Self-Propelling Targeted Magneto-Nanobots for Deep Tumor Penetration and pH-Responsive Intracellular Drug Delivery.

Self-propelling magnetic nanorobots capable of intrinsic-navigation in biological fluids with enhanced pharmacokinetics and deeper tissue penetration implicates promising strategy in targeted cancer therapy. Here, multi-component magnetic nanobot designed by chemically conjugating magnetic Fe3O4 nanoparticles (NPs), anti-epithelial cell adhesion molecule antibody (anti-EpCAM mAb) to multi-walled carbon nanotubes (CNT) loaded with an anticancer drug, doxorubicin hydrochloride (DOX) is reported. Autonomous propulsion of the nanobots and their external magnetic guidance is enabled by enriching Fe3O4 NPs with dual catalytic-magnetic functionality. The nanobots propel at high velocities even in complex biological fluids. In addition, the nanobots preferably release DOX in the intracellular lysosomal compartment of human colorectal carcinoma (HCT116) cells by the opening of Fe3O4 NP gate. Further, nanobot reduce ex vivo HCT116 tumor spheroids more efficiently than free DOX. The multicomponent nanobot's design represents a more pronounced method in targeting tumors with self-assisted anticancer drug delivery for 'far-reaching' sites in treating cancers.

Cell deformation and acquired drug resistance: elucidating the major influence of drug-nanocarrier delivery systems.

Cancer diagnosis and its stage-wise assessment are determined through invasive solid tissue biopsies. Conversely, cancer imaging is enriched through emission tomography and longitudinal high-resolution analysis for the early detection of cancer through altered cell morphology and cell-deformation. Similarly, in post multiple chemo-cycle exposures, the tumor regression and progression thereafter are not well understood. Here, we report chemo-cycles of doxorubicin (Dox) carrying nanoparticles (NPs) to be highly indicative of cell deformation and a progressive indicator of phenotypic expressions of acquired drug resistance (ADR). We designed graphene (G) based nanocarriers by chemically conjugating multiple components: (i) G; (ii) iron oxide (Fe3O4) NPs; and (iii) Dox through a cysteine (Cys) linker (G-Dox and G-Cys-Fe3O4-Dox). Although Dox underwent cell diffusion, the G-based nanocarriers followed a receptor-mediated endocytosis which created a profound impact on the cell membrane integrity. ADR owing to Dox and G-based nanocarriers was analyzed through a cytotoxicity assay, cell morphology deformation parameters and cellular uptake kinetic patterns. Interestingly, after the third chemo-cycle, G-Dox incubated cells showed the greatest decrease in the alteration of the nuclear surface area (NSA) of ∼28%, a ∼40% reduction of the cell surface area (CSA) and a ∼32% increase in the cell roundness (CRd). Our results suggested that the G-based nanocarriers induced the cell deformation process, subsequently resulting in ADR. Although the G-based nanocarriers initiated ADR, G-Dox was most cytotoxic to cancer cells and induced the maximum cell morphology deformation within our scope of study. This outcome implies caution is needed when using G-based nanocarriers and other multi-component nanosystems for Dox delivery as they lead to possible phenotypic expressions of drug resistance in cancer cells.

Cellular regeneration and proliferation on polymeric 3D inverse-space substrates and the effect of doxorubicin.

Spatial arrangement for cells and the opportunity thereof have implications in cell regeneration and cell proliferation. 3D inverse space (3DIS) substrates with micron-sized pores are fabricated under controlled environmental conditions from polymers such as poly(lactic-co-glycolic) acid (PLGA), poly(lactic acid) (PLA) and poly(styrene) (PS). The characterization of 3DIS substrates by optical microscopy, scanning probe microscopy (SPM), etc. shows pores within 1-18 µm diameter and prominent surface roughness extending up to 3.9 nm in height over its base. Conversely, to compare two-dimensional (2D) versus 3DIS substrates, the crucial variables of cell height, cell spreading area and cell volume are compared using lung adenocarcinoma (A549) cells. The results indicate an average cell thickness of ∼6 µm on a glass substrate whereas cells on PLGA 3DIS were ∼12 µm in height, occasionally reaching 20 µm, with a 40% decreased cell spreading area. A549 cells cultured on polymer 3DIS substrates show a cell regeneration growth pattern, dependent on the available spatial volume. Furthermore, PLGA 3DIS cell culture systems with and without graded doxorubicin (DOX) pre-treatment result in potent cell inhibition and cell proliferation, respectively. Additionally, standard DOX administration to A549 cells in the PLGA 3DIS system revealed altered drug sensitivity. 3DIS demonstrates utility in facilitating cellular regeneration and mimicking cell proliferation in defined spatial arrangements.

