Efficacy Interactions of PEG–DOX–N-acetyl Glucosamine Prodrug Conjugate for Anticancer Therapy

Present investigation is exploring structure–biocompatibility interaction of tumour targeted polyethylene glycol (PEG) based drug conjugate of doxorubicin using N-acetyl glucosamine as targeting ligand. The synthesized polymer drug conjugate was evaluated for particle size, zeta potential, molecular weight, haemolysis activity, cytotoxicity, protein binding and in vitro receptor (lectin) binding study. The particle size of synthesized conjugate was observed to be around 30 nm with polydispersability index of 0.213 indicating mono-disperse particles. Fluorescence quenching assay addressed relatively lower binding interactions of polymer drug conjugate to bovine serum albumin in comparison with free doxorubicin which may be governed to the hydrophilicity of polyethylene glycol and N-acetyl glucosamine. The cell compatibility and haemolysis study showed that PEG drug conjugate was nontoxic and biocompatible, which recommends the suitability of polymer drug conjugates for delivering biological active agents systemically. In vitro ligand–lectin receptor binding assays of synthesized targeted polymer conjugate suggest the possibility of promising interaction of N-acetyl glucosamine in vivo. Thus, the study indicated the suitability of N-acetyl glucosamine anchored targeted polymer drug conjugate in delivering bio-therapeutics for specifically targeting to tumour tissues.     

Influence of Plasma Protein Binding on Pharmacodynamics: Estimation of In Vivo Receptor Affinities of β Blockers Using a New Mechanism-Based PK–PD Modelling Approach

The objective of this investigation was to examine in a systematic manner the influence of plasma protein binding on in vivo pharmacodynamics. Comparative pharmacokinetic–pharmacodynamic studies with four β blockers were performed in conscious rats, using heart rate under isoprenaline-induced tachycardia as a pharmacodynamic endpoint. A recently proposed mechanism-based agonist–antagonist interaction model was used to obtain in vivo estimates of receptor affinities (K_B,vivo). These values were compared with in vitro affinities (K_B,vitro) on the basis of both total and free drug concentrations. For the total drug concentrations, the K_B,vivo estimates were 26, 13, 6.5 and 0.89 nM for S(?)-atenolol, S(?)-propranolol, S(?)-metoprolol and timolol. The K_B,vivo estimates on the basis of the free concentrations were 25, 2.0, 5.2 and 0.56 nM, respectively. The K_B,vivo–K_B,vitro correlation for total drug concentrations clearly deviated from the line of identity, especially for the most highly bound drug S(?)-propranolol (ratio K_B,vivo/K_B,vitro ～6.8). For the free drug, the correlation approximated the line of identity. Using this model, for β-blockers the free plasma concentration appears to be the best predictor of in vivo pharmacodynamics. © 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3816–3828, 2009     

Epithelial transport of Noscapine across cell monolayer and influence of absorption enhancers on in vitro permeation and bioavailability: implications for intestinal absorption

Abstract

The purpose of this study was to investigate the permeation of Noscapine (Nos) across the Caco-2 and Madin–Darby canine kidney (MDCK) cell monolayers and to evaluate the influence of absorption enhancers on in vitro and in vivo absorption of Nos. The bidirectional transport of Nos was studied in Caco-2 and MDCK cell monolayers at pH 5.0–7.8. The effect of 0.5% w/v chitosan (CH) or Captisol (CP) on Nos permeability was investigated at pH 5.0 and 5.8. The effect of 1–5% w/v of CP on oral bioavailability of Nos (150?mg/kg) was evaluated in Sprague–Dawley rats. The effective permeability coefficients (P_eff) of Nos across Caco-2 and MDCK cell monolayers was found to be in the order of pH 5.0?>?5.8?>?6.8?>?7.8. The efflux ratios of P_eff?<?2 demonstrated that active efflux does not limit the absorption of Nos. The use of CH or CP have shown significant (***, p?<?0.001) enhancement in P_eff of Nos across cell monolayer compared with the control group. The CP (1–5% w/v) based Nos formulations resulted in significant (***, p?<?0.001) increase in the bioavailability of Nos compared with Nos solution. The use of CP represents viable approach for enhancing the oral bioavailability of Nos and reducing the required dose.     

