Nanoparticle-based drug delivery: case studies for cancer and cardiovascular applications

Nanoparticles (NPs) comprised of nanoengineered complexes are providing new opportunities for enabling targeted delivery of a range of therapeutics and combinations. A range of functionalities can be included within a nanoparticle complex, including surface chemistry that allows attachment of cell-specific ligands for targeted delivery, surface coatings to increase circulation times for enhanced bioavailability, specific materials on the surface or in the nanoparticle core that enable storage of a therapeutic cargo until the target site is reached, and materials sensitive to local or remote actuation cues that allow controlled delivery of therapeutics to the target cells. However, despite the potential benefits of NPs as smart drug delivery and diagnostic systems, much research is still required to evaluate potential toxicity issues related to the chemical properties of NP materials, as well as their size and shape. The need to validate each NP for safety and efficacy with each therapeutic compound or combination of therapeutics is an enormous challenge, which forces industry to focus mainly on those nanoparticle materials where data on safety and efficacy already exists, i.e., predominantly polymer NPs. However, the enhanced functionality affordable by inclusion of metallic materials as part of nanoengineered particles provides a wealth of new opportunity for innovation and new, more effective, and safer therapeutics for applications such as cancer and cardiovascular diseases, which require selective targeting of the therapeutic to maximize effectiveness while avoiding adverse effects on non-target tissues.     

Purification of breast cancer resistance protein ABCG2 and role of arginine-482

Human ABCG2 was efficiently overexpressed in insect cell membranes, solubilized with 3-[(3-cholamidopropyl)dimethyl ammonio]-1-propanesulfonate, and purified through N-terminal hexahistidine tag. Its functionality was assessed by high vanadate-sensitive ATPase activity, and nucleotide-binding capacity. Interestingly, the R482T point mutation increased both maximal hydrolysis rate and affinity for MgATP, and lowered sensitivity to vanadate inhibition. Direct nucleotide binding, as monitored by quenching of intrinsic fluorescence, indicated a mutation-related preference for ATP over ADP. The R482T mutation only produced a limited change, if any, on the binding of drug substrates, indicating that methotrexate, on the one hand, and rhodamine 123 or doxorubicin, on the other hand, bound similarly to wild-type and mutant transporters whether or not they were subject to cellular transport. In addition, the characteristic inhibitors GF120918 and 6-prenylchrysin, which alter mitoxantrone efflux much better for wild-type than mutant ABCG2, bound similarly to purified ABCG2, while the highly-potent Ko143 bound in the nanomolar range also effective in inhibition of drug transport. All results indicate that the role of the arginine-482 mutation on substrate drug transport and inhibitor efficiency is not mediated by changes in drug binding. Received 10 April 2006; received after revision 22 May 2006; accepted 12 June 2006 A. Pozza and J. M. Perez-Victoria contributed equally to this work     

UDP-glucose ceramide glucosyltransferase activates AKT,promoted proliferation,and doxorubicin resistance in breast cancer cells

The UDP-glucose ceramide glucosyltransferase (UGCG) is a key enzyme in the synthesis of glycosylated sphingolipids, since this enzyme generates the precursor for all complex glycosphingolipids (GSL), the GlcCer. The UGCG has been associated with several cancer-related processes such as maintaining cancer stem cell properties or multidrug resistance induction. The precise mechanisms underlying these processes are unknown. Here, we investigated the molecular mechanisms occurring after UGCG overexpression in breast cancer cells. We observed alterations of several cellular properties such as morphological changes, which enhanced proliferation and doxorubicin resistance in UGCG overexpressing MCF-7 cells. These cellular effects seem to be mediated by an altered composition of glycosphingolipid-enriched microdomains (GEMs), especially an accumulation of globotriaosylceramide (Gb3) and glucosylceramide (GlcCer), which leads to an activation of Akt and ERK1/2. The induction of the Akt and ERK1/2 signaling pathway results in an increased gene expression of multidrug resistance protein 1 (MDR1) and anti-apoptotic genes and a decrease of pro-apoptotic gene expression. Inhibition of the protein kinase C (PKC) and phosphoinositide 3 kinase (PI3K) reduced MDR1 gene expression. This study discloses how changes in UGCG expression impact several cellular signaling pathways in breast cancer cells resulting in enhanced proliferation and multidrug resistance.     

