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.Keywords: BBB; drug delivery systems; glioma; stapled RGD; tumor-targeted;

Tumor-homing peptides have been widely used to mediate active targeted drug delivery. l-AE is a reported targeting peptide demonstrating high binding affinity to epidermal growth factor receptor (EGFR) and mutation variant III (EGFRvIII) overexpressed on neovasculature, vasculogenic mimicry, tumor cells, and tumor stem cells. To improve its proteolytic stability, a d-peptide ligand (termed d-AE, the enantiomer of l-AE) was developed. d-AE was confirmed to bind receptors EGFR and EGFRvIII with targeting capability comparable to l-AE. In vivo biodistribution demonstrated the superiority of d-AE in prolonged circulation and enhanced intratumoral accumulation. Furthermore, stabilized peptide modification endowed micelles higher transcytosis efficiency and penetrating capability on blood–brain tumor barrier/U87 tumor spheroids coculture model. When paclitaxel (PTX) was loaded, d-AE-micelle/PTX demonstrated excellent antitumor effect in comparison to Taxol, micelle/PTX, and l-AE-micelle/PTX. These findings indicated that the multitargeted drug delivery system enabled by d-AE ligand provides a promising way for glioma therapy.Keywords: AE; d-peptide; EGFR; EGFRvIII; glioblastoma; multitargeted drug delivery;

Compared to that of other tumors, various barriers, such as the blood–brain barrier (BBB), enzymatic barriers, and the blood–brain tumor barrier, severely impede the successful treatment of gliomas. Peptide ligands were frequently used as targeting moieties to mediate brain tumor-targeted drug delivery. LWSW (SYPGWSW) is a recently reported quorum-sensing (QS) peptide that is able to efficiently cross the BBB. Even though linear LWSW traverses the BBB in vitro, its in vivo targeting ability has been greatly impaired due to proteolysis. Here, we developed a stable peptide, DWSW (DWDSDWDGDPDYDS), using the retro-inverso isomerization technique to achieve an enhanced antiglioma effect. In vitro studies have demonstrated that both the LWSW and DWSW peptides possessed excellent tumor-homing properties and barrier-penetration abilities, whereas DWSW exhibited exceptional stability in serum and maintained its targeting ability after serum preincubation. In vivo, DWSW-modified probes and micelles accumulated more efficiently in the glioma region in comparison with LWSW-modified probes and micelles because of full resistance to proteolysis in blood circulation. As expected, DWSW-modified paclitaxel (PTX)-loaded micelles (DWSW Micelle/PTX) exhibited the longest median survival time among glioma-bearing nude mice. Our results suggested that the QS peptide appears to be a promising targeting moiety, with potential applications in glioma-targeted drug delivery.Keywords: blood−brain barrier; drug-delivery system; glioblastoma; quorum-sensing peptides; stability;

The blood brain barrier separates the circulating blood from the extracellular fluid in the central nervous system and thus presents an essential obstacle to brain transport of therapeutics. Herein, we report on an effective brain-targeted drug delivery system that combines a robust red blood cell membrane-coated nanoparticle (RBCNP) with a unique neurotoxin-derived targeting moiety. The RBCNPs retain the complex biological functions of natural cell membranes while exhibiting physicochemical properties that are suitable for effective drug delivery. CDX peptide is derived from candoxin and shows high binding affinity with nicotinic acetylcholine receptors (nAChRs) expressed on the surface of brain endothelial cells. Through a facile yet robust approach, we successfully incorporate DCDX peptides onto the surface of RBCNPs without compromising the peptide's brain targeting ability. The resulting DCDX-RBCNPs show promising brain targeting efficiency both in vitro and in vivo. Using a glioma mouse model, we demonstrate that doxorubicin-loaded DCDX-RBCNPs have superior therapeutic efficacy and markedly reduced toxicity as compared to the nontargeted drug formulations. While RBCNPs are used as a model system to evaluate the surface modification approach, the reported method can be readily generalized to various types of cell membrane-derived nanocarriers for broad medical applications.Download high-res image (233KB)Download full-size image

