Folic Acid Targeting for Efficient Isolation and Detection of Ovarian

Subscriber access provided by Kent State University Libraries

Applications of Polymer, Composite, and Coating Materials

Folic acid targeting for efficient isolation and detection of ovarian cancer CTCs from human whole blood based on two-step binding strategy Liju Nie, Fulai Li, Xiaolin Huang, Zoraida Aguilar, Yongqiang Andrew Wang, Yonghua Xiong, Fen Fu, and Hengyi Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 05 Apr 2018 Downloaded from http://pubs.acs.org on April 5, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Folic acid targeting for efficient isolation and detection of ovarian cancer CTCs from human whole blood based on two-step binding strategy †‡¶○

†○

†

§

§

Liju Nie,†,‡,¶,○ Fulai Li,†,○ Xiaolin Huang,† Zoraida P. Aguilar,§ Yongqiang Andrew Wang,§ Yonghua Xiong,† Fen Fu,*‡ and Hengyi Xu*† †

State Key Laboratory of Food Science and Technology, Nanchang University, Nanchang

330047, China ‡

The Second Affiliated Hospital of Nanchang University, Nanchang 330006, China

¶

Jiangxi Maternal and Child Health Hospital, Nanchang 330000, China

§

Ocean NanoTech, LLC., San Diego, CA 92126, USA

KEYWORDS: Circulating tumor cells, Ovarian cancer, Multibiotin enhancement, Two-step binding, Folic acid, Magnetic separation

ACS Paragon Plus Environment

1

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 26

ABSTRAT: Studies regarding circulating tumor cells (CTCs) have great significance for cancer prognosis, treatment monitoring, and metastasis diagnosis. However, due to their extremely low concentration in peripheral blood, isolation and enrichment of CTCs are the key steps for early detection. To this end, targeting the folic acid receptors (FR) on the CTCs surface for capture with folic acid (FA) using bovine serum albumin (BSA)-tether for multibiotin enhancement in combination with streptavidin coated magnetic nanoparticles (MNPs-SA) was developed for ovarian cancer CTCs isolation. The streptavidin-biotin system mediated two-step binding strategy was shown to capture CTCs from whole blood efficiently without the need for a pretreatment process. The optimized parameters for this system exhibited an average capture efficiency of 80%, which was 25% higher than FA decorated magnetic nanoparticles (MNPsFA) based one-step CTCs separation method. Moreover, the isolated cells remained highly viable and were cultured directly without detachment from the MNPs-SA~biotin~CTC complex. Furthermore, when the system was applied to the isolation and detection of CTCs in ovarian cancer patients’ peripheral blood samples, it exhibited an 80% correlation with clinical diagnostic criteria. The results indicated that FA targeting in combination with BSA-based multibiotin enhancement magnetic nanoparticle separation is a promising tool for CTCs enrichment and detection of early stage ovarian cancer. 1. Introduction Ovarian cancer (OC) is one of the most dreaded invasive malignancies among women worldwide. 1 According to the American Cancer Society estimates, there would be 22,280 new OC cases in 2016 in the United States with 14,240 deaths.

2

Studies showed that majority of

women with OC are not diagnosed until the disease is in an advanced stage due to vague clinical symptoms. At this advanced stage, the 5-year survival rate is only 10% to 30%,3 while the 5-year


