Co-administration of dual-targeting nanoparticles with penetration enhancement peptide for antiglioblastoma therapy.

Chemotherapy is an indispensable auxiliary treatment for glioma but highly limited by the existence of both blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB). The dysfunctional brain tumor blood vessels and high interstitial pressure in glioma also greatly hindered the accumulation and deep penetration of chemotherapeutics into the glioma. Lactoferrin (Lf), with its receptor overexpressed on both the brain endothelial cells and glioma cells, was here functionalized to the surface of poly(ethylene glycol)-poly(lactic acid) nanoparticles to mediate BBB/BBTB and glioma cell dual targeting. tLyP-1, a tumor-homing peptide, which contains a C-end Rule sequence that can mediate tissue penetration through the neuropilin-1-dependent internalization pathway, was coadministrated with Lf-functionalized nanoparticles (Lf-NP) to enhance its accumulation and deep penetration into the glioma parenchyma. Enhanced cellular association in both BCEC and C6 cells, increased cytotoxicity of the loaded paclitaxel, and deep penetration in the 3D glioma spheroids was achieved by Lf-NP. Following coadministration with tLyP-1, the functionalized nanoparticles obtained improved tumor targeting, glioma vascular extravasation, and antiglioma efficacy. The findings here suggested that the strategy by coadministrating BBB/BBTB and glioma cells dual-targeting nanocarriers with a tumor penetration enhancement peptide represent a promising platform for antiglioma drug delivery.

Activatable cell penetrating peptide-conjugated nanoparticles with enhanced permeability for site-specific targeting delivery of anticancer drug.

Based on the powerful cell-penetrating ability of low molecular weight protamine (LMWP) and the overexpression of matrix metalloproteinases in the tumor sites, we constructed an activatable low molecular weight protamine (ALMWP) and modified it onto the surface of poly(ethylene glycol)-poly(lactic acid) nanoparticles to develop a "smart" drug delivery system with enhanced permeability for facilitating site-specific targeting delivery of anticancer drug. The obtained ALMWP-functionalized nanoparticles (ALMWP-NP) with a particle size of 134.0 ± 4.59 nm and a zeta potential of -34.4 ± 2.7 mV, exhibited an enhanced MMP-dependent accumulation in HT-1080 cells via both energy-independent direct translocation and clathrin-mediated, cytoskeleton-dependent endocytosis. Pharmacokinetic and biodistribution study in HT-1080 tumor-bearing mice showed that ALMWP-NP significantly increased the accumulation of paclitaxel (PTX) in the tumor site but not the nontarget tissues. In addition, intratumor distribution analysis demonstrated that more ALMWP-NP penetrated deeply into the tumor parenchyma. As a result, PTX loaded by ALMWP-NP exhibited improved antitumor efficacy over that by unmodified nanoparticles and LMWP-functionalized nanoparticles. The findings suggested that ALMWP-NP could be used as a safe and effective tumor-targeting drug delivery system and opened a new gateway to the application of cell-penetrating peptides for targeted antitumor therapy.

B6 peptide-modified PEG-PLA nanoparticles for enhanced brain delivery of neuroprotective peptide.

The blood-brain barrier (BBB), which is formed by the brain capillary wall, greatly hinders the development of new drugs for the brain. Over the past decades, among the various receptor-mediated endogenous BBB transport systems, the strategy of using transferrin or anti-transferrin receptor antibodies to facilitate brain drug delivery system is of particular interest. However, the application of large proteins still suffers from the drawbacks including synthesis procedure, stability, and immunological response. Here, we explored a B6 peptide discovered by phase display as a substitute for transferrin, and conjugated it to PEG-PLA nanoparticles (NP) with the aim of enhancing the delivery of neuroprotective drug across the BBB for the treatment of Alzheimer's disease. B6-modified NP (B6-NP) exhibited significantly higher accumulation in brain capillary endothelial cells via lipid raft-mediated and clathrin-mediated endocytosis. In vivo, fluorescently labeled B6-NP exhibited much higher brain accumulation when compared with NP. Administration of B6-NP encapsulated neuroprotective peptide-NAPVSIPQ (NAP)-to Alzheimer's disease mouse models showed excellent amelioration in learning impairments, cholinergic disruption, and loss of hippocampal neurons even at lower dose. These findings together suggested that B6-NP might serve as a promising DDS for facilitating the brain delivery of neuropeptides.

