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Specific On-site Assembly of Multifunctional Magnetic Nano-Cargos Based on Highly Efficient and Parallelized Bioconjugation: Towards Personalized Cancer Targeting Therapy Luyan Sun, Jiaxun Wan, Christian G. Schäfer, Zihao Zhang, Jing Tan, Jia Guo, Limin Wu, and Changchun Wang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00773 • Publication Date (Web): 16 Jan 2017 Downloaded from http://pubs.acs.org on January 16, 2017

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ACS Biomaterials Science & Engineering

Specific On-site Assembly of Multifunctional Magnetic Nano-Cargos Based on Highly Efficient and Parallelized Bioconjugation: Towards Personalized Cancer Targeting Therapy Luyan Sun, † Jiaxun Wan, † Christian G. Schaefer,† Zihao Zhang,† Jing Tan, † Jia Guo, † Limin Wu,‡ Changchun Wang* † ,

†

State Key Laboratory of Molecular Engineering of Polymers, Department of Macromolecular

Science, Laboratory of Advanced Materials, Fudan University, 220 Handan Road, Shanghai, 200433, People’s Republic of China. ‡

Department of Materials Science, Fudan University, 220 Handan Road, Shanghai 200433,

China * Corresponding author. Fax: +86-21-65640293; Tel: +86-21-65642385; E-mail: [email protected] (C.C. Wang)

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ABSTRACT: The rational design of particle-based cancer theranostic agents, combining diagnostic and therapeutic features in a single entity, has emerged as an effective approach towards personalized cancer therapy, however, the ability for a flexible assembly of specific targeting ligands with regard to a broad range of tumor tissues and cells is still challenging. Here we present a convenient and highly variable on-site assembly strategy for the preparation of multifunctional doxorubicin (DOX)-loaded nano-cargos with magnetic supraparticle (MSP) as core and redox-degradable poly(methylacrylic acid-co-N,N-bis(acryloyl) cystamine) (P(MAAco-Cy) as shell, which could be simultaneously modified with multiple targeting ligands through parallelized bioconjugation on the basis of streptavidin-biotin (SA-BT) interaction. Under physiological conditions similar to the cytoplasm of tumor cells, DOX could be controlled released from these nano-cargos to specific tumor sites, while dual-ligand modified nano-cargos showed remarkable proliferation inhibition for the HeLa cells and the SK-OV-3 cells that overexpressed both folate as well as integrin receptors. The experimental results demonstrated that herein described on-site assembly strategy opens access to highly efficient targeting drug delivery systems towards personalized cancer targeting therapy by incorporating functional diversity, which can be easily achieved through highly efficient and parallelized one-step bioconjugation.

KEYWORDS: On-site assembly; parallelized bioconjugation; streptavidin-biotin complex; magnetic nano-cargos; personalized cancer therapy.


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1. INTRODUCTION Cancer is one of the major threats to human health nowadays, and enormous efforts have been made to develop new strategies and technologies that enable highly efficient and personalized cancer therapy. Over the past decades, the development of pharmaceutical carriers,1,2 such as liposomes,3,4 micelles,5,6 nanoemulsions7,8 and polymeric or inorganic nanoparticles,9-11 has attracted a great deal of attention in targeting drug delivery and it has been demonstrated that these systems are particularly valuable to significantly improve the cancer therapy. Among these systems, the use of magnetic nanoparticles as drug carriers went into the focus of current research, due to its outstanding features, such as high drug loading efficacy, low immunogenicity, good accessibility to tumor tissues, and the capability of magnetic manipulation and imaging.10-13 So far, many different structures of magnetic nanoparticles have been synthesized with regard to expand its specific surface area in order to increase the loading capacity of anti-cancer drug and thus to enhance the efficiency to control tumors.11,14-16 In order to further minimize undesirable side effects to normal cells and to improve the accuracy of drug delivery to specific sites, the magnetic stimulus can additionally be used to induce targeting drug release.17 In this way, the drugs loaded in magnetic nanocarriers can be transported to specific tumor sites using an external localized magnetic field, while the drug release can be precisely controlled by the intensity and duration of the magnetic field, and moreover, the magnetic field can be directly removed as soon as the therapy is completed.18,19 The controlled release of drugs in targeted tumor tissues can greatly increase the efficiency of the anti-cancer drugs.16,20 As could be demonstrated by various scientific reports, tumor tissues possess unique environmental conditions compared to normal cells, such as low pH value in tumor