2

Page 3 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


survival rate for early stage OC is about 92%.4 The current available screening methods for OC includes clinical examination, imaging modalities (transvaginal ultrasound), and serum biomarker detection, all of which have limitations and could not deliver the required sensitivity and specificity for accurate diagnosis.3, 5 Thus, seeking an early diagnostic method is extremely important in improving the patients' outcome.6 It is generally recognized that most cancer-related mortalities result from metastatic disease.7, 8 Circulating tumor cells (CTCs) are the cells that detach from the primary tumor and circulate in the bloodstream. These CTCs have potential for malignancy and are able to form overt metastases in distant organs. Moreover, CTCs can be present in peripheral blood prior to detection of the primary tumor via conventional screening methods.9, 10 Therefore, elucidating the presence and number of CTCs in peripheral blood could serve as a real-time “liquid biopsy” that could serve as an indicator for prognosis, evaluation of therapeutic efficiency, disease stage forecasting as well as early diagnosis of metastases.11 CTCs have been demonstrated to have predictive value in breast, colon, and prostate cancers.12, 13 However, CTCs are exceptionally rare in an extremely complex matrix, that is, only up to 102 of CTCs out of >109 hematological cells in 1 mL of blood.14 Thus, highly efficient isolation and enrichment of CTCs are necessary for CTCs to be a useful tool for early stage cancer diagnosis.15, 16 In recent years, numerous approaches had been developed for CTCs isolation and enrichment based on morphological or chemical differences between the target CTCs and normal hematopoietic species.17 These methods could fall into two categories: physical and affinitybased separation methods. Despite the limitations of each method that is due to the heterogeneity of tumor cells, they all have achieved good enrichment effects under certain conditions.16 Among these methods, affinity-based separation method using antibody-modified magnetic nanoparticles


3


Page 4 of 26

was the most widely used strategy for CTCs isolation due to ease of manipulation, high capture efficiency,18 and convenient integration with identification methods.19-21 However, conventional immunomagnetic separation methods for CTCs capture usually consume large amount of costly immunobeads. Moreover, due to the heterogeneity of CTCs, antibody-based capture and isolation using anti-EpCAM antibody, for example, have shown some limitations. Folic acid receptor (FR) are cysteine-rich cell-surface glycoproteins that bind folic acid (FA) with a high affinity constant.22 It has been reported that over 95% of OC over-express the FR, whilst in most healthy tissues, it is expressed at very low levels.23 Thus, FA has received considerable interest as a targeting molecule for cancer cell imaging,24 treatment, separation

26

25

and

. In our previous study, we have reported the use of FA decorated magnetic iron

oxide nanoparticles (MNPs-FA) for OC CTCs separation and nondestructive detection from clinical ovarian cancer patients’ blood directly. 27, 28 However, the level of FR expressed on each CTCs surface varies from cell to cell causing a different binding capacity for FA per cell,

29

which caused a variable number of FA-MNPs anchored on the CTCs from cell to cell. Due to the complexity of the whole blood matrix, low number of FA-MNPs labelled CTCs could not be separated and collected efficiently, which lead to false-negative results for CTCs detection. 27 Therefore, increasing the number of nanoparticles that attach on the CTCs could amplify the magnetic signals and improve CTCs capture efficiency in whole blood. Two-step binding mechanism could be an efficient strategy to improve nanoparticles binding capability of CTCs, it could improve the number of magnetic nanoparticles bound to each CTCs through antibody amplification due to the secondary antibody binding to multiple sites on the primary antibody or the number of secondary antibodies binding per primary antibody. The streptavidin-biotin binding is a widely used amplification system because streptavidin is a


4

Page 5 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


tetrameric protein that could bind to four biotin molecules with high affinity (1015M−1). Therefore, based on the high affinity and multivalent binding characteristics of the streptavidinbiotin system, it could be used as a two-step binding scaffold for cell binding. Haun group’s study suggested that using biotin-avidin based two-step labeling strategy for nanoparticle attachment yielded higher nanoparticle binding to cells with a relative labeling efficacy increase by a factor of 5 compared with direct immunoconjugates.

