Man Ying

Brain tumor-targeted drug delivery strategies.

Despite the application of aggressive surgery, radiotherapy and chemotherapy in clinics, brain tumors are still a difficult health challenge due to their fast development and poor prognosis. Brain tumor-targeted drug delivery systems, which increase drug accumulation in the tumor region and reduce toxicity in normal brain and peripheral tissue, are a promising new approach to brain tumor treatments. Since brain tumors exhibit many distinctive characteristics relative to tumors growing in peripheral tissues, potential targets based on continuously changing vascular characteristics and the microenvironment can be utilized to facilitate effective brain tumor-targeted drug delivery. In this review, we briefly describe the physiological characteristics of brain tumors, including blood–brain/brain tumor barriers, the tumor microenvironment, and tumor stem cells. We also review targeted delivery strategies and introduce a systematic targeted drug delivery strategy to overcome the challenges.

Liposome-based glioma targeted drug delivery enabled by stable peptide ligands

The treatment of glioma is one of the most challenging tasks in clinic. As an intracranial tumor, glioma exhibits many distinctive characteristics from other tumors. In particular, various barriers including enzymatic barriers in the blood and brain capillary endothelial cells, blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB) rigorously prevent drug and drug delivery systems from reaching the tumor site. To tackle this dilemma, we developed a liposomal formulation to circumvent multiple-barriers by modifying the liposome surface with proteolytically stable peptides, (D)CDX and c(RGDyK). (D)CDX is a D-peptide ligand of nicotine acetylcholine receptors (nAChRs) on the BBB, and c(RGDyK) is a ligand of integrin highly expressed on the BBTB and glioma cells. Lysosomal compartments of brain capillary endothelial cells are implicated in the transcytosis of those liposomes. However, both peptide ligands displayed exceptional stability in lysosomal homogenate, ensuring that intact ligands could exert subsequent exocytosis from brain capillary endothelial cells and glioma targeting. In the cellular uptake studies, dually labeled liposomes could target both brain capillary endothelial cells and tumor cells, effectively traversing the BBB and BBTB monolayers, overcoming enzymatic barrier and targeting three-dimensional tumor spheroids. Its targeting ability to intracranial glioma was further verified in vivo by ex vivo imaging and histological studies. As a result, doxorubicin liposomes modified with both (D)CDX and c(RGDyK) presented better anti-glioma effect with prolonged median survival of nude mice bearing glioma than did unmodified liposomes and liposomes modified with individual peptide ligand. In conclusion, the liposome suggested in the present study could effectively overcome multi-barriers and accomplish glioma targeted drug delivery, validating its potential value in improving the therapeutic efficacy of doxorubicin for glioma.

Stabilized Heptapeptide A7R for Enhanced Multifunctional Liposome-Based Tumor-Targeted Drug Delivery.

(L)A7R (ATWLPPR) is a heptapeptide with high binding affinity in vitro to vascular endothelial growth factor receptor 2 (VEGFR2) and neuropilin-1 (NRP-1) overexpressed on glioma, glioma vasculogenic mimicry and neovasculature. However, its tumor targeting efficacy is significantly reduced in vivo due to proteolysis in blood circulation. To improve the in vivo stability and targeting efficacy, the retro inverso isomer of (L)A7R ((D)A7R) was developed for glioma-targeted drug delivery. (D)A7R was expected to have a similar binding affinity to its receptors in vitro (VEGFR2 and NRP-1), which was experimentally confirmed. In vivo, (D)A7R-modified liposomes achieved improved glioma-targeted efficiency than did (L)A7R-modified liposomes. After loading a chemotherapeutic agent (doxorubicin), (D)A7R-modified liposomes significantly inhibited subcutaneous model tumor in comparison to free doxorubicin, plain liposomes and (L)A7R-modified liposomes. In summary, the present study presented the potential of a proteolytically stable d-peptide ligand for in vivo tumor-targeted drug delivery.

Design of Y-shaped targeting material for liposome-based multifunctional glioblastoma-targeted drug delivery