Protein binding of methohexital. Study of parameters and modulating factors using the equilibrium dialysis technique

This paper describes the parameters that characterize methohexital—albumin binding and the influence of physiological or analytical factors on this binding. Two useful and reproducible methods for measuring the free concentration—equilibrium dialysis (ED) and ultrafiltration (UF)—are described and their performances are compared. Methohexital binds exclusively to albumin according to a two-class binding model. The first is a saturable class site of high affinity constant (K_A = 11 200 M⁻¹) and a number of sites per albumin molecule of 1. The second is a non-saturable site of poorer affinity (nK_A = 810 M⁻¹). The bound fraction of methohexital in the therapeutic range and at physiological albumin concentration is 86.7 ± 0.9% in isolated albumin solution. In serum, it ranges from 80 to 84.5%, according to subjects (n = 6). Binding is inhibited by the presence of endogeneous compounds of serum (for a given albumin concentration the bound fraction decreases from 90.3% in isolated albumin solution to 82.6% in serum), probably by free fatty acids. An increase in the bound fraction is observed when the pH is increased from 7 to 9. This phenomenon may be explained by a higher affinity of the drug towards the basic (B-form) conformation of the albumin molecule, in analogy with the close barbiturate thiopental. A decrease in the bound fraction against temperature is shown, as though binding forces diminished with increase in temperature. Indeed, the binding modification is less pronounced in the presence of serum endogenous compounds. As expected, there is no evidence of any effect of heparin anticoagulant on the bound fraction. Methohexital binding is strongly modified by the albumin concentration; the bound fractions change from 67 to 91% in the albumin range 150–900 μM.     

Mechanochemical preparation of triptolide-loaded self-micelle solid dispersion with enhanced oral bioavailability and improved anti-tumor activity

Triptolide (TP), a compound isolated from a Chinese medicinal herb, possesses potent anti-tumor, immunosuppressive, and anti-inflammatory properties, but was clinically limited due to its poor solubility, bioavailability, and toxicity. Considering the environment-friendly, low-cost mechanochemical techniques and potential dissolution enhancement ability of Na₂GA, an amorphous solid dispersion (Na₂GA&TP-BM) consisting of TP and Na₂GA were well-prepared to address these issues. The performance of Na₂GA&TP-BM was improved through ball milling, such as from crystalline state to an amorphous solid dispersion, suitable nano micelle size and surface potential, and increased solubility. This change had a significant improvement of pharmacokinetic behavior in mice and could be able to extend the blood circulation time of the antitumor drug. Moreover, in vitro and in vivo anti-tumor study showed that Na₂GA&TP-BM displayed more potent cytotoxicity to tumor cells. The work illustrated an environment-friendly and safe preparation of the TP formulation, which was promising to enhance the oral bioavailability and antitumor ability of TP, might be considered for efficient anticancer therapy.     

Heme lysis of the bloodstream forms of trypanosoma brucei

The bloodstream forms of Trypanosoma brucei were found to be lysed in vitro by the addition of heme. For each μg/ml of heme, approximately 2 × 10⁵ organisms/ml were killed in 20min at 37°. The mechanism for the lysis is believed to be mediated by heme-induced homolytic cleavage of intracellular H₂O₂ (70 μM) to form hydroxyl radicals. The highly reactive radicals particularly disrupt various membranes and eventually kill the organisms. Heme and several porphyrins which are active in vitro are inactive in vivo due to very tight binding to albumin and other body proteins. Hematoporphyrin, which binds albumin weakly, is curative in vivo (160 mg/kg). Hopefully, new and more effective agents will be found that will enter the trypanosomes and generate free radicals to kill the cells.     