30

Thus, based on streptavidin-biotin

system mediated two-step binding strategy, the potential for CTCs efficient capture and sensitive detection could be enhanced. In this work, a simple and efficient two-step binding method for OC CTCs capture was developed using biotin-BSA-FA targeting in combination with streptavidin coated magnetic nanoparticles (MNPs-SA). For capture of CTCs, BSA was selected as a carrier that was conjugated with FA-PEG2K-NH2 and biotin forming biotin-BSA-FA. At the first step, biotinBSA-FA was attached on the CTCs (forming biotin-BSA-FA~CTC or biotin~CTC) through the recognition between FA and FR that is overexpressed on OC CTCs. We hypothesized that biotin-BSA-FA would bind to CTCs with high affinity through a multivalent FA~FR binding resulting in large number of biotin exposed on the CTCs surface. Then addition of MNPs-SA resulted in the coupling of the MNPs-SA with the biotin~CTC resulting in the formation of MNPs-SA~biotin~CTCs. Finally, MNPs-SA~biotin~CTCs were isolated in an external magnetic field. Using this system, CTCs were successfully isolated from spiked human blood and on OC patients’ peripheral blood samples with significant isolation and enumeration. The results indicated that the use of the biotin-BSA-FA in combination with MNP-SA holds promising application in early stage diagnosis of clinical cases of OC. 2. Materials and Methods


5


Page 6 of 26

2.1. Materials. Iron oxide magnetic nanoparticles (MNPs) and quantum dots (QSH-550-04) were provided by Ocean NanoTech (San Diego, CA). Bovine serum albumin V (BSA) was purchased from Biosharp (Beijing, China). Goat polyclonal antibody anti-HE4 (C-12) was purchased from Santa Cruz Biotechnology and rabbit anti-goat IgG (BA1040) was from Boster Biological Technology. Co. Ltd (Wuhan, China). RPMI-1640 cell culture media was purchased from Solarbio (Beijing, China). Fetal bovine serum and trypsin were obtained from Trans Gen Biotech (Beijing, China). Streptavidin (SA) and biotin-NHS were purchased from Hualan Company

(Shanghai,

China).

Sulfo-N-hydroxysuccinimide

(Sulfo-NHS),

1-ethyl-3,3-

dimethylaminopropyl carbodiimide hydrochloride (EDC), dicyclohexylcarbodiimide (DCC) and Hoechst 33342 dye were purchased from Sigma-Aldrich (St. Louis, Missouri). All other reagents were of reagent grade. 2.2. Cell culture and blood preparation. Human SKOV3 cell line, FR positive, was donated by the Medical Research Center of the First Affiliated Hospital to Nanchang University. A549 cell line, FR negative, was obtained from Jiangxi academy of medical science. All the cell lines were cultured in a flask containing RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum. The flask was placed in a humidified atmosphere with 5% CO2 at 37℃ in a cell culture incubator. The medium was replaced every three days. Whole blood samples were collected in EDTA-coated vacutainer tubes for use within 24 h. These whole blood samples from healthy female volunteers were used as matrix for the studies on spiked samples. 2.3. Preparation of MNPs-SA. SA was conjugated with 25 nm diameter MNPs using a covalent link between the amine group (-NH2) to the carboxyl group (-COOH) of the MNPs.18


6

Page 7 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The hydrodynamic radius and the zeta potential of the MNPs before/after conjugation with the SA were determined using Zetasizer Nano ZS. Transmission electron microscope (TEM) was used to observe the nanoparticles upon synthesis before conjugation to SA to establish size, shape, and monodispersibility. 2.4. Synthesis of biotin-BSA-FA. NH2-PEG2K-FA was synthesized according to our previous study

27

and then conjugated to BSA in the following manner: 17.5 mg of BSA (0.26 µmol)

dissolved in 0.5 mL borate buffer (200 mM, pH 5.0) was activated with 15 mg of EDC and 3 mg of NHSS for 5 min, after which 62.4 mg of FA-PEG2K-NH2 dissolved in 1 mL of borate buffer (200 mM, pH 8.0) was added. The molar ratio of BSA/FA-PEG2K-NH2/EDC/NHSS was 1/100/300/60. The reaction was allowed for 2 h after which all BSA-FA conjugates were purified by dialysis (MWCO 50KDa) against 1 L of 10 mM phosphate buffered saline (PBS) with three changes. The appropriate volume of biotin reagent was added to the BSA-FA solution with a molar ratio of 20:1, the mixture was incubated for 30 min at room temperature and purified using desalting columns or dialysis in ultrapure water. The biotin-BSA-FA was characterized by Fourier transform infrared spectroscopy (FTIR). 2.5. CTCs Capture and separation. Approximately 450 Hoechst 33342-stained SKOV3 cells were spiked in 1 mL whole blood. Approximately 5 µL biotin-BSA-FA was mixed with the separation medium using a vertical mixing device at 12 rpm for 1 h. The MNPs-SA conjugates were then added into the mixed cells suspension and mixed for 1 h. The MNPs-SA~biotin-BSAFA labeled cells adhered on the walls of the tube under a magnetic separator over a 2 h period. The magnetically separated cells, were re-suspended with 50 µL of PBS and the capture efficiency was established by imaging and counting the pre-stained SKOV3 cells under a fluorescence microscope.