extracellular

environment

of

about

pH

6.8

and

in

intracellular 3

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cytoplasm/endosome/lysosome of about pH 6.0-5.0, while the pH value in normal tissue is pH 7.4.21-25 Moreover, the glutathione (GSH) concentration in mitochondria and cytoplasm of tumor cells is much higher than that in normal cells.22,25 Therefore, especially pH and redox stimuliresponsive systems have been shown to be promising candidates for the controlled release of anti-cancer drugs in tumor tissues, while preventing contamination of normal tissues. Tumor cells are reported to overexpress many kinds of cell surface receptors, such as folate receptors (FR),26,27 integrin receptors,28,29 epidermal growth factor receptors (EGFR),30 transferrin receptors31, and so on. In particular, the conjugation of specific targeting ligands to the surface of nanocarriers was widely used to enhance the selectivity of targeting to specific tumor cells, while healthy cells that did not express the targeted receptor were plainly spared. Considering that different receptors are usually expressed on a single kind of tumor cells, some papers reported the successful use of dual-ligand modified nanocarriers, which could synergistically target to the tumor cells, while negative effects to the off-target cells could effectively be avoided.32-36 Saul et al.34 used dual-ligand modified liposomes to target human KB cells that expressed both FR as well as EGFR, and achieved significant toxicity specifically to the targeted cells. Doolittle et al.35 investigated dual-ligand nanocarriers that also could overexpress two target receptors, which could greatly improve the targeting accuracy towards cancer cells. Moreover, they analyzed the effect of spatiotemporal alterations in the expression patterns of the receptors in the cancer sites. However, the reported targeting ligand immobilization was usually irreversible as a result of the conjugation via covalent bonds, and most of the methods reported don’t allow a flexible control over quantity and type of surfaceattached targeting ligands, which strongly restricts the use of nanocarriers for personalized cancer therapy. 4 ACS Paragon Plus Environment

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With regard to a flexible and reversible assembly of targeting ligands, the bioconjugation based on biotin (BT) binding by avidin (AV) proteins has been investigated for many years and has shown to be very promising for various applications, such as immunological assays,37 biorecognition,38 protein isolation,39-41 and so on. The highly specific and very strong noncovalent interaction of AV and BT, characterized by its high dissociation constant of 1015 L/mol,42,43 accompanied with the ability to biotinylate a plethora of different biomolecules and reporters, opens the way to facile and flexible assembly of targeting ligands to nanocarriers. On the opposite side, especially streptavidin (SA), a kind of protein found in the bacterium streptomyces avidinii, is an outstanding candidate for BT-AV bioconjugation, because it possesses four binding sites for biotin per molecule and has an extraordinarily high affinity to biotin. Moreover, the SA-BT complex is highly resistant to organic solvents and extremes of pH and temperature.44 Hence, SA can flexibly bind to a huge variety of different biotinylated moieties under various reaction conditions, which can be used as a modular synthetic toolkit towards the assembly of multifunctional nanocarriers by simple one-step reaction. In the present paper, with the aim to give a flexible assembly of specific targeting ligands to binding sites of particle-based cancer theranostic agents, capable of multiple activities including tumor-targeting, tumor-imaging as well as tumor drug delivery, we present a convenient and variable assembly approach based on highly efficient and parallelizable SA-BT bioconjugation. A new type of SA-modified magnetic nano-cargos composed of magnetic supraparticle cores and redox-degradable poly(methylacrylic acid-co-N,N-bis(acryloyl) cystamine) (P(MAA-co-Cy) shells with high doxorubicin (DOX)-loading efficiency and controlled drug releasing properties under physiological conditions of tumor cells were designed. Herein prepared nano-cargos could be simultaneously modified with multiple biotinylated targeting ligands through parallelized on5 ACS Paragon Plus Environment


