Cellular interactions of therapeutically delivered nanoparticles

INTRODUCTION: Nanoparticles (NPs) are used extensively in drug delivery. They are administered through various routes in the host, and their uptake by the cellular environment has been observed in several pathways. After uptake, NPs interact with cells to different extents, depending on their size, shape, surface properties, ligands tagged to the surface and tumor architecture. Complete understanding of such cellular uptake mechanisms and interactions of NPs is important for their effective use in drug delivery. AREAS COVERED: This article describes the various cellular pathways for NP uptake, and the factors affecting NP uptake and interactions with cells. Understanding these two important aspects will help in the future design of NPs for effective and targeted drug delivery. EXPERT OPINION: Surface charge and ligands tagged on the surface of NPs play a critical role in their uptake and interaction with cells; so surface modifications of NPs can offer increased drug delivery effectiveness, for example, the coupling of ligands on the surface of NPs can increase cellular binding, and NPs in biological fluids can be coated with proteins and as such can exert biological effects. All of the factors affecting NP uptake need to be investigated thoroughly before interpreting any NP-cellular interactions.     

Surface functionalization of polymeric nanoparticles for tumor drug delivery: approaches and challenges

Introduction: For years, injectable polymeric nanoparticles (NPs) have been developed for delivering therapeutic agents to the tumors. Frequently, NPs surface have been modified with different moieties and/or ligands to impart stealth effect and/or elicit specific cellular interactions, both known to dramatically affect the in vivo fate and efficacy of these NPs.

Areas covered: We discuss different types of ligands and molecules used for surface functionalization of polymeric NPs for tumor drug delivery. First, we summarize methods used through the literature for surface modification of polymeric NPs, then discuss challenges that face researchers either in decorating NPs with desired surface functionalities, characterizing functionalized surfaces or achieving intended cellular interactions and in vivo effects.

Expert opinion: Modification of NP surfaces dramatically alters their behavior and favorably enhances their therapeutic efficacy. Choice of surface ligand/functionality should be based on intended therapeutic outcomes, taking into consideration the potential of clinical translation and scale up of the developed systems.     

Nanoparticles in drug delivery: mechanism of action,formulation and clinical application towards reduction in drug-associated nephrotoxicity

Introduction: Over the past few decades, nanoparticles (NPs) have gained immeasurable interest in the field of drug delivery. Various NP formulations have been disseminated in drug development in an attempt to increase efficacy, safety and tolerability of incorporated drugs. In this context, NP formulations that increase solubility, control release, and/or affect the in vivo disposition of drugs, were developed to improve the pharmacokinetic and pharmacodynamic properties of encapsulated drugs.

Areas covered: In this article, important properties related to NP function such as particle size, surface charge and shape are disseminated. Also, the current understanding of how NP characteristics affect particle uptake and targeted delivery is elucidated. Selected NP systems currently used in delivery of drugs in biological systems and their production methods are discussed as well. Emphasis is placed on current NP formulations that are shown to reduce drug-induced adverse renal complications.

Expert opinion: Formulation designs utilizing NP-encapsulated drugs offer alternative pharmacotherapy options with improved safety profiles for current and emerging drugs. NPs have been shown to increase the therapeutic index of several entrapped drugs mostly by decreasing drug localization and side effects on organs. Recent studies on NP-encapsulated chemotherapeutic and antibiotic medications show enhanced therapeutic outcomes by altering drug degradation, increasing systemic circulation and/or enhancing cell specific targeting. They may also reduce the distribution of encapsulated drugs into the kidneys and attenuate drug-associated adverse renal complications. The usefulness of NP formulation in reducing the nephrotoxicity of nonsteroidal anti-inflammatory drugs is an underexplored territory that deserves more attention.     

