In vitro evaluation of novel SN-38 prodrug activated by α-rhamnosidase of exogenous enzyme.

This study introduces the α-rhamnose (Rham)-conjugated prodrug of SN-38 (Rham-SN-38) as a promising alternative to irinotecan. α-rhamnosidase, responsible for SN-38 release from Rham-SN-38, does not express in human cells, minimizing individual variability and side effects. The injection of the α-rhamnosidase into the tumor tissues makes it possible, for the first time, to activate the Rham-SN-38. Furthermore, α-rhamnosidase demonstrates significantly higher activity than carboxylesterase, the specific enzyme activating irinotecan. SN-38 release mediated by α-rhamnosidase completes within 2 h, with a kcat/Km value approximately 5.0 × 104-fold higher than that of irinotecan. The 50% inhibition concentration (IC50) of Rham-SN-38 against three types of cancer cells and one normal cell exceeds 4.5 × 103 nM. The addition of α-rhamnosidase significantly increases cytotoxicity, with IC50 comparable to free SN-38. The QIC50, an index reflecting the difference in cytotoxicity with and without α-rhamnosidase, exceeds approximately 1.0 × 102-fold. Rham-SN-38, synthesized in this study, demonstrates significant potential as a prodrug for cancer therapy.

Oral Administration of PLGA Nanoparticles to Deliver Antisense Oligonucleotides to Inflammatory Lesions in the Gastrointestinal Tract.

In this study, we prepared antisense oligonucleotide (ASO)-encapsulated nanoparticles (NPs) with a suitable profile for oral administration for the treatment of inflammatory bowel disease (IBD). We chose a water-in-oil-in-water (w/o/w) method to prepare the NPs using poly(lactide-co-glycolide) as a matrix and Pluronic as a stabilizer. The obtained NPs had a suitable diameter (158 nm) for the penetration of the mucus layer, endocytic uptake by enterocytes, and accumulation in inflammatory lesions in the intestine. The amount of ASOs in the NPs was relatively large (6.41% (w/w)). When the NPs were stably dispersed in solutions that mimicked gastrointestinal (GI) juice, minimal leakage of ASOs was demonstrated over the required period. The NPs were administered orally to mice with colitis induced by dextran sodium sulfate, which reduced target gene expression in the colons and rectums of the mice, whereas naked ASO administration caused no reduction in gene expression. Thus, the NPs have the potential of promising oral carriers of ASOs for the treatment of IBD that specifically target inflammatory lesions in the GI tract, thereby reducing the non-specific toxic effects of ASOs.

Effective design of PEGylated polyion complex (PIC) nanoparticles for enhancing PIC internalisation in cells utilising block copolymer combinations with mismatched ionic chain lengths.

In nanomedicine, PEGylation of nanomaterials poses a dilemma since it inhibits their interaction with target cells and enables their retention in target tissues despite its biocompatibility and nonspecific internalisation suppression. PEGylated polypeptide-based polyion complexes (PICs) are fabricated via the self-assembly of PEGylated aniomers and homocatiomers based on electrostatic interactions. We propose that various parameters like block copolymer design and PIC domain characteristics can enhance the cell-PEGylated PIC interactions. Remarkably, the properties of the PIC domain were tuned by the matched/mismatched ionomer chain lengths, PIC domain crosslinking degree, chemical modification of cationic species after crosslinking, PIC morphologies (vesicles/micelles) and polyethylene glycol (PEG) chain lengths. Cellular internalisation of the prepared PICs was evaluated using HeLa cells. Consequently, mismatched ionomer chain lengths and vesicle morphology enhanced cell-PIC interactions, and the states of ion pairing, particularly cationic residues, affected the internalisation behaviours of PICs via acetylation or guanidinylation of amino groups on catiomers. This treatment attenuated the cell-PIC interactions, possibly because of reduced interaction of PICs with negatively charged species on the cell-surface, glycosaminoglycans. Moreover, morphology and PEG length were correlated with PIC internalisation, in which PICs with longer and denser PEG were internalised less effectively. Cell line dependency was tested using RAW 264.7 macrophage cells; PIC recognition could be maintained after capping amino groups on catiomers, indicating that the remaining anionic groups were still effectively recognised by the scavenger receptors of macrophages. Our strategy for tuning the physicochemical properties of the PEGylated PIC nanocarriers is promising for overcoming the PEG issue.