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site assembly on the basis of the exceptionally strong SA-BT interaction. In this context, the term “on-site assembly” is basically predicated by the ability to conjugate multiple targeting ligands to the nano-cargos through highly specific and parallelizable bioconjugation with control over nano-cargo/ligand ratio, activity and functionality in an almost quantitative one-step reaction without the need of any purification and post-treatment. As a proof of concept, herein described on-site assembly approach was exemplarily applied to biotinylated arginylglycylaspartic acid (BT-RGD) and biotinylated folic acid (BT-FA), and the experimental results demonstrated that the dual-ligand modified nano-cargos exhibited enhanced uptake behavior and better inhibitive efficacy to SK-OV-3 and HeLa tumor cells than single-ligand modified versions, giving rise that our on-site assembly approach can be used as a modular synthetic toolkit to tailor targeting properties of cancer theranostic agents towards personalized cancer targeting therapy. 2. MATERIALS AND METHODS 2.1 Materials. Iron(III) chloride hexahydrate (FeCl3 · 6H2O), ammonium acetate (NH4OAc), ethylene glycol (EG), methacrylic acid (MAA), N,N-bis(acryloyl) cystamine (Cy), anhydrous acetontrile (AN) and folic acid (FA) were purchased from Shanghai Chemical Reagents Company (China) and used as received. Poly(γ-glutamic acid) (PGA) was purchased from Dingshunyin Biotechnology Company (China). Cyclo (Arg-Gly-Glu-d-Phe-Lys) (RGD) was purchased from Chutai Biotechnology Company (China). Streptavidin (SA), D-biotin (BT), biotin

hydrazide,

rhodamine

B

(Rho-B),

fluorescein

isothiocyanate

(FITC),

methacryloxypropyltrimethoxysilane (MPS), glutathione (GSH), 2,3-dimethylmaleic N-(3dimethylaminopropyl)-N-ethylcarbodiimide

hydrochloride

(EDC·HCl),

(3-

aminopropyl)triethoxysilane and N-hydroxysuccinimide (NHS) were purchased from Aladdin (Shanghai, China). 2,2-Azobisisobutyronitrile (AIBN) was obtained from Sinopharm Chemical 6 ACS Paragon Plus Environment

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Reagent (China). Doxorubicin (DOX), in the form of a hydrochloride salt, was obtained from Beijing Hua Feng United Technology CO., Ltd. and used as received. Dulbecco's modified Eagle's medium (DMEM) without folic acid, fetal bovine serum (FBS), penicillin G, streptomycin, and trypsinase were purchased from Sigma Aldrich. PBS buffer (pH 7.4) was used in all experiments. 2.2 Preparation and MPS functionalization of magnetic supraparticles (MSP). MSP was prepared using the solvothermal method.11 Firstly, 2.7 g (10 mmol) of FeCl3 · 6H2O, 2.0 g of PGA and 7.7 g (0.1 mol) of NH4OAc were dissolved in 140mL ethylene glycol and stirred at 160 °

C. After 1 hour, the mixture was transferred into a Teflon-lined stainless-steel autoclave and

heated to 200 °C. After 16 hours, the mixture was washed with ethanol and separated using a magnet before the product was dried under vacuum.11, 46 For the functionalization of the MSP with MPS, 100 mg of the dried MSP were dispersed in 50 mL ethanol. Then the mixture was under ultrasonication for 10 min. After addition of 2 mL aqueous ammonia and 2.5 mL MPS, the reaction mixture was heated to 80 oC for 2 days. Then the MPS-modified MSP were dried under vacuum. 2.3 Preparation of MSP@P(MAA-Cy). The poly(methylacrylic acid-co-N,N-bis(acryloyl) cystamine) (P(MAA-Cy)) shells was covered onto the MSP cores using distillation-precipitation copolymerization according to our previous report.25 Firstly, 50 mg of MPS-modified MSP were added in 40 mL acetonitrile under ultra-sonication for 15 minutes. Then a mixture of 200 µL MAA, 25 mg Cy and 2.5 mg AIBN was added. The reaction mixture was heated from room temperature to 110 °C within 30 min. During the heating, about 20 mL of acetonitrile were removed by distillation. After 1 h, the product was separated by using a magnet and purified for 7 ACS Paragon Plus Environment


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several times with ethanol and deionized water. The resultant was lyophilized under vacuum for 3 days. 2.4

Preparation

of

MSP@P(MAA-Cy)-SA

and

DOX-MSP@P(MAA-Cy)-SA.

MSP@P(MAA-Cy)-SA was prepared based on the reaction between the carboxylic acid groups in the shell and the amine groups in SA. The carboxylic acid groups were activated with NHS according to the literature.47 In a typical procedure, 1 mg of activated MSP@P(MAA-Cy)-NHS was added to 2 mL SA solution (0.25 mg/mL, pH 7.4) and the mixture was stirred overnight. Subsequently, the solution was washed for three times with PBS by using a magnet to remove excess of SA. The supernatant solution was collected for quantification of the molar ratio of SA to MSP@P(MAA-Cy). In the next step, 0.2 mg of DOX and the obtained MSP@P(MAA-Cy)SA were dispersed in 15 mL PBS buffer and stirred at room temperature for 24 hours. The resulting DOX-MSP@P(MAA-Cy)-SA microsphere were washed with pH 7.4 PBS buffer solution and separated by using a magnet until the supernatant became transparent. The final product was obtained after lyophilisation under vacuum for 3 days. The DOX loaded in the nanocargos was determined by subtracting the amount of DOX in the supernatant from the amount of the drug in the original solution. This was measured by a UV-visible spectrophotometer at 480 nm. The drug loading content (DLC) and drug loading efficiency(DLE) were calculated according to the following equations:

% =

initial weight of DOX − weight of DOX in supernatant × 100% weight of DOX loaded PMAA

#% =

weight of DOX in the PMAA × 100% initial weight of DOX


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2.5 Preparation of biotinylated fluorescent molecules (FITC, FA, Rho-B) and targeting molecules (RGD, FA). For the synthesis of biotinylated FITC (BT-FITC), 100 mg of biotin hydrazide was dissolved in 20 mL PBS solution, before 200 mg FITC was added to the solution. The reaction mixture was stirred at 4 °C for 24 h. The solution was dialyzed by using a dialysis bag (cutoff Mn = 1000) against water to remove excess of FITC and unreacted biotin hydrazide. For the preparation of biotinylated FA (BT-FA), 200 mg FA was dissolved in 20 mL PBS, then 50 mg EDC and 50 mg NHS were added and the reaction mixture was left for FA activation for 2 h. After that, 100 mg biotin hydrazide was added and the reaction mixture was stirred at 4 °C for 24 h. The solution was dialyzed against water by using a dialysis bag (cutoff Mn = 500). Biotinylated Rho-B (BT-Rho) and biotinylated targeting molecules (BT-FA, BT-RGD) were synthesized according to the similar method. 2.6 Surface modification of MSP-DOX@P(MAA-Cy). The conjugation of ligands to MSPDOX@P(MAA-Cy) was realized by streptavidin-biotin binding. First, 1 mg of DOXMSP@P(MAA-Cy)-SA was dispersed in 5 mL PBS buffer (pH 7.4) containing 0.1 mg of biotinylated fluorescent molecules (BT-FITC, BT-FA, BT-Rho) or targeting molecules (BTRGD, BT-FA). The reaction was stirred at 25 °C for 2 h. The resulting dispersion was washed by PBS buffer solution and separated by using a magnet. The final product was obtained after lyophilisation for 3 days. For the parallelized conjugation to MSP-DOX@P(MAA-Cy), different biotinylated molecules were added simultaneously to MSP-DOX@P(MAA-Cy)-SA. 2.7 In vitro cytotoxicity experiments. The in vitro cytotoxicity of DOX-MSP@P(MAA-Cy)FA,

DOX-MSP@P(MAA-Cy)-RGD,

DOX-MSP@P(MAA-Cy)-FA/RGD

and

DOX-

MSP@P(MAA-Cy)-SA was assessed on SK-OV-3 cells, HeLa cells and HEK-293T cells using the CCK8 assay. Typically, a cell suspension at a density of 5×103 cells was added in each well 9 ACS Paragon Plus Environment


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of a 96-well plate and incubated for 24 h to allow attachment. Then the samples with different DOX concentrations were mixed into the fresh DMEM media and added to each wall. After incubating for certain times, the cells were washed with PBS and replaced with the new DMEM solution with 10% of CCK-8 solution. The absorbance of each wall was measured at 450 nm on a micro-plate reader. The cell viability was calculated according to the following equation: Cell viability =

OD450sample − OD450blank ∗ 100% OD450control − OD450blank

2.8 Confocal laser scanning microscopy (CLSM) experiments. SK-OV-3 cells and HeLa cells were separately seeded in φ-15 mm culture plates with a density of 1×105 cells per chamber. After incubation for 24 h to make the cells attach to the plates, each sample with certain concentrations of DOX were mixed with the fresh DMEM. Then the mixture DMEM solution was added to culture the cells for additional times. Afterwards, the cells were rinsed with PBS for three times, then stained with 1 µg/mL Hoechst 33342 for 10 min to make nucleus labelling. The samples were detected by CLSM after cleaning up the surplus Hoechst 33342. The excitation wavelengths were 405 nm and 542 nm. 2.9 The flow cytometer experiments. SK-OV-3 cells, HeLa cells and HEK-293T cells were seeded in 6-well culture chamber with a density of 2×105 cells per well and then incubated for 24 h to make the cells attach to the plate. Then the samples with certain concentration of DOX were mixed into the DMEM without folic acid for further incubation. After incubating for predetermined intervals, the cells were harvested by pancreatic enzyme. Through washing with PBS and centrifuging at 800 rpm to remove excess of DMEM and other fluorescence, the cells were re-suspended in 1 mL PBS. The flow cytometer experiments were performed with the excitation wavelength was 488 nm and the emission wavelength was 580 nm. 10 ACS Paragon Plus Environment

