Selective Targeting and Eradication of Various Human Non-Small Cell Lung Cancer Cell Lines Using Self-Assembled Aptamer-Decorated Nanoparticles.

The leading cause of cancer mortality remains lung cancer (LC), of which non-small cell lung cancer (NSCLC) is the predominant type. Chemotherapy achieves only low response rates while inflicting serious untoward toxicity. Herein, we studied the binding and internalization of S15-aptamer (S15-APT)-decorated polyethylene glycol-polycaprolactone (PEG-PCL) nanoparticles (NPs) by various human NSCLC cell lines. All the NSCLC cell lines were targeted by S15-APT-decorated NPs. Confocal microscopy revealed variable levels of NP binding and uptake amongst these NSCLC cell lines, decreasing in the following order: Adenocarcinoma (AC) A549 cells > H2228 (AC) > H1299 (large cell carcinoma) > H522 (AC) > H1975 (AC). Flow cytometry analysis showed a consistent variation between these NSCLC cell lines in the internalization of S15-APT-decorated quantum dots. We obtained a temperature-dependent NP uptake, characteristic of active internalization. Furthermore, cytotoxicity assays with APT-NPs entrapping paclitaxel, revealed that A549 cells had the lowest IC50 value of 0.03 µM PTX (determined previously), whereas H2228, H1299, H522 and H1975 exhibited higher IC50 values of 0.38 µM, 0.92 µM, 2.31 µM and 2.59 µM, respectively (determined herein). Cytotoxicity was correlated with the binding and internalization of APT-NPs in the various NSCLC cells, suggesting variable expression of the putative S15 target receptor. These findings support the development of APT-targeted NPs in precision nanomedicine for individual NSCLC patient treatment.

Targeted nanomedicine modalities for prostate cancer treatment.

Prostate cancer (PC) is the second most common cause of death amongst men in the USA. Therapy of PC has been transformed in the past decade by introducing novel therapeutics, advanced functional imaging and diagnostic approaches, next generation sequencing, as well as improved application of existing therapies in localized PC. Treatment of PC at the different stages of the disease may include surgery, androgen deprivation therapy (ADT), chemotherapy and radiation therapy. However, although ADT has proven efficacious in PC treatment, its effectiveness may be temporary, as these tumors frequently develop molecular mechanisms of therapy resistance, which allow them to survive and proliferate even under conditions of testosterone deprivation, inhibition of androgen receptor signaling, or cytotoxic drug treatment. Importantly, ADT was found to induce key alterations which frequently result in the formation of metastatic tumors displaying a therapy refractory phenotype. Hence, to overcome these serious therapeutic impediments, novel PC cell-targeted therapeutic strategies are being developed. These include diverse platforms enabling specific enhanced antitumor drug uptake and increased intracellular accumulation. Studies have shown that these novel treatment modalities lead to enhanced antitumor activity and diminished systemic toxicity due to the use of selective targeting and decreased drug doses. The underlying mechanism of targeting and internalization is based upon the interaction between a selective ligand, conjugated to a drug-loaded nanoparticle or directly to an anti-cancer drug, and a specific plasma membrane biomarker, uniquely overexpressed on the surface of PC cells. Another targeted therapeutic approach is the delivery of unique anti-oncogenic signaling pathway-based therapeutic drugs, which are selectively cytotoxic to PC cells. The current paper reviews PC targeted modalities reported in the past 6 years, and discusses both the advantages and limitations of the various targeted treatment strategies.

Targeted Nanoparticles Harboring Jasmine-Oil-Entrapped Paclitaxel for Elimination of Lung Cancer Cells.

