Peptides-mediated Liver Tumor-targeted Diagnosis And Therapy Nano-scaled Delivery System

The incidence of maglinant tumors is gradually increasing, and liver tumor is one of the most common maglinant tumors in China. The incidence of liver tumor ranks the second place in all maglinant tumors. There will add300,000new patients every year in China. Liver tumor has the characteristic of higher malignancy, rapid development and short survival time. The mortality of liver tumor is high, only14.4%patients could live longer than5years.There are many problems in the diagnosis and therapy of liver tumor, which are very difficult to solve. In early stage of liver tumor, there is no evident symptom. So the liver tumor diagnosed is often in advanced and late stage because of the lack of special diagnostic contrast agents. But for advanced and late liver tumor, it is very hard to initiate the surgical treatment, and the postoperative recurrence rate is very high. Thus, for most patients, the treatment should be radiotherapy and chemotherapy. However, evident side effects and poor therapeutic effect exist in these treatments. There is no significant efficient therapeutic drug for now.Targeted nanotechnology has shown the special advantage and great clinical prospect in the diagnosis and therapy of tumor. The goal of this paper was to solve the above difficult problems by constructing peptides-mediated liver tumor-targeted diagnosis and therapy nano-scaled delivery system.The pathological changes of tumor tissues and cells different with normal tissues and cells due to the high metabolic activities and rapid growth of tumor cells,(1) the newly tumor vessels are incomplete, leading to the enhanced permeability (enhanced permeability and retention effect, EPR effect);(2) anaerobic metabolism, cell transformation and the enhanced ability for adapting the transmembrane pH leading to the formation of tumor extracellular acidic microenvironments;(3) the up-regulation of many nutrition-related receptor on the tumor cell surface due to the heavy demand for nutrition。 These different pathological features with normal tissues and cells provide the important basis for the design and application of targeted nanoscale drug delivery system。This paper designed three liver tumor-targeted strategy based on the above pathological features of tumor.The first strategy is the combination of EPR effect and tumor extracellular acidic microenvironments.pH (low) insertion peptide (pHLIP), was derived from the transmembrane helix C of bacteriorhodopsin. The putative transmembrane part of pHLIP peptide was sequenced AEQNPIY WARYADWLFTTPLLLLDLALLV DADEGT. At a slightly acidic pH (6-6.5), this peptide inserts into cell membrane to form a transmembrane helix. A pHLIP-mediated tumor targeting delivery vector (DGL-PEG-pHLIP) was synthesized by conjugating dendrigraft poly-L-lysines (DGL) to the terminus of pHLIP via bi-functional polyethylene glycol (PEG). The nanoscale drug delivery system formed by DGL-PEG-pHLIP loading drugs could target the tumor cells via the EPR effect and pHLIP-mediated pH (low) membrane insertion. The first chapater of this paper used DGL-PEG-pHLIP for earring small interfering RNA. Vascular Endothelial Growth Factor (VEGF) is an important factor for promoting the formation of tumor angiogenic blood vessels. The silencing of VEGF was very efficient in inhibiting the tumor growth. The pHLIP-modified nanoparticles (pHLIP-NP/shVEGF) were constructed based on the electrostatic interactions between gene (shVEGF) and pHLIP-modified DGL. pHLIP-NP/shVEGF showed excellent tumor targeting effect and strong VEGF silencing effect, which leading to the efficient the inhibtion of the formation of tumor angiogenic blood vessels. The accmulation of pHLIP-NP/shVEGF was3-fold higher than that of NP/shVEGF.The second strategy is the combination of EPR effect and receptor-mediated endocytosis.Transferrin (Tf) receptor (TfR) has been reported over-expressed on tumor cells, about100-fold more than that on normal cells, due to the urgent requirement of iron to maintain cellular survival. Binding of iron-loaded Tf to TfR triggers endocytosis and then the Tf-TfR complex enters cells to provide iron. The feasibility of Tf as a targeting ligand has been demonstrated. However, the concentration of endogenous Tf in blood (25μM) is very high, compared with the Kd of Tf binding to TfR. The endogenous Tf may competitively inhibit the binding of Tf-modified drug delivery systems to TfR; in addition, the relatively high molar weight of Tf, about80kDa, makes it difficult for the construction of drug delivery systems. The second chapater of this paper was to focus on the limitation in the application of Tf-modified tumor targeted drug delivery systems and evaluate the feasibility of two novel TfR-binding peptides as tumor targeting ligands. The tumor cellular uptake of peptide-modified polyamidoamine dendrimers (PAMAM) was evaluated. The uptake of PAMAM-PEG-T7was comparable to that of PAMAM-PEG-Tf. However, the Tf at endogenous concentration inhibited the uptake of PAMAM-PEG-Tf but enhanced the uptake of PAMAM-PEG-T7. This data proved that the sites on TfR for T7were different with that of Tf. T7could overcome the limitation in the application of Tf-modified tumor targeted drug delivery systems. Thus, T7was used as the tumor targeting ligand.The third chapater of this paper was to use PAMAM-PEG-T7for conjugating the chelating agent, diethylenetriaminepentaacetic acid (DTPA) and chelate Gd3+to finally obtain the peptide-conjugated nanoscale liver tumor-targeted T1magnetic resonance imaging contrast agent GdDTPA-PAMAM-PEG-T7。