Multivalent Binding and Biomimetic Cell Rolling Improves the Sensitivity and Specificity of Circulating Tumor Cell Capture.

Purpose: We aimed to examine the effects of multivalent binding and biomimetic cell rolling on the sensitivity and specificity of circulating tumor cell (CTC) capture. We also investigated the clinical significance of CTCs and their kinetic profiles in patients with cancer undergoing radiotherapy treatment.Experimental Design: Patients with histologically confirmed primary carcinoma undergoing radiotherapy, with or without chemotherapy, were eligible for enrollment. Peripheral blood was collected prospectively at up to five time points, including before radiotherapy, at the first week, mid-point and final week of treatment, as well as 4 to 12 weeks after completion of radiotherapy. CTC capture was accomplished using a nanotechnology-based assay (CapioCyte) functionalized with aEpCAM, aHER-2, and aEGFR.Results: CapioCyte was able to detect CTCs in all 24 cancer patients enrolled. Multivalent binding via poly(amidoamine) dendrimers further improved capture sensitivity. We also showed that cell rolling effect can improve CTC capture specificity (% of captured cells that are CK+/CD45-/DAPI+) up to 38%. Among the 18 patients with sequential CTC measurements, the median CTC decreased from 113 CTCs/mL before radiotherapy to 32 CTCs/mL at completion of radiotherapy (P = 0.001). CTCs declined throughout radiotherapy in patients with complete clinical and/or radiographic response, in contrast with an elevation in CTCs at mid or post-radiotherapy in the two patients with known pathologic residual disease.Conclusions: Our study demonstrated that multivalent binding and cell rolling can improve the sensitivity and specificity of CTC capture compared with multivalent binding alone, allowing reliable monitoring of CTC changes during and after treatment. Clin Cancer Res; 24(11); 2539-47. ©2018 AACR.

Integration of biomimicry and nanotechnology for significantly improved detection of circulating tumor cells (CTCs).

Circulating tumor cells (CTCs) have received a great deal of scientific and clinical attention as a biomarker for diagnosis and prognosis of many types of cancer. Given their potential significance in clinics, a variety of detection methods, utilizing the recent advances in nanotechnology and microfluidics, have been introduced in an effort of achieving clinically significant detection of CTCs. However, effective detection and isolation of CTCs still remain a tremendous challenge due to their extreme rarity and phenotypic heterogeneity. Among many approaches that are currently under development, this review paper focuses on a unique, promising approach that takes advantages of naturally occurring processes achievable through application of nanotechnology to realize significant improvement in sensitivity and specificity of CTC capture. We provide an overview of successful outcome of this biomimetic CTC capture system in detection of tumor cells from in vitro, in vivo, and clinical pilot studies. We also emphasize the clinical impact of CTCs as biomarkers in cancer diagnosis and predictive prognosis, which provides a cost-effective, minimally invasive method that potentially replaces or supplements existing methods such as imaging technologies and solid tissue biopsy. In addition, their potential prognostic values as treatment guidelines and that ultimately help to realize personalized therapy are discussed.

Intermolecular Structural Change for Thermoswitchable Polymeric Photosensitizer.

We developed a thermoswitchable polymeric photosensitizer (T-PPS) by conjugating PS (Pheophorbide-a, PPb-a) to a temperature-responsive polymer backbone of biocompatible hydroxypropyl cellulose. Self-quenched PS molecules linked in close proximity by π-π stacking in T-PPS were easily transited to an active monomeric state by the temperature-induced phase transition of polymer backbones. The temperature-responsive intermolecular interaction changes of PS molecules in T-PPS were demonstrated in synchrotron small-angle X-ray scattering and UV-vis spectrophotometer analysis. The T-PPS allowed switchable activation and synergistically enhanced cancer cell killing effect at the hyperthermia temperature (45 °C). Our developed T-PPS has the considerable potential not only as a new class of photomedicine in clinics but also as a biosensor based on temperature responsiveness.

