Dual Receptor Targeting Cell Penetrating Peptide Modified Liposome for Glioma and Breast Cancer Postoperative Recurrence Therapy.

PURPOSE: Cell penetrating peptides (CPPs) were widely used as motifs for drug delivery to tumor. In former study, an RGD reverse sequence dGR was used to develop active-targeting liposome R8dGR-Lip, which showed well penetrating ability and treatment efficiency on glioma model. However, recurrence after tumor resection caused by post-operative residual cancer cells was a huge obstacle in tumor treatment. In consideration of the effective anti-cancer effect of PTX-R8dGR-Lip when treating glioma in former study, we decide to evaluate its pharmacodynamics on tumor resection models, which were more invasive and resistant. METHOD: In vitro, the effectiveness of PTX-R8dGR-Lip in reducing tumor initiating cell (TIC) was investigated using mammosphere formation. In vivo, the inhibition efficiency of PTX-R8dGR-Lip on C6 glioma recurrence and 4 T1 breast cancer recurrence model were evaluated, including tumor bioluminescence imaging, survival rate and immumohistochemical staining, etc.. RESULTS: C6 mammosphere formation rate of PTX-R8dGR-Lip group was 48.06 ± 2.72%, and 4 T1 mammosphere formation rate of PTX-R8dGR-Lip group was 39.51 ± 4.02% when PBS group was set as 100%. C6 and 4 T1 bioluminescent tumor resected model were established, then effectiveness of different PTX-loaded preparations were evaluated on these two models. PTX-R8dGR-Lip could obviously inhibit tumor recurrence, prolong survival rate and reduce tumor tissue invasion. CONCLUSION: PTX-R8dGR-Lip could reduce post-operative recurrence rate, prolong survival time, and decrease the proliferation of residual cancer cells through regulating the expression of recurrence-related cytokines.

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

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.

A "dual-guide" bioinspired drug delivery strategy of a macrophage-based carrier against postoperative triple-negative breast cancer recurrence.

Recurrence after tumor resection is mainly caused by post-operative inflammation and residual cancer cells, which is a serious obstacle to breast cancer treatment. Traditional nanoparticles rely primarily on the enhanced permeability and retention (EPR) effect in well-vascularized tumors. In this study, a macrophage-based carrier is designed to enhance the efficiency of targeting to recurrent tumors through a "dual-guide" strategy. After tumor resection, a burst of inflammatory factors occurs in the resection wound, which can recruit monocytes/macrophages rapidly. Combined with the tropism of monocyte chemoattractant protein, a large number of macrophage-mediated carriers will be recruited to surgical recurrence sites. Octaarginine (RRRRRRRR, R8)-modified liposomes in macrophages contain two agents with different pharmacological mechanisms, paclitaxel (PTX) and resveratrol (Res), which have enhanced therapeutic effects. In vitro study demonstrated that macrophage-mediated carriers approach 4 T1 cells through an inflammatory gradient and reach recurrence tumors through a "dual-guide" strategy. Then, membrane fusion and inflammation-triggered release deliver the drug into the recurrent tumor cells. In vivo experiments show that macrophage-based carriers exhibit effective tumor-targeting ability, especially in post-operation situations. More importantly, macrophage-mediated liposomes encapsulated with PTX and Res inhibit tumor recurrence in both ectopic and orthotopic 4 T1 post-operative recurrence models.

Facile strategy by hyaluronic acid functional carbon dot-doxorubicin nanoparticles for CD44 targeted drug delivery and enhanced breast cancer therapy.

