Qianwen Yu

Multifunctional Tandem Peptide Modified Paclitaxel-Loaded Liposomes for the Treatment of Vasculogenic Mimicry and Cancer Stem Cells in Malignant Glioma

The chemotherapy of aggressive glioma is usually accompanied by a poor prognosis because of the formation of vasculogenic mimicry (VM) and brain cancer stem cells (BCSCs). VM provided a transporting pathway for nutrients and blood to the extravascular regions of the tumor, and BCSCs were always related to drug resistance and the relapse of glioma. Thus, it is important to evaluate the inhibition effect of antiglioma drug delivery systems on both VM and BCSCs. In this study, paclitaxel-loaded liposomes modified with a multifunctional tandem peptide R8-c(RGD) (R8-c(RGD)-Lip) were used for the treatment of glioma. An in vitro cellular uptake study proved the strongest targeting ability to be that of R8-c(RGD)-Lip to glioma stem cells. Drug loaded R8-c(RGD)-Lip exhibited an efficient antiproliferation effect on BCSCs and could induce the destruction of VM channels in vitro. The following pharmacodynamics study demonstrated that R8-c(RGD)-modified drug-loaded liposomes achieved both anti-VM and anti-BCSC effects in vivo. Finally, no significant cytotoxicity of the blood system or major organs of the drug-loaded liposomes was observed under treatment dosage in the safety evaluation. In conclusion, all of the results proved that R8-c(RGD)-Lip was a safe and efficient antiglioma drug delivery system.

Dual Receptor Recognizing Cell Penetrating Peptide for Selective Targeting, Efficient Intratumoral Diffusion and Synthesized Anti-Glioma Therapy.

Cell penetrating peptides (CPPs) were widely used for drug delivery to tumor. However, the nonselective in vivo penetration greatly limited the application of CPPs-mediated drug delivery systems. And the treatment of malignant tumors is usually followed by poor prognosis and relapse due to the existence of extravascular core regions of tumor. Thus it is important to endue selective targeting and stronger intratumoral diffusion abilities to CPPs. In this study, an RGD reverse sequence dGR was conjugated to a CPP octa-arginine to form a CendR (R/KXXR/K) motif contained tandem peptide R8-dGR (RRRRRRRRdGR) which could bind to both integrin αvβ3 and neuropilin-1 receptors. The dual receptor recognizing peptide R8-dGR displayed increased cellular uptake and efficient penetration ability into glioma spheroids in vitro. The following in vivo studies indicated the active targeting and intratumoral diffusion capabilities of R8-dGR modified liposomes. When paclitaxel was loaded in the liposomes, PTX-R8-dGR-Lip induced the strongest anti-proliferation effect on both tumor cells and cancer stem cells, and inhibited the formation of vasculogenic mimicry channels in vitro. Finally, the R8-dGR liposomal drug delivery system prolonged the medium survival time of intracranial C6 bearing mice by 2.1-fold compared to the untreated group, and achieved an exhaustive anti-glioma therapy including anti-tumor cells, anti-vasculogenic mimicry and anti-brain cancer stem cells. To sum up, all the results demonstrated that R8-dGR was an ideal dual receptor recognizing CPP with selective glioma targeting and efficient intratumoral diffusion, which could be further used to equip drug delivery system for effective glioma therapy.

Liposomes Combined an Integrin αvβ3-Specific Vector with pH-Responsible Cell-Penetrating Property for Highly Effective Antiglioma Therapy through the Blood–Brain Barrier

Glioma, one of the most common aggressive malignancies, has the highest mortality in the present world. Delivery of nanocarriers from the systemic circulation to the glioma sites would encounter multiple physiological and biological barriers, such as blood-brain barrier (BBB) and the poor penetration of nanocarriers into the tumor. To circumvent these hurdles, the paclitaxel-loaded liposomes were developed by conjugating with a TR peptide (PTX-TR-Lip), integrin αvβ3-specific vector with pH-responsible cell-penetrating property, for transporting drug across the BBB and then delivering into glioma. Surface plasmon resonance (SPR) studies confirmed the very high affinity of TR-Lip and integrin αvβ3. In vitro results showed that TR-Lip exhibited strong transport ability across BBB, killed glioma cells and brain cancer stem cells (CSCs), and destroyed the vasculogenic mimicry (VM) channels. In vivo results demonstrated that TR-Lip could better target glioma, and eliminated brain CSCs and the VM channels in tumor tissues. The median survival time of tumor-bearing mice after administering PTX-TR-Lip (45 days) was significantly longer than that after giving free PTX (25.5 days, p < 0.001) or other controls. In conclusion, PTX-TR-Lip would improve the therapeutic efficacy of brain glioma in vitro and in vivo.