Selectively targeted drug delivery systems are preferable chemotherapeutic platforms, as they specifically deliver the drug cargo into tumor cells, while minimizing untoward toxic effects. However, these delivery systems suffer from insufficient encapsulation efficiency (EE), encapsulation capacity (EC), and premature drug release. Herein, we coencapsulated paclitaxel (PTX) and Jasmine oil (JO) within PEG-PCL nanoparticles (NPs), with an average diameter < 50 nm, selectively targeted to non-small cell lung cancer (NSCLC) cells, via S15-aptamer (APT) decoration. JO was selected as an "adhesive" oily core to enhance PTX entrapment, as JO and PTX share similar hydrophobicity and terpenoid structure. JO markedly enhanced EE of PTX from 23% to 87.8% and EC from 35 ± 6 to 74 ± 8 µg PTX/mg PEG-PCL. JO also markedly increased the residual amount of PTX after 69 h, from 18.3% to 65%. Moreover, PTX cytotoxicity against human NSCLC A549 cells was significantly enhanced due to the co-encapsulation with JO; the IC50 value for PTX encapsulated within JO-containing APT-NPs was 20-fold lower than that for APT-NPs lacking JO. Remarkably, JO-containing APT-NPs displayed a 6-fold more potent cell-killing, relatively to the free-drug. Collectively, these findings reveal a marked synergistic contribution of JO to the cytotoxic activity of APT-NP-based systems, for targeted PTX delivery against NSCLC, which may be readily applied to various hydrophobic chemotherapeutics.

Novel Selectively Targeted Multifunctional Nanostructured Lipid Carriers for Prostate Cancer Treatment.

Prostate cancer (PC) is the most common cancer in men over 50 and the 4th most prevalent human malignancy. PC treatment may include surgery, androgen deprivation therapy, chemotherapy, and radiation therapy. However, the therapeutic efficacy of systemic chemotherapy is limited due to low drug solubility and insufficient tumor specificity, inflicting toxic side effects and frequently provoking the emergence of drug resistance. Towards the efficacious treatment of PC, we herein developed novel selectively PC-targeted nanoparticles (NPs) harboring a cytotoxic drug cargo. This delivery system is based upon PEGylated nanostructured lipid carriers (NLCs), decorated with a selective ligand, targeted to prostate-specific membrane antigen (PSMA). NPs loaded with cabazitaxel (CTX) displayed a remarkable loading capacity of 168 ± 3 mg drug/g SA-PEG, encapsulation efficiency of 67 ± 1%, and an average diameter of 159 ± 3 nm. The time-course of in vitro drug release from NPs revealed a substantial drug retention profile compared to the unencapsulated drug. These NPs were selectively internalized into target PC cells overexpressing PSMA, and displayed a dose-dependent growth inhibition compared to cells devoid of the PSMA receptor. Remarkably, these targeted NPs exhibited growth-inhibitory activity at pM CTX concentrations, being markedly more potent than the free drug. This selectively targeted nano-delivery platform bears the promise of enhanced efficacy and minimal untoward toxicity.

Selective eradication of human non-small cell lung cancer cells using aptamer-decorated nanoparticles harboring a cytotoxic drug cargo.

Targeted cancer therapy is currently the leading modality to enhance treatment selectivity and efficacy, as well as to minimize untoward toxicity to healthy tissues. Herein, we devised and studied nanoparticles (NPs) composed of the biocompatible block-copolymer PEG-PCL entrapping the hydrophobic chemotherapeutic drug paclitaxel (PTX), which are targeted to human non-small cell lung cancer (NSCLC) cells. To achieve selective NSCLC targeting, these NPs were decorated with single-stranded oligonucleotide-based S15 aptamers (S15-APTs), which we have recently shown to serve as efficient tumor cell targeting ligands. Prepared without using surfactants, these 15 nm PEG-PCL/PTX NPs entered NSCLC cells via clathrin-mediated endocytosis. These NPs demonstrated efficient encapsulation of PTX, high selectivity to- and potent eradication of human A549 NSCLC cells, with a remarkable half maximal inhibitory concentration (IC50) of 0.03 µM PTX. In contrast, very high IC50 values of 1.7, 4.2, 43, 87, and 980 µM PTX were obtained towards normal human bronchial epithelial BEAS2B, cervical carcinoma HeLa, colon adenocarcinoma CaCo-2, neonatal foreskin fibroblast FSE, and human embryonic kidney HEK-293 cells, respectively. These results demonstrate 2-5 orders of magnitude difference in the selective cytotoxicity towards NSCLCs, reflecting a potentially outstanding therapeutic window. Moreover, the dual utility of aptamer-decorated NPs for both drug stabilization and selective tumor targeting was studied by increasing APT concentrations during NP "decoration". The optimal aptamer density on the surface of NPs for selective targeting, for high fluorescence diagnostic signal and for maintaining small particle size to enable endocytosis, was achieved by using 30 nM APTs during NP decoration. Collectively, our findings suggest that these APT-decorated NPs hold great preclinical promise in selective targeting and eradication of human NSCLC cells without harming normal tissues.