&NA; Since the treatment of glioma in clinic has been hindered by the blood‐brain barrier (BBB) and blood‐brain tumor barrier (BBTB), multifunctional glioma‐targeted drug delivery systems that can circumvent both barriers have received increasing scrutiny. Despite recent research efforts have been made to develop multifunctional glioma‐targeted liposomes by decorating two or more ligands, few successful trials have been achieved due to the limitation of ligand density on the surface of liposomes. In this study, we designed a Y‐shaped multifunctional targeting material c(RGDyK)‐pHA‐PEG‐DSPE, in which cyclic RGD (c(RGDyK)) and p‐hydroxybenzoic acid (pHA) were linked with a short spacer. Since c(RGDyK) and pHA could respectively circumvent the BBTB and BBB, c(RGDyK)‐pHA‐PEG‐DSPE‐incorporated liposomes could achieve multifunctional glioma‐targeted drug delivery with maximal density of both functional moieties. c(RGDyK)‐pHA‐PEG‐DSPE‐incorporation enhanced cellular uptake of liposomes in bEnd.3, HUVECs and U87 cells, and increased cytotoxicity of doxorubicin (DOX) loaded liposomes on glioblastoma cells. c(RGDyK)‐pHA‐PEG‐DSPE‐incorporated liposomes (c(RGDyK)‐pHA‐LS) could deeply penetrate the 3D glioma spheroids after crossing the BBB and BBTB models in vitro. Moreover, in vivo fluorescence imaging showed the highest tumor distribution of c(RGDyK)‐pHA‐LS than did plain liposomes (no ligand modification) and liposomes modified with a single ligand (either c(RGDyK) or pHA). When loaded with DOX, c(RGDyK)‐pHA‐LS displayed the best anti‐glioma effect with a median survival time (36.5 days) significantly longer than that of DOX loaded plain liposomes (26.5 days) and liposomes modified with a single ligand (28.5 days for RGD and 30 days for pHA). These results indicated that design of Y‐shaped targeting material was promising to maximize the multifunctional targeting effects of liposomes on the therapy of glioma. Graphical abstract Figure. No caption available.

Stapled RGD Peptide Enables Glioma-Targeted Drug Delivery by Overcoming Multiple Barriers

Malignant glioma, the most frequent and aggressive central nervous system (CNS) tumor, severely threatens human health. One reason for its poor prognosis and short survival is the presence of the blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB), which restrict the penetration of therapeutics into the brain at different stages of glioma. Herein, inspired by the peptide stapling technique, we designed a cyclic RGD ligand via an all-hydrocarbon staple (stapled RGD, sRGD) to facilitate BBB penetration while retaining the capacity of BBTB penetration and targeting ability to glioma cells. As expected, sRGD-modified micelles were able to penetrate the in vitro BBB model while retaining the glioma targeted capability. The results of the in vivo imaging studies further revealed that this nanocarrier could not only efficiently transverse the intact BBB of normal mice, but also could specifically target glioma cells of intracranial glioma-bearing nude mice. Furthermore, Paclitaxel-loaded sRGD-modified micelles exhibited improved antiglioma efficacy in vitro and significantly prolonged survival time of glioma-bearing nude mice. Overall, this sRGD peptide showed potency for glioma-targeted drug delivery by overcoming multiple barriers.

A stabilized peptide ligand for multifunctional glioma targeted drug delivery

Peptide ligands consisting of l-amino acids are subject to proteolysis in vivo. When modified on the surface of nanocarriers, those peptide ligands would readily degrade and the targeting efficacy is significantly attenuated. It has received increasing scrutiny to design stable peptide ligands for targeted drug delivery. Here, we present the design of a stable peptide ligand by the formation of a head-to-tail amide bond as an example. Even though the linear l-peptide A7R (termed LA7R) can bind specifically to vascular endothelial growth factor receptor 2 (VEGFR2) and neuropilin-1 (NRP-1) that are overexpressed on glioma cells, neovasculature and glioma vasculogenic mimicry (VM), the tumor-homing capacity of LA7R is greatly impaired in vivo due to proteolysis (e.g. in the serum). A cyclic A7R (cA7R) peptide was identified by computer-aided peptide design and synthesized with high yield by combining solid phase peptide synthesis and native chemical ligation. The binding of cA7R to both receptors was theoretically and experimentally assessed. In our simulated model hydrophobic and ionic interactions dominated the binding of LA7R to receptors. It is very interesting that cA7R adopting a different structure from LA7R retained high binding affinities to receptors without affecting the hydrophobic and ionic interactions. After head-to-tail cyclization by the formation of an amide bond, cA7R exhibited exceptional stability in mouse serum. Either cA7R or LA7R was conjugated on the surface of doxorubicin (DOX) loaded liposomes (cA7R-LS/DOX or LA7R-LS/DOX). The results of in vitro cellular assays indicated that cA7R-LS/DOX not only displayed stronger anti-proliferative effect against glioma cells, but also demonstrated to be more efficient in destruction of VM and HUVEC tubes in comparison to LA7R-LS/DOX and plain liposomes (LS/DOX, without peptide conjugation). cA7R conjugation could achieve significantly higher accumulation of liposomes in glioma than did LA7R conjugation, which in turn, cA7R-LS/DOX could substantially suppress subcutaneous tumor growth when compared with other DOX formulations (free DOX, LS/DOX and LA7R-LS/DOX). The designed cyclic A7R exhibited the capability of targeting glioma cells, neovasculature and VM simultaneously in vivo. Considering the ease of synthesis, high binding affinity to receptors and increased stability of cA7R peptide in the present study, the design of head-to-tail cyclized peptides by the formation of amide bond based on computer-aided peptide design presents an alternative method to identify proteolytically stable peptide ligands.