Dynamic frustrated charge hotspots created by charge density modulation sequester globular proteins into complex coacervates.

This study presents a simple strategy for the sequestration of globular proteins as clients into synthetic polypeptide-based complex coacervates as a scaffold, thereby recapitulating the scaffold-client interaction found in biological condensates. Considering the low net charges of scaffold proteins participating in biological condensates, the linear charge density (σ) on the polyanion, polyethylene glycol-b-poly(aspartic acids), was reduced by introducing hydroxypropyl or butyl moieties as a charge-neutral pendant group. Complex coacervate prepared from the series of reduced-σ polyanions and the polycation, homo-poly-l-lysine, could act as a scaffold that sequestered various globular proteins with high encapsulation efficiency (>80%), which sometimes involved further agglomerations in the coacervates. The sequestration of proteins was basically driven by electrostatic interaction, and therefore depended on the ionic strength and charges of the proteins. However, based on the results of polymer partitioning in the coacervate in the presence or absence of proteins, charge ratios between cationic and anionic polymers were maintained at the charge ratio of unity. Therefore, the origin of the electrostatic interaction with proteins is considered to be dynamic frustrated charges in the complex coacervates created by non-neutralized charges on polymer chains. Furthermore, fluorescence recovery after photobleaching (FRAP) measurements showed that the interaction of side-chains and proteins changed the dynamic property of coacervates. It also suggested that the physical properties of the condensate are tunable before and after the sequestration of globular proteins. The present rational design approach of the scaffold-client interaction is helpful for basic life-science research and the applied frontier of artificial organelles.

A human cell orthogonal enzyme ß-D-galacturonidase for sensitive detection of antigen proteins.

Enzymes are used to amplify signals for detection of antigen proteins in biological samples. However, the enzymes conventionally used for this purpose have limitations, such as the presence of the same (i.e., endogenous) activity in human cells and difficulty in simultaneous use of multiple enzymes because of differences in their required reaction conditions. In this report, we identify an enzyme that can overcome these problems: ß-D-galacturonidase (GalUAase) from Eisenbergiella tayi. GalUAase activity was confirmed to be absent from human cells. The substrate of GalUAase, galacturonic acid, is highly hydrophilic because of its anionic carboxylate group; high substrate hydrophilicity is an ideal characteristic for the substrate of an enzyme used for detection because it decreases nonspecific adsorption to biological samples. We show that E. tayi GalUAase could be used in the detection of antigen proteins on live human cells with lower background signal than the conventionally used enzyme ß-D-galactosidase. The combinatorial use of GalUAase with ß-D-galactosidase enabled simultaneous detection of two antigens on live cells.

[Development of New Sublingual Formulation for Transmucosal Delivery of Peptides].

Since oral bioavailability of peptides is extremely low, self-injectable and intranasal formulations have been developed; however, these treatments have problems such as storage and discomfort. The sublingual route is considered suitable for peptide absorption because there is less peptidase and it is not subject to hepatic first-pass effects. In this study, we attempted to develop a new jelly formulation for sublingual delivery of peptides. Gelatins with molecular weights of 20000 and 100000 were used as the jelly base. The gelatin was dissolved in water with a small amount of glycerin and air-dried for at least 1 d to form a thin jelly formulation. A mixed base of locust bean gum and carrageenan was used as the outer layer of the two-layer jelly. Jelly formulations with various compositions were prepared, and we evaluated the dissolution time of the jelly formulations and urinary excretion. It was found that the dissolution time of the jelly became slower as the amount of gelatin and the molecular weight increased. Using cefazolin as a model drug, urinary excretion after sublingual administration was measured, and it was found that urinary excretion tended to increase when using a two-layer jelly covered with a mixed base of locust bean gum and carrageenan compared to oral administration of an aqueous solution. Our findings suggest that sublingual drug absorption could be improved by allowing the drug eluted from the jelly formulation to remain in sublingual region for a longer time.

Engineered macrophages acting as a trigger to induce inflammation only in tumor tissues.