Acidic pH-Triggered Drug-Eluting Nanocomposites for Magnetic Resonance Imaging-Monitored Intra-arterial Drug Delivery to Hepatocellular Carcinoma.

Transcatheter hepatic intra-arterial (IA) injection has been considered as an effective targeted delivery technique for hepatocellular carcinoma (HCC). Recently, drug-eluting beads (DEB) were developed for transcatheter IA delivery to HCC. However, the conventional DEB has offered relatively modest survival benefits. It can be difficult to control drug loading/release from DEB and to monitor selective delivery to the targeted tumors. Embolized DEBs in hepatic arteries frequently induce hypoxic and low pH conditions, promoting cancer cell growth. In this study, an acidic pH-triggered drug-eluting nanocomposite (pH-DEN) including superparamagnetic iron oxide nanocubes and pH-responsive synthetic peptides with lipid tails [octadecylamine-p(API-l-Asp)10] was developed for magnetic resonance imaging (MRI)-monitored transcatheter delivery of sorafenib (the only FDA-approved systemic therapy for liver cancer) to HCC. The synthesized sorafenib-loaded pH-DENs exhibited distinct pH-triggered drug release behavior at acidic pH levels and highly sensitive MR contrast effects. In an orthotopic HCC rat model, successful hepatic IA delivery and distribution of sorafenib-loaded pH-DEN was confirmed with MRI. IA-delivered sorafenib-loaded pH-DENs elicited significant tumor growth inhibition in a rodent HCC model. These results indicate that the sorafenib-pH-DENs platform has the potential to be used as an advanced tool for liver-directed IA treatment of unresectable HCC.

Photosensitizer-Conjugated Hyaluronic Acid-Shielded Polydopamine Nanoparticles for Targeted Photomediated Tumor Therapy.

Photodynamic therapy (PDT) is a widely used clinical option for tumor therapy. However, the clinical utilization of conventional small-molecule photosensitizers (PSs) for PDT has been limited by their low selectivity for disease sites, and undesirable photoactivation. To overcome these limitations, we demonstrated a tumor-specific and photoactivity-controllable nanoparticle photomedicine based on a combination of PS-biomacromolecule conjugates and polydopamine nanoparticles (PD-NP) for an effective tumor therapy. This novel photomedicine consisted of a PD-NP core and a PS-conjugated hyaluronic acid (PS-HA) shell. The PD-NP and the PS-HA play roles as a quencher for PSs and a cancer targeting moiety, respectively. The synthesized PS-HA-shielded PD-NPs (PHPD-NPs) had a relatively narrow size distribution (approximately 130 nm) with uniform spherical shapes. In response to cancer-specific intracellular enzymes (e.g., hyaluronidase), the PHPD-NPs exhibited an excellent singlet oxygen generation capacity for PDT. Furthermore, an efficient photothermal conversion ability for photothermal therapy (PTT) was also shown in the PHPD-NPs system. These properties provide superior therapeutic efficacy against cancer cells. In mice tumor model, the photoactive restorative effects of the PHPD-NPs were much higher in cancer microenvironments compared to that in the normal tissue. As a result, the PHPD-NPs showed a significant antitumor activity in in vivo mice tumor model. The nanoparticle photomedicine design is a novel strategy for effective tumor therapy.

Recent advances in nanotechnology-based detection and separation of circulating tumor cells.

Although circulating tumor cells (CTCs) in blood have been widely investigated as a potential biomarker for diagnosis and prognosis of metastatic cancer, their inherent rarity and heterogeneity bring tremendous challenges to develop a CTC detection method with clinically significant specificity and sensitivity. With advances in nanotechnology, a series of new methods that are highly promising have emerged to enable or enhance detection and separation of CTCs from blood. In this review, we systematically categorize nanomaterials, such as gold nanoparticles, magnetic nanoparticles, quantum dots, graphenes/graphene oxides, and dendrimers and stimuli-responsive polymers, used in the newly developed CTC detection methods. This will provide a comprehensive overview of recent advances in the CTC detection achieved through application of nanotechnology as well as the challenges that these existing technologies must overcome to be directly impactful on human health.