Budding trends in integrated pest management using advanced micro- and nano-materials: Challenges and perspectives.

One of the most vital supports to sustain human life on the planet earth is the agriculture system that has been constantly challenged in terms of yield. Crop losses due to insect pest attack even after excessive use of chemical pesticides, are major concerns for humanity and environment protection. By the virtue of unique properties possessed by micro and nano-structures, their implementation in Agri-biotechnology is largely anticipated. Hence, traditional pest management strategies are now forestalling the potential of micro and nanotechnology as an effective and viable approach to alleviate problems pertaining to pest control. These technological innovations hold promise to contribute enhanced productivity by providing novel agrochemical agents and delivery systems. Application of these systems engages to achieve: i) control release of agrochemicals, ii) site-targeted delivery of active ingredients to manage specific pests, iii) reduced pesticide use, iv) detection of chemical residues, v) pesticide degradation, vi) nucleic acid delivery and vii) to mitigate post-harvest damage. Applications of micro and nano-technology are still marginal owing to the perception of low economic returns, stringent regulatory issues involving safety assessment and public awareness over their uses. In this review, we highlight the potential application of micro and nano-materials with a major focus on effective pest management strategies including safe handling of pesticides.

Self-propelled carbon nanotube based microrockets for rapid capture and isolation of circulating tumor cells.

Here, we report a non-invasive strategy for isolating cancer cells by autonomously propelled carbon nanotube (CNT) microrockets. H2O2-driven oxygen (O2) bubble-propelled microrockets were synthesized using CNT and Fe3O4 nanoparticles in the inner surface and covalently conjugating transferrin on the outer surface. Results show that self-propellant microrockets can specifically capture cancer cells.

Bio-physical evaluation and in vivo delivery of plant proteinase inhibitor immobilized on silica nanospheres.

Recombinant expression of Capsicum annuum proteinase inhibitors (CanPI-13) and its application via synthetic carrier for the crop protection is the prime objective of our study. Herein, we explored proteinase inhibitor peptide immobilization on silica based nanospheres and rods followed by its pH mediated release in vitro and in vivo. Initial studies suggested silica nanospheres to be a suitable candidate for peptide immobilization. Furthermore, the interactions were characterized biophysically to ascertain their conformational stability and biological activity. Interestingly, bioactive peptide loading at acidic pH on nanospheres was found to be 62% and showed 56% of peptide release at pH 10, simulating gut milieu of the target pest Helicoverpa armigera. Additionally, in vivo study demonstrated significant reduction in insect body mass (158 mg) as compared to the control insects (265 mg) on 8th day after feeding with CanPI-13 based silica nanospheres. The study confirms that peptide immobilized silica nanosphere is capable of affecting overall growth and development of the feeding insects, which is known to hamper fecundity and fertility of the insects. Our study illustrates the utility and development of peptide-nanocarrier based platform in delivering diverse biologically active complexes specific to gut pH of H. armigera.

Calcium phosphate nanocapsule crowned multiwalled carbon nanotubes for pH triggered intracellular anticancer drug release.

We report calcium phosphate (CaP) nanocapsule crowned multiwalled carbon nanotubes (CNT-GSH-G4-CaP) as a novel platform for intracellular delivery of an anticancer drug. As a proof-of-concept, CNT-GSH-G4-CaP demonstrates release of anticancer drug doxorubicin hydrochloride (DOX) within intracellular lysosomes from the interior cavity of CNT upon pH triggered CaP dissolution. Importantly, we found that the CNT with a CaP nanolid can efficiently prevent untimely drug release at physiological pH but promotes DOX release at increased acidic milieu as observed in subcellular compartments such as lysosomes (∼5.0). This "zero premature release" characteristic is of clinical significance in delivering cytotoxic drugs, by reducing systemic toxicity and thus beneficial for the effective anticancer treatment. We envision that this pH triggered CaP crowned CNT nanosystem would lead to a new generation of self-regulated platforms for intracellular delivery of a variety of anticancer drugs.