Tandem Peptide Based on Structural Modification of Poly-Arginine for Enhancing Tumor Targeting Efficiency and Therapeutic Effect

The nonselectivity of cell penetrating peptides had greatly limited the application in systemic administration. By conjugating a dGR motif to the C-terminal of octa-arginine, the formed tandem peptide R8-dGR had been proved to specifically recognize both integrin αvβ3 and neuropilin-1 receptors. However, the positive charge of poly-arginine would still inevitably lead to rapid clearance in the circulation system. Therefore, in this study, we tried to reduce the positive charge of poly-arginine by decreasing the number of arginine, to thus achieve improved tumor targeting efficiency. We had designed several different Rx-dGR peptides (x = 4, 6, and 8) modified liposomes and investigated their tumor targeting and penetrating properties both in vitro and in vivo. Among all the liposomes, R6-dGR modified liposomes exhibited a long circulation time similar to that of PEGylated liposomes while they retained strong penetrating ability into both tumor cells and tumor tissues, and thus had displayed the most superior tumor targeting efficiency. Then, paclitaxel and indocyanine green coloaded liposomes were prepared, and R6-dGR modified coloaded liposomes also exhibited enhanced antitumor effect on C6 xenograft tumor bearing mice. Therefore, we suggest R6-dGR as a potential tumor targeting ligand with both strong penetrating ability and improved pharmacokinetic behavior, which could be further used for efficient antitumor therapy.

Integrin αvβ3 targeting activity study of different retro-inverso sequences of RGD and their potentiality in the designing of tumor targeting peptides

Retro-inverso peptide represented the isomer of a parent peptide in which the direction of the sequence was reversed and the chirality of each amino acid residue was inverted. Generally, retro-inverso peptides possessed equal or even higher activities compared to the original peptide. RGD was a commonly used ligand for tumor and vascular targeting due to its affinity to integrin αvβ3 receptors. The biological activity study of the isomers of RGD would indeed provide useful suggestions for the design of tumor targeting peptides. Therefore, the tumor targeting activities of octa-arginine which was modified with different retro-inverso sequences of RGD peptide were investigated in this study. Three different tandem peptides (R8-GDGR, R8-GdGr and R8-GdGR) were designed on the basis of R8-GRGD. The tumor targeting activities of these tandem peptides were evaluated both in vitro and in vivo. Finally, R8-GdGR displayed selective binding affinity to integrin αvβ3 at the cellular level, and exhibited efficient tumor homing and penetrating capabilities in vivo. Meanwhile, R8-GdGR also showed stronger neovessel targeting ability compared to the others. In conclusion, all the results demonstrated that dGR possessed similar biological activity to RGD and was a potential ligand for further designing of tumor targeting peptides.

Enhanced glioma therapy by synergistic inhibition of autophagy and tyrosine kinase activity

Autophagy is a lysosomal degradation pathway that acts as a cytoprotective mechanism causing treatment resistance in various cancer cells. Recent studies showed that hydroxychloroquine can inhibit the latter step of autophagy and therefore enhance the anti-glioma efficiency of ZD6474, a tyrosine kinase inhibitor. However, the nonselective distribution of ZD6474 in vivo and the low penetrating ability of hydroxychloroquine when crossing the blood-brain barrier restrict their clinical use in glioma therapy. Here we coencapsulated ZD6474 and hydroxychloroquine into R6dGR peptide-modified liposomes (R6dGR-Lip) which can specifically recognize both integrin αvβ3 and neuropilin-1 receptors that are highly expressed on the endothelial cells and glioma cells. R6dGR significantly enhanced the brain targeting and overcame the blood-brain barrier. Our results confirmed that loading hydroxychloroquine into R6dGR-Lip blocked autophagic flux more efficiently than free hydroxychloroquine in glioma cells and significantly sensitized glioma cells to ZD6474-induced cell death in vitro and in vivo. The coencapsulated R6dGR-modified liposomes (ZD6474/HCQ-R6dGR-Lip) prolonged the medium survival time of intracranial C6 glioma bearing mice by 1.2-fold compared with ZD6474-R6dGR-Lip, 1.5-fold compared with free ZD6474/HCQ, and 1.8-fold compared with free ZD6474, exhibiting a synergistic therapeutic effect. Therefore, ZD6474/HCQ-R6dGR-Lip is presented as a potential strategy which could be further used for efficient anti-glioma therapy.

Losartan loaded liposomes improve the antitumor efficacy of liposomal paclitaxel modified with pH sensitive peptides by inhibition of collagen in breast cancer

Abstract The dense collagen network in tumors restricts the penetration of drugs into tumors. Free losartan could inhibit collagen, but it would cause hypotension at the dosage of 10 mg/kg/d. In this study, losartan was encapsulated in liposomes (LST-Lip) and the collagen inhibition ability of LST-Lip was investigated. Our results showed the blood pressure was not affected by LST-Lip at the dosage of 2.5 mg/kg every other day. The amount of Evans Blue in tumor in LST-Lip group was 1.98 times of that in control group. Confocal laser scanning microscopy images showed that prior injection of LST-Lip could inhibit collagen and further improve the tumorous accumulation of liposomes modified with TH peptides (AGYLLGHINLHHLAHL(Aib)HHIL-NH2) (TH-Lip) in 4T1 tumors. Compared with control group, the tumor inhibition rate of combined strategy of LST-Lip and paclitaxel loaded TH-Lip (PTX-TH-Lip) was 41.73%, while that of group only treated with PTX-TH-Lip was 14.94%. Masson’s trichrome staining confirmed that collagen was inhibited in LST-Lip group. Thus, the administration of LST-Lip in advance could inhibit the collagen in tumors effectively and did not affect the blood pressure, then PTX-TH-Lip injected subsequently could exert enhanced antitumor efficacy. In conclusion, this combined strategy might be promising for breast cancer therapy.