Cholesterol–PEG comodified poly (N-butyl) cyanoacrylate nanoparticles for brain delivery: in vitro and in vivo evaluations

This study investigated cholesterol–polyethylene glycol (PEG) comodified poly (ethyleneglycol)-poly (lactide) nanoparticles (CLS-PEG NPs) as a novel, biodegradable brain drug delivery system and included an evaluation of its in vitro and in vivo properties. To this end, coumarin-6 (C6), a fluorescent probe, was encapsulated into CLS-PEG NPs by an emulsion polymerization method. We reported that the use of CLS-PEG NPs led to a sustained drug release in vitro. Additionally, cell viability experiments confirmed their safety. The uptake and transport of CLS-PEG NPs, by bEnd.3 cells (an immortalized mouse brain endothelial cell line), was significantly higher than that of a control C6 solution. An investigation of the uptake mechanisms of different NP formulations demonstrated that cholesterol modifications may be the primary way to improve the efficiency of cellular uptake, wherein macropinocytosis may be the most important endocytic pathway in this process. An investigation of the transport mechanisms of CLS-PEG NPs also implicated macropinocytosis, energy and cholesterol in bEnd.3 cells lines. Following an intravenous (IV) administration to rats, pharmacokinetic experiments indicated that C6-loaded CLS-PEG NPs achieved sustained release for up to 12?h. In addition, IV delivery of CLS-PEG NPs appeared to significantly improve the ability of C6 to pass through the blood–brain barrier: the concentration of C6 found in the brain increased nearly 14.2-fold when C6 CLS-PEG NPs were used rather than a C6 solution. These in vitro and in vivo results strongly suggest that CLS-PEG NPs are a promising drug delivery system for targeting the brain, with low toxicity.     

Cellular uptake and transport of gold nanoparticles incorporated in a liposomal carrier

Recent interest in using gold nanoparticles (Au NPs) for therapy in radiation medicine has motivated development of a liposome-based system to enhance their delivery to cells. In this study, liposomes were demonstrated to perform like a “Trojan Horse” to deliver small (1.4 nm) Au NPs into tumor cells by overcoming the energetically unfavorable endocytosis process for small NPs. The results reveal that the liposomal approach provides a thousand-fold enhancement in the cellular uptake of the small Au NPs. Real-time intracellular tracking of the Au NP–liposomes revealed an average speed of 12.48 ± 3.12 μm/hr for their intracellular transport. Analysis of the time-dependent intracellular spatial distribution of the Au NP–liposomes demonstrated that they reside in lysosomes (final degrading organelles) within 40 minutes of incubation. Knowledge gained in these studies opens the door to pursuing liposomes as a viable strategy for delivery of Au NPs in radiation therapy applications.From the Clinical EditorGold nanoparticles (Au NPs) as part of an optimized liposome-based delivery system have been proposed for therapy in radiation medicine. The approach resulted in a thousand-fold enhancement in the cellular uptake of Au NPs compared to conventional delivery methods, with the nanoparticles residing in lysosomes within 40 minutes of incubation.     

Concanavalin A-conjugated poly(ethylene glycol)–poly(lactic acid) nanoparticles for intranasal drug delivery to the cervical lymph nodes

Abstract

Concanavalin A (ConA)-conjugated poly(ethylene glycol)–poly(lactic acid) nanoparticles (ConA-NPs) were prepared for targeted drug delivery to the cervical lymph nodes after intranasal administration. ConA, a lectin specifically binding to α-mannose and α-glucose, was covalently conjugated on NPs without loss of its carbohydrates binding bioactivity. In vitro cellular uptake experiment demonstrated that NPs could be uptaken by Calu-3 cells in a time- and concentration-dependent manner, and conjugation of ConA on NPs could significantly increase the rate and amount of cellular uptake. ConA-NP showed no obvious toxicity to Calu-3 cells in vitro or to the nasal cilia of rats in vivo. Compared with NPs without ConA, ConA-NP is more effective in targeting drugs to the deep cervical lymph nodes, as evidenced by 1.36–2.52 times increase of targeting efficiency, demonstrating that ConA-NP is a potential carrier for targeted drug delivery to the cervical lymph nodes via nasal route.     

Effects of poly(ethylene glycol) grafting density on the tumor targeting efficacy of nanoparticles with ligand modification

Abstract

To evaluate the effects of poly(ethylene glycol) (PEG) grafting density on the tumor targeting efficacy of nanoparticles (NPs) with ligand modification, various amounts of PEG were conjugated to linoleic acid and poly(β-malic acid) double grafted chitosan (LMC) NPs bearing similar substitution degree of folate (FA). Increased particle size, decreased surface charge, reduced contact angle, retarded drug release and suppressed protein adsorption of LMC NPs were detected after surface modification. Compared to LMC NPs, FA-modified LMC NPs (FA-LMC NPs) remarkably enhanced tumor specificity. For PEG-modified FA-LMC NPs, increased drug accumulation in tumor tissues and reduced cellular uptake were observed with the increase of PEG grafting density. In regard to in vivo antitumor efficacy, FA-LMC NPs with moderate PEG grafting density (8.9%) significantly outperformed FA-LMC NP. Therefore, PEG modification with moderate grafting density could be a promising approach to coordinating with the tumor targeting efficacy of ligand-modified NPs.     