A facile approach was developed to synthesize an innovative hyaluronic acid-modified carbon dot-doxorubicin nanoparticles drug delivery platform. CD44 targeted HA-modified carbon dots (HA-CDs) were synthesized as carrier by one-step hydrothermal treatment within one hour with citric acid and branch-PEI as core carbon source. HA not only functioned as carbon dot component but also as hydrophilic group and targeting ligand of this system. The as-prepared HA-CDs were then loaded with doxorubicin (HA-CD@p-CBA-DOX) via an acid-cleavable bond, which released drug in a pH-responsive manner. In in vitro experiments, HA-CD@p-CBA-DOX displayed good hemocompatibility and serum stability, while exhibited high cytotoxicity on 4T1 cells. The confocal laser scanning microscopy and flow cytometry results demonstrated that DOX-loaded nanoparticles were internalized by 4T1 cells via HA-mediated CD44-targeting effect. The enhanced in vivo tumor accumulation of HA-CD@p-CBA-DOX was testified by live imaging. Compared with free DOX, superior in vivo anti-tumor efficacy of HA-CD@p-CBA-DOX was observed in both heterotopic and orthotopic 4T1 cell tumor models. Furthermore, blood hematology and blood biochemistry analysis demonstrated that HA-CD@p-CBA-DOX did not induce noticeable toxicity, which further confirmed the good biocompatibility of HA-CD@p-CBA-DOX. The formulated HA-CD@p-CBA-DOX provides an alternative strategy for targeted breast cancer therapy.

Synergistic cytotoxicity and co-autophagy inhibition in pancreatic tumor cells and cancer-associated fibroblasts by dual functional peptide-modified liposomes.

Pancreatic ductal adenocarcinoma (PDA) is a highly fatal disease with 5-year survival of ∼8.5%. Nanoplatforms such as nab-paclitaxel and nanoliposomal irinotecan demonstrate superiority and utility in treating different progressions of PDA by prolonging the median overall survival by only a few months. Due to the dense surrounding stroma and the high autophagy in pancreatic cancer, integrin ɑvß3 targeting, acid environmental sensitive, TR peptide-modified liposomal platforms loaded with combined autophagy inhibiting hydroxychloroquine (HCQ), and cytotoxic paclitaxel (PTX) were designed (TR-PTX/HCQ-Lip) to accomplish the aim of synergistically killing tumor cells while inhibiting stroma fibrosis. The results showed that TR peptide-modified liposomes (TR-Lip) have superior targeting and penetrating effects both in vitro and in vivo. TR-PTX/HCQ-Lip efficiently inhibited autophagy in pancreatic cells and surrounding cancer-associated fibroblasts. The synergistic anti-fibrosis roles were also confirmed both in vitro and in vivo, all of which contributes to the enhanced curative effects of TR-PTX/HCQ-Lip in both heterogenetic and orthotopic pancreatic cancer models. STATEMENT OF SIGNIFICANCE: Autophagy plays a significant role in pancreatic ductal adenocarcinoma, especially in activating cancer associated fibroblasts which is also related to collagen generation that promotes the formation of dense stroma, which hinder the cytotoxic drugs to target and kill cancer cells. In this study, we designed integrin ɑvß3 targeting, acid environmental sensitive liposomal platforms to co-loaded paclitaxel and the autophagy inhibitor hydroxychloroquine. The results showed that the muti-functional liposomes can target to the pancreatic tumor and efficiently kill tumor cells and inhibit stroma fibrosis, thus improve the therapeutic effect in orthotopic pancreatic cancer models.

A stabilized retro-inverso peptide ligand of transferrin receptor for enhanced liposome-based hepatocellular carcinoma-targeted drug delivery.

The application of tumor targeting ligands to the treatment of cancer holds promise for improving efficacy and reducing toxicity. LT7 (L(HAIYPRH)) peptide, a phage display-selected peptide, exhibited high binding affinity to transferrin receptor (TfR) overexpressed on tumor cells. However, its in vivo tumor targeting efficiency was impaired due to enzymatic degradation in blood circulation. To improve the stability and targeting ability, a retro-inverso analogue of LT7 peptide, named DT7 peptide (D(HRPYIAH)), was designed for targeted therapy of hepatocellular carcinoma. The result of computer simulation predicted that DT7 bound to TfR protein more efficiently than LT7, and this prediction was confirmed experimentally by surface plasmon resonance (SPR). Ex vivo stability experiment demonstrated that DT7 possessed stronger ability against proteolysis than LT7 in fresh mouse serum. We further prepared DT7-, LT7-, and transferrin (Tf)-modified liposomes (DT7-LIP, LT7-LIP, and Tf-LIP, respectively). DT7-LIP showed a significantly stronger in vitro targeting ability than LT7-LIP and Tf-LIP under normal condition and simulated biological condition. In addition, the in vitro antitumor effect of DTX-loaded DT7-LIP was markedly enhanced in comparison to DTX-loaded LT7-LIP and DTX-loaded Tf-LIP. In vivo imaging indicated that DT7-LIP had better tumor accumulation than LT7-LIP and Tf-LIP. For in vivo antitumor studies, the tumor growth rate of mice treated with DTX-loaded DT7-LIP was significantly inhibited compared to that in mice treated with DTX-loaded LT7-LIP and DTX-loaded Tf-LIP. Overall, this study verified the potential of the stable DT7 peptide in improving the efficacy of docetaxel in the treatment of hepatocellular carcinoma. STATEMENT OF SIGNIFICANCE: A phage display library-selected LT7 (L(HAIYPRH)) peptide exhibited high affinity to transferrin receptor (TfR). However, its bioactivity was impaired in vivo as L-peptides are susceptible to degradation by proteolytic enzymes. Here, we designed a retro-inverso peptide DT7(D(HRPYIAH)) and demonstrated its increased serum stability and higher binding affinity to TfR. A stabilized targeted drug delivery system was further constructed by modified DT7 peptide on the surface of liposomes. The data indicated that DT7 peptide-modified liposomes exhibited higher targeting ability in vitro and in vivo. More importantly, DT7-modified liposomes demonstrated positive preclinical significance in enhancing the therapeutic effects against hepatocellular carcinoma.