Developing Body-Components-Based Theranostic Nanoparticles for Targeting Ovarian Cancer.

Ovarian cancer mortality is the highest among gynecologic malignancies. Hence, the major challenges are early diagnosis and efficient targeted therapy. Herein, we devised model theranostic nanoparticles (NPs) for combined diagnostics and delivery of chemotherapeutics, targeted to ovarian cancer cells. These NPs were made of natural biocompatible and biodegradable body components: hyaluronic acid (HA) and serum albumin (SA). The hydrophilic HA served as the targeting ligand for cancer cells overexpressing CD44, the HA receptor. SA, the natural carrier of various ligands through the blood, served as the hydrophobic block of the self-assembling block copolymeric Maillard-conjugates. We show the successful construction of fluorescently-labeled SA-HA conjugate-based theranostic NPs, their loading with paclitaxel (PTX) (association constant (8.6 ± 0.8) × 103 M-1, maximal loading capacity of 4:1 PTX:BSA, and 96% encapsulation efficiency), selective internalization and cytotoxicity to CD44-overexpressing ovarian cancer cells (IC50: 26.4 ± 2.3 nM, compared to 115.0 ± 17.4 of free PTX, and to 58.6 ± 19.7 nM for CD44-lacking cognate ovarian cancer cells). Fluorescein isothiocyanate (FITC) was used for in vitro imaging, whereas long wavelength fluorophores or other suitable tracers would be used for future in vivo diagnostic imaging. Collectively, our findings demonstrate that fluorescent HA-SA NPs harboring a cytotoxic drug cargo can specifically target, label CD44-expressing ovarian cancer cells and efficiently eradicate them.

ß-Casein micelles for oral delivery of SN-38 and elacridar to overcome BCRP-mediated multidrug resistance in gastric cancer.

Gastric cancer is the third leading cause of cancer-related mortality worldwide. A dominant hindrance towards curative cancer therapy is multidrug resistance (MDR) mediated by ATP-dependent efflux pumps. We have previously demonstrated the ability of ß-casein (ß-CN) micelles and re-assembled casein micelles to serve as nanovehicles for oral delivery and target-activated release of hydrophobic chemotherapeutics in the stomach, and to overcome P-glycoprotein-dependent MDR in gastric cancer. Herein we investigated the modularity and versatility of this ß-CN-based delivery system using a different synergistic drug duo to treat MDR gastric cancer cells overexpressing the breast cancer resistance protein (BCRP). The chemotherapeutic drug SN-38, a BCRP transport substrate, and the BCRP efflux transport inhibitor, elacridar, exhibited high binding affinity to ß-CN, as demonstrated by spectrophotometry and spectrofluorometry. Furthermore, light microscopy and dynamic light scattering confirmed that ß-CN solubilized these drugs and suppressed drug crystal growth. In vitro cytotoxicity against MDR human gastric carcinoma cells overexpressing BCRP revealed a synergistic activity of this drug combination and a complete MDR reversal. Hence, our findings highlight the great promise of casein-based nanovehicles, harboring hydrophobic synergistic drug combinations, as a modular and versatile oral delivery system for local drug release in the stomach to overcome chemoresistance in gastric cancer.

Cancer cell-selective, clathrin-mediated endocytosis of aptamer decorated nanoparticles.

Lung cancer is the leading cause of cancer mortality worldwide, resulting in 88% deaths of all diagnosed patients. Hence, novel therapeutic modalities are urgently needed. Single-stranded oligonucleotide-based aptamers (APTs) are excellent ligands for tumor cell targeting. However, the molecular mechanisms underlying their internalization into living cells have been poorly studied. Towards the application of APTs for active drug targeting to cancer cells, we herein studied the mechanism underlying S15-APT internalization into human non-small cell lung cancer A549 cells. We thus delineated the mode of entry of a model nanomedical system based on quantum dots (QDs) decorated with S15-APTs as a selective targeting moiety for uptake by A549 cells. These APT-decorated QDs displayed selective binding to, and internalization by target A549 cells, but not by normal human bronchial epithelial BEAS2B, cervical carcinoma (HeLa) and colon adenocarcinoma CaCo-2 cells, hence demonstrating high specificity. Flow cytometric analysis revealed a remarkably low dissociation constant of S15-APTs-decorated QDs to A549 cells (Kd = 13.1 ± 1.6 nM). Through the systematic application of a series of established inhibitors of known mechanisms of endocytosis, we show that the uptake of S15-APTs proceeds via a classical clathrin-dependent receptor-mediated endocytosis. This cancer cell-selective mode of entry could possibly be used in the future to evade plasma membrane-localized multidrug resistance efflux pumps, thereby overcoming an important mechanism of cancer multidrug resistance.