Toward comprehension of multiple human cells uptake of engineered nano metal oxides: quantitative inter cell line uptake specificity (QICLUS) modeling

To address the nanomaterial exposure threat, it is imperative to understand how nanomaterials are recognized, internalized, and distributed within diverse cell systems. Targeting of nanomaterials to a specific cell type is generally attained through the modification of the nanoparticle (NP) surface leading to required cellular uptake. The enhanced cellular uptake to normal cells can direct to the higher interaction of NPs with subcellular organelles resulting the provocation of various signaling pathways. The successes of NPs rely on the prospect for the synthesis of functionalized NPs with necessary properties and their enhanced potential for cellular uptake for specific targeting. In the present study, we have modeled the cellular uptake of 109 surface modifiers of metal oxide nanoparticles (MNPs) for three different cell lines: HUVEC (Human endothelial cells), U937 (human macrophage cells), and PaCa2 (cancer cell lines). Along with the quantitative structure–activity relationship (QSAR) models, for the very first time we have developed and performed quantitative inter cell line uptake specificity (QICLUS) modeling to identify the physicochemical properties, as well as majorly structural fragments responsible for cellular uptake differences between two specific cell lines. The present work provides a comprehensive understanding of the cellular uptake of MNPs and the underlying structural parameters controlling the nano-cellular interactions. This phenomenon has also been analyzed from the QSAR and QICLUS models that concluded the functional groups of surface modifiers like amine, anhydride, halogen atoms, nitro group, acids have the dominating roles for the uptake of MNPs into the cell lines. Thus, the developed models may be used for designing of novel surface modifiers of MNPs of desired characteristics for proper cell–NPs interactions, as well as in the context of virtual screening aspect. Moreover, the MNP–cell interactions can give some idea about the toxicity for target-specific drug delivery treatment as higher cellular uptake is required for specific cells to treat the disease and lower uptake to the neighboring cells for lower toxicity.     

Lactose-modified DNA tile nanostructures as drug carriers

Background: DNA hybridization allows the preparation of nanoscale DNA structures with desired shape and size. DNA structures using simple base pairing can be used for the delivery of drug molecules into the cells. Since DNA carries multiple negative charges, their cellular uptake efficiency is low. Thus, the modification of the DNA structures with molecules that may enhance the cellular internalization may be an option.

Objective: The objective of this study is to construct DNA-based nanocarrier system and to investigate the cellular uptake of DNA tile with/without lactose modification.

Methods: Doxorubicin was intercalated to DNA tile and cellular uptake of drug-loaded DNA-based carrier with/without lactose modification was investigated in vitro. HeLa, BT-474, and MDA-MB-231 cancer cells were used for cellular uptake studies and cytotoxicity assays. Using fluorescence spectroscopy, flow cytometry, and confocal microscopy, cellular uptake behavior of DNA tile was investigated. The cytotoxicity of DNA tile structures was determined with WST-1 assay.

Results: The results show that modification with lactose effectively increases the intracellular uptake of doxorubicin loaded DNA tile structure by cancer cells compared with the unmodified DNA tile.

Conclusion: The findings of this study suggest that DNA-based nanostructures modified with carbohydrates can be used as suitable multifunctional nanocarriers with simple chemical modifications.     

Functionalized gold nanoparticles improve afatinib delivery into cancer cells

Objectives: A drug delivery system based on colloidal pegylated gold nanoparticles (PEGAuNPs) conjugated with the tyrosine kinase inhibitor afatinib was designed and tested for enhancing the drug activity against pancreatic and NSCLC cells.

Methods: PEGAuNPs were synthesized and characterized physicochemically. Confocal imaging was performed to evaluate the nanoparticle (NP) internalization in cancer cells. For cell-cycle distribution analysis, conjugated NPs and afatinib alone were incubated with cells and alterations on the cell-cycle profile subsequently analyzed by total DNA staining. Cancer cell survival and growth inhibition following incubation with afatinib and PEGAuNPs–afatinib (concentrations between 0.007 and 0.500 µM afatinib) were evaluated.