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2.10 Characterizations. Transmission electron microscopy (TEM) images were taken on a H600 (Hitachi, Japan) transmission electron microscope at an accelerating voltage of 75 kV. Samples dispersed at an appropriate concentration were cast onto a carbon-coated copper grids. Magnetic characterization was carried out using a vibrating-sample magnetometer (VSM) Model 6000 (Quantum Design, USA) at 300 K. Fourier transform infrared (FT-IR) spectra were recorded on a Magna-550 (Nicolet, USA) spectrometer. For FT-IR measurements, the samples were dried, pulverized, mixed with KBr, and pressed to a pellet. Thermogravimetric analysis (TGA) data were obtained on a Pyris-1 (Perkin Elmer, USA) thermal analysis system under nitrogen atmosphere in the temperature range from 100 °C to 800 °C and at a heating rate of 20 °C/min. The fluorescence emission spectra were recorded on a RF-5301PC spectrometer (PTI, USA) by using an excitation wavelength of 365 nm, 480 nm or 520 nm, respectively. Ultravioletvisible (UV-vis) absorption spectra were obtained by using a UV-3150 (Shimadzu, Japan) UVvis spectrophotometer. The confocal laser scanning microscopy (CLSM) images were recorded on a FV1000 confocal laser scanning microscope. 3. RESULTS AND DISCUSSION 3.1 Preparation of streptavidin modified magnetic nano-cargos. For the synthesis of multifunctional magnetic nano-cargos with simultaneous targeting, imaging and drug delivery capability, magnetic supraparticles (MSP) stabilized by poly(γ-glutamic acid) (PGA) were used as core material, because they offer three main advantages: first, the MSP are highly biocompatible and biodegradable,17,18 second, they provide a fast separation and enrichment of the nano-cargos under an applied magnetic field,19 and third, the nano-cargos can potentially be used for MRI imaging as well as for magnetically targeting delivery.9,18 On the basis of the MSP, uniform core/shell-structured nano-cargos were prepared in three main steps.26 11 ACS Paragon Plus Environment


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In order to form a redox-degradable poly(methylacrylic acid-co-N,N-bis(acryloyl) cystamine) (P(MAA-Cy)) shell onto the MSP core in the first step by using precipitation-distillation polymerization

of

MAA

and

Cy

monomer,

the

MSP

were

functionalized

with

methacryloxypropyltrimethoxysilane (MPS), which served as a grafting anchor for the polymer shell growth. The successful generation of a crosslinked P(MAA-Cy) shell onto the MSP core was verified by transmission electron microscopy (TEM). In Figure 1a and b, TEM images of the particles before and after the formation of the polymer shell are given, demonstrating that uniform core/shell microspheres were obtained. The average diameters of the microspheres were measured to be 180 nm for the MSP cores and 220 nm for the final MSP@P(MAA-Cy) core/shell particles, respectively, as determined by TEM measurements (Figure 1a and b). In order to accurately examine the final polymer content of the MSP@P(MAA-Cy) particles, they were additionally investigated by TGA measurements (Figure 1c). A comparison of the TGA measurements of the initial MSP and the MSP@P(MAA-Cy) particles in Figure 1c revealed a polymer shell content of about 40 wt%, which was in excellent agreement with expectations. Magnetic measurements of the MSP and the MSP@P(MAA-Cy) particles were conducted using a vibrating-sample magnetometer (VSM). The VSM results in Figure 1d displayed that the MSP cores showed a very high saturation magnetization of 72.4 emu/g and that the MSP@P(MAACy) microspheres still maintained superparamagnetic behavior, although the value of the saturation magnetization decreased to 35.6 emu/g.11,13 In the next step of the synthetic procedure, the streptavidin (SA) modified particles (DOXMSP@P(MAA-Cy)-SA) were prepared through the reaction between the amino groups of SA and the carboxylic acid groups of MAA, which were activated with N-hydroxysuccinimide (NHS), to form stable amide bonds. The successful generation of DOX-MSP@P(MAA-Cy)-SA