Reactive oxygen species responsive drug releasing nanoparticle based on chondroitin sulfate-anthocyanin nanocomplex for efficient tumor therapy.

To develop a reactive oxygen species (ROS) sensitive drug carrier, a chondroitin sulfate (CS)-anthocyanin (ATC) based nanocomplex was developed. Doxorubicin hydrochloride (DOX) was loaded in the CS-ATC nanocomplex (CS-ATC-DOX) via intermolecular stacking interaction. The nanocomplex was fabricated by a simple mixing method in the aqueous phase. The morphology and size of CS-ATC-DOX were determined by ATC content. In the group with 1.5mg/ml of ATC loaded CS-ATC-DOX (CS-ATC2-DOX), the drug content and loading efficiency were 8.5% and 99.1%, respectively. The ROS sensitive drug release of CS-ATC2-DOX was confirmed under in vitro physiological conditions. The results demonstrated that 1.67 times higher DOX release occurred in CS-ATC2-DOX for 48h compared to CS-DOX (ATC absent sample). Drug release and nanocomplex destruction were induced by ROS mediated ATC degradation. We determined that 66.7% of ROS was scavenged by CS-ATC2-DOX. Additionally, an HCT-116 tumor bearing animal model was used to confirm ROS sensitive therapeutic effects of CS-ATC2-DOX. The results indicate that DOX was released from the intravenously injected CS-ATC2-DOX in the tumor tissue. Thus, nuclei shrinkage and dead cells were observed in H&E staining and TUNEL assay, respectively. These data suggest that the tumor growth was effectively inhibited. This study means that CS-ATC2-DOX has potential in improving tumor therapy.

Vitamin Bc -Bearing Hydrophilic Photosensitizer Conjugate for Photodynamic Cancer Theranostics.

The accurate diagnosis and proper therapy for cancer are essential to improve the success rate of cancer treatment. Here, we demonstrated that the vitamin Bc -bearing hydrophilic photosensitizer conjugate folic acid-polyethylene glycol-pheophorbideA (FA-PEG-PheoA) has been synthesized for the intracellular diagnosis and photodynamic therapy of a tumor. The synthesized vitamin Bc -bearing hydrophilic photosensitizer conjugate has been characterized for the folic acid receptor expressing the ability to target tumor cells, which is facilitated by the chemical conjugation with folic acid. The vitamin Bc -bearing hydrophilic photosensitizer conjugate internalization mechanism was identified through a competitive inhibition test with free folic acid. We optimized the laser-sensitive, cytotoxicity changeable, vitamin Bc -bearing hydrophilic photosensitizer conjugate concentration, which is non-cytotoxic under normal conditions and specifically cytotoxic toward cancer cells (maximum 69.15%) under laser irradiation conditions used for theranostic agents. The cancer therapeutic and diagnosis effects of synthesized conjugate were confirmed in MDA-MB-231 cells and MDA-MB-231-bearing mice. As a result, the vitamin Bc -bearing hydrophilic photosensitizer conjugate exhibited a highly photodynamic therapeutic effect, which enabled the selective detection of a folic acid receptor expressing cancer using optical imaging.

Multifunctional tumor pH-sensitive self-assembled nanoparticles for bimodal imaging and treatment of resistant heterogeneous tumors.