A comparative study of the anticlastogenic effects of chlorophyllin on N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or 7,12-dimethylbenz (α) anthracene (DMBA) induced micronuclei in mammalian cells in vitro and in vivo

Chlorophyllin (CHL), a water soluble derivative of chlorophyll has been shown to have both anticarcinogenic and antigenotoxic properties. We evaluated the protective effects of CHL (25 μM in vitro, 4 and 100 mg/kg. b.w.) on the clastogenic action of two model carcinogens, MNNG and DMBA (25 μM and 2 μM respectively) in vitro on human hepatoma cells (HepG2) and (40 mg and 25 mg/Kg/b.w. respectively) in vivo on bone marrow of mice, using the frequencies of induced micronuclei as the end point. Pre-, post- and simultaneous treatments with CHL and the carcinogen were carried out in vitro. With MNNG, only simultaneous treatment with CHL was effective in reducing the frequencies of MN, suggesting a direct interaction between CHL and MNNG. A statistically significant reduction in of DMBA induced MN was found by pre-or post treatment with CHL while a reduction (not significant) was observed by simultaneous treatment. In in vivo experiments, CHL pre-treatment did not affect the frequencies of MN in PCEs of bone marrow induced by MNNG or DMBA. However, increased the toxic effect of DMBA (reduction in percent of PCEs) was accompanied by a reduction in the induced frequencies of MN. CHL was not clastogenic in both in vitro and in vivo tests. It can be concluded that (a) CHL has a protective effect against MNNG and DMBA. This effect is dependent upon the protocol employed in in vitro experiments. In vivo, CHL did not have a protective effect against MNNG and DMBA. A protective effect of CHL against DMBA was evident only at high toxic levels.     

Antigenotoxicity properties of Copaifera multijuga oleoresin and its chemical marker,the diterpene (−)-copalic acid

In view of the biological activities and growing therapeutic interest in oleoresin obtained from Copaifera multijuga, this study aimed to determine the genotoxic and antigenotoxic potential of this oleoresin (CMO) and its chemical marker, diterpene (?)-copalic acid (CA). The micronucleus (MN) assay in V79 cell cultures and the Ames test were used for in vitro analyses, as well as MN and comet assays in Swiss mice for in vivo analyses. The in vitro genotoxicity/mutagenicity results showed that either CMO (30, 60, or 120 µg/ml-MN assay; 0.39–3.12 mg/plate-Ames test) or CA (2.42; 4.84, or 9.7 µg/ml-MN assay; 0.39–3.12 mg/plate-Ames test) did not induce a significant effect on the frequency of MN and number of revertants, demonstrating an absence of genotoxic and mutagenic activities, respectively, in vitro. In contrast, these natural products significantly reduced the frequency of MN induced by methyl methanesulfonate (MMS), and exerted a marked inhibitory effect against indirect-acting mutagens in the Ames test. In the in vivo test system, animals treated with CMO (6.25 mg/kg b.w.) exhibited a significant decrease in rate of MN occurrence compared to those treated only with MMS. An antigenotoxic effect of CA was noted in the MN test (1 and 2 mg/kg b.w.) and the comet assay (0.5 mg/kg b.w.). Data suggest that the chemical marker of the genus Copaifera, CA, may partially be responsible for the observed chemopreventive effect attributed to CMO exposure.

Abbreviations: 2-AA, 2-anthramine; 2-AF, 2-aminofluorene; AFB₁, aflatoxin B₁; B[a]P, benzo[a]pyrene; BOD, biological oxygen demand; BPDE, benzo[a]pyrene-7,8-diol-9,10-epoxide; CA, (?)-copalic acid; CMO, oleoresin of Copaifera multijuga, DMEM, Dulbecco`s Modified Eagles`s Medium; DMSO, dimethylsulfoxide; EMBRAPA, Brazilian agricultural research corporation; GC–MS, gas chromatography–mass spectrometry; HAM-F10, nutrient mixture F-10 Ham; HPLC, high performance liquid chromatography; LC–MS, liquid chromatography–mass spectrometry; MI, mutagenic index; MMC, mitomycin C; MMS, methyl methanesulfonate; MN, micronucleus; MNPCE, micronucleated polychromatic erythrocyte; NCE, normochromatic erythrocyte; NDI, nuclear division index; NMR, nuclear magnetic resonance; NPD, 4-nitro-o-phenylenediamine; PBS, phosphate-buffered saline; PCE, polychromatic erythrocyte; SA, sodium azide; V79, Chinese hamster lung fibroblast.     