Targeted nanomedicine for cancer therapeutics: Towards precision medicine overcoming drug resistance.

Intrinsic anticancer drug resistance appearing prior to chemotherapy as well as acquired resistance due to drug treatment, remain the dominant impediments towards curative cancer therapy. Hence, novel targeted strategies to overcome cancer drug resistance constitute a key aim of cancer research. In this respect, targeted nanomedicine offers innovative therapeutic strategies to overcome the various limitations of conventional chemotherapy, enabling enhanced selectivity, early and more precise cancer diagnosis, individualized treatment as well as overcoming of drug resistance, including multidrug resistance (MDR). Delivery systems based on nanoparticles (NPs) include diverse platforms enabling a plethora of rationally designed therapeutic nanomedicines. Here we review NPs designed to enhance antitumor drug uptake and selective intracellular accumulation using strategies including passive and active targeting, stimuli-responsive drug activation or target-activated release, triggered solely in the cancer cell or in specific organelles, cutting edge theranostic multifunctional NPs delivering drug combinations for synergistic therapy, while facilitating diagnostics, and personalization of therapeutic regimens. In the current paper we review the recent findings of the past four years and discuss the advantages and limitations of the various novel NPs-based drug delivery systems. Special emphasis is put on in vivo study-based evidences supporting significant therapeutic impact in chemoresistant cancers. A future perspective is proposed for further research and development of complex targeted, multi-stage responsive nanomedical drug delivery systems for personalized cancer diagnosis and efficacious therapy.

Albumin and Hyaluronic Acid-Coated Superparamagnetic Iron Oxide Nanoparticles Loaded with Paclitaxel for Biomedical Applications.

Super paramagnetic iron oxide nanoparticles (SPION) were augmented by both hyaluronic acid (HA) and bovine serum albumin (BSA), each covalently conjugated to dopamine (DA) enabling their anchoring to the SPION. HA and BSA were found to simultaneously serve as stabilizing polymers of Fe3O4·DA-BSA/HA in water. Fe3O4·DA-BSA/HA efficiently entrapped and released the hydrophobic cytotoxic drug paclitaxel (PTX). The relative amount of HA and BSA modulates not only the total solubility but also the paramagnetic relaxation properties of the preparation. The entrapping of PTX did not influence the paramagnetic relaxation properties of Fe3O4·DA-BSA. Thus, by tuning the surface structure and loading, we can tune the theranostic properties of the system.

Hyaluronic acid-serum albumin conjugate-based nanoparticles for targeted cancer therapy.

Multiple carcinomas including breast, ovarian, colon, lung and stomach cancer, overexpress the hyaluronic acid (HA) receptor, CD44. Overexpression of CD44 contributes to key cancer processes including tumor invasion, metastasis, recurrence, and chemoresistance. Herein, we devised novel targeted nanoparticles (NPs) for delivery of anticancer chemotherapeutics, comprised of self-assembling Maillard reaction-based conjugates of HA and bovine serum albumin (BSA). HA served as the hydrophilic block, and as the ligand for actively targeting cancer cells overexpressing CD44. We demonstrate that Maillard reaction-based covalent conjugates of BSA-HA self-assemble into NPs, which efficiently entrap hydrophobic cytotoxic drugs including paclitaxel and imidazoacridinones. Furthermore, BSA-HA conjugates stabilized paclitaxel and prevented its aggregation and crystallization. The diameter of the NPs was < 15 nm, thus enabling CD44 receptor-mediated endocytosis. These NPs were selectively internalized by ovarian cancer cells overexpressing CD44, but not by cognate cells lacking this HA receptor. Moreover, free HA abolished the endocytosis of drug-loaded BSA-HA conjugates. Consistently, drug-loaded NPs were markedly more cytotoxic to cancer cells overexpressing CD44 than to cells lacking CD44, due to selective internalization, which could be competitively inhibited by excess free HA. Finally, a CD44-targeted antibody which blocks receptor activity, abolished internalization of drug-loaded NPs. In conclusion, a novel cytotoxic drug-loaded nanomedicine platform has been developed, which is based on natural biocompatible biopolymers, capabale of targeting cancer cells with functional surface expression of CD44.