PEG-PLA nanoparticles modified with APTEDB peptide for enhanced anti-angiogenic and anti-glioma therapy.

Tumor neovasculature and tumor cells dual-targeting chemotherapy can not only destroy the tumor neovasculature, cut off the supply of nutrition and starve the tumor cells, but also directly kill tumor cells, holding great potential in overcoming the drawbacks of anti-angiogenic therapy only and improving the anti-glioma efficacy. In the present study, by taking advantage of the specific expression of fibronectin extra domain B (EDB) on both glioma neovasculature endothelial cells and glioma cells, we constructed EDB-targeted peptide APTEDB-modified PEG-PLA nanoparticles (APT-NP) for paclitaxel (PTX) loading to enable tumor neovasculature and tumor cells dual-targeting chemotherapy. PTX-loaded APT-NP showed satisfactory encapsulated efficiency, loading capacity and size distribution. In human umbilical vein endothelial cells, APT-NP exhibited significantly elevated cellular accumulation via energy-dependent, caveolae and lipid raft-involved endocytosis, and improved PTX-induced apoptosis therein. Both in vitro tube formation assay and in vivo matrigel angiogenesis analysis confirmed that APT-NP significantly improved the antiangiogenic ability of PTX. In U87MG cells, APT-NP showed elevated cellular internalization and also enhanced the cytotoxicity of the loaded PTX. Following intravenous administration, as shown by both in vivo live animal imaging and tissue distribution analysis, APT-NP achieved a much higher and specific accumulation within the glioma. As a result, APT-NP-PTX exhibited improved anti-glioma efficacy over unmodified nanoparticles and Taxol(®) in both subcutaneous and intracranial U87MG xenograft models. These findings collectively indicated that APTEDB-modified nanoparticles might serve as a promising nanocarrier for tumor cells and neovasculature dual-targeting chemotherapy and hold great potential in improving the efficacy anti-glioma therapy.

Lactoferrin-modified PEG-co-PCL nanoparticles for enhanced brain delivery of NAP peptide following intranasal administration.

Development of effective non-invasive drug delivery systems is of great importance to the treatment of Alzheimer's diseases and has made great progress in recent years. In this work, lactoferrin (Lf), a natural iron binding protein, whose receptor is highly expressed in both respiratory epithelial cells and neurons is here utilized to facilitate the nose-to-brain drug delivery of neuroprotection peptides. The Lf-conjugated PEG-PCL nanoparticle (Lf-NP) was constructed via a maleimide-thiol reaction with the Lf conjugation confirmed by CBQCA Protein Quantitation and XPS analysis. Other important parameters such as particle size distribution, zeta potential and in vitro release of fluorescent probes were also characterized. Compared with unmodified nanoparticles (NP), Lf-NP exhibited a significantly enhanced cellular accumulation in 16HBE14o-cells through both caveolae-/clathrin-mediated endocytosis and direct translocation. Following intranasal administration, Lf-NP facilitated the brain distribution of the coumarin-6 incorporated with the AUC0-8h in rat cerebrum (with hippocampus removed), cerebellum, olfactory tract, olfactory bulb and hippocampus 1.36, 1.53, 1.70, 1.57 and 1.23 times higher than that of coumarin-6 carried by NP, respectively. Using a neuroprotective peptide - NAPVSIPQ (NAP) as the model drug, the neuroprotective and memory improvement effect of Lf-NP was observed even at lower dose than that of NP in a Morris water maze experiment, which was also confirmed by the evaluation of acetylcholinesterase, choline acetyltransferase activity and neuronal degeneration in the mice hippocampus. In conclusion, Lf-NP may serve as a promising nose-to-brain drug delivery carrier especially for peptides and proteins.

PEG-co-PCL nanoparticles modified with MMP-2/9 activatable low molecular weight protamine for enhanced targeted glioblastoma therapy.