Involvement of Rat Organic Cation Transporter 2 in the Renal Uptake of Jatrorrhizine

Jatrorrhizine is a protoberberine alkaloid derived from Coptis chinensis concentrated extremely in rat kidney. In the present study, the involvement of rat organic cation transporter 2 (rOCT2) in the renal uptake of jatrorrhizine in rat was investigated through in vitro and in vivo experiments. Saturable and nonsaturable uptakes of jatrorrhizine were observed in rat kidney slices and rOCT2–Madin–Darby canine kidney (MDCK) cells. Michaelis–Menten constants of 677.8 and 21.0 μM, maximum uptake rate of 123 (pmol/min)/mg kidney and 13.7 (pmol/min)/mg protein, and nonsaturable uptake clearance of 0.054 (μL/min)/mg kidney and 0.032 (μL/min)/mg protein were observed in rat kidney slices and rOCT2–MDCK cells, respectively. As inhibitors of rOCT2, corticosterone, verapamil, and cimetidine can inhibit jatrorrhizine uptake in rat kidney slices and rOCT2–MDCK cells. Their median inhibitory concentration in rat kidney slices was 7, 78, and 538 μM, whereas that in rOCT2–MDCK cells was 1.07, 86.5, and 151.8 μM. Coadministration with 20 mg/kg corticosterone, a selective inhibitor of rOCT2, reduced the jatrorrhizine concentration in the cortex and medulla in the in vivo experiment. Thus, rOCT2 is mainly responsible for the renal uptake of jatrorrhizine in rat and in the regulation of jatrorrhizine concentration in the kidney.     

Anti-tumor activity of paclitaxel through dual-targeting lipoprotein-mimicking nanocarrier

In the present study, we devised a strategy that paclitaxel (PTX) with lipid and octadecylamine were prepared to lipid nanoparticle (PTX-LNP) with positive charge, folic acid-modified bovine serum albumin (FB)-coated surface of PTX-LNP through electrostatic attraction and generated the lipoprotein-mimicking nanocomplex (FB-PTX-LNP) for dual-targeting therapy. Bovine serum albumin (BSA) was used as the protein model due to its specific targeting to tumor by increased transendothelial gp60-mediated transport and increased intratumoral accumulation as a result of the secreted protein, acidic and rich in cysteine (SPARC)–albumin interaction. The further conjugating folic acid to BSA achieved the dual active targeting. In vitro cytotoxicity tests suggested FB-PTX-LNP and BSA-PTX-LNP exhibited significantly higher cytotoxic activity against MCF-7 and HepG2 cells compared to PTX-LNP. The cellular uptake experiments indicated that FB-coumarin-6-LNP modified with dual-targeting had a faster and greater cellular uptake when compared to BSA-coumarin-6-LNP and coumarin-6-LNP by MCF-7 cells. Thus, both BSA and FA did play roles in in vitro cytotoxicity and cellular uptake. Furthermore, the targeting ability and therapeutic efficacy of FB-PTX-LNP were assessed in vivo. FB-PTX-LNP produced very marked targeting ability and anti-tumor activity in MDA-MB-231 tumor-bearing mice. These results indicate the protein–lipid nanocomplex FB-PTX-LNP is a potential nanocarrier for Paclitaxel dual-targeting to tumor.     

Long-circulating self-assembled cholesteryl albumin nanoparticles enhance tumor accumulation of hydrophobic anticancer drug