Results: A higher cellular uptake of PEGAuNPs was observed by cancer cells. Our data suggest an efficient conjugation of PEGAuNPs with the drug, enhancing the afatinib activity in comparison with afatinib alone. In fact, IC₅₀ and GI₅₀ results obtained show that the PEGAuNPs–afatinib conjugate is ca. 5 and 20 times more potent than afatinib alone in S2-013 and A549 cell lines, respectively.

Conclusions: Conjugating PEGAuNPs with afatinib is a promising antitumor delivery system for cancer therapy as it improves drug efficacy, allowing a reduction in drug dose used and minimizing possible toxicity-related side effects.     

Extracellular stability of nanoparticulate drug carriers

Nanoparticulate (NP) drug carrier systems are attractive vehicles for selective drug delivery to solid tumors. Ideally, NPs should evade clearance by the reticuloendothelial system while maintaining the ability to interact with tumor cells and facilitate cellular uptake. Great effort has been made to fulfill these design criteria, yielding various types of functionalized NPs. Another important consideration in NP design is the physical and functional stability during circulation, which, if ignored, can significantly undermine the promise of intelligently designed NP drug carriers. This commentary reviews several NP examples with stability issues and their consequences, ending in a discussion of experimental methods for reliable prediction of NP stability.     

Polydopamine-Based Surface Modification for the Development of Peritumorally Activatable Nanoparticles

Purpose

To create poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), where a drug-encapsulating NP core is covered with polyethylene glycol (PEG) in a normal condition but exposes a cell-interactive TAT-modified surface in an environment rich in matrix metalloproteinases (MMPs).

Methods

PLGA NPs were modified with TAT peptide (PLGA-pDA-TAT NPs) or dual-modified with TAT peptide and a conjugate of PEG and MMP-substrate peptide (peritumorally activatable NPs, PANPs) via dopamine polymerization. Cellular uptake of fluorescently labeled NPs was observed with or without a pre-treatment of MMP-2 by confocal microscopy and flow cytometry. NPs loaded with paclitaxel (PTX) were tested against SKOV-3 ovarian cancer cells to evaluate the contribution of surface modification to cellular delivery of PTX.

Results

While the size and morphology did not significantly change due to the modification, NPs modified with dopamine polymerization were recognized by their dark color. TAT-containing NPs (PLGA-pDA-TAT NPs and PANPs) showed changes in surface charge, indicative of effective conjugation of TAT peptide on the surface. PLGA-pDA-TAT NPs and MMP-2-pre-treated PANPs showed relatively good cellular uptake compared to PLGA NPs, MMP-2-non-treated PANPs, and NPs with non-cleavable PEG. After 3 h treatment with cells, PTX loaded in cell-interactive NPs showed greater toxicity than non-interactive ones as the former could enter cells during the incubation period. However, due to the initial burst drug release, the difference was not as clear as microscopic observation.

Conclusions

PEGylated polymeric NPs that could expose cell-interactive surface in response to MMP-2 were successfully created by dual modification of PLGA NPs using dopamine polymerization.     

Surfactant protein D (SP-D) alters cellular uptake of particles and nanoparticles

Abstract

Surfactant protein D (SP-D) is primarily expressed in the lungs and modulates pro- and anti-inflammatory processes to toxic challenge, maintaining lung homeostasis. We investigated the interaction between NPs and SP-D and subsequent uptake by cells involved in lung immunity. Dynamic light scattering (DLS) and scanning electron microscopy (SEM) measured NP aggregation, particle size and charge in native human SP-D (NhSP-D) and recombinant fragment SP-D (rfhSP-D). SP-D aggregated NPs, especially following the addition of calcium. Immunohistochemical analysis of A549 epithelial cells investigated the co-localization of NPs and rfhSP-D. rfhSP-D enhanced the co-localisation of NPs to epithelial A549 cells in vitro. NP uptake by alveolar macrophages (AMs) and lung dendritic cells (LDCs) from C57BL/6 and SP-D knock-out mice were compared. AMs and LDCs showed decreased uptake of NPs in SP-D deficient mice compared to wild-type mice. These data confirmed an interaction between SP-D and NPs, and subsequent enhanced NP uptake.     