D-SP5 Peptide-Modified Highly Branched Polyethylenimine for Gene Therapy of Gastric Adenocarcinoma.

Peptide-mediated targeting of tumors has become an effective strategy for cancer therapy. Retro-inverso peptides resist protease degradation and maintain their bioactivity. We used the retro-inverso peptide D(PRPSPKMGVSVS) (D-SP5) as a targeting ligand to develop gene therapy for gastric adenocarcinoma. D-SP5 has a higher affinity for human gastric adenocarcinoma (SGC7901) cells compared with that of its parental peptide, L(SVSVGMKPSPRP) (L-SP5). Polyethylenimine (PEI)/pDNA, polyethylene glycol (mPEG)-PEI/pDNA and D-SP5-PEG-PEI/pDNA were prepared for further study. Quantitative luciferase assays showed the transfection efficiency of D-SP5-PEG-PEI/pGL(4.2) was larger compared with that of mPEG-PEI/pGL(4.2). Flow cytometry assays revealed that the apoptosis rates of SGC7901 cells treated with D-SP5-PEG-PEI/pTRAIL were larger than mPEG-PEI/pTRAIL. Western blot assays indicated that the expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) protein in SGC7901 cells treated with D-SP5-PEG-PEI/pTRAIL was higher compared with that in cells treated with mPEG-PEI/pTRAIL. In vivo pharmacodynamics study revealed that D-SP5-PEG-PEI/pTRAIL could inhibit the growth of gastric adenocarcinoma SGC7901 xenografts in nude mice. Our results demonstrate that D-SP5-PEG-PEI is a safe and efficient gene delivery vector with potential applications in antitumor gene therapy.

Glioma-Targeted Drug Delivery Enabled by a Multifunctional Peptide

The rapid proliferation of glioma relies on vigorous angiogenesis for the supply of essential nutrients; thus, a radical method of antiglioma therapy should include blocking tumor neovasculature formation. A phage display selected heptapeptide, the glioma-initiating cell peptide GICP, was previously reported as a ligand of VAV3 protein (a Rho GTPase guanine nucleotide exchange factor), which is overexpressed on glioma cells and tumor neovasculature. Therefore, GICP holds potential for the multifunctional targeting of glioma (tumor cells and neovasculature). We developed GICP-modified micelle-based paclitaxel delivery systems for antiglioma therapy in vitro and in vivo. GICP and GICP-modified PEG-PLA micelles (GICP-PEG-PLA) could be significantly taken up by U87MG cells, a human cell line derived from malignant gliomas and human umbilical vein endothelial cells (HUVECs). Furthermore, GICP-PEG-PLA micelles demonstrated enhanced penetration in a tumor spheroid model in vitro in comparison to unmodified micelles. In vivo, DiR-loaded GICP-PEG-PLA micelles exhibited superior accumulation in the tumor region by targeting neovasculature and glioma cells in nude mice bearing subcutaneous glioma. When loaded with paclitaxel, GICP-PEG-PLA micelles could more effectively suppress tumor growth and neovasculature formation than unmodified micelles in vivo. Our results indicated that GICP could serve as a promising multifunctional ligand for glioma targeting.

N-trimethyl chitosan nanoparticles and CSKSSDYQC peptide: N-trimethyl chitosan conjugates enhance the oral bioavailability of gemcitabine to treat breast cancer

