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Studies on Tumor Targeting of RGD Modified Doxorubicin Loaded PEG-PAMAM Conjugates

Author: ZhuSaiJie
Tutor: PeiYuanYing;JiangZuoZuo
School: Fudan University
Course: Pharmacy
Keywords: RGD peptides polyethylene glycol polyamidoamine dendrimer doxorubicin cis-acotonic anhydride acid sensitive tumor targeting
CLC: R944
Type: PhD thesis
Year: 2010
Downloads: 822
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Abstract


This study is based on the combined use of pharmaceutics, macromolecular chemistry, pharmacology and molecular biology. For the construction of this polymeric drug delivery system, Polyamidoamine (PAMAM) dendrimer was used as the scaffold which was first modified with Polyethylene glycol (PEG) to produce PEG-PAMAM conjugates with different PEGylation degree. Doxorubicin (DOX) was then conjugated to each PEG-PAMAM either by acid sensitive cis-aconityl linkage or acid insensitive succinic linkage to produce the final product of PPCD and PPSD conjugates, respectively. By evaluating the in vitro and in vivo antitumor activity, PPCD 32/1 which had the highest PEGylation and acid sensitive linkage was found to be the most potent product. To further improve the antitumor activity of PPCD 32/1, RGD peptide was used to modify this polymeric drug delviery system via bifunctional MAL-PEG-NHS. By controlling the feed ratio of starting materials, RGD-PPCD was synthesized with similar PEGylation degree and drug laoding with PPCD 32/1. Subsquent evaluation was performed to study the effects of RGD modification on the antitumor activity of PPCD 32/1 and the underlying mechanism.The first chapter focused on the synthesis and characterization of PEG-PAMAM-DOX conjugates. With MeO-PEG-NH2 (M.W.4834) and G4 PAMAM denderimer as the starting materials, PEG-PAMAM conjugates with three different PEGylation degree were prepared, namely PEG-PAMAM 4/1,16/1 and 32/1. The conjugated number of PEG molecules per PAMAM dendrimer was calculated to be 3.8,12.9 and 20.1 respectively. TLC, UV,’H-NMR, FTIR, GPC were used to confirm the conjugation between PEG and PAMAM dendrimer. DOX was then converted to its cis-aconityl and succinic derivatives by ammonolysis reaction to produce CAD and SAD respectively. The two derivatives were subsequently activated and conjugated to PEG-PAMAM with different PEGylation degree to obtain the final products of PPCD and PPSD conjugates. The conjugated number of DOX molecule was controlled to be about 14, DOX loading percentage was in the range of 6.18-18.18% by weight, and the content of free CAD/SAD was less than 0.6% by mole. The particle of PEG-PAMAM 4/1 increased significantly from -7nm to -80 nm after conjugation of CAD/SAD, which might be due to the aggregation of these particles, while PEG-PAMAM 16/1 and 32/1 displayed slight increase in particle size after DOX conjugation, with 16/1 larger than 32/1. The Zeta potential of PEG-PAMAM-DOX decreased with increasing PEGylation degree, and PPSD showed higher Zeta potential than PPCD conjugates at the same PEGylation degree.The second chapter was about the in vitro and in vivo evaluation of PEG-PAMAM-DOX conjugates. In the first section, we first evaluated the in vitro release of DOX from PPCD and PPSD conjugates. PPCD was characteristic of acid sensitive DOX release, with DOX release increasing with increasing PEGylation and decreasing pH value, while PPSD was acid insensitive with no DOX release at any pH condition. Cathespin B, a major protease in lysosomes, had no effect on drug release of PEG-PAMAM-DOX, suggesting the stability of amide bond between PAMAM and CAD/SAD. When evaluating the heamolytic toxicity of these conjugates, we found significantly reduced heamolytic toxicity after conjugation of PEG and CAD/SAD as compared with G4 PAMAM, which ensured their further i.v. administration. Cellular level experimented were carried out using SKOV-3 and B16 cells as the model cell lines. The cytotoxicity of PEG-PAMAM conjugates decreased with increasing PEGylation, while PPCD conjugates showed the opposite trend, which might be due to the release of free DOX. For the same reason, PPCD were more cytotoxic than PPSD. As to cellular uptake, both cell lines showed decreased uptake of PEG-PAMAM-DOX with increasing PEGylation degree. Further investigation on the internalization mechanism by SKOV-3 cells indicated that PEG-PAMAM-DOX mainly interacted with plasma membrane by electrostatic interaction, and then was internalized by clathrin-mediated endocytosis. The results of intracellular distribution by confocal laser microscopy showed that the slightly acid condition in lysosomes triggered DOX release from PPCD 32/1, and enable the following entry into nucleus, while PPSD 32/1 release no free DOX although it was also located in lysosomes.In