Herein, we report engineered macrophages, termed "MacTrigger," acting as a trigger to induce an inflammatory environment only in tumor tissues. This led to intensive anti-tumor effects based on the removal potential of foreign substances. The strength of this study is the utilization of two unique functions of macrophages: (1) their ability to migrate to tumor tissues and (2) polarization into the anti-inflammatory M2 phenotype in the presence of tumor tissues. The MacTrigger accelerated the release of inflammatory cytokines, tumor necrosis factor-alpha (TNF-α), when it was polarized to the M2 phenotype. When the MacTrigger was administered to tumor-bearing mice, tumor growth was significantly inhibited compared with the non-treatment group, the un-transfected macrophages group, and the group with engineered macrophages capable of randomly releasing TNF-α. Additionally, the ratio of the M1 phenotype to the M2 phenotype in tumor tissues was >1 only in the MacTrigger group. Moreover, the ratios of natural killer cells and CD8+T cells in tumor tissues were increased compared with other groups. These results indicate that MacTrigger can induce inflammation in tumor tissues, leading to effective anti-tumor effects. In normal tissues, especially the liver, notable side effects were not observed. This is because, in the liver, the MacTrigger was not polarized to the M2 phenotype and could not induce inflammation. These results suggest that the MacTrigger is a "trigger" that can induce inflammation only in tumor tissues, then allowing the body to attack tumor tissues through the innate immunity system.

Engineered macrophages acting as a trigger to induce inflammation only in tumor tissues based on arginase 1-responsive TNF-α accelerated release.

Herein, we report engineered macrophages, termed "MacTrigger," acting as a trigger to induce an inflammatory environment only in tumor tissues. This led to intensive anti-tumor effects based on the removal potential of foreign substances. The strength of this study is the utilization of two unique functions of macrophages: (1) their ability to migrate to tumor tissues and (2) polarization into the anti-inflammatory M2 phenotype in the presence of tumor tissues. The MacTrigger accelerated the release of inflammatory cytokines, tumor necrosis factor-alpha (TNF-α), when it was polarized to the M2 phenotype. When the MacTrigger was administered to tumor-bearing mice, tumor growth was significantly inhibited compared with the non-treatment group, the un-transfected macrophages group, and the group with engineered macrophages capable of randomly releasing TNF-α. Additionally, the ratio of the M1 phenotype to the M2 phenotype in tumor tissues was >1 only in the MacTrigger group. Moreover, the ratios of natural killer cells and CD8+T cells in tumor tissues were increased compared with other groups. These results indicate that MacTrigger can induce inflammation in tumor tissues, leading to effective anti-tumor effects. In normal tissues, especially the liver, notable side effects were not observed. This is because, in the liver, the MacTrigger was not polarized to the M2 phenotype and could not induce inflammation. These results suggest that the MacTrigger is a "trigger" that can induce inflammation only in tumor tissues, then allowing the body to attack tumor tissues through the innate immunity system.

Characterization of polypropyleneimine as an alternative transfection reagent.

Polypropyleneimine (PPI) was examined as a transfection reagent comparing with most widely used polymer, polyethyleneimine (PEI). PPI had better responsiveness to the endosomal pH and showed more condensation ability of plasmid DNA than PEI. Although the cytotoxicity of PPI was somewhat higher than PEI, the transfection efficacy of PPI was comparable with PEI or higher than PEI in some cell line. Thus, PPI would be an alternative transfection reagent.

Facile preparation of hexagonal nanosheets via polyion complex formation from α-helical polypeptides and polyphosphate-based molecules.

The polyion complex-based supramolecular self-assembly of hexagonal nanosheets was achieved via the complexation of a PEGylated block catiomer with ATP and other polyphosphate-containing small molecules. The formation of hexagonal nanosheets required the presence of a polyethylene glycol block and α-helix formation in the catiomer block, which was induced by complexation with the polyphosphate moiety.

A Mouse Model for Tuberculosis Combined With Inhalable Imiquimod-PLGA Nanocomposite Particles Based on Macrophage Phenotype.