By taking advantage of the dramatically upregulated expression of matrix metalloproteinases MMP-2 and MMP-9 in glioblastomas and the powerful transport ability of low molecular weight protamine (LMWP), we constructed an activatable low molecular weight protamine (ALMWP) and conjugated it to PEG-PCL nanoparticles (NP) to develop a 'smart' drug delivery system for enhanced targeted glioblastoma therapy. Important parameters such as particle size distribution, zeta potential and surface content were determined, which confirmed the conjugation of ALMWP to the surface of nanoparticle. ALMWP-NP loaded with paclitaxel (PTX) exhibited a desirable pharmacokinetic and biodistribution profiles for anti-glioblastoma drug delivery. Cellular experiments showed that ALMWP-NP exhibited significantly elevated MMP-dependent cellular accumulation in C6 cells via lipid raft-mediated endocytosis and energy-dependent macropinocytosis, and improved the cytotoxicity of PTX. In vitro C6 tumor spheroid uptake confirmed the tumor penetrating ability of ALMWP-NP, in vivo imaging and glioma distribution justified its specific accumulation in the glioma. The improved glioma-targeting and tumor penetration led to an anticipated enhanced in vivo anti-glioblastoma effect: animals (nude mice bearing intracranial C6 glioma) treated with ALMWP-NP-PTX survive significantly longer than those treated with saline, Taxol(®) NP-PTX and LMWP-NP-PTX. The findings here offered strong evidence for the glioblastoma-targeting therapy of ALMWP-NP-PTX, and could also lead to a significant advancement in the application of CPPs for targeted therapy of glioma.

Glioma therapy using tumor homing and penetrating peptide-functionalized PEG-PLA nanoparticles loaded with paclitaxel.

By taking advantage of the excessively upregulated expression of neuropilin (NRP) on the surface of both glioma cells and endothelial cells of angiogenic blood vessels, the ligand of NRP with high affinity - tLyp-1 peptide, which also contains a CendR motif ((R/K)XX(R/K)), was functionalized to the surface of PEG-PLA nanoparticles (tLyp-1-NP) to mediate its tumor homing, vascular extravasation and deep penetration into the glioma parenchyma. The tLyp-1-NP was prepared via a maleimide-thiol coupling reaction with uniformly spherical shape under TEM and particle size of 111.30 ± 15.64 nm. tLyp-1-NP exhibited enhanced cellular uptake in both human umbilical vein endothelial cells and Rat C6 glioma cells, increased cytotoxicity of the loaded PTX, and improved penetration and growth inhibition in avascular C6 glioma spheroids. Selective accumulation and deep penetration of tLyp-1-NP at the glioma site was confirmed by in vivo imaging and glioma distribution analysis. The longest survival was achieved by those mice bearing intracranial C6 glioma treated with PTX-loaded tLyp-1-NP. The findings here strongly indicate that tLyp-1 peptide-functionalized nanoparticulate DDS could significantly improve the efficacy of paclitaxel glioma therapy.

F3 peptide-functionalized PEG-PLA nanoparticles co-administrated with tLyp-1 peptide for anti-glioma drug delivery.

The development of a drug delivery strategy which can mediate efficient tumor targeting together with high cellular internalization and extensive vascular extravasation is essential and important for glioma treatment. To achieve this goal, F3 peptide that specifically bind to nucleolin, which is highly expressed on the surface of both glioma cells and endothelial cells of glioma angiogenic blood vessels, is utilized to decorate a nanoparticulate drug delivery system to realize glioma cell and neovasculature dual-targeting and efficient cellular internalization. Tumor homing and penetrating peptide, tLyp-1 peptide, which contains the motif of (R/K)XX(R/K) and specially binds to neuropilin is co-administrated to improve the penetration of the nanoparticles across angiogenic vasculature into glioma parenchyma. The F3 conjugation via a maleimide-thiol coupling reaction was confirmed by XPS analysis with 1.03% nitrogen detected on the surface of the functionalized nanoparticles. Enhanced cellular interaction with C6 cells, improved penetration in 3D multicell tumor spheroids, and increased cytotoxicity of the loaded paclitaxel were achieved by the F3-functionalized nanoparticles (F3-NP). Following co-administration with tLyp-1 peptide, F3-NP displayed enhanced accumulation at the tumor site and deep penetration into the glioma parenchyma and achieved the longest survival in mice bearing intracranial C6 glioma. The findings here clearly indicated that the strategy by co-administrating a tumor homing and penetrating peptide with functionalized nanoparticles dual-targeting both glioma cells and neovasculature could significantly improve the anti-glioma drug delivery, which also hold a great promise for chemotherapy of other hard-to-cure cancers.