Nanoparticles can be enriched at tumor site and improve the therapeutic efficacy of many chemotherapy drugs with the well-known enhanced permeability and retention (EPR) effect. While conventional preparations of materials for nanoscale drug delivery system mainly focused on chemical synthesis, recently the combination of synthetic carrier and natural biomimetic carrier has gained more and more attention. As a new generation of biomimetic nanoparticles, cell membrane-coated nanoparticles combine the complex biological functions of natural membranes and the physicochemical properties of synthetic nanomaterials for a more effective drug delivery. Herein, we briefly review the recent advances on cell membrane-coated nanoparticles for tumor-targeted drug delivery via the prolonging systemic circulation lifetime and the active targeting effect. Since the preferential accumulation of cell membrane-coated nanoparticles in tumor site, they are able to improve the therapeutic efficacy of conventional chemotherapy drugs in antitumor treatment as well as to reduce the systemic toxicity. We also introduce a systematic targeted strategy for the promising application of this platform on brain tumors.由于EPR效应, 纳米粒子能够在肿瘤部位浓集并提高许多化疗药物的治疗指数. 传统纳米递药系统的载体材料主要通过化学合成方 法制备, 而目前将化学合成载体与天然仿生载体相结合的策略得到越来越多的关注. 细胞膜包覆纳米粒子作为新一代仿生纳米制剂, 它将 细胞膜特有的生物学功能与化学合成材料的理化性质相结合, 形成更有效的递药系统. 本文就细胞膜包覆纳米粒子的长循环效果及主动 靶向作用在肿瘤靶向治疗中的研究进行综述, 并就其在脑部肿瘤治疗中的应用前景进行了展望.

GRP78, a specific cancer cell-surface marker, is implicated in cancer cells proliferation, apoptosis resistance, metastasis and drug resistance. l-VAP (SNTRVAP) is a tumor homing peptide exhibiting high binding affinity in vitro to GRP78 protein overexpressed on glioma, glioma stem cells, vasculogenic mimicry and neovasculature. Even though short peptides are often non-immunogenic and demonstrate high affinity to tumor cells, their targeting efficacy is always undermined by rapid blood clearance and enzymatic degradation. In the present study, two d peptides RI-VAP (retro inverso isomer of l-VAP) and d-VAP (retro isomer of l-VAP) were developed by structure-guided peptide design and retro-inverso isomerization technique for glioma targeting. RI-VAP and d-VAP were predicted to bind their receptor GRP78 protein with similar binding affinity, which was experimentally confirmed. The results of in vivo imaging demonstrated that RI-VAP and d-VAP had remarkably advantage over l-VAP for tumor accumulation. In addition, RI-VAP and d-VAP modified paclitaxel-loaded polymeric micelle had better anti-tumor efficacy in comparison to taxol, paclitaxel-loaded plain micelles and l-VAP modified micelles. Overall, the VAP modified micelles suggested in the present study could effectively achieve glioma-targeted drug delivery, validating the potential of the stable VAP peptides in improving the therapeutic efficacy of paclitaxel for glioma.Download high-res image (162KB)Download full-size image

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.Download high-res image (289KB)Download full-size image

Melittin, the major component of the European bee venom, is a potential anticancer candidate due to its lytic properties. However, in vivo applications of melittin are limited due to its main side effect, hemolysis, especially when applied through intravenous administration. The polyethylene glycol-stabilized lipid disk is a novel type of nanocarrier, and the rim of lipid disks has a high affinity to amphiphilic peptides. In our study, a c(RGDyK) modified lipid disk was developed as a tumor targeted drug delivery system for melittin. Cryo-TEM was used to confirm the shape and size of lipid disks with or without c(RGDyK) modification. In vitro and in vivo hemolysis analyses revealed that the hemolysis effect significantly decreased after melittin associated with lipid disks. Importantly, the results of our in vivo biodistribution and tumor growth inhibitory experiments showed that c(RGDyK) modification increased the distribution of lipid disks in the tumor and the anticancer efficacy of melittin loaded lipid disks. Thus, we successfully achieved a targeted drug delivery system for melittin and other amphiphilic peptides with a good therapeutic effect and low side effects.