Chemotherapy priming of the Pancreatic Tumor Microenvironment Promotes Delivery and Anti-Metastasis Efficacy of Intravenous Low-Molecular-Weight Heparin-Coated Lipid-siRNA Complex.

Pancreatic ductal adenocarcinoma (PDAC) is a type of malignant tumor with high lethality. Its high tumor cell-density and large variety of extracellular matrix (ECM) components present major barriers for drug delivery. Methods: Paclitaxel-loaded PEGylated liposomes (PTX-Lip) were used as a tumor-priming agent to induce tumor cell apoptosis and decrease the abundance of ECM to promote cellular uptake and tumor delivery of nanodrugs. Paclitaxel exerts anti-cancer effects but, paradoxically, exacerbates cancer metastasis and drug resistance by increasing the expression of apoptotic B-cell lymphoma-2 protein (BCL-2). Thus, low-molecular-weight heparin-coated lipid-siRNA complex (LH-Lip/siBCL-2) was constructed to inhibit cancer metastasis and silence BCL-2 by BCL-2 siRNA (siBCL-2). Results: Significant tumor growth inhibition efficacy was observed, accompanied by obvious inhibition of cancer metastasis in vivo. Conclusion: These results suggested our sequential delivery of PTX-Lip and LH-Lip/siBCL-2 might provide a practical approach for PDAC or other ECM-rich tumors.

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

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 pH 6.8, 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.

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 αvß3 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.

Efficient siRNA transfer to knockdown a placenta specific lncRNA using RGD-modified nano-liposome: A new preeclampsia-like mouse model.

Preeclampsia is one of the most serious pregnancy complications. Many animal models have already been developed by researchers to study the pathogenesis and treatment of preeclampsia. However, most of these animal models were established by systemic administration or by surgery in the uterine cavity, which could lead to unwanted systemic toxicity or operative wounds and affect the accuracy of the results. Because of the high expression level of integrin αvß3 on the placenta, arginine-glycine-aspartic acid peptide (RGD) modified PEGylated cationic liposome (RGD-Lip) was designed as a novel gene delivery system to target the placenta safely and efficiently, and a new animal model of preeclampsia was established through targeting of long noncoding RNA (lncRNA). The results of cellular uptake and endosomal localization showed that RGD-Lip enhanced cellular uptake and endosomal escape of small interfering RNA (siRNA) on HTR-8/SVneo. In vivo imaging revealed that RGD-Lip was selectively delivered to the placenta. Additionally, H19x siRNA was efficiently transferred into the placenta of C57BL/6 mice via the injection of H19x siRNA-loaded RGD-Lip, which could result in the occurrence of preeclampsia-like symptoms. In summary, RGD-Lip provided a platform to efficiently deliver siRNA to the placenta, and a new preeclampsia-like mouse model was developed targeting placenta enriched/specific genes, including noncoding RNAs.

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 αvß3 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.

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

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-ß1. 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.

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 αvß3 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.

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.

Liposomes Combined an Integrin αvß3-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 αvß3-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 αvß3. 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.