Biophysical interactions of polyamidoamine dendrimer coordinated Fe3O4 nanoparticles with insulin.

Advanced delivery systems, such as nano/micro carriers have not been studied significantly for their molecular interactions with serum proteins and other biologically relevant macromolecules. Here, we investigated the effect of surface chemistry of iron oxide (Fe3O4) nanoparticles on molecular interactions with human insulin by fluorescence, XRD and FTIR spectroscopy. Nanoparticles of Fe3O4 were chemically modified as Fe3O4-glutathione (GSH) and Fe3O4-GSH-polyamidoamine generation 4 (PAMAM G4) dendrimer. Our results demonstrate that, Fe3O4 and its conjugates such as Fe3O4-GSH, Fe3O4-GSH-G4 quenched insulin fluorescence, indicating strong interactions between insulin protein molecule and Fe3O4. The fluorescence quenching constants Ksv were obtained as 0.0367 x 10(3), 0.0303 x 10(3) and 0.0131 x 10(3) M and the binding constant K were found to be 27.095, 8.404 and 6.026 mM for Fe3O4, Fe3O4-GSH and Fe3O4-GSH-PAMAM G4, respectively. Both the Ksv and K (binding constant) values revealed that the interaction of Fe3O4 with insulin to be stronger over to dendrimer conjugates. In addition, the FTIR spectra suggested that the presence of nanoparticles results in secondary structure alteration in the insulin conformation. The study implies the critical evaluation of new delivery systems in establishing the biocompatibility, especially when delivered by systemic route.

Transferrin-mediated rapid targeting, isolation, and detection of circulating tumor cells by multifunctional magneto-dendritic nanosystem.

A multicomponent magneto-dendritic nanosystem (MDNS) is designed for rapid tumor cell targeting, isolation, and high-resolution imaging by a facile bioconjugation approach. The highly efficient and rapid-acting MDNS provides a convenient platform for simultaneous isolation and high-resolution imaging of tumor cells, potentially leading towards an early diagnosis of cancer.

Design, synthesis and evaluation of N-acetyl glucosamine (NAG)-PEG-doxorubicin targeted conjugates for anticancer delivery.

Efficacy of anticancer drug is limited by the severe adverse effects induced by drug; therefore the crux is in designing delivery systems targeted only to cancer cells. Toward this objectives, we propose, synthesis of poly(ethylene glycol) (PEG)-doxorubicin (DOX) prodrug conjugates consisting N-acetyl glucosamine (NAG) as a targeting moiety. Multicomponent system proposed here is characterized by (1)H NMR, UV spectroscopy, and HPLC. The multicomponent system is evaluated for in vitro cellular kinetics and anticancer activity using MCF-7 and MDA-MB-231 cells. Molecular modeling study demonstrated sterically stabilized conformations of polymeric conjugates. Interestingly, PEG-DOX conjugate with NAG ligand showed significantly higher cytotoxicity compared to drug conjugate with DOX. In addition, the polymer drug conjugate with NAG and DOX showed enhanced internalization and retention effect in cancer cells, compared to free DOX. Thus, with enhanced internalization and targeting ability of PEG conjugate of NAG-DOX has implication in targeted anticancer therapy.

PEG-conjugated highly dispersive multifunctional magnetic multi-walled carbon nanotubes for cellular imaging.