The objective of this study was to develop an albumin nanoparticle with improved stability and drug loading capacity. Generation of nanomaterials having physiologically stable and high potential for drug delivery is still challenging. Herein we synthesized cholesteryl albumin conjugate using N,N-disuccinimidyl carbonate coupling reagent and prepared paclitaxel-loaded cholesteryl albumin nanoparticle (PTX-Chol-BSA) by self-assembly with the mean hydrodynamic diameter of 147.6 ± 1.6 nm and with high loading capacity. PTX-Chol-BSA nanoparticle showed much higher colloidal stability than a simple complex of PTX and BSA (PTX–BSA) and sustained release profile. PTX-Chol-BSA nanoparticles exhibited greater cellular uptake and cytotoxicity in B16F10 and MCF-7 cancer cell lines, as compared with PTX in Cremophor EL/ethanol (PTX-Cre/EtOH) and PTX–BSA formulations. A pharmacokinetic study in tumor-bearing mice showed that the area under the concentration–time curve (AUC_{0–8 h}) following the administration of PTX-Chol-BSA was 1.6–2-fold higher than those following the administration of PTX-Cre/EtOH and PTX–BSA. In addition, the tumor AUC_{0–8 h} of PTX-Chol-BSA was around 2-fold higher than that of PTX–BSA. Furthermore, in vivo antitumor efficacy results revealed that PTX-Chol-BSA nanoparticles have greater antitumor efficacy. In conclusion, we demonstrated the potential of PTX-Chol-BSA nanoparticles for anti-tumor chemotherapy, with enhanced in vitro and in vivo behaviors, as compared to PTX–BSA and PTX-Cre/EtOH.     

MDCK (Madin-Darby Canine Kidney) Cells: A Tool for Membrane Permeability Screening

The goal of this work was to investigate the use of MDCK (Madin–Darby canine kidney) cells as a possible tool for assessing the membrane permeability properties of early drug discovery compounds. Apparent permeability (P_app) values of 55 compounds with known human absorption values were determined using MDCK cell monolayers. For comparison, P_app values of the same compounds were also determined using Caco‐2 cells, a well‐characterized in vitro model of intestinal drug absorption. Monolayers were grown on 0.4‐µm Transwell‐COL membrane culture inserts. MDCK cells were seeded at high density and cultured for 3 days, and Caco‐2 cells were cultured under standard conditions for 21 to 25 days. Compounds were tested using 100 µM donor solutions in transport medium (pH 7.4) containing 1% DMSO. The P_app values in MDCK cells correlated well with those in Caco‐2 cells (r² = 0.79). Spearman's rank correlation coefficient for MDCK P_app and human absorption was 0.58 compared with 0.54 for Caco‐2 P_app and human absorption. These results indicate that MDCK cells may be a useful tool for rapid membrane permeability screening.     

Study of curcumin and genistein interactions with human serum albumin

Curcumin, the yellow pigment from the rhizoma of Curcuma longa, is a widely studied polyphenolic compound which has a variety of biological activity as anti-inflammatory and antioxidative agent. Genistein one of the flavonoids found in soybean and chickpeas inhibits DNA strand breaks acting as a direct scavenger of reactive oxygen species. Human serum albumin (HSA) with high affinity binding sites is a major transporter for delivering several endogenous compounds and drugs in vivo. The aim of this study was to examine the interactions of curcumin and genistein with human serum albumin at physiological conditions, using constant protein concentration and various pigment contents. FTIR, UV–Visible, CD and fluorescence spectroscopic methods were used to analyse drug binding mode, the binding constant and the effects of pigment complexation on HSA stability and conformation. Structural analysis showed that curcumin and genistein bind HSA via polypeptide polar groups with overall binding constants of K_curcumin = 5.5 (±0.8) × 10⁴ M⁻¹ and K_genistein = 2.4 (±0.40) × 10⁴ M⁻¹. The number of bound pigment (n) is 1.33 for curcumin and 1.49 for genistein. The HSA conformation was altered by pigment complexation with reduction of α-helix and increase of random coil and turn structures suggesting a partial protein unfolding.     

PEGylated dendritic polyglycerol conjugate targeting NCAM-expressing neuroblastoma: Limitations and challenges

Neural cell adhesion molecule (NCAM) is found to be a stem-cell marker in several tumor types and its overexpression is known to correlate with increased metastatic capacity. To combine extravasation- and ligand-dependent targeting to NCAM overexpressing-cells in the tumor microenvironment, we developed a PEGylated NCAM-targeted dendritic polyglycerol (PG) conjugate. Here, we describe the synthesis, physico-chemical characterization and biological evaluation of a PG conjugate bearing the mitotic inhibitor paclitaxel (PTX) and an NCAM-targeting peptide (NTP). PG-NTP-PTX-PEG was evaluated for its ability to inhibit neuroblastoma progression in vitro and in vivo as compared to non-targeted derivatives and free drug. NCAM-targeted conjugate inhibited the migration of proliferating endothelial cells, suggesting it would be able to inhibit tumor angiogenesis. The targeting conjugate provided an improved binding and uptake on IMR-32 cells compared to non-targeted control. However, these results did not translate to our in vivo model on orthotopic neuroblastoma bearing mice.     

