Studies On Glioma Targeting Of RGD Modified Doxorubicin Loaded PEG-PAMAM Conjugates

This study is based on the combined use of pharmaceutics, macromolecular chemistry, pharmacology and molecular biology. According to our previous studies, Polyamidoamine (PAMAM) dendrimer was used as the scaffold which was first modified with Polyethylene glycol (PEG) to produce PEG-PAMAM conjugates. Doxorubicin (DOX) was then conjugated to PEG-PAMAM either by acid sensitive cis-aconityl linkage or acid insensitive succinic linkage to produce the final product of PEG-PAMAM-DOX conjugate. By controlling the feed ratio of starting materials, we further conjugated RGDyC to PEG-PAMAM-DOX by covalent modification to produce active targeting RGD-PEG-PAMAM-DOX conjugates with similar PEGylation degree and DOX loadding to PEG-PAMAM-DOX. Baced on the above studies, we prepared RGD-PEG-PAMAM-DOX, PEG-PAMAM-DOX solution and implants and subsquent, in vitro and in vivo evaluations were performed to study the effects of them on the C6 glioma.PEG-PAMAM and RGD-PEG-PAMAM conjugates were synthesized by covalent modification of G4 PAMAM denderimer With MeO-PEG-NHS (M.W. 5000), bifunctional MAL-PEG-NHS and cyclic RGDyC peptide (RGDyC).'H-NMR was used to confirm the conjugation between RGD/PEG and PAMAM dendrimer. The conjugated number of PEG molecules per PAMAM dendrimer was calculated to be 18.9 (PEG-PAMAM) and 19 (RGD-PEG-PAMAM) respectively. The conjugated number of RGD molecules per PAMAM dendrimer was 10.1 (RGD-PEG-PAMAM). 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 and RGD-PEG-PAMAM to obtain the final products of acid sensitive PPCD, RGD-PPCD and acid insensitive PPSD, RGD-PPSD conjugates. Acid hydrolysis method was used to determine DOX content of the four conjugates. The conjugated number of DOX molecules was controlled to be about 13. The particle size of the conjugates was about 17～20nm.Cellular level experimented were carried out using C6 cells as the model cell lines. The cytotoxicity of PEG-PAMAM conjugates decreased compared with PAMAM, while RGD-modified PEG-PAMAM showed higher cytotoxicity than PEG-PAMAM conjugates. PPCD and RGD-PPCD were more cytotoxic than PPSD and RGD-PPSD which might be due to the release of free DOX. IC50 of PPCD,RGD-PPCD,PPSD and RGD-PPSD were 1.38,1.25,4.51 and 3.34μmol/L respectively. As to cellular uptake, C6 cell line showed increased total uptake and internalization of RGD-PEG-PAMAM-DOX compared with PEG-PAMAM-DOX (P<0.05)An orthotopic murine model of C6 glioma was prepared and the tissue biodistribution and in vivo antitumor activity of PEG-PAMAM-DOX and RGD-PEG-PAMAM-DOX solution agent were evaluated in C6 tumor bearing mice by i.v. administration of the conjugates. The results demonstrated that DOX displayed significant longer blood retention time, with prolonged AUC, T1/2 and MRT, and reduced Vc (9.0～25.4% of the DOX) and CL (0.5～3.3% of the DOX) after conjugation with PEG-PAMAM and RGD-PEG-PAMAM conjugates. The blood retention time of RGD-modified conjugates was less than RGD-unmodified ones. Biodistribution profiles of DOX was also significantly changed. Brain tumor accumulation of conjugates was increased significantly. Total DOX AUC of PPCD,RGD-PPCD,PPSD and RGD-PPSD in tumor was increased by 5.6,8.0,16.5 and 21.2 times as compared with DOX solution. The total DOX deposition in heart had no significant difference between DOX conjugates and DOX. PPCD and RGD-PPCD showed obviously accumulation in liver and spleen. The accumulation of RGD-modified conjugates in the spleen of intracranial glioma animals was much higher than those unmodified ones. RGD-modified conjugates exhibited longer survival times compared with RGD-unmodified ones. The median time of RGD-PPCD,RGD-PPSD,PPCD and PPSD treatment groups were 50,26,16 and 23 days. RGD-PPCD exhibited the longest survival times among all treatment groups.The results of the tissue biodistribution and in vivo antitumor activity of PEG-PAMAM-DOX and RGD-PEG-PAMAM-DOX conjugates in an orthotopic murine model of C6 glioma by i.v. administration of the conjugates demonstrated that tumor accumulation of the conjugates was increased and RGD-PPCD was efficient targeting to the glioma. In the study, some organs of reticuloendothelial system (RES) drug significant accumulation was found, particularly liver and spleen. In order to decrease the normal tissue accumulation, we evaluated the distribution of DOX conjugates and DOX solution in an orthotopic murine model of C6 glioma by injection of PPCD and DOX solution through intracerebral route in this study. The results of the study demonstrated that drug concentration increased in the tumor and PPCD solution displayed shorter tumor retention time and less tumor accumulation compared with DOX. It is much easier for PPCD to be absorbed into the blood compared with DOX.In order to increase the tumor accumulation and prolong the tumor retention time of DOX conjugates, We further prepared DOX conjugates implants. We use solvent-volatilizing method to prepare sustained-release implants of PEG-PAMAM-DOX and RGD-PEG-PAMAM-DOX conjugates. Polylactide-co-glycolide copolymer (PLGA 75:25) and polylactide (PLA) (Mw 10000～25000) were chosen for carrier, and PEG4000 as regulators to prepare the DOX and DOX conjugate implants. After single factor studies, we choose a better prescription of the ratio of PLGA (75:25) to PLA being3:1, including 1%(W/W) PEG4000 and 5% (W/W) drug loading to prepare implants. The results of in vitro conjugates release study demonstrated that the release curves of all the four conjugates implants were similar to each other, and the percentage of accumulative drug release was about 50% in 45d and about more than 55% in 60d.A subcutaneous murine model of C6 glioma was prepared and the tissue biodistribution and in vivo antitumor activity of PEG-PAMAM-DOX and RGD-PEG-PAMAM-DOX implants were evaluated in C6 tumor bearing mice. The result of the tissue distribution demonstrated that Tumor accumulation of DOX conjugates and DOX implants increased significantly as compared with other normal tissue accumulation. Tumor accumulation and the tumor retention time of DOX conjugates implants increased compared with DOX implant. The antitumor activity of the DOX conjugates implants was tested in C6 glioma bearing mice by local treatment of the tumor. All the DOX formulations implants were effective in preventing tumor growth compared with the carrier. Acid-sensitive conjugates, PPCD and RGD-PPCD demonstrated more effecient antitumor activity than acid-insensitive conjugates (PPSD and RGD-PPSD) (Inhibitory rate of tumor of acid-sensitive conjugates 35 days after the implants implantation was more than 50%, acid-insensitive was about 30%). And the atitumor activity of RGD-modified conjugates implant increased compared with RGD-unmodified ones. RGD-PPCD demonstrated the greatest inhibitory rate of tumor (IRT%) 35 days after the implants implantation (IRT 65.2%), PPCD ranked the second among all the treatment groups with 50.1% inhibitory rate of tumor 35 days after the implants implantation.