their low toxicity in vitro and in vivo allowed their use as delivery agent for conventional anticancer drugs, but also antibodies and nucleic acids. Early studies by Szoka et al. showed that dendrimers above generation 4 (G4) can efficiently transfect different tumor cell lines in vitro [2]. Further improved transfection properties of PAMAM dendrimers were found when utilizing dendrimers partially degraded at the amide linkage [3]. The latter is already commercialized since several years (Superfect™ from Qiagen). The ability of PAMAM dendrimers to transfect tumor cells in vivo was already demonstrated earlier when delivering oligonucleotides with PAMAM dendrimers to ascitic tumors by intraperitoneal injection [4] or plasmid DNA by intratumoral injection [5]. Here we demonstrate the intrinsic tumor targeting properties of PAMAM dendrimers generation 4 and 5 when applying PAMAM/plasmid DNA polyplexes intravenously in two different subcutaneous tumor models in mice. The plasmid type strongly influences strength and duration of transgene expression in the tumor tissue.

Section snippets

Materials

PAMAM-EDA dendrimer G4 and G5 (MW 14.215 Da and 28.826 Da, respectively; 5 mg/ml in methanol) were purchased from Sigma-Aldrich (Madrid, Spain), PAMAM-BDA dendrimer G5 (10% w/v in methanol) from Dendritic Nanotechnologies (Mount Pleasant, MI, USA). Methanol was removed by evaporation under vacuum (speedvac) and the residue dissolved at a final concentration of 4 mg/ml in sterile HBG (HEPES buffered glucose; 20 mM HEPES pH 7.5, 5% glucose (w/v)). BPEI (Sigma) was dispersed in distilled water and the

Results and discussion

PAMAM G4 and G5 both lead to complete condensation of plasmid DNA and protect DNA from enzymatic degradation [[11], [14]]. When transfecting murine Neuro2A neuroblastoma cells in vitro in the presence of serum with a plasmid expressing luciferase under the control of a CMV promoter [8], both PAMAM-EDA dendrimers G4 and G5 were able to induce significant transgene expression levels 24 h after transfection in a similar range as branched PEI (25 kDa) (Supplemental Fig. S 1A); G5 PAMAM polyplexes

Acknowledgements

This work was supported by FUN (University of Navarra Foundation), Government of Navarra (Department of Education) and Bancaja Foundation, the EC funded project GIANT, DFG SFB486 and SPP1230, and the DFG Excellence Cluster “Nanosystems Initiative Munich” (NIM). Melinda Kiss is gratefully acknowledged for technical assistance.

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