Dopamine-functionalized hyaluronic acid microspheres for effective capture of CD44-overexpressing circulating tumor cells

Highlights

• Dopamine-functionalized hyaluronic acid microspheres were prepared based on microfluidics.

• HA-DA microspheres were used to capture CD44-overexpressing CTCs.

• A microfluidic filter was designed for the isolation of “microspheres-CTCs” from blood.

Abstract

As one of the biomarkers of liquid biopsy, circulating tumor cells (CTCs) provides important clinical information for cancer diagnosis. However, accurate separation and identification of CTCs remains a great deal of challenge. In present work, we developed novel dopamine-functionalized hyaluronic acid microspheres (HA-DA microspheres) to capture CD44-overexpressing CTCs. The dopamine was grafted onto the hyaluronic acid chain, which was polymerized and cross-linked by oxidation of the catechol groups. Afterwards, a facile microfluidic chip was designed and developed to fabricate the HA-DA microspheres with a diameter of about 45 μm. Our results showed that the CD44+ cells (i.e., HeLa, HepG2, A549, MCF-7 and DU-145 cells) could be selectively captured. Then a double-layer microfluidic filter (DLMF) was fabricated for dynamic isolation and detection of CTCs in blood samples. Many slit openings with 15 μm in height were designed to allow white blood cells to clear away, while the microspheres with CTCs were intercepted in the DLMF, which achieved effective separation of CTCs from blood cells. The approach exhibited high capture efficiency even at the cell density as low as 10 cells/mL. We believe the DLMF integrated with HA-DA microspheres could be a promising approach for isolation and detection of CD44-overexpressing CTCs, which is useful for prognosis and early metastasis of cancer patients.

Introduction

Cancer has become a major disease threatening human life and metastasis is the leading cause responsible for cancer-related deaths [1]. Circulating tumor cells (CTCs) are essential for establishing metastasis [2], which have been shown to have significant predictive and prognostic information in cancer diagnosis and precision medicine [3,4]. Unfortunately, CTCs detection is faced with a grand challenge due to the extremely low density of CTCs in the patient's blood [5,6]. Generally, CTCs could be separated from blood depending on the different physical and biological properties [4,7], such as the differences in size, deformability, hydrodynamics [8,9] and the expression of surface protein markers [10,11].

Microfluidics have attracted considerable attention in the past few years due to the advantages of small sample volume consumption, low cost, high detection sensitivity, high throughput and automatic operation [12]. To date, various microfluidic approaches have been developed for isolation of CTCs, such as physical properties-based isolation by microchannels [[13], [14], [15]], antibody recognition by microarray platform [16,17] and immunomagnetic enrichment by microfluidic chips with applied magnetic field [18]. While many microfluidic devices have been reported for separation of CTCs by differences in the physical size and deforming force [15], there are still chances that false negative or positive results would be generated as it is possible that cancer cells are deformed and squeezed out of the physical traps or the white blood cells with diverse sizes would be remained in the microfluidic device. The combination of microparticles and microfluidics [19] provide a strategy to amplify the size difference for better separation. Epithelial cell adhesion molecules (EpCAM) are often used as surface biomarkers for CTCs capture, but epithelial-mesenchymal transformation (EMT) occurs during cancer metastasis [20]. Based on this, a series of highly specific recognition method have been developed. For example, the combination of the dual antibodies (i.e., anti-EpCAM and anti-N-Cadherin or anti-CD146) was used to capture epithelial and mesenchymal CTCs [21,22]. Unfortunately, the method based on antibodies recognition is challenging because of the high cost and strict storage conditions. Therefore, a cheaper and more stable non-antibody-based recognition strategy would be in demand [23].

CD44, the main site of HA binding on cell surface, is highly expressed in many tumor cells [24], which is closely related to the cancer occurrence and metastasis. The process of HA targeting CD44 is that around 100 amino acids on CD44 protein can recognize repeated disaccharide structural units on HA to form the “link module” [25]. CD44 has become a valuable biomarker for early cancer detection and for discriminating between tumor and normal cells [26]. In particular, many studies have demonstrated the value of HA in cancer drug therapy [[27], [28], [29]].

Herein, a novel approach for preparing HA microspheres has been developed for the first time to capture CD44-overexpressing CTCs. In the traditional methods, the HA microspheres prepared by microemulsion technique with divinyl sulfone (DVS) as crosslinker had low monodispersity [30], while the existing methods of preparing HA microspheres with high monodispersity by microfluidics required the synthesis of a series of complex UV crosslinked substrates [31]. In this study, dopamine-functionalized hyaluronic acid microspheres (HA-DA microspheres) were prepared by grafting DA onto the HA chain, which was polymerized and cross-linked by oxidation of the catechol groups. Then, a facile microfluidic chip was designed and developed to fabricate the HA-DA microspheres with higher monodispersity. In theory, the specific surface area of the microspheres is larger, which can expose more CD44 binding sites. The diameter of HA-DA microspheres was about 45 μm, effectively amplifying the cell size and much larger than that of white blood cells (i.e., 15−20 μm for monocytes, 10−15 μm for granulocytes and 6−15 μm for lymphocytes) [32]. Further, a double-layer microfluidic filter (DLMF) was fabricated for dynamic cell isolation. Many slit openings (15 μm in height) were designed to allow white blood cells to clear away, while the HA-DA microspheres with cancer cells were intercepted in the DLMF, which achieved effective separation of tumor cells from blood cells. On this basis, we believe this research can provide important implications for CTCs capture, tumor diagnosis and treatment in the future.