ß-casein nanovehicles for oral delivery of chemotherapeutic Drug combinations overcoming P-glycoprotein-mediated multidrug resistance in human gastric cancer cells.

Multidrug resistance (MDR) is a primary obstacle to curative cancer therapy. We have previously demonstrated that ß-casein (ß-CN) micelles (ß-CM) can serve as nanovehicles for oral delivery and target-activated release of hydrophobic drugs in the stomach. Herein we introduce a novel nanosystem based on ß-CM, to orally deliver a synergistic combination of a chemotherapeutic drug (Paclitaxel) and a P-glycoprotein-specific transport inhibitor (Tariquidar) individually encapsulated within ß-CM, for overcoming MDR in gastric cancer. Light microscopy, dynamic light scattering and zeta potential analyses revealed solubilization of these drugs by ß-CN, suppressing drug crystallization. Spectrophotometry demonstrated high loading capacity and good encapsulation efficiency, whereas spectrofluorometry revealed high affinity of these drugs to ß-CN. In vitro cytotoxicity assays exhibited remarkable synergistic efficacy against human MDR gastric carcinoma cells with P-glycoprotein overexpression. Oral delivery of ß-CN - based nanovehicles carrying synergistic drug combinations to the stomach constitutes a novel efficacious therapeutic system that may overcome MDR in gastric cancer.

Rationally designed nanovehicles to overcome cancer chemoresistance.

Drug resistance is a primary hindrance towards curative cancer chemotherapy. Nanotechnology holds great promise in establishing efficacious and innovative strategies to overcome chemoresistance, and markedly facilitate complementary treatments and cancer diagnostics. Various nanomedical devices are being introduced and evaluated, demonstrating encouraging results. While stealth liposomes serve as a benchmark, astonishing progress is witnessed in polymeric nanovehicles, sometimes combined with low molecular weight surfactants, some of which inhibit drug resistance in addition to solubilizing drugs. Cutting edge multifunctional or quadrugnostic nanoparticles currently developed offer simultaneous targeted delivery of chemotherapeutics and chemosensitizers or drug-resistance gene silencing cargo, along with diagnostic imaging agents, like metallic NPs. Viral and cellular components offer exciting new routes for cancer targeting and treatment. Targeting intracellular compartments is another challenging frontier spawning pioneering approaches and results. To further enhance rational design of nanomedicine for overcoming drug resistance, we review the latest thoughts and accomplishments in recent literature.

ß-Casein nanoparticle-based oral drug delivery system for potential treatment of gastric carcinoma: stability, target-activated release and cytotoxicity.

We studied a potential drug delivery system comprising the hydrophobic anticancer drug paclitaxel entrapped within ß-casein (ß-CN) nanoparticles and its cytotoxicity to human gastric carcinoma cells. Paclitaxel was entrapped by stirring its dimethyl sulfoxide (DMSO) solution into PBS containing ß-CN. Cryo-TEM analysis revealed drug nanocrystals, the growth of which was blocked by ß-CN. Entrapment efficiency was nearly 100%, and the nanovehicles formed were colloidally stable. Following encapsulation and simulated digestion with pepsin (2 hours at pH=2, 37 °C), paclitaxel retained its cytotoxic activity to human N-87 gastric cancer cells; the IC(50) value (32.5 ± 6.2 nM) was similar to that of non-encapsulated paclitaxel (25.4 ± 2.6 nM). Without prior simulated gastric digestion, ß-CN-paclitaxel nanoparticles were non-cytotoxic, suggesting the lack of untoward toxicity to bucal and esophageal epithelia. We conclude that ß-CN shows promise to be useful for target-activated oral delivery of hydrophobic chemotherapeutics in the treatment of gastric carcinoma, one of the leading causes of cancer mortality worldwide.

Nanomedicine for targeted cancer therapy: towards the overcoming of drug resistance.