The influence of the penetrating peptide iRGD on the effect of paclitaxel-loaded MT1-AF7p-conjugated nanoparticles on glioma cells.

Low permeability across the blood-brain tumor barrier (BTB) and poor penetration into the glioma parenchyma represent key obstacles for anti-glioblastoma drug delivery. In this study, MT1-AF7p peptide, which presents high binding affinity to membrane type-1 matrix metalloproteinase (MT1-MMP) that over-expressed on both angiogenic blood vessels and glioma cells, was employed to decorate the paclitaxel-loaded PEG-PLA nanoparticles (MT1-NP-PTX) to mediate glioblastoma targeting. Tumor-homing and penetrating peptide iRGD was co-administrated to further facilitate nanoparticles extravasation from the tumor vessels and penetration into the glioma parenchyma. MT1-NP-PTX showed satisfactory encapsulated efficiency, loading capacity and size distribution. In C6 glioma cells, MT1-NP was found to exhibit significantly enhanced cellular accumulation than that of unmodified NP via both energy-dependent macropinocytosis and lipid raft-mediated endocytosis. The anti-proliferative and apoptosis-induction activity of PTX was significantly enhanced following its encapsulation in MT1-NP. In vivo imaging and glioma distribution together confirmed that MT1-AF7p functionalization and iRGD co-administration significantly improved the nanoparticles extravasation across BTB and accumulation in glioma parenchyma. Furthermore, in vitro C6 glioma spheroid assays evidenced that MT1-NP effectively penetrated into the glioma spheroids and significantly improved the growth inhibitory effects of loaded PTX on glioma spheroids. More importantly, the median survival time of those nude mice bearing intracranial C6 glioma received MT1-NP-PTX and iRGD combination regimen was 60 days, significantly longer than that of other groups. The findings suggested that the BTB/glioma cells dual-targeting DDS co-administrated with iRGD peptide might provide a both practical and feasible solution to highly efficient anti-glioblastoma drug delivery.

Preparation and characterization of paclitaxel-loaded DSPE-PEG-liquid crystalline nanoparticles (LCNPs) for improved bioavailability.

Lipid-based liquid crystalline nanoparticles (LCNPs) have attracted growing interest as a new drug nanocarrier system for improving bioavailability for both hydrophilic and hydrophobic drugs. In this study, self-assembled LCNPs based on soy phosphatidyl choline and glycerol dioleate, which was known possessing low toxicity and negligible hemolysis, were prepared using poly(ethylene glycol)-grafted 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine (DSPE-PEG) as the dispersing agent. Paclitaxel (PTX) was used as a model hydrophobic drug. The particle size of the optimized DSPE-PEG-LCNPs and PTX-loaded DSPE-PEG-LCNPs were around 70nm. Crossed polarized light microscopy was used to characterize the phase behavior of liquid crystalline (LC) matrices, which showed a fan-like birefringent texture in dark background indicating the coexistence of reversed cubic and hexagonal phase in the optimized LC matrix. Transmission electron microscopy and cryo-field emission scanning electron microscopy revealed its internal water channel and "twig-like" surface morphology. PTX-loaded DSPE-PEG-LCNPs exhibited a biphasic drug sustained release pattern with a relatively fast release at the initial stage and a sustained release afterwards. PTX-loaded DSPE-PEG-LCNPs presented higher AUC (410.942±72.522µg/Lh) when compared with commercial product Taxol (212.670±41.396µg/Lh). These results indicated that DSPE-PEG-LCNPs might serve as a potential sustained release system for poorly water-soluble agents.

Penetratin-functionalized PEG-PLA nanoparticles for brain drug delivery.