BACKGROUND/AIM: In vivo models of tuberculosis are effective tools for developing new drugs. The objective of this study was to prepare in vivo models for tuberculosis by utilizing nanocomposite particles (NCPs) containing imiquimod-loaded poly(lactic-co-glycolic acid) nanoparticles. MATERIALS AND METHODS: NCPs were prepared from dichloromethane with imiquimod and poly(lactic-co-glycolic acid) using a spray dryer. Mice were treated with NCPs in the lungs by inhalation, and then infection with Mycobacterium bovis bacille Calmette-Guerin was performed (treatment groups). The concentrations of the pro-inflammatory cytokines, tumor necrosis factor-α and interferon-γ were measured in bronchoalveolar lavage fluid using an enzyme-linked immunosorbent assay. RESULTS: When animals were treated with NCPs, the concentrations of tumor necrosis factor-α and interferon-γ in bronchoalveolar lavage fluid were significantly higher than in animals not treated with NCPs. In addition, high bacterial counts and circular granuloma were observed. CONCLUSION: NCPs prepared in this study enhanced the level of inflammation in the lungs and support the preparation of in vivo models of tuberculosis.

Nanostructure Control of an Antibiotic-Based Polyion Complex Using a Series of Polycations with Different Side-Chain Modification Rates.

Developing nanovehicles for delivering antibiotics is a promising approach to overcome the issue of antibiotic resistance. This study aims to utilize a polyion complex (PICs) system for developing novel nanovehicles for polymyxin-type antibiotics, which are known as last resort drugs. The formation of antibiotic-based PIC nanostructures is investigated using colistimethate sodium (CMS), an anionic cyclic short peptide, and a series of block catiomers bearing different amounts of guanidinium moieties on their side chains. In addition, only the modified catiomer, and not the unmodified catiomer, self-assembles with CMS, implying the importance of the guanidine moieties for enhancing the interaction between the catiomer and CMS via the formation of multivalent hydrogen bonding. Moreover, micellar and vesicular PIC nanostructures are selectively formed depending on the ratio of the guanidine residues. Size-exclusion chromatography reveals that the encapsulation efficiency of CMS is dependent on the guanidinium modification ratio. The antimicrobial activity of the PIC nanostructures is also confirmed, indicating that the complexation of CMS in the PICs and further release from the PICs successfully occurs.

Preparation of Micelles Encapsulating Doxorubicin and Their Anticancer Effect in Combination With Tranilast in 3D Spheroids and In Vivo.

BACKGROUND/AIM: The objective of this study was to prepare doxorubicin encapsulated in micelles (DOX-micelles) using poly(hexadecanyloxyethylene glycol-lactate phosphate), which we recently synthesized, and to evaluate the anticancer effect of DOX-micelles in vitro and in vivo. MATERIALS AND METHODS: To evaluate the anticancer effect of DOX-micelles in vitro, three-dimensional spheroids composed of B16 mouse melanoma cells and fibroblasts were prepared by changing the ratio of cancer cells to fibroblasts. In addition, for efficient doxorubicin treatment of the cells present in the center of the spheroids, tranilast, an anti-fibrotic drug was added to the spheroids before treatment with DOX-micelles, then the amount of doxorubicin and cell viability of spheroids were evaluated. Moreover, to assess the effects of the combination of DOX-micelles with tranilast in vivo, relative tumor volume was investigated in a mouse model of melanoma. RESULTS: The mean diameter and doxorubicin content of DOX-micelles were 93.3 nm and 3.5%, respectively. When the ratio of cancer cells to fibroblasts was 20:80, spheroids with spherical and rigid shapes were obtained. In addition, the amount of doxorubicin in the spheroids was increased by tranilast treatment, and an efficient anticancer effect was also observed. The anticancer effect of the combination of tranilast and DOX-micelles was confirmed in vivo. CONCLUSION: Micelles encapsulating doxorubicin are promising for cancer therapy, and their anticancer effect is improved by tranilast pretreatment in 3D spheroids in vivo.

Poly(Alkyloxyethylene-Lactate Phosphate) for Delivery of Doxorubicin: Synthesis and Characterization.

BACKGROUND/AIM: Serious side effects are associated with the use of doxorubicin. Nanoparticles as carriers for anticancer drugs are useful for reducing side effects and improving therapeutic effects. In this study, a polymer for preparing doxorubicin-containing nanoparticles was developed. Using a novel strategy, a biodegradable poly(oxyethylene glycol lactate H-phosphonate) based on dimethyl H-phosphonate and poly(ethylene glycol)-lactate (PEG-lactate) was synthesized. MATERIALS AND METHODS: Poly(hexadecanyloxyethylene - lactate phosphate) was obtained via chlorination of poly(oxyethylene glycol - lactate H-phosphonate) with trichloroisocyanuric acid and the addition of 1-hexadecanol. The polymer was characterized by 1H NMR and 31P NMR. RESULTS: The results of 1H NMR and 31P NMR showed that the polymer was successfully synthesized, and the yield was 46.9%. CONCLUSION: Poly(hexadecanyloxyethylene - lactate phosphate) has potential as a drug carrier.

