| Tumor is one of the major diseases that threatens the health of people, with high recurrence rate and high mortality. The standard treatment for tumors includes surgical resection, radiotherapy and chemotherapy, but therapeutic effect is not satisfactory. In addition to traditional chemotherapy drugs, targeted cancer therapy, including small molecule targeted drugs and nano-medicine, has been applied clinically in recent years. In contrast to conventional chemotherapy, targeted cancer therapy possess specific anticancer effects. However, there are some defects for the application of targeted therapies, such as the poor penetration ability into the tumor parenchyma. Recently, a tumor-targeting and tumor-penetrating cyclic peptide, internalizing RGD peptide (iRGD, CRGDK/RGPD/EC) was reported to increase drug penetration into extravascular tumor tissue, as compared with conventional RGD. In our previous study, PEGylated PAMAM dendrimer (G4) with DOX conjugated by acid-sensitive cis-aconityl linkage (PEG-PAMAM-cis-aconityl-DOX, PPCD) was modified by a RGD cyclopeptide (RGDyC). The RGD-PPCD conjugate could increase tumor targeting by binding with the integrin receptors and showed high anti-glioma efficacy in vitro and in vivo. In order to improve the tumor penetration ability of RGD-PPCD conjugate, a CRGDKGPDC cyclopeptide was used to instead of RGDyC to produce iRGD-PPCD in the present study. In vivo and in vitro studies were performed so as to compare the differences between the RGDs-mediated PPCD systems (including iRGD/RGD conjugated and co-administrated with PPCD). In addition, local administration is another effective method to increase drug accumulation in the tumor tissue. Our group have prepared DOX and DOX-polymer implants for long-term intratumoral release, this study will explore its behavior in vitro and in vivo.iRGD-PEG-PAMAM conjugate was synthesized by covalent modification of G4 PAMAM denderimer with bifunctional MAL-PEG-NHS and iRGD peptide. DOX was then converted to its cis-aconityl derivatives by ammonolysis reaction to produce CAD. CAD was subsequently activated and conjugated to iRGD-PEG-PAMAM to obtain the final products of iRGD-PPCD. The conjugates were characterized and the results showed that iRGD-PPCD contained 20.5 molecules of PEG,10.1 molecules of iRGD and 11.9 molecules of CAD in each PAMAM molecule. The particle size and zeta potential of iRGD-PPCD were 20.11±1.07 nm and 2.45±0.25 mV, respectively. The cumulative release percentage of iRGD-PPCD within 60 days in pH 4.5, pH 5.5, pH 6.5 and pH 7.4 was 58.74±3.76%、15.03±2.67%、6.88±1.51% and 2.64± 0.66%, respectively, indicating the characteristic of acid sensitive DOX release. In addition, the preparations of RGD-PPCD and PPCD were repeated using the previously reported method. The characterization results of the three conjugates were consistent with each other, which provides the rational comparative study of the follow-up.αv integrins and NPR-1 were expressed in different degree in four tumor cell lines, including C6, F98, U87 and B16. C6 cells displayed the highest expression of both av integrins and NPR-1, thus C6 was selected as cell model for the further study. The IC50 values of iRGD-PPCD,RGD-PPCD,PPCD were 0.96μM,1.21μM, and 2.40μM, respectively, indicating RGDs-modified conjugates significantly enhanced anti-tumor activity as compared to the unmodified ones. As compared to PPCD, the total cellular uptake of iRGD-PPCD and RGD-PPCD increased by 29% and 26%, the internalization increased by 67% and 59%, respectively. The results also showed that there was no significant difference between RGD-PPCD and iRGD-PPCD in cytotoxicity and cellular uptake.The co-incubation of PPCD with RGD or iRGD did not change in vitro activity of PPCD.Besides, the cellular uptake of RGD-PPCD and iRGD-PPCD were significantly inhibited by free RGD and iRGD,indicating that RGDs peptides could specifically bind to C6 cells by ligand-receptor interaction. In vitro avascular C6 glioma spheroid model was established to evaluate the penetration tumor ability of RGDs-mediated PPCD. the penetration ability of free DOX in the spheroid was the strongest among all treatment groups owing to its liposolubility and low molecular weight, the penetration tumor ability of PPCD and RGD+PPCD was poor. Confocal images showed that RGD-PPCD, iRGD-PPCD and iRGD+PPCD penetrated into C6 spheroids with a depth of 115.0 μm,144 μm and 150 μm. The fluorescence intensity