7


Page 8 of 26

2.6. Confirmation of CTCs with fluorescence immunocytochemistry (ICC). The captured cells were fixed with 4% (v/v) formaldehyde for 15 min at room temperature on a slide followed by washing immediately with Tris-buffered saline (TBS) containing 1% (w/v) BSA. The cells were permeabilized with 0.1% (v/v) Triton X-20 in TBS for 20 min, washed and blocked for 1 h with PBS containing 0.1% (w/v) casein and 5% (w/v) BSA. The slides were washed and incubated with primary goat polyclonal anti-HE4 (1:100 dilution) antibodies for 2 h. After washing three times with TBS-BSA, a mixture of the secondary rabbit anti-goat antibody conjugated quantum dots (which was prepared like the MNPs-SA) was applied on the slides as well as stained with Hoechst 33342 for 30 min followed by PBS washing at least three times. The control was prepared without incubating with the primary goat polyclonal antibody antiHE4. The captured CTCs was confirmed under a fluorescence microscope. 2.7. Cell viability analysis. To evaluate whether the cell proliferation was affected by contact with MNPs after magnetic separation, the cells were counted and directly plated onto 12-well plates. The same number of MNPs-isolated cells and non-MNPs exposed control cells, 1.0 × 104 cells/well, were similarly seeded onto the 12-well cell culture plates. The cells were cultured for up to three days and observed under a fluorescence microscope daily to evaluate cell proliferation on the plate wells. 2.8. Capture of CTCs from clinical blood samples. To evaluate the applicability of the newly developed CTCs magnetic separation method, 20 samples of whole blood from OC patients and 100 samples from healthy females were tested. The captured cells were identified with ICC. 3. Results and Discussion


8

Page 9 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


3.1. The theory and principle of CTCs separation. For the magnetic separation of CTCs, the trajectories of the MNPs labeled CTCs in the blood could be determined by the magnetic force (Fm), drag force (Fd), buoyancy force, and gravity. However, due to buoyancy force and gravity which are perpendicular to the Fm and Fd, the buoyancy force and gravity were neglected. Thus, the Fm and Fd were considered as the two dominant forces that would significantly affect CTCs capture. To capture CTCs efficiently, in this study, magnetic nanoparticles were allowed to attach on the CTCs using a two-step binding strategy. First, BSA was selected as a carrier of the FA that was needed to bind with the FR on the CTCs surface. BSA was conjugated to biotin and FA creating the biotin-BSA-FA complex. The FA, which was tethered on the BSA through a PEG2K-linker, could bind on the CTCs surface through the high population of FRs forming the biotin-BSA-FA~FR(CTCs) or simply biotin~CTC. When these cells were exposed to MNPs-SA, the high affinity between SA and biotin would result in the formation of the MNPsSA~biotin~CTCs. The magnetic force, Fm2, acting on the magnetically labeled CTCs, could be represented as follows (1) 31: ‫ܨ‬௠ଶ = (݊ଵ θଵ λଵ )(݊ଶ θଶ λଶ )݊ଷ ‫ܨ‬௣

(1)

where Fm2 represented the magnetic force acting on a magnetically labeled CTCs using streptavidin-biotin system mediated two-step binding manner; Fp represented the magnetic force acting on one magnetic nanoparticle, MNPs-SA; n1 is the number of antigen binding sites (FR) per CTC, θ1 is the fraction of FR molecules on the cell surface bound by FA in the biotin-BSAFA, λ1 represented the valence of the biotin-BSA-FA complex binding with CTC; n2 is the number of binding sites on biotin-BSA-FA recognized by MNPs-SA, θ2 was the fraction of binding sites on the biotin-BSA-FA that were bound to MNPs-SA, λ2 represented the valence of the SA~biotin binding; and n3 represented the number of streptavidin conjugated to the magnetic


