Over recent years, cell surface nucleolin function as an anticancer target has attracted tons of attentions. To improve the antitumor efficacy, we developed a nucleolin targeting protein nanoparticle (NTPN) delivery system, in which human serum albumin (HSA) was used as drug carrier and a DNA aptamer named AS1411, which had high affinity to nucleolin, was used as targeting bullet. The HSA nanoparticles (NPs-PTX) were fabricated by a novel self-assembly method and then modified with AS1411 (Apt-NPs-PTX). The resulted Apt-NPs-PTX was spherical and PTX was dispersed as amorphous state. Fluorescence microscopy and flow cytometry showed that the uptake of Apt-NPs-PTX was much higher than NPs-PTX in MCF-7 cells, while in non-tumor cell lines MCF-10A and 3T3,the fluorescence intensity was lower when treated with Apt-NPs-PTX than that of NPs-PTX. In cytotoxicity study, Apt-NPs-PTX displayed an enhanced cytotoxicity in MCF-7 tumor cells and showed no cytotoxicity in MCF-10A normal cells. After the addition of endostatin, a nucleolin inhibitor, the internalization of Apt-NPs-PTX in cells was decreased markedly, suggesting nucleolin did mediate the transmembrane process of Apt-NPs-PTX. Our AS1411 modified NTPN delivery system displayed a high efficiency in target drug delivery and its antitumor ability was also outstanding.

Stearic acid-grafted chitosan oligosaccharide (CS-SA) micelle has been demonstrated as an effective gene carrier in vitro and in vivo. Although being advantageous for DNA package, protection and excellent cellular internalization, CS-SA based delivery system may lead to difficulties in the dissociation of polymer/DNA complexes in intracells. In this research, bovine serum albumin (BSA) with different isoelectric point value (4.7, 6.0 and 9.3) was synthesized and incorporated into CS-SA based gene delivery system. CS-SA/DNA binary complexes and CS-SA/BSA/DNA ternary complexes were then prepared and characterized. The binding ability of the CS-SA vector with DNA was not affected by the incorporation of BSA. However referring to the transfection activity, BSA of different isoelectric point value (pI) had distinct influence on the CS-SA/BSA/DNA complexes. CS-SA/BSA (4.7)/DNA and CS-SA/BSA (6.0)/DNA complexes had better transfection efficiency than binary complexes, especially CS-SA/BSA (4.7)/DNA complexes showed the highest transfection efficiency. On the contrary, CS-SA/BSA (9.3)/DNA complexes had undesirable performances. Interestingly, the incorporation of BSA (4.7) in CS-SA/DNA complexes significantly enhanced the dissociation of polymer/DNA complexes and improved the release of DNA intracellular without influencing their cellular uptake. The aforementioned results indicated that the acid group in protein played an important role in enhancing the transfection efficiency of CS/BSA/DNA complexes and the study provided the guideline in design of an efficient vector for DNA transfection.

Multidrug resistance (MDR) remains one of the major challenges for successful chemotherapy. Herein, we tried to develope a mitochondria targeted teniposide loaded self-assembled nanocarrier based on stearylamine (SA-TSN) to reverse MDR of breast cancer. SA-TSN was nanometer-sized spherical particles (31.59 ± 3.43 nm) with a high encapsulation efficiency (99.25 ± 0.21%). The MDR in MCF-7/ADR cells was obviously reduced by SA-TSN, which mainly attributed to the markedly reduced expression of P-gp, increased percentages in G2 phase, selectively accumulation in mitochondria, decrease of mitochondrial membrane potential, and greatly improved apoptosis. The plasma concentration of teniposide was greatly improved by SA-TSN, and the intravenously administered SA-TSN could accumulate in the tumor site and penetrate into the inner site of tumor in MCF-7/ADR induced xenografts. In particular, the in vivo tumor inhibitory efficacy of SA-TSN in MCF-7/ADR induced models was more effective than that of teniposide loaded self-assembled nanocarrier without stearylamine (TSN) and teniposide solution (TS), which verified the effectiveness of SA-TSN in reversal of MDR. Thereby, SA-TSN has potential to circumvent the MDR for the chemotherapy of breast cancer.

We have developed a new method for assembling targeted nanoparticles that utilizes the complexation between targeting agents that contain boronic acids and polymer-drug conjugates that possess diols. Here, we report the first in vivo, antitumor results of a nanoparticle formed via this new assembly methodology. A nanoparticle consisting of a mucic acid polymer conjugate of camptothecin (CPT), MAP-CPT; and containing on average one Herceptin antibody is investigated in nude mice bearing HER2 overexpressing BT-474 human breast cancer tumors. Nontargeted MAP-CPT and antibody-containing MAP-CPT nanoparticles of ca. 30-40 nm diameter and slightly negative zeta potential show prolonged in vivo circulation and similar biodistributions after intravenous tail vein injections in mice. The maximum tolerated dose (MTD) of the nontargeted and Herceptin-containing MAP-CPT nanoparticles are found to be 10 and 8 mg CPT/kg, respectively, in mice. Mice bearing BT-474 human breast tumors treated with nontargeted MAP-CPT nanoparticles at 8 mg CPT/kg show significant tumor growth inhibition (mean tumor volume of 63 mm(3)) when compared to Irinotecan at 80 mg/kg (mean tumor volume of 575 mm(3)) and CPT at 8 mg/kg (mean tumor volume of 808 mm(3)) at the end of the study. Herceptin antibody treatment at 5.9 mg/kg results in complete tumor regressions in 5 out of 8 mice, with a mean tumor volume of 60 mm(3) at the end of the study. Mice treated with MAP-CPT nanoparticles at 1 mg CPT/kg do not show tumor inhibition. However, all mice receiving administrations of MAP-CPT nanoparticles (1 mg CPT/kg) that contain on average a single Herceptin molecule per nanoparticle (5.9 mg Herceptin equivalent/kg) show complete tumor regression by the end of the study. These results demonstrate that the antitumor efficacy of nanoparticles carrying anticancer drugs can be enhanced by incorporating on average a single antibody.