Anticancer drug resistance almost invariably emerges and poses major obstacles towards curative therapy of various human malignancies. In the current review we will distinguish between mechanisms of chemoresistance that are predominantly mediated by ATP-driven multidrug resistance (MDR) efflux transporters, typically of the ATP-binding cassette (ABC) superfamily, and those that are independent of such drug efflux pumps. In recent years, multiple nanoparticle (NP)-based therapeutic systems have been developed that were rationally designed to overcome drug resistance by neutralizing, evading or exploiting various drug efflux pumps and other resistance mechanisms. NPs are being exploited for selective drug delivery to tumor cells, to cancer stem/tumor initiating cells and/or to the supportive cancer cell microenvironment, i.e. stroma or tumor vasculature. Some of these NPs are currently undergoing preclinical in vivo studies as well as advanced stages of clinical evaluation with promising results. Nanovehicles harboring a payload of therapeutic drug combinations for the selective targeting and elimination of tumor cells as well as the simultaneous overcoming of mechanisms of drug resistance are a subject of intense research efforts, some of which are expected to enter clinical trials in the near future. In the present review we highlight novel approaches to selectively target cancer cells and overcome drug resistance phenomena, through the use of various nanometric drug delivery systems. In the near future, it is anticipated that innovative theragnostic nanovehicles will be developed which will harbor four major components: (1) a selective targeting moiety, (2) a diagnostic imaging aid for the localization of the malignant tumor and its micro- or macrometastases, (3) a cytotoxic, small molecule drug(s) or novel therapeutic biological(s), and (4) a chemosensitizing agent aimed at neutralizing a resistance mechanism, or exploiting a molecular "Achilles hill" of drug resistant cells. We propose to name these envisioned four element-containing nanovehicle platform, "quadrugnostic" nanomedicine. This targeted strategy holds promise in paving the way for the introduction of highly effective nanoscopic vehicles for cancer therapeutics while overcoming drug resistance.

Beta-casein nanoparticles as an oral delivery system for chemotherapeutic drugs: impact of drug structure and properties on co-assembly.

PURPOSE: To develop a novel oral drug delivery system comprising a hydrophobic chemotherapeutic drug entrapped within beta casein (ß-CN), a major milk protein, which self-associates into micelles in aqueous solutions. The efficient gastric digestibility of ß-CN suggests possible targeting to gastric cancers. METHODS: Antitumor drug entrapment was performed by stirring its dimethyl-sulfoxide solution into a phosphate-buffered saline containing ß-CN. The association of drugs to ß-CN was characterized by spectrophotometry and Trp143 fluorescence quenching; particle-size by dynamic light scattering, and colloidal stability by zeta potential. RESULTS: The optimal drug-to-ß-CN molar loading-ratios for paclitaxel and vinblastine at 1 mg/ml ß-CN were found to be 7.3 ± 1.2 and 5.3 ± 0.6 and the association constants were (6.3 ± 1.0) x 10(3) M(-1) and (2.0 ± 0.3) x 10(4) M(-1), respectively. Zeta potential analysis suggested that nanoencapsulation by ß-CN stabilized all studied drugs in aqueous solution. The initial drug-ß-CN association was apparently governed by hydrophobic interactions and at higher drug concentrations, also by electrostatic interactions. Up to the optimal drug:ß-CN loading-ratio, ~80% of the particles were below 100 nm in diameter. At higher drug concentrations, particle diameter increased, and bi- or tri-modal particle distributions were observed. CONCLUSIONS: Beta-CN forms colloidally-stable nanovehicles of hydrophobic anticancer drugs, and may be used for oral-delivery of chemotherapeutics.

Beta-casein-based nanovehicles for oral delivery of chemotherapeutic drugs: drug-protein interactions and mitoxantrone loading capacity.