Strategies Using Gelatin Microparticles for Regenerative Therapy and Drug Screening Applications.

Gelatin, a denatured form of collagen, is an attractive biomaterial for biotechnology. In particular, gelatin particles have been noted due to their attractive properties as drug carriers. The drug release from gelatin particles can be easily controlled by the crosslinking degree of gelatin molecule, responding to the purpose of the research. The gelatin particles capable of drug release are effective in wound healing, drug screening models. For example, a sustained release of growth factors for tissue regeneration at the injured sites can heal a wound. In the case of the drug screening model, a tissue-like model composed of cells with high activity by the sustained release of drug or growth factor provides reliable results of drug effects. Gelatin particles are effective in drug delivery and the culture of spheroids or cell sheets because the particles prevent hypoxia-derived cell death. This review introduces recent research on gelatin microparticles-based strategies for regenerative therapy and drug screening models.

Efficient cell transplantation combining injectable hydrogels with control release of growth factors.

INTRODUCTION: The objective of this study is to investigate the effect of gelatin microspheres incorporating growth factors on the therapeutic efficacy in cell transplantation. The strength of this study is to combine gelatin hydrogel microspheres incorporating basic fibroblast growth factor and platelet growth factor mixture (GM/GF) with bioabsorbable injectable hydrogels (iGel) for transplantation of adipose-derived stem cells (ASCs). METHODS: The rats ASCs suspended in various solutions were transplanted in masseter muscle. Rats were euthanized 2, 7, 14 days after injection for measurement of the number of ASCs retention in the muscle and morphological evaluation of muscle fibers and the inflammation of the injected tissue by histologic and immunofluorescent stain. RESULTS: Following the injection into the skeletal muscle, the GM/GF allowed the growth factors to release at the injection site over one week. When ASCs were transplanted into skeletal muscle using iGel incorporating GM/GF (iGel+GM/GF), the number of cells grafted was significantly high compared with other control groups. Moreover, for the groups to which GM/GF was added, the cells transplanted survived, and the Myo-D expression of a myoblast marker was observed at the region of cells transplanted. CONCLUSIONS: The growth factors released for a long time likely enhance the proliferative and differentiative capacity of cells. The simple combination with iGel and GM/GF allowed ASCs to enhance their survival at the injected site and consequently achieve improved therapeutic efficacy in cell transplantation.

Biomaterial-Assisted Regenerative Medicine.

This review aims to show case recent regenerative medicine based on biomaterial technologies. Regenerative medicine has arousing substantial interest throughout the world, with "The enhancement of cell activity" one of the essential concepts for the development of regenerative medicine. For example, drug research on drug screening is an important field of regenerative medicine, with the purpose of efficient evaluation of drug effects. It is crucial to enhance cell activity in the body for drug research because the difference in cell condition between in vitro and in vivo leads to a gap in drug evaluation. Biomaterial technology is essential for the further development of regenerative medicine because biomaterials effectively support cell culture or cell transplantation with high cell viability or activity. For example, biomaterial-based cell culture and drug screening could obtain information similar to preclinical or clinical studies. In the case of in vivo studies, biomaterials can assist cell activity, such as natural healing potential, leading to efficient tissue repair of damaged tissue. Therefore, regenerative medicine combined with biomaterials has been noted. For the research of biomaterial-based regenerative medicine, the research objective of regenerative medicine should link to the properties of the biomaterial used in the study. This review introduces regenerative medicine with biomaterial.

Immunosuppressive mesenchymal stem cells aggregates incorporating hydrogel microspheres promote an in vitro invasion of cancer cells.