Nanoparticle-based diagnosis-therapy integrative systems represent an emerging approach to cancer treatment. However, the diagnostic sensitivity, treatment efficacy, and bioavailability of nanoparticles as well as the heterogeneity and drug resistance of tumors pose tremendous challenges for clinical implementation. We herein report on the fabrication of tumor pH-sensitive magnetic nanogrenades (termed PMNs) composed of self-assembled iron oxide nanoparticles and pH-responsive ligands. These PMNs can readily target tumors via surface-charge switching triggered by the acidic tumor microenvironment, and are further disassembled into a highly active state in acidic subcellular compartments that "turns on" MR contrast, fluorescence and photodynamic therapeutic activity. We successfully visualized small tumors implanted in mice via unique pH-responsive T1MR contrast and fluorescence, demonstrating early stage diagnosis of tumors without using any targeting agents. Furthermore, pH-triggered generation of singlet oxygen enabled pH-dependent photodynamic therapy to selectively kill cancer cells. In particular, we demonstrated the superior therapeutic efficacy of PMNs in highly heterogeneous drug-resistant tumors, showing a great potential for clinical applications.

Endolysosomal environment-responsive photodynamic nanocarrier to enhance cytosolic drug delivery via photosensitizer-mediated membrane disruption.

The endolysosome is a major barrier for the effective intracellular delivery by conventional nanocarriers. Herein, we demonstrate that endolysosome environment-responsive photodynamic nanocarriers (EPNs) are capable of encapsulation of the hydrophobic drug paclitaxel (PTX) and photosensitizer (PS)-mediated ELB disruption for effective cancer therapy. EPNs were self-assembled from PS (chlorin e6, Ce6) or Black Hole Quencher-3 (BHQ3) conjugated covalently to polypeptide-based amphiphilic copolymers [monomethoxy polyethylene glycol-block-poly(ß-benzyl-L-aspartic acid), mPEG-pBLA]. EPNs have a spherical shape and a unimodal size distribution below 100 nm. Photoquenching of the EPNs was dependent on the molar ratio of mPEG-pBLA-BHQ3/mPEG-pBLA-Ce6. However, in the presence of the endolysosomal enzyme (e.g., esterase), the benzyl ester bond is cleaved which leads to the structural collapse of EPNs, thus triggering drug release and restoring photoactivity. Live cell imaging studies demonstrated that PS-mediated lipid peroxidation significantly increased the ability of model drug (i.e., Nile red) to overcome the ELB. In comparison with PTX treatment alone, the combined treatment of PTX encapsulated EPNs with laser irradiation synergistically induced the death of HeLa and drug-resistant HCT-8 cells in vitro, and suppressed CT-26 tumor growth in vivo. These results suggest that this approach is a promising platform for cancer treatment. Furthermore, this EPN system offers significant potential for effective cytosolic delivery of chemical and biological therapeutics.

Photo-activatable ternary complex based on a multifunctional shielding material for targeted shRNA delivery in cancer treatment.

A photo-activatable ternary complex (PTC) consisting of multifunctional shielding material (MSM) with photosensitizer (PS)-conjugated chondroitin sulfate and polyethyleneimine based binary complexes containing epidermal growth factor receptor (EGFR)-shRNA delivery for CD44 targeted cancer therapy has been developed. The PTC has a negative surface charge of -37.9 mV, and a size of approximately 90 nm, and the ternary complexes were found to be stable against plasma proteins. The stable nanostructure of PTC especially could enable CD44-receptor mediated tumor-targeted delivery and PS-mediated endosomal disruption for efficient gene silencing, and an enhanced rate of cancer cell death was achieved both in vitro and in vivo. This suggests that PTC could represent a promising strategy for the delivery of other therapeutic genes for cancer therapy.

Photo-mediated internalization of nanocomplex for effective gene delivery to adipose tissue-derived stem cells.