Irinotecan is a powerful anti-cancer drug with severe systemic side effects that limit its clinical application. Drug-targeted delivery with non-invasive methods is required to enhance the drug concentration locally and to reduce these undesirable events. Microbubble-assisted ultrasound has become a promising method for non-invasive targeted drug delivery. The aim of this study is to evaluate the therapeutic effectiveness of in-vitro and in-vivo irinotecan delivery based on the combination of ultrasound and microbubbles. In the present study, in-vitro results showed that the irinotecan treatment with microbubble-assisted ultrasound induced a significant decrease in cell viability of human glioblastoma cells. Moreover, using subcutaneous glioblastoma xenografts, the in-vivo preclinical study in nude mice demonstrated that this therapeutic protocol led to a decrease in tumor growth and perfusion and an increase of tumor necrosis. The conclusions drawn from this study demonstrate the promising potential of this therapeutic approach for the anti-cancer targeted therapy.

Today, early characterization of drug properties by the Biopharmaceutics Classification System (BCS) has attracted significant attention in pharmaceutical discovery and development. In this direction, the present report provides a systematic study of the development of a BCS-based provisional classification (PBC) for a set of 322 oral drugs. This classification, based on the revised aqueous solubility and the apparent permeability across Caco-2 cell monolayers, displays a high correlation (overall 76%) with the provisional BCS classification published by World Health Organization (WHO). Current database contains 91 (28.3%) PBC class I drugs, 76 (23.6%) class II drugs, 97 (31.1%) class III drugs, and 58 (18.0%) class IV drugs. Other approaches for provisional classification of drugs have been surveyed. The use of a calculated polar surface area with a labetalol value as a high permeable cutoff limit and aqueous solubility higher than 0.1 mg/mL could be used as alternative criteria for provisionally classifying BCS permeability and solubility in early drug discovery. To develop QSPR models that allow screening PBC and BCS classes of new molecular entities (NMEs), 18 statistical linear and nonlinear models have been constructed based on 803 0-2D Dragon and 126 Volsurf+ molecular descriptors to classify the PBC solubility and permeability. The voting consensus model of solubility (VoteS) showed a high accuracy of 88.7% in training and 92.3% in the test set. Likewise, for the permeability model (VoteP), accuracy was 85.3% in training and 96.9% in the test set. A combination of VoteS and VoteP appropriately predicts the PBC class of drugs (overall 73% with class I precision of 77.2%). This consensus system predicts an external set of 57 WHO BCS classified drugs with 87.5% of accuracy. Interestingly, computational assignments of the PBC class reasonably correspond to the Biopharmaceutics Drug Disposition Classification System (BDDCS) allocations of drugs (accuracy of 63.3-69.8%). A screening assay has been simulated using a large data set of compounds in different drug development phases (1, 2, 3, and launched) and NMEs. Distributions of PBC forecasts illustrate the current status in drug discovery and development. It is anticipated that a combination of the QSPR approach and well-validated in vitro experimentations could offer the best estimation of BCS for NMEs in the early stages of drug discovery.

Management of a lipophilic-hydrophilic balance is a key element in drug design to achieve desirable pharmacokinetic characters. Therefore we have created unique modular molecules, symmetrically branched oligoglycerols (BGL), as an alternative way to endow hydrophobic molecules with sufficient hydrophilicity. We have successfully demonstrated amelioration of the water-solubility and thermal stability of several hydrophobic agents by covalent conjugation to BGL so far. However, it has not been clarified whether the molecular modification by BGL also improves the pharmacological and/or pharmacokinetic properties indeed. Recently, we synthesized a novel BGL-prodrug derivative of fenofibrate, which is an anti-hyperlipidemic agent and one of the most hydrophobic medicinal compound currently used clinically, by conjugating fenofibric acid to symmetrically branched glycerol trimer (BGL003), the simplest BGL. We have previously demonstrated that the hydrophilicity and water-solubility of fenofibrate are improved more than 2,000 times just by conjugation to the BGL003. To verify our hypothesis that the prodrug strategy with BGL should improves pharmacological efficacy and pharmacokinetic properties of extremely hydrophobic agents such as fenofibrate by the rise in hydrophilicity, we evaluated BGL003-prodrug derivative of fenofibrate (FF-BGL) using rodent models. Here we demonstrate that the lipid-lowering effects of fenofibrate are much potentiated by chemical conjugation to BGL003 without exhibiting significant toxicity. Plasma concentration of fenofibric acid, an active metabolite of fenofibrate, after single oral administration of FF-BGL was more than 3-times higher than that of fenofibrate, in accordance. In fasting rats, plasma concentration of fenofibric acid after fenofibrate administration was curtailed into less than half of that in ad libitum-fed rats, while FF-BGL showed about the same plasma level even in the starving rats. This is the first report showing that BGL-prodrug improves pharmacological and pharmacokinetic properties as well as hydrophilicity of highly hydrophobic compounds. Furthermore, prodrug strategy using BGL suggests the possibility of diminishing the food-drug interaction effects, which should be advantageous for promoting drug compliance. BGL will be a suitable prodrug strategy to ameliorate physical, pharmacological and pharmacokinetic characteristics of extremely hydrophobic compounds.