9


Page 10 of 26

nanoparticles in MNPs-SA. The combined term n1θ1λ1 would be equivalent to the commonly used term “antibody binding capacity” of a cell population.31 In equation (1), “antibody binding capacity” could be understood as the number of biotin-BSA-FA binding to a CTC. The n2θ2λ2 could be combined into one overall term, ψ, which represented the signal amplification due to the MNPs-SA binding to multiple biotin sites on the biotin-BSA-FA or the number of MNPs-SA binding per biotin-BSA-FA. Thus, Fm2 in equation (1) could be defined as equation (2): ‫ܨ‬௠ଶ = (݊ଵ θଵ λଵ )߰݊ଷ ‫ܨ‬௣ =ܰ௣ଶ ‫ܨ‬௣

(2)

where Np2 represented the number of magnetic nanoparticles bound to the CTCs. The two-step multivalent binding strategy could increase the number of magnetic nanoparticles (Np2) bound per cell (ψ= (n2θ2λ2) ˃1, with amplification), thus, the magnetic force acting on nanoparticles labeled CTCs based on two-step binding strategy (Fm2) could be amplified compared with FAMNPs based one-step binding manner (Fm1) (Fm ∝ Np, Np2˃Np1, Fm2˃Fm1). In addition, this interaction could significantly reduce the probability for the MNPs-SA to fall-off from the MNPs-SA~biotin~cell complex in subsequent cell capture. Amplification of the magnetic force could improve the CTCs capture efficiency and shorten the magnetic separation time (Scheme 1). Based on these considerations, the two-step binding strategy using BSA mediated multibiotin enhancement integrated with the biotin-SA biological amplification system could capture CTCs more efficiently from whole blood.


10

Page 11 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Scheme 1. The mechanical analysis of CTCs separation from whole blood using two-step binding strategy. Fm represented the magnetic force acting on a magnetically labeled CTCs using FA-MNPs based one-step binding manner (Fm1) or using streptavidin-biotin system mediated two-step binding manner (Fm2); Np represented the number of MNPs-SA bound to CTCs using FA-MNPs based one-step binding manner (Np1) and based on the two-step binding manner (Np2); ψ represented the signal amplification due to the MNPs-SA binding to multiple biotin sites on the biotin-BSA-FA or the number of MNPs-SA binding per biotin-BSA-FA; Fd represented the drag force on one-step labeled CTCs (Fd1) and two-step labeled CTCs (Fd2); µ0 represented the magnetic permeability of free space, ∆χ was the difference in magnetic susceptibility between the magnetic material and the surrounding medium, Vp was the volume of each nanoparticle, and B was the magnetic flux density; η was the viscosity of the blood, ν was the magnetically induced flow velocity on the magnetic labeled cell in one-step binding manner (ν1) and two-step binding manner (ν2) in the magnetic field, and Dc was the diameter of the cell. 3.2. Characterization of MNPs-SA and biotin-BSA-FA. The covalent conjugation of MNPs with SA was a simple process that had been demonstrated in multiple studies

32

. Successful

conjugation was confirmed with the changes in the zeta potential and hydrodynamic size of the MNPs. As shown in Figure 1A, SA-MNPs conjugates had an average diameter of 34.4 nm, which was bigger than that of the unconjugated MNPs at 24.8 nm. The zeta potential of SAMNPs was -27.0 mV while the MNPs was at -41.6 mV. This increase in zeta potential and hydrodynamic size confirmed that SA was conjugated on the surface of the MNPs. The TEM images revealed that the SA-MNPs were well dispersed without aggregation with a uniform size distribution without significant changes in morphology after conjugation (Figure 1B). MNPs