We report synthesis of a highly versatile multicomponent nanosystem by covalently decorating the surface of multiwalled carbon nanotubes (CNTs) by magnetite nanoparticles (Fe(3)O(4)), poly(ethylene glycol) (PEG), and fluorophore fluorescein isothiocyanate (FITC). The resulting Fe(3)O(4)-PEG-FITC-CNT nanosystem demonstrates high dispersion ability in an aqueous medium, magnetic responsiveness, and fluorescent capacity. Transmission electron microscopy images revealed that Fe(3)O(4) nanoparticles were well anchored onto the surfaces of the CNT. In vitro time kinetic experiments using confocal microscopy demonstrated a higher uptake of the Fe(3)O(4)-PEG-FITC-CNT nanosystem localized at the perinuclear region of MCF7 cells compared to the free FITC. In addition, the CNT nanosystem demonstrated no evidence of toxicity on cell growth. Surface conjugation of multicomponents, combined with in vitro non-toxicity, enhanced cellular uptake for FITC and site specific targeting ability makes this fluorescent Fe(3)O(4)-PEG-FITC-CNT nanosystem an ideal candidate for bioimaging, both in vitro and in vivo.

Multifunctional tumor-targeted polymer-peptide-drug delivery system for treatment of primary and metastatic cancers.

PURPOSE: In order to improve drug delivery to drug-resistant ovarian tumors, we constructed a multifunctional polymer-peptide-drug conjugate (PPDC) system for effective treatment of primary and metastatic ovarian cancers. METHODS: The PPDC consists of the poly(Ethylene Glycol) (PEG) polymeric carrier conjugated via citric acid spacers to anticancer drug (Camptothecin, CPT), tumor targeting moiety (LRHR, a synthetic analog of luteinizing hormone-releasing hormone) and a suppressor of cellular antiapoptotic defense (BH3 peptide). To test the conjugates in vitro and in vivo, cancer cells were isolated from tissue samples obtained from patients with ovarian primary tumor and metastatic malignant ascites. RESULTS: It was found that cells isolated from malignant ascites were more aggressive in terms of tumor growth and more resistant to chemotherapy when compared with those isolated from primary tumors. PPDC containing two copies of drugs and peptides was most efficient in treatment of primary tumors and intraperitoneal metastases. Multiple treatments with this PPDC led to almost complete regression of primary tumor and prevented growth of malignant ascites. CONCLUSION: The proposed multifunctional polymeric delivery system which consists of multiple copies of the drug and peptides demonstrated significantly higher antitumor activity in primary and metastatic cancers when compared with drug alone and PEG-CPT conjugate.

LHRH-targeted nanoparticles for cancer therapeutics.

Synthesis and evaluation of a novel cancer cell's receptor-targeted internally quaternized and surface neutral poly(amidoamine) (PAMAM) generation four dendrimer as well as PAMAM-paclitaxel conjugate are described. The advantages of developed nanocarriers include but are not limited to (1) internal cationic charges for the complexation with small interfering RNA or antisense oligonucleotides and their protection from the degradation in systemic circulation; (2) neutral-modified surface for low cytotoxicity of empty unloaded dendrimers; (3) efficient internalization by cancer cells; and (4) preferential accumulation in the tumor and the prevention of adverse side effects of chemotherapy.

Nonviral nanoscale-based delivery of antisense oligonucleotides targeted to hypoxia-inducible factor 1 alpha enhances the efficacy of chemotherapy in drug-resistant tumor.

PURPOSE: To enhance the efficacy of cancer treatment, we propose a complex approach: simultaneous delivery to the tumor of a chemotherapeutic agent and a suppressor of hypoxia-inducible factor 1 alpha (HIF1A). EXPERIMENTAL DESIGN: The novel complex liposomal drug delivery system was developed and evaluated in vitro and in vivo on nude mice bearing xenografts of multidrug-resistant human ovarian carcinoma. The proposed novel complex drug delivery system consists of liposomes as a nanocarrier, a traditional anticancer drug (doxorubicin) as a cell death inducer, and antisense oligonucleotides targeted to HIF1A mRNA as a suppressor of cellular resistance and angiogenesis. RESULTS: The system effectively delivers active ingredients into tumor cells, multiplies the cell death signal initiated by doxorubicin, and inhibits cellular defensive mechanisms and angiogenesis by down-regulating BCL2, HSP90, and vascular endothelial growth factor proteins. This, in turn, activates caspases, promotes apoptosis, necrosis, and tumor shrinkage. The proposed novel complex multipronged approach enhances the efficiency of chemotherapy. CONCLUSIONS: The proposed combination therapy prevents the development of resistance in cancer cells, and thus, increases the efficacy of chemotherapy to an extent that cannot be achieved by individual components applied separately. It could form the foundation for a novel type of cancer therapy based on simultaneous delivery of an anticancer drug and a suppressor of HIF1A.