This study proposes use of the phase separation of immiscible polymer blends as a formulation approach to improve the stabilization and solubilisation of amorphous molecular dispersions of poorly soluble drugs. This approach uses the phase separation and different drug solubilisation properties of the two immiscible polymers in the blend to optimize drug loading and stabilisation. The model system tested in this study is a EUDRAGIT® E PO-PVP-VA 50/50 (w/w) blend loaded with felodipine via hot melt extrusion. The phase separation behavior of the polymer blend and drug loaded polymer blend formulations were characterized using a range of thermal (MTDSC), spectroscopic (ATR-FTIR) and imaging (AFM and nano-thermal imaging) techniques. The polymer blend formulations demonstrated superior performance in drug release as well as stabilisation against stressed temperature, stressed humidity and mechanical milling in comparison to the drug-polymer binary systems. This is attributed to the configuration of the phase separated micro-structure of the polymer blend formulations where the hydrophilic polymer domains host high concentrations of molecularly dispersed drug which are protected from moisture induced recrystallisation on aging by the hydrophobic polymer domains. Additionally drug incorporation as a molecular dispersion in different polymer phases reduces the drug recrystallisation tendency on aging under high temperatures and during milling.

Therapeutic efficacy of glycoproteins is affected by many factors, including molecular size and net charge; both are influenced by the presence and composition of glycan structures. Human alpha 1-antitrypsin (A1AT) was cloned and expressed in human embryonic kidney cells (HEK293) that are capable of mammalian glycosylation. Utilizing PCR based site directed mutagenesis new A1AT variants were created with single, double, or triple additional N glycosylation sites to the three naturally occurring N glycosylation sites. Due to the supplementary N glycans, the A1AT variants exhibited an increased molecular weight. Retention of inhibitory activity was shown via trypsin inhibitory assay. The A1AT variants were treated with PNGase F and the resulting N glycans were analyzed by MALDI-TOF mass spectrometry. The N glycan profile of the recombinant A1AT variants was mostly composed of monofucosylated bi-, tri- and tetraantennary complex type N glycans, with a tendency towards higher antennary structures compared to the wild type. The relevance of N glycosylation in A1AT for the circulatory serum half life was demonstrated in CD1 mice. The A1AT neoglycoprotein with an additional N glycosylation site at position N123 exhibited a 62 % increase in serum half-life. Additionally, using a two-compartment model, the A1AT variants exhibited increased α-phase values, especially N123 (223 %) and N201 (255 %). The results suggest the recombinant A1AT neoglycoprotein as a serious alternative to A1AT derived from human plasma.

The human Organic Cation/ Ergothioneine Transporter1 (hOCTN1, gene symbol SLC22A4) is responsible for the cellular uptake of substances, such as L-ergothioneine, which is an important antioxidant in mammalian cells. The common-function-altered variant L503F-hOCTN1 has been associated with susceptibility to Crohn's disease in certain populations. Previously we identified eight novel nonsynonymous single nucleotide polymorphisms (SNPs) in the SLC22A4 gene in the Chinese and Indian populations of Singapore. The present study evaluated the impact of these novel SNPs on hOCTN1 transport function in HEK-293 cells. Transport uptake assays with L-ergothioneine were used to assess the function of the variant transporters. Cell surface biotinylation and western blot analysis were used to characterize cellular transporter expression. Comparative modelling was used to locate amino acid substitutions in the topology of hOCTN1 in order to account for altered transport function. Transporter activity was markedly impaired in four of the naturally occurring hOCTN1 variants (R63H, R83P, G482D and I500N). Multiple glycosylated isoforms of hOCTN1 proteins were identified in the plasma membrane and in the whole cell. Either the total cellular or membrane expression of the functionally deficient transporter variants was lower than that of the wild-type hOCTN1. The underlying mechanism involves both impaired transporter-substrate binding affinity and turn-over rate. Considered together, several naturally occurring SNPs in the SLC22A4 gene encode variant hOCTN1 transporters that may impact on the cellular uptake of L-ergothioneine and other substrates, with the potential to influence the antioxidant capacity of human cells.