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.

LA7R (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 LA7R (DA7R) was developed for glioma-targeted drug delivery. DA7R was expected to have a similar binding affinity to its receptors in vitro (VEGFR2 and NRP-1), which was experimentally confirmed. In vivo, DA7R-modified liposomes achieved improved glioma-targeted efficiency than did LA7R-modified liposomes. After loading a chemotherapeutic agent (doxorubicin), DA7R-modified liposomes significantly inhibited subcutaneous model tumor in comparison to free doxorubicin, plain liposomes and LA7R-modified liposomes. In summary, the present study presented the potential of a proteolytically stable d-peptide ligand for in vivo tumor-targeted drug delivery.

As the most aggressive brain tumor, chemotherapy of malignant glioma remains to be extremely challenging in clinic. The blood–brain barrier (BBB) and blood–brain tumor barrier (BBTB) are physiological and pathological barriers preventing therapeutic drugs from reaching the glioma region. In addition, vasculogenic mimicry (VM) formed by invasive glioma cells instead of endothelial cells and angiogenesis are very common in glioma, leading to the poor prognosis and recurrence of glioma. An ideal drug delivery system for glioma chemotherapy needs to traverse the BBB and BBTB and then target VM, angiogenesis, and glioma cells. Herein we developed a liposome-based drug delivery system with the modification of proteolytically stable d-peptide ligands (dCDX/dA7R-LS). dCDX is a d-peptide ligand of nicotine acetylcholine receptors (nAChRs) capable of circumventing the BBB, and dA7R is a d-peptide ligand of vascular endothelial growth factor receptor 2 (VEGFR2) and neuropilin-1 (NRP-1) overexpressed on angiogenesis, VM, and glioma, presenting excellent glioma-homing property. dCDX/dA7R-LS could efficiently internalize into the brain capillary endothelial cells, glioma cells, tumor neovascular endothelial cells, and tumor spheroids and cross the in vitro BBB and BBTB models. Ex vivo imaging and in vivo immunofluorescence assays confirmed the superiority of dCDX/dA7R-LS in targeting intracranial glioma in comparison to plain liposomes or liposomes modified with an individual d-peptide ligand (either dCDX or dA7R). When loaded with doxorubicin, dCDX/dA7R-LS achieved the best antiglioma, antiangiogenesis, and anti-VM effects among all tested formulations. These results suggested that systemic glioma-targeted drug delivery enabled by all-d peptide ligands was promising for the antiglioma therapy.Keywords: dA7R; dCDX; glioma; liposomes; systemic targeted drug delivery

Antagonizing MDM2 and MDMX to activate the tumor suppressor protein p53 is an attractive therapeutic paradigm for the treatment of glioblastoma multiforme (GBM). However, challenges remain with respect to the poor ability of p53 activators to efficiently cross the blood–brain barrier and/or blood–brain tumor barrier and to specifically target tumor cells. To circumvent these problems, we developed a cyclic RGD peptide-conjugated poly(ethylene glycol)-co-poly(lactic acid) polymeric micelle (RGD-M) that carried a stapled peptide antagonist of both MDM2 and MDMX (sPMI). The peptide-carrying micelle RGD-M/sPMI was prepared via film-hydration method with high encapsulation efficiency and loading capacity as well as ideal size distribution. Micelle encapsulation dramatically increased the solubility of sPMI, thus alleviating its serum sequestration. In vitro studies showed that RGD-M/sPMI efficiently inhibited the proliferation of glioma cells in the presence of serum by activating the p53 signaling pathway. Further, RGD-M/sPMI exerted potent tumor growth inhibitory activity against human glioblastoma in nude mouse xenograft models. Importantly, the combination of RGD-M/sPMI and temozolomide — a standard chemotherapy drug for GBM increased antitumor efficacy against glioblastoma in experimental animals. Our results validate a combination therapy using p53 activators with temozolomide as a more effective treatment for GBM.

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, DCDX and c(RGDyK). DCDX 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 DCDX 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.