Receptor targeted polymers, dendrimers, liposomes: which nanocarrier is the most efficient for tumor-specific treatment and imaging?

To compare the influence of different characteristics of nanocarriers on the efficacy of chemotherapy and imaging, we designed, characterized, and evaluated three widely used nanocarriers: linear polymer, dendrimer and liposome in vitro and in vivo. These nanocarriers delivered the same anticancer drug (paclitaxel) and/or imaging agent (Cy5.5). A synthetic analog of LHRH peptide targeted to receptors overexpressed on the membrane of cancer cells was attached to the nanocarriers as a tumor targeting moiety. Significant differences were found between various studied non-targeted carriers in their cellular internalization, cytotoxicity, tumor and organ distribution and anticancer efficacy. LHRH peptide substantially enhanced intratumoral accumulation and anticancer efficacy of all delivery systems and minimized their adverse side effects. For the first time, the present study revealed that the targeting of nanocarriers to tumor-specific receptors minimizes the influence of the architecture, composition, size and molecular mass of nanocarriers on the efficacy of imaging and cancer treatment.

Targeted sialic acid-doxorubicin prodrugs for intracellular delivery and cancer treatment.

PURPOSE: To evaluate a novel targeted anticancer prodrug consisting of several copies of sialic acid (SA, targeting moiety), doxorubicin (DOX), citric acid (multifunctional spacer) and poly(ethylene glycol) (PEG, carrier). METHODS: alpha, omega bis carboxyl PEG was covalently conjugated with multiple copies of SA and DOX through a citric acid spacer and characterized by proton nuclear magnetic resonance ((1)HNMR), matrix-assisted laser desorption/ionization-time of flight (MALDI/TOF), and high-performance liquid chromatography (HPLC). The molecular models of conjugates were established using ChemDraw software. Stability, spontaneous and esterase-stimulated drug release was analyzed by HPLC. Cellular internalization (fluorescence microscopy) and cytotoxicity [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay] of free DOX and prodrugs were evaluated. RESULTS: (1)HNMR, MALDI/TOF, and HPLC showed the formation of the PEG prodrug conjugates. More than 40% of the drug was released from its conjugate in the presence of esterase enzyme, whereas the conjugate was stable at pH 7.4 in the absence of enzyme. Molecular modeling studies showed stable conformations of conjugates. The targeted prodrug conjugates with two copies of SA and DOX showed enhanced cytotoxicity when compared with non-targeted prodrugs and free DOX. CONCLUSIONS: Targeting of the conjugate to cancer cells by SA with increased copies of targeting moiety and anticancer drug enhanced prodrug uptake by cancer cells and cytotoxicity of the prodrug.

Dendrimer versus linear conjugate: Influence of polymeric architecture on the delivery and anticancer effect of paclitaxel.

The relative difference in polymeric architectures of dendrimer and linear bis(poly(ethylene glycol)) (PEG) polymer in conjugation with paclitaxel has been described. Paclitaxel, a poorly soluble anticancer drug, was covalently conjugated with PAMAM G4 hydroxyl-terminated dendrimer and bis(PEG) polymer for the potential enhancement of drug solubility and cytotoxicity. Both conjugates were characterized by 1NMR, HPLC, and MALDI/TOF. In addition, molecular conformations of dendrimer, bis(PEG), paclitaxel, and its polymeric conjugates were studied by molecular modeling. Hydrolysis of the ester bond in the conjugate was analyzed by HPLC using esterase hydrolyzing enzyme. In vitro cytotoxicity of dendrimer, bis(PEG), paclitaxel, and polymeric conjugates containing paclitaxel was evaluated using A2780 human ovarian carcinoma cells. Cytotoxicity increased by 10-fold with PAMAM dendrimer-succinic acid-paclitaxel conjugate when compared with free nonconjugated drug. Data obtained indicate that the nanosized dendritic polymer conjugates can be used with good success as anticancer drug carriers.