A novel αVβ3 ligand-modified HPMA copolymers for anticancer drug delivery

The integrin α_Vβ₃ receptor emerged as one of the most promising targets owing to its high expression on the surface of various malignant tumour cells and tumour angiogenesis endothelial cells, but with little expression in mature endothelial cells and the majority of normal cells. Here, we report a new targeting ligand FQSIYPpIK (FQS) with high affinity to integrin α_Vβ₃ receptor. To take the advantage of the particular interaction between FQS and integrin α_Vβ₃ receptor, FQS was linked to N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers. A model drug doxorubicin (DOX) was simultaneously conjugated to the same HPMA copolymers via pH-sensitive hydrazone linkages (FQS–HPMA–DOX). In in vitro study, FQS–HPMA–DOX could be internalised into α_Vβ₃ receptor-overexpressed B16F10 cells via a highly specific ligand???receptor pathway (5.0 times and 4.5 times higher cellular internalisation than HPMA–DOX and a scrambled peptide (s)-FQS (sequence: SYFIPKQIp)-modified copolymers ((s)-FQS–HPMA–DOX)). It is worth noting that compared with the classical α_Vβ₃ ligand cRGDfK-modified HPMA copolymers (cRGDfK–HPMA–DOX), FQS–HPMA–DOX also showed superior targeting efficiency. In in vivo study in the B16F10 melanoma bearing mice model showed the antitumour efficiency of FQS–HPMA–DOX (83.9%) were significantly higher than HPMA–DOX (44.9%) and cRGDfK–HPMA–DOX (77.5%). These results suggest that FQS peptide can act as an effective targeting ligand for the delivery of therapeutic agents.     

Adult age and ex vivo protein binding of lorazepam, oxazepam and temazepam in healthy subjects

AIM

To see if adult age correlates with ex vivo protein binding of lorazepam, oxazepam and temazepam in healthy subjects.

METHODS

Sixty healthy drug free subjects were recruited in the age groups 18–39, 40–64 and ≥65 years. Plasma albumin concentrations were determined. Ex vivo unbound fractions (f_u) were assessed by spiking samples and measuring the free and total concentrations.

RESULTS

No correlation of age with f_u was seen. The study was powered to demonstrate a change in f_u of ≥7–10%. A decline in plasma albumin concentration of ∼0.03 g l⁻¹ year⁻¹ was seen with increasing age (P = 0.032) and was associated with increased f_u of lorazepam (P = 0.009) and oxazepam (P = 0.014).

CONCLUSIONS

There was no association of adult age with ex vivo f_u of lorazepam, oxazepam or temazepam in healthy subjects.     

Galactosylated chitosan triptolide nanoparticles for overcoming hepatocellular carcinoma: Enhanced therapeutic efficacy,low toxicity,and validated network regulatory mechanisms

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths worldwide. Current therapies present significant limitations. Triptolide (TP) is highly effective against multiple cancers including HCC. However, high toxicity, low water solubility, and unknown therapeutic targets limit its clinical application. Herein, we designed galactosylated-chitosan-TP-nanoparticles (GC-TP-NPs) with high drug loading capacities for targeted delivery to HCC. In addition to a sustained release pattern, an efficient asialoglycoprotein receptor mediated cellular uptake in vitro, and high liver tumor accumulation in vivo, GC-TP-NPs showed lower systemic and male reproductive toxicities than free TP. Importantly, GC-TP-NPs retained the anti-cancer activities of the free TP, exerting the same pro-apoptotic and anti-proliferative effects on HCC cells in vitro, and displayed higher efficacies in reducing tumor sizes in vivo. Further investigation revealed that GC-TP-NPs induced cancer cell apoptosis via blocking TNF/NF-κB/BCL2 signaling. Collectively, GC-TP-NP represents a promising candidate in halting liver cancer progression while minimizing systemic toxicity.     