The blood–brain barrier (BBB) prevents most drugs from reaching the site of central nervous system (CNS) diseases, intensively confining the therapeutic efficiency. Angiopep-2 (here termed LAngiopep), which is a 19-mer peptide derived from human Kunitz domain, can trigger transcytosis and traverse the BBB by recognizing low density lipoprotein-related protein 1 (LRP-1) expressed on the brain capillary endothelial cells. Various enzymes in the blood and the BBB, however, present multiple metabolic barriers to peptide-inspired brain-targeted drug delivery. Here we designed a retro-inverso isomer of LAngiopep, termed DAngiopep, to inspire brain-targeted drug delivery. Both DAngiopep and LAngiopep displayed high uptake capacity in LRP-1 overexpressed cells, including bEnd.3 and U87 cells. DAngiopep demonstrated lower uptake efficiency in both cell lines than did LAngiopep, suggestive of lower binding affinity to LRP-1 of the d-peptide. DAngiopep was resistant to proteolysis in fresh rat blood serum, while more than 85% of LAngiopep disappeared within 2 h. Endocytosed DAngiopep and LAngiopep were found to be colocalized with lysosomal compartments of bEnd.3 cells, indicating that susceptibility to proteolysis of LAngiopep in the BBB may further attenuate its transcytosis efficiency. In vivo, DAngiopep modified PEG-DSPE micelles displayed high distribution in normal brain and intracranial glioblastoma. Due to the expression of LRP-1 on the BBB and glioblastoma cells, proteolytically stable DAngiopep holds much potential for designing two-order brain tumor targeted delivery systems.Keywords: Angiopep-2; brain-targeted drug delivery; glioblastoma; PEG-DSPE micelle; retro-inverso isomer;

Currently, the inefficient transport of liposomes in tumor tissue hinders their clinical application. Tumor-penetrating peptides (TPP) are a series of targeting peptides with the function of penetrating tumor blood vessels and tumor stroma. This work aimed to improve the penetration of liposomes in tumor tissues by TPP modification, thereby enhancing the antitumor effect. First, RPARPAR, a TPP, was modified to the surface of liposomes loaded with doxorubicin. The RPARPAR-modified liposomes (RPA-LP) and unmodified liposomes (LP) showed spherical morphology with average sizes about 90 nm. RPA-LP exhibited remarkably increased cellular accumulation by PC-3 tumor cells than LP as evidenced by the cellular uptake test. The in vivo imaging study confirmed that RPARPAR modification significantly increased the liposome accumulation in subcutaneous tumor tissues. RPA-LP could penetrate through tumor blood vessels and tumor stroma and into the deep tumor tissues as evidence by the immunofluorescence staining analysis. The cytotoxicity of RPARPAR-modified doxorubicin liposomes (RPA-LP-DXR) is considerably increased compared with that of doxorubicin liposomes (LP-DXR). The RPA-LP-DXR also showed significantly (p < 0.005) stronger growth-inhibiting effect on tumor than LP-DXR, possibly due to the tumor-penetrating ability of RPA-LP and targeted killing of tumor cells. This study proved that TPP mediation may be an effective strategy for improving the transport of liposomes in tumor tissue.Keywords: doxorubicin; liposomes; targeting; transport in tumor tissue; tumor-penetrating peptide;

Peptide retro-inverso isomerization is thought to be functionally neutral and has been widely used as a tool for designing proteolytically stable d-isomers to recapitulate biological activities of their parent l-peptides. Despite success in a wide range of applications, exceptions amply exist that clearly defy this rule of thumb when parent l-peptides adopt an α-helical conformation in their bound state. The detrimental energetic effect of retro-inverso isomerization of an α-helical l-peptide on its target protein binding has been estimated to be 3.0–3.4 kcal/mol. To better understand how the retro-inverso isomer of a structured protein works at the molecular level, we chemically synthesized and functionally characterized the retro-inverso isomer of a rationally designed miniature protein termed stingin of 18 amino acid residues, which adopts an N-terminal loop and a C-terminal α-helix stabilized by two intra-molecular disulfide bridges. Stingin emulated the transactivation peptide of the p53 tumor suppressor protein and bound with high affinity and via its C-terminal α-helix to MDM2 and MDMX—the two negative regulators of p53. We also prepared the retro isomer and d-enantiomer of stingin for comparative functional studies using fluorescence polarization and surface plasmon resonance techniques. We found that retro-inverso isomerization of l-stingin weakened its MDM2 binding by 720 fold (3.9 kcal/mol); while enantiomerization of l-stingin drastically reduced its binding to MDM2 by three orders of magnitude, sequence reversal completely abolished it. Our findings demonstrate the limitation of peptide retro-inverso isomerization in molecular mimicry and reinforce the notion that the strategy works poorly with biologically active α-helical peptides due to inherent differences at the secondary and tertiary structural levels between an l-peptide and its retro-inverso isomer despite their similar side chain topologies at the primary structural level.1