Uptake and bioconversion of stereoisomeric dipeptide prodrugs of ganciclovir by nanoparticulate carriers in corneal epithelial cells

Purpose: The objective of this study is to investigate cellular uptake of prodrug-loaded nanoparticle (NP). Another objective is to study bioconversion of stereoisomeric dipeptide prodrugs of ganciclovir (GCV) including L-Val-L-Val-GCV (LLGCV), L-Val-D-Val-GCV (LDGCV) and d-Val-l-Val-GCV (DLGCV) in human corneal epithelial cell (HCEC) model.

Methods: Poly(_D,L-lactic-co-glycolic acid) (PLGA) NP encapsulating prodrugs of GCV were formulated under a double emulsion method. Fluorescein isothiocyanate isomer–PLGA conjugates were synthesized to fabricate biocompatible fluorescent PLGA NP. Intracellular uptake of FITC-labeled NP was visualized by a fluorescent microscope in HCEC cells.

Results: Fluorescent PLGA NP and non-fluorescent NP display similar hydrodynamic diameter in the range of 115–145?nm with a narrow particle size distribution and zeta potentials around ?13 mV. Both NP types showed identical intracellular accumulation in HCEC cells. Maximum uptake (around 60%) was noted at 3?h for NP. Cellular uptake and intracellular accumulation of prodrugs are significantly different among three stereoisomeric dipeptide prodrugs. The microscopic images show that NPs are avidly internalized by HCEC cells and distributed throughout the cytoplasm instead of being localized on the cell surface. Following cellular uptake, prodrugs released from NP gradually bioreversed into parent drug GCV. LLGCV showed the highest degradation rate, followed by LDGCV and DLGCV.

Conclusion: LLGCV, LDGCV and DLGCV released from NP exhibited superior uptake and bioreversion in corneal cells.     

Extracellularly activatable nanocarriers for drug delivery to tumors

Introduction: Nanoparticles (NPs) for drug delivery to tumors need to satisfy two seemingly conflicting requirements: they should maintain physical and chemical stability during circulation and be able to interact with target cells and release the drug at desired locations with no substantial delay. The unique microenvironment of tumors and externally applied stimuli provide a useful means to maintain a balance between the two requirements.

Areas covered: We discuss nanoparticulate drug carriers that maintain stable structures in normal conditions but respond to stimuli for the spatiotemporal control of drug delivery. We first define the desired effects of extracellular activation of NPs and frequently used stimuli and then review the examples of extracellularly activated NPs.

Expert opinion: Several challenges remain in developing extracellularly activatable NPs. First, some of the stimuli-responsive NPs undergo incremental changes in response to stimuli, losing circulation stability. Second, the applicability of stimuli in clinical settings is limited due to the occasional occurrence of the activating conditions in normal tissues. Third, the construction of stimuli-responsive NPs involves increasing complexity in NP structure and production methods. Future efforts are needed to identify new targeting conditions and increase the contrast between activated and nonactivated NPs while keeping the production methods simple and scalable.     

The interaction of nanoparticles with plasma proteins and the consequent influence on nanoparticles behavior

Introduction: Plasma protein binding with nanoparticles (NPs) occurs immediately upon their introduction into a physiological environment and is affected by the characteristics of NPs, including their composition, size, shape and surface properties. According to their specific functions, adsorbed proteins can be divided into opsonins and dysopsonins. Opsonins often induce the rapid blood clearance of NPs, while dysopsonins benefit prolonged blood circulation.

Areas covered: This review discusses the influential factors that are involved in the interaction between NPs and plasma proteins. The influence of this interaction on distribution of NPs was reviewed followed by the function and influence of ligand modification.

Expert opinion: Protein adsorption is a key element that influences biological responses, such as endocytosis and biodistribution, and also contributes to the characteristics of NPs and the physiological environment. By contrast, the surface modification of ligands is a common and useful method to functionalize NPs to provide an engineered targeting effect. The protein adsorption of ligand-modified NPs is even more important and requires in-depth discussion. Differences between modified and unmodified NPs lead to varying degrees of opsonization, which greatly affects targeting and may result in opposing effects. Understanding these influences is necessary to improve targeting effects and reduce defects in protein adsorption, which are crucial for drug delivery.     