To deliver efficiently osteogenic, chondrogenic or adipogenic induction genes, such as Runx2, SOX9 and C/EBP-α, to adipose tissue-derived stem cells (ADSCs), a photo-mediated nanocomplex internalization gene delivery system was designed using chlorin e6 as a photosensitizer (PS) and polyethyleneimine (PEI) as a gene delivery carrier. In this system, gene delivery efficacy was significantly increased in ADSCs by photo irradiation. The gene transfection efficiency of Runx2, SOX9 and C/EBP-α was increased by 8.6-, 6.7- and 9.3-fold, respectively, by applying 0.7J/cm(2) of irradiation. Osteogenic, chondrogenic and adipogenic differentiation was confirmed by differentiation-related markers and histological analysis. ADSCs transfected with Runx2, SOX9 and C/EBP-α genes via photo irradiation indicated enhanced differentiation in comparison to the non-irradiated cells. These findings demonstrate that photo-mediated internalization is a promising system for efficient gene delivery and differentiation in ADSCs.

Biodegradable cationic nanoparticles loaded with an anticancer drug for deep penetration of heterogeneous tumours.

To enhance limited drug penetration that mediated drug resistance and heterogeneity within the tumour microenvironment, we designed a paclitaxel (PTX) loaded degradable cationic nanogel (DpNG) consisted with acetylated pullulan and low molecular weight polyethyleneimine ((Low)bPEI, 1.8 kDa). The restoration of cationic charge on the DpNG was achieved via HA degradation by hyaluronidase which is secreted in tumour. The size and surface charge of HA-coated DpNG loaded with PTX (HA/DpNG-PTX) was 200-250 nm and 0 mV, respectively. The DpNG-PTX was showed significant cytotoxicity in heterogeneous cancer cells. The IC50 value of DpNG-PTX was 100 times less than that of free PTX. The growth of heterogeneous tumour in Balb/c mice was inhibited via intravenous injection of HA/DpNG-PTX. Furthermore, the invasive distance and amount of HA/DpNG-PTX localised within the deep tissue regions were increased two times than that of PA-PTX. Therefore, the DpNG based drug delivery system could be useful for treatment of heterogeneous tumour.

Hyaluronic acid-conjugated graphene oxide/photosensitizer nanohybrids for cancer targeted photodynamic therapy.

Hyaluronic acid (HA)-graphene oxide (GO) conjugates, with a high loading of photosensitizers (PS; Ce6), were developed as a cancer cell targeted and photoactivity switchable nanoplatform for photodynamic therapy (PDT). HA-GO conjugates with size below 100 nm were first prepared by the chemical conjugation between ADH-modified HA and fractionated GO sheets with size relevant for drug delivery. Before evaluating the drug delivery efficacies, their chemical structure, morphology, and biocompatibility were characterized by 1H NMR, UV, TGA, AFM, DLS and MTT assays. The physical adsorption of Ce6 onto HA-GO nanocarriers was mainly due to the π-π stacking as well as hydrophobic interactions. It was demonstrated by CLSM and FACS that the cellular internalization of the HA-GO/Ce6 nanohybrids was much more effective when compared with free Ce6, which was also found to be significantly influenced by the co-treatment with an excess amount of HA polymers, illustrating their active targeting to HA receptors overexpressed on cancer cells. The photoactivity of Ce6 adsorbed on HA-GO nanocarriers was mostly quenched in aqueous solution to ensure biocompatibility, but was quickly recovered after the release of Ce6 from HA-GO nanocarriers upon cellular uptake. As a result, the PDT efficiency of the HA-GO/Ce6 nanohybrids was remarkably improved ∼10 times more than that of free Ce6, as well demonstrated in both MTT and LIVE/DEAD assays.

The transfection efficiency of photosensitizer-induced gene delivery to human MSCs and internalization rates of EGFP and Runx2 genes.