The dengue capsid protein C is a highly basic alpha-helical protein of ∼100 amino acid residues that forms an emphipathic homodimer to encapsidate the viral genome and to interact with viral membranes. The solution structure of dengue 2 capsid protein C (DEN2C) has been determined by NMR spectroscopy, revealing a large dimer interface formed almost exclusively by hydrophobic residues. The only acidic residue (Glu87) conserved in the capsid proteins of all four serotypes of dengue virus forms a salt bridge with the side chains of Lys45 and Arg55′. To understand the structural and functional significance of this conserved salt bridge, we chemically synthesized an N-terminally truncated form of DEN2C (WTDEN2C) and its salt bridge-void analog E87ADEN2C using the native chemical ligation technique developed by Kent and colleagues. Comparative biochemical and biophysical studies of these two synthetic proteins using circular dichroism spectroscopy, fluorescence polarization, protein thermal denaturation, and proteolytic susceptibility assay demonstrated that the conserved salt bridge contributed to DEN2C dimerization and stability as well as its resistance to proteolytic degradation. Our work provided insight into the role of a fully conserved structural element of the dengue capsid protein C and paved the way for additional functional studies of this important viral protein.

To investigate a fatty acid-based strategy for efficient brain targeted gene delivery and to understand mechanism(s) of this small molecule-mediated brain gene delivery strategy.A series of fatty acids (FAs) were conjugated with polyethylenimine (PEI25k). A near-infrared fluorescence probe, IR820, was used to study in vivo and ex vivo brain targeting ability of these fatty acid-PEI conjugates (FA-PEIs). Brain uptake of FA-PEI25k/rhodamine-6-isothiocyanate (RITC)-labeled DNA nanoparticles was investigated via a fluorescence imaging method. Moreover, pEGFP was used as a model gene to study in vitro and in vivo transfection effect of the ideal FA-PEI25k conjugate.FA modification did not have interference with the complexation between DNA and the PEI25k. The FA-PEI25k conjugates showed excellent brain targeting ability compared with unmodified PEI25k. Among these FA-PEI25k conjugates studied, myristic acid (MC)-PEI25k showed sustained brain distribution profile and higher brain DNA uptake. Furthermore, MC-PEI25k/pEGFP nanoparticles was able to achieve efficient in vitro and in vivo gene transfection. GFP expression was observed at different brain regions in vivo.These results demonstrated that the small molecule fatty acid, particularly myristic acid-based brain gene delivery strategy, is promising to mediate efficient gene transfection in the brain.