INTRODUCTION: The objective of this study is to design a co-culture system of cancer cells and three-dimensional (3D) mesenchymal stem cells (MSC) aggregates for the in vitro evaluation of cancer invasion. METHODS: First, the MSC of an immunosuppressive phenotype (MSC2) were prepared by the MSC stimulation of polyriboinosinic polyribocytidylic acid. By simple mixing MSC2 and gelatin hydrogel microspheres (GM) in a U-bottomed well of 96 well plates which had been pre-coated with poly (vinyl alcohol), 3D MSC2 aggregates incorporating GM were obtained. The amount of chemokine (C-C motif) ligand 5 (CCL5) secreted from the MSC2 aggregates incorporating GM. Finally, an invasion assay was performed to evaluate the cancer invasion rate by co-cultured cancer cells and the 3D MSC2 incorporating GM. RESULTS: The amount of CCL5 secreted for the 3D MSC2 aggregates incorporating GM was significantly higher than that of two-dimensional (2D) MSC, 2D MSC2, and 3D MSC aggregates incorporating GM. When MDA-MB-231 human breast cancer cells were co-cultured with the 3D MSC2 aggregates incorporating GM, the invasion rate of cancer cells was significantly high compared with that of 2D MSC or 2D MSC2 and 3D MSC aggregates incorporating GM. In addition, high secretion of matrix metalloproteinase-2 was observed for the 3D MSC2 aggregates/cancer cells system. CONCLUSIONS: It is concluded that the co-culture system of 3D MSC2 aggregates incorporating GM and cancer cells is promising to evaluate the invasion of cancer cells in vitro.

Three-Dimensional Culture System of Cancer Cells Combined with Biomaterials for Drug Screening.

Anticancer drug screening is one of the most important research and development processes to develop new drugs for cancer treatment. However, there is a problem resulting in gaps between the in vitro drug screening and preclinical or clinical study. This is mainly because the condition of cancer cell culture is quite different from that in vivo. As a trial to mimic the in vivo cancer environment, there has been some research on a three-dimensional (3D) culture system by making use of biomaterials. The 3D culture technologies enable us to give cancer cells an in vitro environment close to the in vivo condition. Cancer cells modified to replicate the in vivo cancer environment will promote the biological research or drug discovery of cancers. This review introduces the in vitro research of 3D cell culture systems with biomaterials in addition to a brief summary of the cancer environment.

A Co-Culture System of Three-Dimensional Tumor-Associated Macrophages and Three-Dimensional Cancer-Associated Fibroblasts Combined with Biomolecule Release for Cancer Cell Migration.

The objective of this study is to design a cancer invasion model by making use of cancer-associated fibroblasts (CAF) or tumor-associated macrophages (TAM) and gelatin hydrogel microspheres (GM) for the sustained release of drugs. The GM containing adenosine (A) (GM-A) were prepared and cultured with TAM to obtain three-dimensional (3D) TAM aggregates incorporating GM-A (3D TAM-GM-A). The GM-A incorporation enabled TAM to enhance the secretion level of vascular endothelial growth factor. When co-cultured with HepG2 liver cancer cells in an invasion assay, the 3D TAM-GM-A promoted the invasion rate of cancer cells. In addition, the E-cadherin expression level decreased to a significantly greater extent compared with that co-cultured with TAM aggregates incorporating GM, whereas the significantly higher expression of N-cadherin and Vimentin was observed. This indicates that the epithelial-mesenchymal transition event was induced. The GM containing transforming growth factor-ß1 (TGF-ß1) were prepared to incorporate into 3D CAF (3D CAF-GM-TGF-ß1). Following a co-culture of mixed 3D CAF-GM-TGF-ß1 and 3D TAM-GM-A and every HepG2, MCF-7 breast cancer cell, or WA-hT lung cancer cell, the invasion rate of every cancer cell enhanced depending on the mixing ratio of 3D TAM-GM-A and 3D CAF-GM-TGF-ß1. The amount of matrix metalloproteinase-2 (MMP-2) secreted also enhanced, and the enhancement was well corresponded with that of cancer cell invasion rate. The higher MMP secretion assists the breakdown of basement membrane, leading to the higher rate of cancer cell invasion. This model is a promising 3D culture system to evaluate the invasion ability of various cancer cells in vitro. Impact statement This study proposes a cell culture system to enhance the tumor-associated macrophage function based on the combination of three-dimensional (3D) cell aggregates and gelatin hydrogel microspheres (GM) for adenosine delivery. An additional combination of 3D cancer-associated fibroblasts incorporating GM containing transforming growth factor-ß1 allowed cancer cells to enhance their invasion rate. This co-culture system is promising to evaluate the ability of cancer cell invasion for anticancer drug screening.