11


Page 12 of 26

before/after conjugation with SA were also demonstrated through a gel electrophoresis. As shown in Figure 1C, the unconjugated MNPs showed faster mobility compared with the SAMNPs. This was indicative of the conjugation of SA on the surface of the MNPs resulting in a higher molecular weight of the SA-MNPs than the MNPs and a less negative zeta potential that influenced the speed of the nanoparticle migration towards the positive end of the electric field. The conjugation of the biotin to the BSA-FA was confirmed by FTIR spectroscopy. A representative spectrum showed the presence of the amide bonds which exhibited the reaction between the biotin-NHS and the carboxyl groups on BSA as shown in Figure 1D. Biotin was covalently attached to the amine-functional groups on the surface of BSA forming the bands that are unique to conjugated biotin at 1540 and~1660 cm-1 that are indicative of amide II (N-H bend + C-N stretch) and amide I (C=O stretch) vibrational modes, respectively. These bands indicated that the amide bonds formed between biotin and BSA forming biotin-BSA. Bands unique to unreacted NHS-biotin were at 1741, 1788, and 1818 cm-1 assigned to the asymmetric and symmetric stretches of the succinimide carbonyls and the ester of the NHS moiety, respectively 33

(which were absent in Figure 1D-(b)). The characteristic peak (1606 cm-1) of cyclobenzene

loops which were present in FA were shown in Figure 1D-(c), but absent in Figure 1D-(a)

34

,

which indicated that the biotin-BSA-FA was successfully prepared.


12

Page 13 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. Characterization of MNPs-SA conjugates and biotin-BSA-FA. (A) Hydrodynamic size of MNPs and MNPs-SA conjugates. (B) TEM image of MNPs-SA. (C) Agarose gel electrophoresis of biocompatible water soluble MNPs (right) and MNPs-SA (left). (D) FTIR spectrum of biotin (a), BSA-FA (b), and biotin-BSA-FA (c). 3.3. CTCs capture in different medium. The CTCs capture capability of the biotin-BSA-FA in combined with MNPs-SA was investigated in different medium. The samples were prepared by spiking stained SKOV3 cells in human whole blood. Similarly, for comparison, capture efficiencies were also examined in a matrix of PBS and lysed blood. The capture efficiency for SKOV3 cells were 98.0 ± 2.0%, 83.5 ± 6.4%, and 80.0 ± 7.8%, respectively in PBS, lysed blood, and whole blood (Figure 2). The capture efficiency in the PBS was the highest, suggesting that complex matrix conditions affected the CTCs enrichment and capture. However, in the lysed blood and whole blood, high capture efficiency was also observed without any significant difference. These results indicated that FA targeting with BSA-based multibiotin enhanced


13


Page 14 of 26

magnetic separation system was able to capture the CTCs efficiently in whole blood without any pretreatment.

Figure 2. Capture efficiency of SKOV3 cells in PBS, lysed blood and whole blood. 3.4. Optimization of the magnetic separation parameters for isolating CTCs in whole blood. Since the two-step binding based magnetic separation for CTCs isolation were very crucial factors for real clinical sample applications, attempts were made to optimize the magnetic signal amplification conditions and meticulous selection of magnetic separation parameters including the use of different molar ratios of MNPs-SA and biotin-BSA-FA. The results shown in Figure S1 indicated that the capture was optimum when biotin-BSA-FA was added at a molar ratio of 10 times that of the number of FR expressed on 450 SKOV3 cells. Other optimized conditions were MNPs-SA at a molar ratio of 4:1 to that of the biotin-BSA-FA (Figure S1-C), the MNPs-SA conjugation was optimum at a molar ratio of SA to MNPs at 10:1 (Figure S1-A) while the biotin-BSA-FA was optimized at a molar ratio of biotin to BSA-FA at 40:1 (Figure S1B). These optimized conditions provided the maximum capture efficiency.