Chemotherapies for brain diseases have been hampered due to the inability of transport of drug across the blood-brain barrier (BBB). In order to overcome the barrier, p-hydroxybenzoic acid (p-HA), a small molecule of benzamide analogue, was used as a ligand for brain-targeted drug delivery. The p-HA was conjugated to PEG-DSPE to form p-HA-PEG-DSPE. Docetaxel-loaded polymeric micelles were prepared by a thin-film hydration method using methoxy-poly(ethylene glycol)-distearoylphosphatidylethanolamine (mPEG2000-DSPE) as a carrier and the p-HA-PEG-DSPE as a brain targeted material. The prepared micelles showed spherical with a mean diameter of (18±3) nm. Encapsulation efficiency and drug loading were (83.49±1.3)%, (7.7±1.2)% for unmodified micelles and (80.65±1.6)%, (7.47±1.8)% for p-HA-modified micelles, respectively. In vitro cellular uptake experiments showed that the p-HA-modified micelles increased BCECs cellular uptake by 1.2 times compared to the unmodified micelles. Exvivo near-infrared fluorescence imaging showed that brain uptake of the p-HA-modified micelles was 1.3–1.8 times higher than that of the unmodified micelles. In vitro cytotoxicity assay against glioblastoma cell U87 MG showed that inhibition rate of the p-HA-modified micelles increased by 1.2 times compared to that of the unmodified micelles and 1.7 times compared to that of DTX. Survival time of nude mice bearing intracranial glioblastoma showed that the lifetime of saline group, Taxotere group, mPEG-DSPE/DTX micelles group and p-HA-PEG-DSPE/DTX micelles group was 22, 27, 32 and 45.8 d, representively, which indicated that anti-glioblastoma activity of DTX could be significantly enhanced by the p-HA-modified polymeric micelles. These results demonstrated that the p-HA-modified micelles could be a promising brain-targeted drug delivery system for hydrophobic drugs against glioblastoma.

The use of glioblastoma-targeted drug delivery system facilitates efficient delivery of chemotherapeutic agents to malignant gliomas in the central nervous system while minimizing high systemic doses associated with debilitating toxicities. To employ the high binding affinity of a cyclic RGD peptide (c(RGDyK), cyclic Arginine–Glycine–Aspartic acid-d-Tyrosine-Lysine) with integrin αvβ3 over-expressed on tumor neovasculature and U87MG glioblastoma cells, we prepared paclitaxel-loaded c(RGDyK)-Poly(ethylene glycol)-block-poly(lactic acid) micelle (c(RGDyK)-PEG-PLA-PTX). In vitro physicochemical characterization of these novel micelles showed satisfactory encapsulated efficiency, loading capacity and size distribution. In vitro cytotoxicity studies proved that the presence of c(RGDyK) enhanced the anti-glioblastoma cell cytotoxic efficacy by 2.5 folds. The binding affinity of c(RGDyK)-PEG-PLA micelle with U87MG cells was also investigated. The competitive binding IC50 value of c(RGDyK)-PEG-PLA micelle was 26.30 nM, even lower than that of c(RGDyK) (56.23 nM). In U87MG glioblastoma-bearing nude mice model, biodistribution of 125I-radiolabeled c(RGDyK)-PEG-PLA or DiR encapsulated micelles and anti-glioblastoma pharmacological effect was investigated after intravenous administration. c(RGDyK)-PEG-PLA micelle accumulated in the subcutaneous and intracranial tumor tissue, and when loaded with PTX (c(RGDyK)-PEG-PLA-PTX), exhibited the strongest tumor growth inhibition among the studied paclitaxel formulations. The anti-glioblastoma effect of c(RGDyK)-PEG-PLA-PTX micelle was also reflected in the median survival time of mice bearing intracranial U87MG tumor xenografts where the median survival time of c(RGDyK)-PEG-PLA-PTX micelle-treated mice (48 days) was significantly longer than that of mice treated with PEG-PLA-PTX micelle (41.5 days), Taxol® (38.5 days) or saline (34 days). Therefore, our results suggested that c(RGDyK)-PEG-PLA micelle may be a potential drug delivery system in the treatment of integrin αvβ3 over-expressed glioblastoma.Paclitaxel-loaded c(RGDyK)-PEG-PLA micelle was developed. It was much effective to the subcutaneous and intracranial glioblastoma models.