Nanoparticulate drug delivery system possesses distinct advantages for brain drug delivery. However, its amount that reach the brain is still not satisfied. Cell-penetrating peptides (CPPs), short peptides that facilitate cellular uptake of various molecular cargo, would be appropriate candidates for facilitating brain delivery of nanoparticles. However, such effect could be deprived by the rapid systemic clearance of CPPs-functionalized nanoparticles due to their positive surface charge. Penetratin (CPP with relatively low content of basic amino acids) was here functionalized to poly(ethylene glycol)-poly(lactic acid) nanoparticles (NP) to achieve desirable pharmacokinetic and biodistribution profiles for brain drug delivery. The obtained penetratin-NP showed a particle size of 100 nm and zeta potential of -4.42 mV. The surface conjugation of penetratin was confirmed by surface chemical compositions analysis via X-ray photo electron spectroscopy. In MDCK-MDR cell model, penetratin-NP presented enhanced cellular accumulation via both lipid raft-mediated endocytosis and direct translocation processes with the involvement of Golgi apparatus, lysosome and microtubules. In vivo pharmacokinetic and biodistribution studies showed that penetratin-NP exhibited a significantly enhanced brain uptake and reduced accumulation in the non-target tissues compared with low-molecular-weight protamine (CPP with high arginine content)-functionalized nanoparticles. These data strongly implicated that penetratin-NP might represent a promising brain-targeting drug delivery system. The findings also provided an important basis for the optimization of brain drug delivery systems via surface charge modulation.

Lipid-based liquid crystalline nanoparticles as oral drug delivery vehicles for poorly water-soluble drugs: cellular interaction and in vivo absorption.

BACKGROUND: Lipid-based liquid crystalline nanoparticles (LCNPs) have attracted growing interest as novel drug-delivery systems for improving the bioavailability of both hydrophilic and hydrophobic drugs. However, their cellular interaction and in vivo behavior have not been fully developed and characterized. METHODS: In this study, self-assembled LCNPs prepared from soy phosphatidylcholine and glycerol dioleate were developed as a platform for oral delivery of paclitaxel. The particle size of empty LCNPs and paclitaxel-loaded LCNPs was around 80 nm. The phase behavior of the liquid crystalline matrix was characterized using crossed polarized light microscopy and small-angle X-ray scattering, and showed both reversed cubic and hexagonal phase in the liquid crystalline matrix. Transmission electron microscopy and cryofield emission scanning electron microscopy analysis revealed an inner winding water channel in LCNPs and a " ball-like"/"hexagonal" morphology. RESULTS: Cellular uptake of LCNPs in Caco-2 cells was found to be concentration-dependent and time-dependent, with involvement of both clathrin and caveolae/lipid raft-mediated endocytosis. Under confocal laser scanning microscopy, soy phosphatidylcholine was observed to segregate from the internalized LCNPs and to fuse with the cell membrane. An in vivo pharmacokinetic study showed that the oral bioavailability of paclitaxel-loaded LCNPs (13.16%) was 2.1 times that of Taxol(®) (the commercial formulation of paclitaxel, 6.39%). CONCLUSION: The findings of this study suggest that this LCNP delivery system may be a promising candidate for improving the oral bioavailability of poorly water-soluble agents.

Determination of sulphasalazine and its main metabolite sulphapyridine and 5-aminosalicylic acid in human plasma by liquid chromatography/tandem mass spectrometry and its application to a pharmacokinetic study.