Survival of docetaxel-resistant prostate cancer cells in vitro depends on phenotype alterations and continuity of drug exposure

We evaluated in vitro the effect of paclitaxel and docetaxel on PC-3 and DU-145 prostate cancer cell lines to understand better the downstream events in drug-induced tumor cell death. Taxane treatments of DU-145 cells induced rapid cell death by apoptosis, but in PC-3 cells, treatments achieved growth arrest, followed by extensive karyokinesis resulting in multinucleation, giant-cell formation and delayed cell death. To determine if the giant multinucleated cells were able to produce proliferating and drug-resistant survivors, we first delineated the kinetics of drug activity and cytotoxic dose range. Analysis of both lines by colorimetric and cell viability assays demonstrated improved cytotoxicity of taxanes applied continuously. Selected doses and schedules of docetaxel were used to induce giant multinucleated cells that gave rise to docetaxel-resistant survivors, which remained sensitive to paclitaxel and other chemotherapeutics. Growth and morphology of the recovered clones was similar to parental cells. The resistant phenotype of these clones determined by immunofluorescence and immunoblot was associated with transient expression of the β-tubulin IV isoform and was independent of P-glycoprotein, bcl-2 and bcl-xL. Resistant clones will be useful to model progression of resistance to taxanes and to identify unknown and clinically important molecular mechanisms of cell death and resistance. Received 15 March 2002; received after revision 25 April 2002; accepted 27 May 2002     

Know your enemy: understanding the role of PfCRT in drug resistance could lead to new antimalarial tactics

The prevention and treatment of malaria is heavily dependent on antimalarial drugs. However, beginning with the emergence of chloroquine (CQ)-resistant Plasmodium falciparum parasites 50 years ago, efforts to control the disease have been thwarted by failed or failing drugs. Mutations in the parasite's 'chloroquine resistance transporter' (PfCRT) are the primary cause of CQ resistance. Furthermore, changes in PfCRT (and in several other transport proteins) are associated with decreases or increases in the parasite's susceptibility to a number of other antimalarial drugs. Here, we review recent advances in our understanding of CQ resistance and discuss these in the broader context of the parasite's susceptibilities to other quinolines and related drugs. We suggest that PfCRT can be viewed both as a 'multidrug-resistance carrier' and as a drug target, and that the quinoline-resistance mechanism is a potential 'Achilles' heel' of the parasite. We examine a number of the antimalarial strategies currently undergoing development that are designed to exploit the resistance mechanism, including relatively simple measures, such as alternative CQ dosages, as well as new drugs that either circumvent the resistance mechanism or target it directly.     

An overview of cancer multidrug resistance: a still unsolved problem

Although various mechanisms involved in anticancer multidrug resistance (MDR) can be identified, it remains a major problem in oncology. Beyond that, the introduction of new “targeted” drugs have not solved the problem. On the contrary, it has been demonstrated that the “classical” MDR-associated mechanisms are similar or identical to those causing resistance to these novel agents. These mechanisms include the enhanced activity of drug pumps, i.e. ABC or alternative transporters; modulation of cellular death pathways; alteration and repair of target molecules; and various less common mechanisms. Together they build a complex network of cellular pathways and molecular mechanisms mediating an individual MDR phenotype. Although the application of new high throughput “-omics” technologies have identified multiple new gene-/protein expression signatures or factors associated with drug resistance, so far none of these findings has been useful for creating improved diagnostic assays, for prediction of individual therapy response, or for development of updated chemosensitizers. Received 05 March 2008; received after revision 21 May 2008; accepted 23 May 2008     

Expression of the anti-apoptotic gene survivin correlates with taxol resistance in human ovarian cancer

Stable transfection of human ovarian carcinoma cells with survivin cDNA caused a four- to sixfold increase in cell resistance to taxotere and taxol (two-sided Student's t test, p < 0.05), with a concomitant reduction in the apoptotic response to taxol, but did not affect cell sensitivity to cisplatin or oxaliplatin. Such findings were indirectly supported by similar observations obtained with clinical tumours. In fact, high levels of survivin protein expression (>30% positive cells), detected by immunohistochemistry in 90/124 (73%) advanced ovarian carcinomas, were significantly associated with clinical resistance to a taxol/platinum-based regimen but unrelated to tumour shrinkage following cisplatin-including combinations (non-taxol based). In the 95 patients receiving a taxol/platinum-based regimen, survivin overexpression correlated with a lower clinical or pathologic complete remission rate than absent/low protein expression (43 vs 75%, p = 0.0058 by logistic regression adjusted for tumour stage, histological grade and p53 expression). Conversely, in the 29 cases treated with cisplatin-containing regimens (not taxol based), survivin expression was unrelated to tumour response. Cellular studies and clinical data suggest a direct link between survivin expression and tumour cell susceptibility to taxol.     