A novel conjugate, Folate-PEG-CKK2-DTPA, was designed and prepared as a carrier for lymphatic metastasized tumor imaging diagnosis and targeting therapy.Folate-PEG-CKK2-DTPA was synthesized and characterized by analysis High Performance Liquid Chromatography, Size Exclusive Chromatography and 1H-NMR. 99mTc-labeled conjugation was prepared, and in vivo quantitative biodistribution and SPECT imaging were studied after subcutaneously injected into the rats and rabbits, respectively. Cell uptake study was carried in a KB cell line using fluorescent methods. In vivo and ex vivo fluorescent imaging study was carried in tumor-bearing nude mouse to evaluate its targeting ability.Folate-PEG-CKK2-DTPA was synthesized with high purity. Both in vivo biodistribution study and SPECT imaging study show the rapid direction and high distribution of the conjugation to the lymph nodes. The uptake of fluorescence-labeled Folate-PEG-CKK2-DTPA in human oral epidermis carcinoma cells was observed. In vivo and ex vivo fluorescent imaging study indicated it could accumulate in tumor region after vein tail injection in nude mouse.All these findings suggested Folate-PEG-CKK2-DTPA as a novel and dependable carrier for tumor diagnosis and therapy, especially for lymph-metastasized tumors.

The oncoproteins MDM2 and MDMX negatively regulate the activity and stability of the tumor suppressor protein p53, conferring tumor development and survival. Antagonists targeting the p53-binding domains of MDM2 and MDMX kill tumor cells both in vitro and in vivo by reactivating the p53 pathway, promising a class of antitumor agents for cancer therapy. Aided by native chemical ligation and mirror image phage display, we recently identified a D-peptide inhibitor of the p53-MDM2 interaction termed DPMI-α (TNWYANLEKLLR) that competes with p53 for MDM2 binding at an affinity of 219 nM. Increased selection stringency resulted in a distinct D-peptide inhibitor termed DPMI-γ (DWWPLAFEALLR) that binds MDM2 at an affinity of 53 nM. Structural studies coupled with mutational analysis verified the mode of action of these D-peptides as MDM2-dependent p53 activators. Despite being resistant to proteolysis, both DPMI-α and DPMI-γ failed to actively traverse the cell membrane and, when conjugated to a cationic cell-penetrating peptide, were indiscriminately cytotoxic independently of p53 status. When encapsulated in liposomes decorated with an integrin-targeting cyclic-RGD peptide, however, DPMI-α exerted potent p53-dependent growth inhibitory activity against human glioblastoma in cell cultures and nude mouse xenograft models. Our findings validate D-peptide antagonists of MDM2 as a class of p53 activators for targeted molecular therapy of malignant neoplasms harboring WT p53 and elevated levels of MDM2.

Tumor Necrosis Treatment (TNT) was developed to target solid tumors using monoclonal antibodies such as the chimeric TNT-3 monoclonal antibody (chTNT-3), which bind to degenerating cells located in necrotic regions of tumors. Since biotinylated chTNT-3 showed shorter circulating time and more uptakes in tumors than unmodified chTNT-3, we designed the two-step pretargeting approach composed of administering biotinylated chTNT-3 and 24 h later administering streptavidin modified liposomes encapsulating doxorubicin (DOX) to deliver DOX to the tumor site. The preservation of immunoreactivity of biotinylated chTNT-3 was confirmed by ELISA. The biological half-life of total DOX in two-step pretargeting approach was longer than that of free DOX but shorter than that of sterically stabilized liposomes in Sprague-Dawley rats. The two-step pretargeting approach regimen displayed good tumor targeting with a gradual process in biodistribution study. At 4 h and 24 h after administering DOX-loaded liposomes a highest DOX level of the two-step pretargeting approach was observed. The best antitumor efficacy was observed 3 days after the second treatment in Balb/c nude mice bearing H460 tumors. These results suggested the two-step pretargeting approach regimen may be a new form for delivering anticancer drugs to tumor necrotic regions.