&NA; Gemcitabine is a nucleoside analogue effective against a number of cancers. However, the full potential of this drug has not been realised, in part due to low oral bioavailability and frequent dosing requirements. This study reports the synthesis, in‐vitro, ex‐vivo and in‐vivo evaluation of trimethyl chitosan (TMC) ‐ CSKSSDYQC (CSK) peptide conjugates capable of enhancing the oral bioavailability of gemcitabine due to the ability to target intestinal goblet cells and promote intestinal cellular uptake. TMC was synthesized by a novel two‐step methylation method to improve quanternization and yield. The CSK‐TMC conjugates were prepared by ionic gelation to achieve particles sized at 173.6 ± 6.8 nm, zeta potential of +18.5 ± 0.2 mV and entrapment efficiency of 66.4 ± 0.1%, capable of sustained drug release. By encapsulating gemcitabine into CSK‐TMC conjugates, an increased amount of drug permeated through porcine intestinal epithelial membranes compared with the unconjugated TMC nanoparticles (NPs). The rate of cellular uptake of drug loaded conjugates into HT29‐MTX‐E12 intestinal goblet cells, was time‐ and concentration‐dependant. The conjugates underwent active transport associated with adsorptive mediated, clathrin and caveolae mediated endocytosis. In cellular transport studies, drug loaded conjugates had greater drug transport capability compared with drug solution and TMC NPs over the co‐cultured Caco‐2/HT29‐MTX‐E12 cell monolayer. The drug loaded conjugates exhibited electrostatic interaction with the intestinal epithelial cells. Both P‐glycoprotein (P‐gp) and multiple resistance protein‐2 (MRP2) efflux affected the cellular transport of the conjugates. Importantly, during the pharmacokinetic studies, the orally administrated drug loaded into TMC NPs showed an improved oral bioavailability of 54.0%, compared with gemcitabine solution of 9.9%. Notable, the CSK‐TMC conjugates further improved oral bioavailability to 60.1% and reduced the tumour growth rate in a BALB/c nude mouse model, with a 5.1‐fold and 3.3‐fold reduction compare with the non‐treated group and gemcitabine solution group. Furthermore, no major evidence of toxicity was discernible on histologic studies of selected organs. In conclusion, the presented CSK‐TMC conjugates and TMC nanoparticles both significantly improve the oral bioavailability of gemcitabine and have the potential for the treatment of breast cancer.

Multifunctional targeted liposomal drug delivery for efficient glioblastoma treatment

Glioblastoma multiforme (GBM) has been considered to be the most malignant brain tumors. Due to the existence of various barriers including the blood–brain barrier (BBB) and blood–brain tumor barrier (BBTB) greatly hinder the accumulation and deep penetration of chemotherapeutics, the treatment of glioma remains to be the most challenging task in clinic. In order to circumvent these hurdles, we developed a multifunctional liposomal glioma-targeted drug delivery system (c(RGDyK)/pHA-LS) modified with cyclic RGD (c(RGDyK)) and p-hydroxybenzoic acid (pHA) in which c(RGDyK) could target integrin αvβ3 overexpressed on the BBTB and glioma cells and pHA could target dopamine receptors on the BBB. In vitro, c(RGDyK)/pHA-LS could target glioblastoma cells (U87), brain capillary endothelial cells (bEnd.3) and umbilical vein endothelial cells (HUVECs) through a comprehensive pathway. Besides, c(RGDyK)/pHA-LS could also increase the cytotoxicity of doxorubicin encapsulated in liposomes on glioblastoma cells, and was able to penetrate inside the glioma spheroids after traversing the in vitro BBB and BBTB. In vivo, we demonstrated the targeting ability of c(RGDyK)/pHA-LS to intracranial glioma. As expected, c(RGDyK)/pHA-LS/DOX showed a median survival time of 35 days, which was 2.31-, 1.76- and 1.5-fold higher than that of LS/DOX, c(RGDyK)-LS/DOX, and pHA-LS/DOX, respectively. The findings here suggested that the multifunctional glioma-targeted drug delivery system modified with both c(RGDyK) and pHA displayed strong antiglioma efficiency in vitro and in vivo, representing a promising platform for glioma therapy.Glioblastoma multiforme (GBM) has been considered to be the most malignant brain tumors. Due to the existence of various barriers including the blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB) greatly hinder the accumulation and deep penetration of chemotherapeutics, the treatment of glioma remains to be the most challenging task in clinic. In order to circumvent these hurdles, we developed a multifunctional liposomal glioma-targeted drug delivery system (c(RGDyK)/pHA-LS) modified with cyclic RGD (c(RGDyK)) and p-hydroxybenzoic acid (pHA) in which c(RGDyK) could target integrin αvβ3 overexpressed on the BBTB and glioma cells and pHA could target dopamine receptors on the BBB. In vitro, c(RGDyK)/pHA-LS could target glioblastoma cells (U87), brain capillary endothelial cells (bEnd.3) and umbilical vein endothelial cells (HUVECs) through a comprehensive pathway. Besides, c(RGDyK)/pHA-LS could also increase the cytotoxicity of doxorubicin encapsulated in liposomes on glioblastoma cells, and was able to penetrate inside the glioma spheroids after traversing the in vitro BBB and BBTB. In vivo, we demonstrated the targeting ability of c(RGDyK)/pHA-LS to intracranial glioma. As expected, c(RGDyK)/pHA-LS/DOX showed a median survival time of 35 days, which was 2.31-, 1.76- and 1.5-fold higher than that of LS/DOX, c(RGDyK)-LS/DOX, and pHA-LS/DOX, respectively. The findings here suggested that the multifunctional glioma-targeted drug delivery system modified with both c(RGDyK) and pHA displayed strong antiglioma efficiency in vitro and in vivo, representing a promising platform for glioma therapy.