the second section, we evaluated the biodistribution and in vivo antitumor activity of these PEG-PAMAM-DOX conjugates. In vivo fluorescence imaging was first used to evaluate the tumor accumulation in SKOV-3 tumor bearing nude mice. The results demonstrated that tumor accumulation increased with increasing PEGylation and PPSD conjugates showed higher tumor accumulation than PPCD conjugates. FLD-HPLC method determining the total and free DOX was established to evaluate the biodistribution of these conjugates in B16 tumor bearing mice. After conjugation with PEG-PAMAM conjugates, DOX displayed significant longer blood retention time, with prolonged AUC, T1/2βand MRT, and reduced Vc and CL. Biodistribution profiles of DOX was also significantly changed. Total DOX AUC of PPSD 4/1, PPSD 16/1, PPSD 32/1, PPCD 4/1, PPCD 16/1 and PPCD 32/1 in tumor was increased by 16.6、51.4、75.8、11.6、21.9 and 37.5 times as compared with DOX solution. Significant RES uptake was observed at lower PEGylation degree, which could be improved at higher PEGylation degree. As to free DOX concentration, AUCs in normal tissues were smaller than that of DOX solution. PPCD conjugates displayed more tumor accumulation of free DOX than PPSD conjugates, with the AUCs of PPCD 32/1 and 16/1 even larger than DOX solution, reaching 35.0 and 15.5 h-μg/g, respectively.In the third chapter, we were intended to modify PPCD 32/1, the most powerful PEG-PAMAM-DOX conjugate, with RGD peptides to prepare active targeting drug delivery system. We first evaluated the receptor binding affinity of the newly designed cyclic RGDyC peptide by molecular simulation. With the obtained positive results, we further conjugated RGDyC to PAMAM dendrimer by bifunctional MAL-PEG-NHS, followed by the coupling of CAD. The final product of RGD-PPCD contained 21.2 molecules of PEG,16.8 molecules of RGDyC and 14.2 molecules of CAD in each PAMAM molecule. The particle size and Zeta potential of RGD-PPCD were 17.02±0.13 nm and 2.31±0.15 mV, respectively. In conclusion, RGD-PPCD had similar PEGylation degree, drug loading, particle size and Zeta potential with PPCD, thus could be used to evaluate the effects of RGD modification on the in vitro and in vivo antitumor activities of PPCD conjugate.In vitro and in vivo evaluation of RGD-PPCD was carried out in the fourth chapter. In the first section, it was demonstrated that RGD modification had no influence on the acid sensitive release of DOX from PPCD conjugates. Besides, RGD-PPCD displayed no heamolytic toxicity which permitted its further i.v. administration. Human Umbilical Vein Endothelial Cells (HUVEC) was included into the cellular level experiments as the endothelial cell model of tumor neovasculature. Cytotoxicity of RGD-PPCD against HUVEC, SKOV-3 and B16 cells was enhanced as compared with PPCD. RGD-PPCD also displayed significant higher cellular uptake than PPCD (P<0.01), with 1.57-fold increase in total uptake by HUVEC cells, 1.32-and 1.58-fold increase in total uptake and internalization respectively by SKOV-3 cells, and 1.38- and 1.85-fold increase in total uptake and internalization respectively by B16 cells. Internalization mechanism studies revealed that RGD-PPCD interacted with plasma membrane both by non-specific electrostatic interaction and specific recognition between integrinαvβ3 and RGD peptides, and subsequently be internalized mainly by clathrin-mediated endocytosis, which was related with the cellular movement involving actin filaments and microtubule cytoskeleton, and also with complex of integrinαvβ3 and RGD peptides. All the three cell lines were able to internalize RGD-PPCD and deliver it to the lysosomes where the acidic condition permitted the release of free DOX and the entry into nucleus.In the second section, we first evaluated the tissue distribution of RGD-PPCD in SKOV-3 tumor bearing nude mice by in vivo fluorescence imaging. Fluorescent intensity of RGD-PPCD in tumor site was 1.52 times higher than that of PPCD 48 h post injection (P<0.05), which could be significatnly reduced but not fully inhibited by free RGDyK. Pharmacokinetics in B16 tumor bearing mice demonstrated that modification of RGD peptides led to reduced t1/2β, AUC and MRT, and increased Vc and CL. RGD-PPCD displayed 1.46- (by total DOX) and 2.36-(by free DOX) fold higher AUC in tumor site than PPCD. On the other hand, RGD-PPCD not only induced apoptosis of tumor cell, but also remarkably induced apoptosis of tumor neovasculature endothelial cell. Accordingly, RGD-PPCD demonstrated higher antitumor activity than PPCD, with greater MST, Median and ILS. The results of HE staining and TUNEL revealed that RGD-PPCD exhibited almost no toxicity to normal organs, which was a great improvement in safeness compared with DOX solution.

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