This study aimed to investigate the pharmacokinetic characteristics of total and free mycophnolic acid (MPA) and its 7-O-glucuronide metabolite (MPAG) in Chinese renal transplant recipients. In addition, limited sampling strategies were developed to estimate the individual area under concentration curve (AUC) of total and free MPA.Total and free MPA and MPAG concentrations were determined by high performance liquid chromatography. Whole 12-h pharmacokinetic profiles were obtained on the 10th day after operation in 12 adult Chinese de novo renal transplant recipients administrated with mycophenolate mofetil (MMF, 750 mg bid), cyclosporine and corticosteroids. Limited sampling strategies with jackknife technique, a resampling method, and Bland-Altman analysis were employed to develop equations to estimate total and free MPA AUC.The pattern of total and free MPA and MPAG plasma concentration-time curves in the cohort of patients taking lower doses of MMF was consistent with previous reports of Caucasian patients taking MMF 1 g bid, except that dose-normalized exposure of total and free MPAG was much lower in the current study than in those of the Caucasians. The mean Cmax and AUC0–12h of total and free MPA were 9.4 ± 3.4 mg/L, 20.2 ± 6.5 mg·h/L and 0.4 ± 0.4 mg/L, 0.7 ± 0.5 mg·h/L, respectively, whereas mean Cmax and AUC0–12h of total and free MPAG were 97.3 ± 32.6 mg/L, 656.0 ± 148.0 mg·h/L and 29.9 ± 8.5 mg/L, 222.0 ± 58.1 mg·h/L respectively. The mean fractions of free MPA and MPAG were 3.5 ± 2.0 and 34.6 ± 8.0%, respectively. No determinant was identified to influence the pharmacokinetics of total and free MPA and MPAG or the free fraction of MPA and MPAG. The combinations of C2h−C4h and C1h-C2h-C3h were the best to estimate free and total MPA AUC0–12h respectively, whereas the combination of C2h-C3h-C4h and C1h-C2h-C4h was the best to estimate both simultaneously.This is the first time that the pharmacokinetics profile of total and free MPA and its main metabolite MPAG has been examined in Chinese adult renal transplant patients. The limited sampling strategies proposed to estimate individual free and total MPA AUC could be useful in optimizing patient care.

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.In this review, we briefly describe physiological and pathological conditions of brain tumors, including blood–brain/brain tumor barriers, tumor microenvironment and tumor stem cells; we review the corresponding targeting delivery strategies and introduce a systematic targeted drug delivery strategy to overcome current challenges. Download full-size image

Virus-like particles (VLPs) have been exploited for various biomedical applications, such as the monitoring, prevention, diagnosis and therapy of disease. In this study, a novel multifunctional VLPs nanocarrier (TK-VLPs) was prepared and used for tumor-targeted delivery. The SPR and cell uptake results indicated that the TK peptide is a “bi-functional ligand” with high affinity for Caco-2, HRT-18 and HUVEC cells through the integrin α6β1 and integrin αvβ3 receptors. The results of the direct immunofluorescence, SDS-PAGE and western blot assays demonstrated that the TK-VLPs were successfully prepared using the baculovirus expression system. Confocal laser scanning microscopy and the flow cytometry analysis validated that the TK-VLPs could target to Caco-2, HRT-18 and HUVEC cells. An in vivo study further confirmed that the TK-VLPs could target and efficiently deliver fluorescein to tumor cells and the tumor vasculature in mice bearing subcutaneous tumors. TK-VLPs-DOX displayed a uniform, spherical shape and an average size of approximately 28 nm. The results of the cell uptake and cytotoxicity assays indicated that TK-VLPs-DOX could enhance the selectivity for colorectal cancer cells. Together, our studies provide strong evidence that TK-VLPs could target colon tumor cells and tumor angiogenesis with enhanced permeability and retention effects, suggesting that the TK-VLPs are a multifunctional nanocarrier with potential applications in a colon tumor-targeted drug delivery system.Download high-res image (154KB)Download full-size image

Protein and peptide toxins offer an invaluable source for the development of actively targeted drug delivery systems. They avidly bind to a variety of cognate receptors, some of which are expressed or even up-regulated in diseased tissues and biological barriers. Protein and peptide toxins or their derivatives can act as ligands to facilitate tissue- or organ-specific accumulation of therapeutics. Some toxins have evolved from a relatively small number of structural frameworks that are particularly suitable for addressing the crucial issues of potency and stability, making them an instrumental source of leads and templates for targeted therapy. The focus of this review is on protein and peptide toxins for the development of targeted drug delivery systems and molecular therapies. We summarize disease- and biological barrier-related toxin receptors, as well as targeted drug delivery strategies inspired by those receptors. The design of new therapeutics based on protein and peptide toxins is also discussed.Download high-res image (136KB)Download full-size image