14

Page 15 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Incubation time has always been a very important factor for CTCs isolation. We investigated the capture efficiency for the SKOV3 cells in whole blood for different durations of incubation. As Figures S2(A-C) showed, 2 h incubation of biotin-BSA-FA with the SKOV3 cells and 1 h incubation of MNPs-SA conjugates with biotin~CTCs enabled the system to capture the SKOV3 cells in whole blood efficiently in a 2 h magnetic separation process. The biotin-BSA-FA in combination with SA-MNPs significantly shortened the magnetic separation time compared with our previous study using only the MNPs-FA.27 This new two-step system comprised of a combination of the cell isolation with the biotin-BSA-FA and separation of the captured cells using MNPs- SA demonstrated a significantly improved CTCs capture efficiency in a shorter magnetic collection time. 3.5. Specificity test. The specificity of the system was evaluated using A549 cells, which were FR negative. In addition, conjugates of SA-MNPs, biotin-BSA-MNPs (or BSA-MNPs), FAMNPs were prepareds. The results showed that the conjugates of the MNPs-SA and biotin-FA binding based magnetic separation system exhibited the highest capture efficiency followed by the one-step based MNPs-FA separation process. The capture efficiency with MNPs-SA and MNPs-BSA which both did not have the FA were significantly lower (Figure 3B). Without the biotin-BSA-FA, the MNPs-SA conjugates could hardly capture SKOV3 cells (Figure 3A), which indicated the FA targeting with BSA-based multibiotin enhanced magnetic separation system was the most effective and most specific for the OC CTCs isolation.


15


Page 16 of 26

Figure 3. Specificity of the CTCs capture system. (A) Capture efficiency of A549 cells, FR negative, based on two-step binding capture system; (B) Capture efficiency of SKOV3 cells with various MNPs. 3.6. Cell viability and ICC identification of the captured cells. The viability of the isolated CTCs has been very important for further investigation of their malignant nature and to assess the invasive potential of individual CTCs in clinical studies.35 Thus, the captured cells were cultured on a plate to examine their bioactivity. As shown in Figure 4(A-B), after cell enrichment and incubation overnight, the captured cells were viable, propagated, and adhered to the culture dish, without detectable changes in morphology and behavior compared with the same number of healthy untreated cells. The bioactivity of the captured cells indicated the biocompatibility of the newly developed multibiotin enhanced CTCs magnetic isolation system. Additional studies are necessary to further explore the proliferation properties of the cancer cells that were isolated from the whole blood of cancer patients. After the CTCs were isolated, a conventional ICC method was applied to identify and enumerate the CTCs from non-specifically trapped blood cells. A goat polyclonal antibody against HE4 (anti-HE4), a marker for SKOV3 cells and QSH550-labeled rabbit anti-goat IgG as well as Hoechst 33342 nuclear staining were also used. As shown in Figure 4C, CTCs exhibited


16

Page 17 of 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


strong QDs signals in response to the goat polyclonal antibody (anti-HE4). In contrast, in the absence of the goat polyclonal antibody against HE4 there were insignificant signals. Hoechst 33342 staining validated that the captured cells retained intact nuclei maintaining cell viability that allowed cell propagation.

Figure 4. Viability analyses and ICC identification of the isolated SKOV3 cells. (A) Microscopic images of tumor cells without magnetic separation. (B) Microscopic images of the isolated CTCs. (C) Fluorescence microscopic image of the captured cells stained with Hoechst.33342 and QSH550-labeled rabbit anti-goat IgG. Nucleus (Hoechst.33342): excitation 405 nm, emission 447 ± 30 nm band-pass. HE4 (QSH550): excitation 488 nm, emission 525 ± 25 nm band-pass. Merged: nucleus (DAPI) and HE4 (QSH550). 3.7. Detection of CTCs in cancer patients’ peripheral blood samples. The optimized parameters from the previous sections were applied to detect CTCs in whole blood samples from 20 OC patients and 100 healthy females. The isolated cells were identified with ICC as described above. The cells positive for HE4-QD and Hoechst (Hoechst+ and HE4-QD+, cell sizes ranging


17


Page 18 of 26

from 10 µm to 40 µm) from blood cells (cell sizes

Folic Acid Targeting for Efficient Isolation and Detection of Ovarian

Recommend Documents