Intracellular localization of DR5 and related regulatory pathways as a mechanism of resistance to TRAIL in cancer

TNF-related apoptosis-inducing ligand (TRAIL) is a prominent cytokine capable of inducing apoptosis. It can bind to five different cognate receptors, through which diverse intracellular pathways can be activated. TRAIL’s ability to preferentially kill transformed cells makes it a promising potential weapon for targeted tumor therapy. However, recognition of several resistance mechanisms to TRAIL-induced apoptosis has indicated that a thorough understanding of the details of TRAIL biology is still essential before this weapon can be confidently unleashed. Critical to this aim is revealing the functions and regulation mechanisms of TRAIL’s potent death receptor DR5. Although expression and signaling mechanisms of DR5 have been extensively studied, other aspects, such as its subcellular localization, non-signaling functions, and regulation of its membrane transport, have only recently attracted attention. Here, we discuss different aspects of TRAIL/DR5 biology, with a particular emphasis on the factors that seem to influence the cell surface expression pattern of DR5, along with factors that lead to its nuclear localization. Disturbance of this balance apparently affects the sensitivity of cancer cells to TRAIL-mediated apoptosis, thus constituting an eligible target for potential new therapeutic agents.     

Herbicide resistance and supersensitivity in photosystem II

Resistance to triazine herbicides in higher plants was first observed in 1970. A mutation in the photosystem II reaction center D1 protein at position Ser264 --> Gly is responsible for this resistance. So far, 37 single mutants, 16 double mutants, 5 triple mutants and 5 deletion/insertion mutants in the D1 protein have been obtained by randomly induced and site-directed mutagenesis in cyanobacteria and algae. The influence of these mutations on the binding affinities of different classes of herbicides will be discussed. Because a sufficiently high resolution X-ray structure of photosystem II does not yet exist, the reaction center of purple photosynthetic bacteria, which is homologous to photosystem II, served as a model. In the bacterial reaction center a total of 25 single and 3 double herbicide-resistant mutants have been generated.     

Antibiotic resistance in microbes

The treatment of infectious disease is compromised by the development of antibiotic-resistant strains of microbial pathogens. A variety of biochemical processes are involved that may keep antibiotics out of the cell, alter the target of the drug, or disable the antibiotic. Studies have shown that resistance determinants arise by either of two genetic mechanisms: mutation and acquisition. Antibiotic resistance genes can be disseminated among bacterial populations by several processes, but principally by conjugation. Thus the overall problem of antibiotic resistance is one of genetic ecology and a better understanding of the contributing parameters is necessary to devise rational approaches to reduce the development and spread of antibiotic resistance and so avoid a critical situation in therapy--a return to a pre-antibiotic era.     

Griseofulvin resistance in dermatophytes

Zusammenfassung Häufigkeit und Eigenschaften der griseofulvinresistenten Mutanten vonMicrosporum gypseum wurden geprüft. 2 Loci, durch welche die Resistenz gesteuert wird, wurden in verschiedenen Kopplungsgruppen identifiziert und im Vergleich mit dem Ausgangsstamm die Resistenz bei allen Mutanten hundertfach vergrössert.     

Disease resistance in farm animals

Genetic variations in disease resistance of farm animals can be observed at all levels of defence against infectious agents. In most cases susceptibility to infections has polygenic origins. In domestic animals only a few instances of a single genetic locus responsible for disease resistance are known. A well-examined example is the Mx1 gene product of certain mice strains conferring selective resistance to influenza virus infections. Attempts to improve disease resistance by gene transfer of different gene constructs into farm animals include the use of monoclonal antibody gene constructs, transgenes consisting of antisense RNA genes directed against viruses and Mx1 cDNA containing transgenes.     

Disease resistance in farm animals

Genetic variations in disease resistance of farm animals can be observed at all levels of defence against infectious agents. In most cases susceptibility to infections has polygenic origins. In domestic animals only a few instances of a single genetic locus responsible for disease resistance are known. A well-examined example is the Mx1 gene product of certain mice strains conferring selective resistance to influenza virus infections. Attempts to improve disease resistance by gene transfer of different gene constructs into farm animals include the use of monoclonal antibody gene constructs, transgenes consisting of antisense RNA genes directed against viruses and Mx1 cDNA containing transgenes.     

Antiparkinsonian drug doses and neuroleptic receptors

Summary The clinical potency of 3 drugs, apomorphine, N-propylnorapomorphine, and bromocryptine, have been found to be closely correlated to their potencies in competing for³H-haloperidol and³H-spiroperidol both of which label the dopamine receptor. This correlation indicates that the direct binding assay may be used to predict clinical potencies of anti-parkinsonian drugs, and indicates that agonists as well as antagonists compete potently for³H-neuroleptic binding.This work was supported by the Ontario Mental Health Foundation, the W. Garfield Weston Foundation and the Medical Research Council of Canada.