Effect of scorpion (Leiurus Quinquestriatus H and E) venom on lipid metabolism.

Injection of rats with a sublethal dose (200 μg per kg) of Leiurus quinquestriatus venom, induces an increase in the total lipid, cholesterol and phospholipid content of the liver. In the serum an increase in the free and esterified cholesterol, esterified fatty acids, phosphatidylcholine, phosphatidylethanolamine and a decrease in free fatty acids, sphingomylin and lysolecithin was demonstrated. An increase in the albumin, alpha₁-and alpha₂-globulins, and a decrease in the beta and gamma globulins of the serum was also observed.     

pH-Sensitive PEGylated Liposomal Silybin: Synthesis,In Vitro and In Vivo Anti-Tumor Evaluation

The drug delivery systems improve the efficacy of chemotherapeutics through enhanced targeting and controlled release however, biological barriers of tumor microenvironment greatly impede the penetration of nanomedicine within the tumor. We report herein the fabrication of a PEG-detachable silybin (SLB) pH-sensitive liposome decorated with TAT-peptide. For this, Acyl hydrazide-activated PEG₂₀₀₀ was prepared and linked with ketone-derivatized DPPE via an acid-labile hydrazone bond to form mPEG₂₀₀₀-HZ-DPPE. TAT peptide was conjugated with a shorter -PEG₁₀₀₀-DSPE spacer and post-inserted into PEGylated liposome (DPPC: mPEG₂₀₀₀–DSPE: Chol). To prepare nanoliposomes (around 100 nm), first, a novel method was used to prepare SLB-Soya PC (SLB-SPC) complex, then this complex was incorporated into nanoliposomes. The pH-sensitivity and shielding effect of long PEG chain on TAT peptide was investigated using DiI liposome and FACS analysis. Pre-treatment to the lowered pH enhanced cellular association of TAT-modified pH-sensitive liposome due to the cleavage of hydrazone bond and TAT exposure. Besides, TAT-modified pH-sensitive liposomes significantly reduced cell viability compared to the plain liposome. In vivo results were very promising with pH-sensitive liposome by detaching PEG moieties upon exposure to the acidic tumor microenvironment, enhancing cellular uptake, retarding tumor growth, and prolonging the survival of 4T1 breast tumor-bearing BALB/c mice. TAT modification of pH-sensitive liposome improved cancer cell association and cytotoxicity and demonstrated potential intracellular delivery upon exposure to acidic pH. However, in in vivo studies, TAT as a targeting ligand significantly decreased the therapeutic efficacy of the formulation attributed to an inefficient tumor accumulation and higher release rate in the circulation. The results of this study indicated that pH-sensitive liposome containing SLB, which was prepared with a novel method with a significant SLB loading efficiency, is very effective in the treatment of 4T1 breast tumor-bearing BALB/c mice and merits further investigation.     

Brain Delivery of NAP with PEG-PLGA Nanoparticles Modified with Phage Display Peptides

Purpose

A phage-displayed peptide TGN was used as a targeting motif to help the delivery of NAP-loaded nanoparticles across the blood–brain barrier (BBB), which sets an obstacle for brain delivery of NAP in vivo.

Methods

Intracerebroventricular injection of Aβ_1-40 into mice was used to construct in vivo model of Alzheimer’s disease. The water maze task was performed to evaluate the effects of the NAP formulations on learning and memory deficits in mice. The neuroprotective effect was tested by detecting acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activity and conducting histological assays.

Results

Intravenous administration of NAP-loaded TGN modified nanoparticles (TGN-NP/NAP) has shown better improvement in spatial learning than NAP solution and NAP-loaded nanoparticles in Morris water maze experiment. The crossing number of the mice with memory deficits recovered after treatment with TGN-NP/NAP in a dose dependent manner. Similar results were also observed in AChE and ChAT activity. No morphological damage and no detectable Aβ plaques were found in mice hippocampus and cortex treated with TGN-NP/NAP.

Conclusions

TGN modified nanoparticles could be a promising drug delivery system for peptide and protein drug such as NAP to enter the brain and play the therapeutic role.