A simple and sensitive liquid chromatography/positive-ion electrospray ionization mass spectrometry (LC-ESI-MS/MS) method has been developed for the simultaneous determination of sulphasalazine (SASP) and its main metabolite sulphapyridine (SP) and 5-aminosalicylic acid (5-ASA) with 100 µL of human plasma using dimenhydrinate as the internal standard (I.S.). The API-3000 LC-MS/MS was operated under the multiple reaction-monitoring mode (MRM) using the electrospray ionization technique. Protein precipitation process was used to extract SASP, SP, 5-ASA and I.S. from human plasma. The total run time was 9.0 min and the elution of SASP, SP and 5-ASA was at 4.8 min, 2.5 min and 2.0 min, respectively. The separation was achieved with a mobile phase consisting of 0.2% formic acid, 2 mM ammonium acetate in water (mobile phase A) and 0.2% formic acid, 2 mM ammonium acetate in methanol (mobile phase B) by using gradient elution on a XBP Phenyl column (100 mm × 2.1 mm, 5 µm). The developed method was validated in human plasma with a lower limit of quantitation of 10 ng/mL for SASP, SP and 5-ASA, respectively. A linear response function was established for the range of concentrations 10-10,000 ng/mL (r>0.99) for SASP and 10-1000 ng/mL (r>0.99) for SP and 5-ASA. The intra and inter-day precision values for SASP, SP and 5-ASA met the acceptance as per FDA guidelines. SASP, SP and 5-ASA were stable during stability studies, i.e., long term, auto-sampler and freeze/thaw cycles. The method was successfully applied for the evaluation of pharmacokinetics of SASP, SP and 5-ASA after single oral doses of 250 mg SASP to 10 healthy volunteers.

Aptamer-functionalized PEG-PLGA nanoparticles for enhanced anti-glioma drug delivery.

Targeted delivery of therapeutic nanoparticles in a disease-specific manner represents a potentially powerful technology especially when treating infiltrative brain tumors such as gliomas. We developed a nanoparticulate drug delivery system decorated with AS1411 (Ap), a DNA aptamer specifically binding to nucleolin which was highly expressed in the plasma membrane of both cancer cells and endothelial cells in angiogenic blood vessels, as the targeting ligand to facilitate anti-glioma delivery of paclitaxel (PTX). Ap was conjugated to the surface of PEG-PLGA nanoparticles (NP) via an EDC/NHS technique. With the conjugation confirmed by Urea PAGE and XPS, the resulting Ap-PTX-NP was uniformly round with particle size at 156.0 ± 54.8 nm and zeta potential at -32.93 ± 3.1 mV. Ap-nucleolin interaction significantly enhanced cellular association of nanoparticles in C6 glioma cells, and increased the cytotoxicity of its payload. Prolonged circulation and enhanced PTX accumulation at the tumor site was achieved for Ap-PTX-NP, which eventually obtained significantly higher tumor inhibition on mice bearing C6 glioma xenografts and prolonged animal survival on rats bearing intracranial C6 gliomas when compared with PTX-NP and Taxol(®). The results of this contribution demonstrated the potential utility of AS1411-functionalized nanoparticles for a therapeutic application in the treatment of gliomas.

Low molecular weight protamine-functionalized nanoparticles for drug delivery to the brain after intranasal administration.

The development of new strategies for enhancing drug delivery to the brain is of great importance in diagnostics and therapeutics of central nervous diseases. Low-molecular-weight protamine (LMWP) as a cell-penetrating peptide possesses distinct advantages including high cell translocation potency, absence of toxicity of peptide itself, and the feasibility as an efficient carrier for delivering therapeutics. Therefore, it was hypothesized that brain delivery of nanoparticles conjugated with LMWP should be efficiently enhanced following intranasal administration. LMWP was functionalized to the surface of PEG-PLA nanoparticles (NP) via a maleimide-mediated covalent binding procedure. Important parameters such as particle size distribution, zeta potential and surface content were determined, which confirmed the conjugation of LMWP to the surface of nanoparticle. Using 16HBE14o- cells as the cell model, LMWP-NP was found to exhibit significantly enhanced cellular accumulation than that of unmodified NP via both lipid raft-mediated endocytosis and direct translocation processes without causing observable cytotoxic effects. Following intranasal administration of coumarin-6-loaded LMWP-NP, the AUC(0-8 h) of the fluorescent probe detected in the rat cerebrum, cerebellum, olfactory tract and olfactory bulb was found to be 2.03, 2.55, 2.68 and 2.82 folds, respectively, compared to that of coumarin carried by NP. Brain distribution analysis suggested LMWP-NP after intranasal administration could be delivered to the central nervous system along both the olfactory and trigeminal nerves pathways. The findings clearly indicated that the brain delivery of nanoparticles could be greatly facilitated by LMWP and the LMWP-functionalized nanoparticles appears as a effective and safe carrier for nose-to-brain drug delivery in potential diagnostic and therapeutic applications.