of iRGD+PPCD and iRGD-PPCD at 190μm of spheroids was 1.54- and 1.38-fold higher than RGD-PPCD. The results confirmed that iRGD-mediated conjugates penetrated deeper and distributed more extensive in the spheroids than RGD-PPCD,The tumor xenograft-bearing mice were established and fibered confocal fluorescence microscopy (FCFM) was used to evaluate the tumor vascular morphology and permeability. Tumor vascular permeability study displayed that the vascular images displayed an obvious difference among seven groups. All conjugate groups showed a relatively higher vascular penetration than saline and DOX group. iRGD-PPCD and iRGD+ PPCD significantly increased vascular penetration, as compared to RGD-PPCD group. As to tumor vascular morphology study, the RGD-PPCD group displayed an obvious decrease in vascular density and average vascular diameter compared with the saline group, while the phenomena were not found for the DOX, PPCD and RGD+ PPCD groups. Furthermore, as compared to RGD-PPCD, iRGD+ PPCD, and iRGD-PPCD decreased the vascular density by 30.8% and 24.0%, reduced the vascular diameter by 29.1% and 21.3%, respectively. The results confirmed that iRGD-mediated PPCD significantly inhibited the tumor angiogenesis.An orthotopic murine model of C6 glioma was prepared and the tissue biodistribution and in vivo antitumor activity of DOX and DOX-polymer conjugates were evaluated in C6 tumor bearing mice. The DOX conjugates significantly increased The area under the concentration-time curve (AUC)and prolonged elimination half-life (T1/2P) and mean residence time (MRT). The AUC values of DOX in tumor for PPCD, RGD+ PPCD, RGD-PPCD, iRGD-PPCD and iRGD+ PPCD were 8.2-,8.8-,10.5-13.7-, and 14.7-fold higher than that of free drug, respectively. Moreover, the values of iRGD+ PPCD and iRGD-PPCD were 1.4-and 1.3-fold higher than that of RGD-PPCD, respectively. Glioma penetration displayed that the red fluorescence distribution of iRGD+ PPCD and iRGD-PPCD groups was wider and deeper from the tumor vessels, suggesting that iRGD-mediated PPCD possessed a significantly higher penetration and wider distribution ability than RGD-PPCD. The median survival time of iRGD+PPCD, iRGD-PPCD and RGD-PPCD treatment groups were 61,57.5 and 43.5 days. Median ILS (increase in life span) of iRGD+PPCD and iRGD-PPCD over RGD-PPCD were 40.2% and 32.2%, respectively. Compared to other groups, the number of tumor cells decreased, nucleus shrank and the area of endochylema increased with the treatment of iRGD+ PPCD and iRGD-PPCD.Based on our previous study, DOX and its conjugate implants were prepared by solvent-volatilizing method using biodegradable polymer PLGA/PLA/PEG as matrix materials. The behavior of the implants in vivo and in vitro was studied. The in vitro release study displayed that the total drug release was dependent on neither the loaded drugs (DOX or DOX conjugates) nor the pH of the release media in this study. All of the release curves in both pH media displayed consistent features which were fell into three phases, including a lag-period, the second phase which was consistent with zero-order model followed by a plateau. The cumulative release percentage reached an average of 63.4% in 60 days, indicating that the implants had obviously sustained release characteristics.the amount of free DOX in different release media was directly detected and the results confirmed that the DOX-polymer conjugates in implants kept stable and still possessed the property of acid-sensitive drug release. Then-fluorescent imaging was performed to visualize the drug in vivo distribution. It was found that the fluorescent area at d 7, d 30 and d 45 was 1.31-,1.69-and 2.26-fold higher than the initial value at d 0, respectively, indicating that drug did release from the implants and penetrated in tumor tissues gradually.To facilitate efficient anti-tumor drug delivery, a new drug delivery system, iRGD-mediated DOX-polymer conjugates, was developed in present study, the comparative study was performed between RGD and iRGD mediated PPCD. The studies confirmed that iRGD-mediated PPCD showed the obvious advantages over RGD ones.It was mainly reflected in:increasing tumor vascular permeability, inhibiting tumor vascular growth, enhancing intratumoral drug accumulation and consequently achieving the longer survival. In addition, the behavior and the mechanism of the implants for long-term intratumoral release was studied in vitro and in vivo. |