Suppression for lung metastasis by depletion of collagen I and lysyl oxidase via losartan assisted with paclitaxel-loaded pH-sensitive liposomes in breast cancer

Abstract Tumor metastasis would seriously impair the efficacy of chemotherapy. Our previous studies showed losartan combined with paclitaxel-loaded pH-sensitive cleavable liposomes (PTX-Cl-Lip) facilitated paclitaxel accumulation and led to enhanced antitumor efficacy in 4T1 bearing mice. Since losartan could inhibit the level of collagen I which was related to tumor metastasis, this strategy was further applied to suppress tumor metastasis this time. Our in vivo anti-metastatic study manifested losartan could lower the colonies occupied in lungs by 76.4% compared with that of saline group. When losartan and PTX-Cl-Lip were combined, anti-metastatic efficiency reached to 88.2%, which was the best among all the groups. In vitro 3D tumor spheroids studies proved losartan could significantly suppress the invasion of tumor cells. Losartan plus PTX-Cl-Lip could further weaken the metastasis of tumor cells. Mechanism study showed the declination of collagen I level via losartan was caused by inhibition of active transforming growth factor-β1. Western-blot study showed losartan could decrease the level of lysyl oxidase, then inhibit the cross-linking of collagen I, finally weakened the cell signaling transmit via integrin and the metastasis of tumor cells was restrained. All above studies illustrated this combined tactic could achieve favorable effect on suppression of lung tumor metastasis.

pH-sensitive folic acid and dNP2 peptide dual-modified liposome for enhanced targeted chemotherapy of glioma

ABSTRACT Effective chemotherapy for clinical glioma treatment is still lacking due to the poor penetration of blood‐brain barrier (BBB) and the poor internalization into tumor cells. To facilitate the transmigration across the BBB as well as the glioma targeting of chemotherapeutics, we constructed cell penetrating peptide dNP2 and tumor microenvironment‐cleavable folic acid (FA) dual modified, paclitaxel (PTX) loaded liposome for the targeted delivery of glioma. The modification of dNP2 significantly enhanced the transmigration across the BBB in an in vitro BBB model. The acid‐cleavable cFd‐Lip/PTX exhibited sensitive cleavage of FA at pH6.8, which led to enhanced cellular uptake mediated by both cell penetrating peptide dNP2 and the interaction between FA and folate receptor (FR) on the glioma cells. After intravenous injection, compared with non‐cleavable Fd‐Lip and single modified liposomes, cFd‐Lip enhanced the accumulation in orthotropic glioma and improved the anti‐tumor effect of glioma‐bearing mice. The dual modified liposomes also facilitated deep penetration into tumor cells and consequently enhanced the cytotoxicity of PTX‐loaded liposomes. The acid‐cleavable dual modified strategy retained the BBB penetrating and tumor targeting ability, meanwhile, the cleavage of FA further maximized the cell permeability of dNP2, exhibiting enhanced tumor targeting effect. The multi‐targeting strategy provides a promising approach towards targeted chemotherapy for glioma.

Cell-penetrating peptides induce apoptosis and necrosis through specific mechanism and cause impairment of Na + –K + -ATPase and mitochondria

Cell-penetrating peptides (CPPs) are widely used in the development of various drug delivery systems because of their ability of penetrating plasma membrane. However, the safety of their application remains largely unknown. In this study, we found that the incubation of two main kinds of CPPs with human normal liver cells could cause the occurrence of apoptosis and necrosis, then the detailed apoptosis-related protein were detected out. To discover the specific way which leads to these results, several methods were used in this study. Several cytokines, such as Caspase3 and Bcl-2, were detected to prove that the damage happened after treated with different CPPs. Then shielding the positive charge of TAT and R8, depletion of Na+ in culturing medium and addition of several inhibitors of specific ATPase site were used to investigate whether the cytotoxicity were charge-dependent and ATPase-related. Furthermore, the membrane potential of mitochondria and the leakage of mitochondrial cytochrome c were detected after treated with CPPs to investigate the damage on mitochondria. In general, our results assess the cytotoxicity caused by two main kinds of CPPs and reveal the clear mechanism of how it occurs. This study reveals the essence of cytotoxicity caused by CPPs, and the methods we followed can be used to evaluate the biocompatibility of new-designed CPPs, which makes the application of CPPs better and safer.