To improve the transfection efficiency of non-viral gene vectors to human mesenchymal stem cells (hMSCs), a photosensitizer (PS)-induced gene delivery system was designed by using pheophorbide-a (pheo-a) as a PS. In FACS results, this system showed excellent gene transfection efficiency depending on irradiation power. The result was strongly supported by western blot and real-time quantitative PCR (RT-qPCR) assays. The protein and mRNA expression of enhanced green fluorescent protein (EGFP) in hMSCs treated with 0.9 J/cm(2) irradiation increased 9.8- and 8.7-fold compared with non-irradiated hMSCs, respectively. Furthermore, the internalization of PEI/pDNA complexes in hMSCs was enhanced by light irradiation even under conditions that inhibited endocytosis. The hemolytic activity of PS with irradiation (0.9 J/cm(2)) significantly increased to 55%. Thus, PS with light irradiation facilitated both the internalization and endosomal escape of gene complexes. For osteogenic induction, the Runt-related transcription factor 2 (Runx2) gene was transferred to hMSCs via PS-induced transfection. Von Kossa staining indicated that Runx2 overexpression significantly enhanced the osteogenesis of hMSCs. Therefore, this PS-induced gene delivery method has potential value for stem cell therapy via gene delivery.

The controlled photoactivity of nanoparticles derived from ionic interactions between a water soluble polymeric photosensitizer and polysaccharide quencher.

In order to design a water soluble polymeric photosensitizer (WPS) with controllable photoactivity, a nano-photosensitizer (NPS) was prepared from a polyelectrolyte complex between polyethylene glycol-polyethylenimine-chlorine e6 conjugate (PEG-PEI-Ce6) and Black Hole Quencher-3 chondroitin sulfate conjugate (BHQ-3-CS). NPSs have a unimodal size distribution below 100 nm. Photoquenching of the NPS was dependent on the weight ratio of BHQ-3-CS/WPS. This phenomenon was maintained in a salt condition up to 300 mm, indicating that the photoactivity of the NPS disappears in the normal blood stream of the body. The quenched photoactivity was restored by the enzyme degradation of BHQ-3-CS after esterase treatment. In a HCT-116 (human colon cancer) cell test, the rapid cellular internalization of the NPS without any other ligands was observed by confocal imaging. Upon light irradiation after internalization, phototoxicity was detected via MTT colorimetric assay. Also, when the NPS was subcutaneously injected in both tumoral and normal regions of HCT-116 tumor-bearing mice, the fluorescence signal in the tumors rapidly increased compared to the normal region due to the enzymatic-triggered dissociation of the NPS in vivo. These results suggest that the NPS can provide both tumor diagnosis and therapy simultaneously, and has great potential for biological studies and clinical treatments of various tumors.

Potential of self-organizing nanogel with acetylated chondroitin sulfate as an anti-cancer drug carrier.

In order to obtain feasibility data regarding the possibility of using chondroitin sulfate (CS) in an anti-cancer drug delivery system, CS was chemically modified by a one-step process with acetic anhydride. Although 3 samples with different degrees of acetylation were synthesized, only the sample with the highest degree of acetylation (AC-CS3) was tested as a nanogel because the others (AC-CS1 and 2) dissolved in distilled water (DW) in the test range (1-10 mg/ml). The AC-CS3 nanogel was characterized by fluorescence probe and dynamic light scattering (DLS) techniques. Its critical aggregation concentration (CAC) was <2.0 x 10(-2) mg/ml at 25 degrees C. The partition equilibrium constant, K(v), of the nanogel (7.88 x 10(5)) was similar to that of polymeric micelles, which means that the acetyl group may act as a hydrophobic core controlling pharmacokinetic behavior. The higher surface charge value in the nanogel, above - 40 due to carboxyl and sulfate groups in CS, explains its good stability. The anticancer drug doxorubicin (DOX) loading efficiency of the AC-CS3 nanogel was also superior, at above 90%. Changes in the size of the polydispersion index (PDI) of nanogels loaded with DOX over a 3-week period were negligible. The nanogels interacted with HeLa cells and were internalized together with the entrapped drug within the cytoplasm, probably via an endocytic mechanism exploited by sugar receptors. Based on these results, the AC-CS3 nanogel is expected to prove useful as an anti-cancer drug carrier for chemotherapy.