Brain-targeted delivery of paclitaxel using glutathione-coated nanoparticles for brain cancers

Paclitaxel is not effective for treatment of brain cancers because it cannot cross the blood–brain barrier (BBB) due to efflux by P-glycoprotein (P-gp). In this work, glutathione-coated poly-(lactide-co-glycolide) (PLGA) nanoparticles (NPs) of paclitaxel were developed for brain targeting for treatment of brain cancers. P-gp ATPase assay was used to evaluate the NP as potential substrates. The NP showed a particle size suitable for BBB permeation (particle size around 200?nm) and higher cellular uptake of the NP was demonstrated in RG2 cells. The P-gp ATPase assay suggested that the NP were not substrate for P-gp and would not be effluxed by P-gp present in the BBB. The in vitro release profile of the NP exhibited no initial burst release and showed sustained drug release. The proposed coated NP showed significantly higher cytotoxicity in RG2 cells compared with uncoated NP (p?≤?0.05). Tubulin immunofluorescent study showed higher cell death by the NP due to increased microtubule stabilization. In vivo brain uptake study in mice showed higher brain uptake of the NP containing coumarin-6 compared with solution. The proposed brain-targeted NP delivery of paclitaxel could be an effective treatment for the brain cancers.     

SN-38 active loading in poly(lactic-co-glycolic acid) nanoparticles and assessment of their anticancer properties on COLO-205 human colon adenocarcinoma cells

Abstract

SN-38 is a highly effective drug against many cancers. The development of an optimal delivery system for SN-38 is extremely challenging due to its low solubility and labile lactone ring. Herein, SN-38 encapsulated in poly(d,l-lactide-co-glycolide) nanoparticles (NPs) is introduced to enhance its solubility, stability and cellular uptake. SN-38-loaded NPs prepared by spontaneous emulsification solvent diffusion (SESD) method had an average diameter of 310?nm, a zeta potential of ?9.69?mV and a loading efficiency of 71%. They were able to protect the active lactone ring of SN-38 against inactivation under physiological condition. A colorectal adenocarcinoma cell line (COLO-205) was used to assess the NPs effects on cytotoxicity and cellular uptake. Result showed a significant decreased cell proliferation and cell apoptosis. These results suggest that these SN-38-loaded NPs can be an effective delivery system for the treatment of colon cancer and potentially for other types of cancers.     

Design of a novel curcumin-soybean phosphatidylcholine complex-based targeted drug delivery systems

Recently, the global trend in the field of nanomedicine has been toward the design of combination of nature active constituents and phospholipid (PC) to form a therapeutic drug-phospholipid complex. As a particular amphiphilic molecular complex, it can be a unique bridge of traditional dosage-form and novel drug delivery system. In thisarticle, on the basis of drug-phospholipid complex technique and self-assembly technique, we chose a pharmacologically safe and low toxic drug curcumin (CUR) to increase drug-loading ability, achieve controlled/sustained drug release and improve anticancer activity. A novel CUR-soybean phosphatidylcholine (SPC) complex and CUR-SPC complex self-assembled nanoparticles (CUR-SPC NPs) were prepared by a co-solvent method and a nanoprecipitation method. DSPE-PEG-FA was further functionalized on the surface of PEG-CUR-SPC NPs (designed as FA-PEG-CUR-SPC NPs) to specifically increase cellular uptake and targetability. The FA-PEG-CUR-SPC NPs showed a spherical shape, a mean diameter of about 180?nm, an excellent physiological stability and pH-triggered drug release. The drug entrapment efficiency and drug-loading content was up to 92.5 and 16.3%, respectively. In vitro cellular uptake and cytotoxicity studies demonstrated that FA-PEG-CUR-SPC NPs and CUR-SPC NPs presented significantly stronger cellular uptake efficacy and anticancer activity against HeLa cells and Caco-2 cells compared to free CUR, CUR-SPC NPs and PEG-CUR-SPC NPs. More importantly, FA-PEG-CUR-SPC NPs showed the prolonged systemic circulation lifetime and enhanced tumor accumulation compared with free CUR and PEG-CUR-SPC NPs. These results suggest that the FA targeted PEGylated CUR-SPC complex self-assembled NPs might be a promising candidate in cancer therapy.