The liver tumor uptake of PAMAM-PEG-T7was significant higher than that of PAMAM-PEG. The relaxivity of the GdDTPA-PAMAM-PEG-T7was2.2-fold that of GdDTPA. The contrast efficiency for liver tumor by GdDTPA-PAMAM-PEG-T7was2.9-fold that by GdDTPA-PAMAM-PEG,4.1-fold that by GdDTPA. In addition, GdDTPA-PAMAM-PEG-T7could specially target liver tumor and prolong the imaging time.The interior region of dendrimers can be employed to encapsulate hydrophobic antitumor drugs. The nanometer structure and simple modification make PAMAM a kind of ideal vector for passive and active tumor-targeting studies. The fourth chapater of this paper was to use PAMAM-PEG-T7to encapsulate the antitumor drug doxorubicin (DOX) to form the peptide-conjugated tumor targeted DOX delivery system, PAMAM-PEG-T7/DOX. The size of this system was about10nm. Based on the pH-sensitivity of PAMAM [36], DOX was released at a faster rate in acidic conditions, while keeped stable in normal conditions. PAMAM-PEG-T7/DOX could significantly enhance the DOX uptake by tumor cells in the presence of endogenous Tf. The IC50for tumor cells by PAMAM-PEG-T7/DOX in the presence of Tf was3-fold that without Tf. The accmulation of PAMAM-PEG-T7/DOX in liver rumor was1.7-fold that of PAMAM-PEG/DOX, and5.3-fold that of free DOX, leading to the strongest antitumor effect.DOX could intercalate within the DNA strand to form complex. The complex of DNA and DOX (DNA-DOX) is very stable without any DOX leakage before leaving the bloodstream. Based on these findings, in the fourth chapater of this paper we tried using PAMAM-PEG-T7for loading the complex of genes and chemotherapeutic drugs to combinedly treat tumor. For cancer therapy, single medication could not obtain optimal efficacy because cancer cells often exhibit major resistance to one kind of chemotherapeutic drugs. The human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) could induce apoptosis in a wide range of malignant cells. The therapeutic gene used in this work was an expression vector containing TRAIL open reading frame (pORF-hTRAIL). In addition, TRAIL-mediated apoptosis of cancer cells can be enhanced by DOX. The fifth chapater of this paper ustilized PAMAM-PEG-T7as gene delivery vector for loading the complex of pORF-hTRAIL and DOX to form the pORF-hTRAIL and DOX combined tumor targeting delivery system, PAMAM-PEG-T7/pORF-hTRAIL-DOX. The goal of this chapater was to treat tumor at many targets based on different mechanisms. About375DOX molecules were bound to pORF-hTRAIL. The release rate of DOX from DNA-DOX complex was not influenced by condensation effect of PAMAM. PAMAM-PEG-T7/pORF-hTRAIL-DOX significantly concentrated in tumor, mediated the expression of pORF-hTRAIL and induced the apoptosis of tumor cells, compared with PAMAM-PEG/pORF-hTRAIL-DOX. In addition, the antitumor efficiency by PAMAM-PEG-T7//pORF-hTRAIL-DOX was even higher than that by clinical free DOX with dose of30-fold.The third strategy is the combination of EPR effect, tumor extracellular acidic environments and receptor-mediated endocytosis.Many ligand-modified delivery systems based on these receptors have been demonstrated to be very efficient for enhancing accumulation of anti-tumor drug in tumor tissues.TfR is also expressed in normal tissues including liver, lung, spleen, kidney and brain besides in tumor. It could be predicted that serious side effects in patients would be inevitable when T7-modified delivery systems used for cancer therapy.Comprehensively considered the pathological features of tumor, based on the combination of EPR effect, tumor extracellular acidic environments and receptor-mediated endocytosis, the sixth chapater of this paper designed tumor-acidity controlled active tumor targeting nanoscale delivery system. DTPA was utilized as the shield molecule to modify the targeting ligand T7because its hydrophilic nature and negative charge could prevent the contact of NPs with the cell membrane and the binding terminus of T7could be masked or exposed with the hydrazone linkage. dependent on extracellular environment pH. The up-regulation of cadherin (CDH17) is invovled with the tumor metastasis.The silencing of CDH17was very efficient in inhibiting the tumor growth and metastasis. This chapater utilized DTPA-modifed gene carrier for loading the therapeutic gene containing the interfering RNA and DOX as combined drugs to reduce or even avoid the uptake by healthy cells and to keep the tumor targeting delivery efficiency.