Beta-casein (beta-CN), a major milk protein, is amphiphilic and self-associates into micelles in aqueous solutions. We have recently introduced a novel oral drug delivery system based on beta-CN nanoparticles. The current research builds on and complements this work by studying the interactions of mitoxantrone (MX) and beta-CN as they co-assemble into nanoparticles, using absorption and emission spectra, static and dynamic light scattering, and fluorescent emission of both MX and tryptophan 143 (Trp143) of beta-CN. The optimal loading molar ratio was 3.3 MX/beta-CN at 1 mg/mL beta-CN, and the association constant was (2.45 +/- 1.76) x 10(5) M(-1) based on beta-CN Trp143 fluorescence; independent MX fluorescence results provided supporting values. In these conditions a bimodal particle distribution was obtained (174.4 nm, 45.9%; 485.1 nm, 54.1%). The gastric digestibility of beta-CN suggests possible targeting to stomach tumors. Hence, beta-CN nanoparticles have potential to serve as effective vehicles of hydrophobic drugs for oral delivery preparations. From the clinical editor: Beta-casein (b-CN) is an amphiphilic milk protein that self-associates into micelles in aqueous solutions and can be utilized as a novel oral drug delivery system. This study investigates the basic properties of a mitoxantrone delivery system based on the above principles.

Arabinogalactan-folic acid-drug conjugate for targeted delivery and target-activated release of anticancer drugs to folate receptor-overexpressing cells.

Folic acid (FA) is a high affinity ligand (K(d) = 0.1-1 nM) of folate receptors (FRs) responsible for cellular uptake of folates via receptor-mediated endocytosis. FRs are frequently overexpressed in malignant epithelial cells including ovary, brain, kidney, breast, colon, and lung. FR has emerged as a target for the differential-delivery of anticancer chemotherapeutics with several FA-linked therapeutic agents currently undergoing clinical trials. Here we show that by tethering both FA and the anticancer drug methotrexate (MTX) to arabinogalactan (AG), a highly branched natural polysaccharide with unusual water solubility, a targeted biomacromolecular nanovehicle is formed, which can differentially deliver a cytotoxic cargo into FR-overexpressing cells. Moreover, by linking MTX via an endosomally cleavable peptide (GFLG), we demonstrate a target-activated release mechanism. This FA-AG-GFLG-MTX drug conjugate displayed 6.3-fold increased cytotoxic activity to FR-overexpressing cells compared to their FR-lacking counterparts. These findings establish a novel FA-tethered polymeric nanoconjugate for the targeted delivery of antitumor agents into cancer cells overexpressing FR.

Beta-casein nanovehicles for oral delivery of chemotherapeutic drugs.

Bovine beta-casein (beta-CN) is an abundant milk protein that is highly amphiphilic and self-assembles into stable micellar structures in aqueous solutions. Here we introduce a drug-delivery system comprising a model hydrophobic anticancer drug, mitoxantrone (MX), entrapped within beta-CN-based nanoparticles. This novel drug-delivery system allows hydrophobic drugs to be thermodynamically stable in aqueous solutions for oral-delivery applications aimed at treatment of various disorders. The gastric digestibility of beta-CN suggests possible targeting to stomach tumors. Dimethyl sulfoxide (DMSO)-dissolved MX was entrapped in beta-CN nanoparticles by stirring this solution into phosphate-buffered beta-CN solution. High-affinity MX-beta-CN association was found (K(a) = [2.15 +/- 0.30] x 10(6) M(-1)). The optimal nanovehicle formation conditions were 1 mg/mL beta-CN,

Specificity of disulfide bond formation during thermal aggregation in solutions of beta-lactoglobulin B and kappa-casein A.

Heat-induced (90 degrees C, 10 min, pH 6.7) intermolecular disulfide bond formation in 1:1 mixtures of beta-lactoglobulin B (beta-Lg) and kappa-casein A (kappa-CN) was studied by enzymatic digestion with trypsin or glu-C, reverse-phase HPLC, and MALDI-TOF-MS. Observed masses were compared to theoretically calculated masses of disulfide-bonded peptide dimers and trimers, and the number of different masses matching peptide combinations involving each bond was used as a measure of confidence of identification. The beta-Lg cysteine residues 121 or 119 were involved in bonds with both cysteines of kappa-CN and all cysteines of beta-Lg. This agrees with the supposed initiatory role of beta-C121 in heat-induced SH/SS interchange. The largest numbers of matches corresponded to bonds linking beta-C119/C121 with kappa-C11 or with beta-C66. Multiple matches were recorded for beta-C119/C121 bonding with beta-C119/C121, with beta-C160, or with kappa-C88. However, beta-C106 was observed only in bonds with beta-C119/C121 and did not appear to bond to kappa-CN, suggesting it remains buried in the core of the protein.