New generation of liposomal drugs for cancer.

This review is focused on liposomes as a delivery system for anticancer agents and more specifically on the advantages of using liposomes as drug nanocarrier in cancer chemotherapy. The main advantages of liposomal drugs over the non-encapsulated drugs include: (1) improved pharmacokinetics and drug release, (2) enhanced intracellular penetration, (3) tumor targeting and preventing adverse side effects and (4) ability to include several active ingredients in one complex liposomal drug delivery system (DDS). The review also includes our recent data on advanced liposomal anticancer drug delivery systems. As a conclusion we propose a novel liposomal DDS which includes inhibitors of pump resistance combined in one liposomal drug delivery system with an inhibitor of antiapoptotic cellular defense, an apoptosis inducer (a traditional anticancer drug) and a targeting moiety. The proposed drug delivery system utilizes a novel three tier approach, simultaneously targeting three molecular targets: (1) extracellular receptors or antigen expressed on the surface of plasma membrane of cancer cells in order to direct the whole system specifically to the tumor, preventing adverse side effects on healthy tissues; (2) drug efflux pumps in order to inhibit them and enhance drug retention by cancer cells, increasing intracellular drug accumulation and thereby limiting the need for prescribed high drug doses that cause adverse drug side effects; and (3) intracellular controlling mechanisms of apoptosis in order to suppress cellular antiapoptotic defense.

JNK1 as a molecular target to limit cellular mortality under hypoxia.

Many pathological conditions and environmental impacts lead to a decrease in tissue oxygen supply and severe cellular hypoxia. This secondary hypoxia can disturb cellular homeostasis, limiting the efficacy of the prescribed treatment for the primary lesion, eventually leading to cellular and organismal death. Jun N-terminal kinase 1 (JNK1) plays a major role in the hypoxic cellular damage. Therefore, we hypothesized that suppression of JNK1 activity will decrease cellular mortality under hypoxia and might increase the efficacy of traditional treatment of many pathological conditions. These investigations are aimed at studying the influence of the suppression of JNK1 activity on the development of cellular hypoxic damage. We used antisense oligonucleotides (ASO) and small interfering RNA (siRNA) targeted to JNK1 mRNA to inhibit the protein synthesis. Experiments were carried out on a cell culture under normoxia and hypoxic conditions that led to the death of approximately 50% of cells. ASO or siRNA was delivered by neutral or cationic liposomes. Intracellular localization of ASO and liposomes and mechanisms of apoptosis were studied. We found that the suppression of JNK1 activity by liposomal antisense oligonucleotides or siRNA limits the caspase-dependent apoptosis signaling pathway and decreases cellular mortality after severe hypoxia. JNK1 protein might be an attractive target for antihypoxic therapy in increasing resistance to many pathological conditions and diseases, leading to the oxygen deficit.

In vitro and in vivo intracellular liposomal delivery of antisense oligonucleotides and anticancer drug.

The specific aims of this investigation were (1) to show that conventional and PEGylated liposomes can penetrate cancer cells in vitro and in vivo; (2) to demonstrate that liposomes can be successfully used both for cytoplasmic and nuclear delivery of therapeutics, including anticancer drugs and antisense oligonucleotides; (3) to examine the specific activity of anticancer drugs and nucleotides delivered inside tumor cells by PEGylated liposomes; and (4) to confirm that simultaneous inhibition of pump and nonpump cellular resistance by liposomal ASO can substantially enhance the antitumor activity of traditional well established anticancer drugs in mice bearing xenografts of human multidrug resistant ovarian carcinoma. Experimental results show that PEGylated liposomes are capable of penetrating directly into tumor cells after systemic administration in vivo and do successfully provide cytoplasmic and nuclear delivery of encapsulated anticancer drug (doxorubicin, DOX) and antisense oligonucleotides (ASO). Encapsulation of DOX and ASO into liposomes substantially increased their specific activity. Simultaneous suppression of pump and nonpump resistance dramatically enhanced the ability of DOX for inducing apoptosis leading to higher in vitro cytotoxicity and in vivo antitumor activity.