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particles was followed by FTIR spectroscopy, as illustrated in Figure 2a. The emerging peak at 1680 cm-1 confirmed the formation of the amide bond, and hence furnishing proof for the successful conjugation of SA to the shell of DOX-MSP@P(MAA-Cy) particles (Figure 2a).47 The successful conjugation was additionally verified by monitoring and comparing the UV-vis spectra of the SA stock solution (maximum absorption at 280 nm) and the supernatant solution after purification by using a magnet (Figure 2b). Comparison of the UV-vis spectra in Figure 2b clearly showed that the majority of SA was conjugated to the surface of the polymer shell, providing further evidence for the efficient immobilization of SA.

Figure 1. TEM images of (a) MSP and (b) MSP@P(MAA-Cy); (c) TGA curves of (i) MSP and (ii) MSP@P(MAA-Cy); (d) VSM curves of (i) MSP and (ii) MSP@P(MAA-Cy). In the last step, the anti-cancer drug doxorubicin (DOX) was loaded as a guest molecule into the polymer shell network based on electrostatic interaction between the carboxylic acid groups in the P(MAA-Cy) shell and the amine groups in DOX.25 Recently, we were able to show that 13 ACS Paragon Plus Environment


the typical absorption band of DOX with a maximum absorption at 280 nm can be used in order to determine the drug loading capacity and drug loading efficiency of DOX by using UV/vis spectroscopy.16 The loading capacity of DOX was determined to be 18.2 wt%, while the drug loading efficiency was 78.2 %. Compared to drug loading capacities of methoxypoly(ethylene glycol)-block-polylactide (mPEG-PLA) reported in the literature,2 PMAA shows a significantly higher loading efficiency, suggesting the great potential for use as drug nanocarriers.

(a)

(b) Absorbance(O.D)

(i)

Absorbance(a.u)

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(ii)

C=O(amide)

(iii)

3500 3000 2500 2000 1500 1000 500

Wavenumber(cm-1)

0.20

0.15

(i)

0.10 (ii)

0.05

0.00 260

280

300

320

340

360

380

Wavelength(nm)

Figure 2. (a) FT-IR spectra of (i) MSP@P(MAA-Cy), (ii) DOX-MSP@P(MAA-Cy) and (iii) DOX-MSP@P(MAA-Cy)-SA; (b) UV-vis spectra of (i) the streptavidin (SA) stock solution and (ii) its supernatant solution after immobilization. 3.2 Immobilization of fluorescent probe molecules onto magnetic nano-cargos. Beginning with the concept of the specific on-site assembly of required molecular elements on the asprepared magnetic nano-cargos, the reliability with regard to accessibility, stability, specificity and efficiency of the streptavidin (SA)-biotin (BT) assembly was investigated. In order to verify the accessibility of the SA binding sites, the attachment of biotinylated fluorescent probes was used to enable detection. For this purpose, three kinds of biotinylated probe molecules, labelled with fluorescein isothiocyanate (BT-FITC), folic acid (BT-FA) and rhodamine B (BT-Rho), 14 ACS Paragon Plus Environment

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respectively, were synthesized and assembled to the surface by mixing with an aqueous dispersion of MSP@P(MAA-Cy)-SA. Finally, the conjugated nano-cargos were repeatedly washed by magnetic separation for at least five times in order to ensure that all physisorbed molecules were completely removed. The successful immobilization of the fluorescent probes was verified by fluorescence emission spectroscopy. The corresponding fluorescence emission spectra are shown in Figure 3a-c.

(b) MSP@P(MAA-Cy)-SA-BT-FA

40.0k

Intensity

Intensity

9.0k

6.0k

3.0k

(c)

440.0

460.0

480.0

500.0

24.0k 16.0k

0.0 500

Wavelength(nm)

(d)

MSP@P(MAA-Cy)-SA-BT-Rho

32.0k

32.0k

24.0k 16.0k 8.0k

8.0k 0.0 420.0

40.0k

MSP@P(MAA-Cy)-SA-BT-FITC

Intensity

(a) 12.0k

520 540 560 Wavelength(nm)

580

0.0 540

560

580 600 Wavelength(nm)

620

12000

MSP-FA-FITC-Rho-520nm

MSP-FA-FITC-Rho-488nm

MSP-FA-FITC-Rho-360nm 10000

Intensity

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


8000 6000 4000 2000 0 420

440

460

480

Wavelength(nm)

500 500

520

540

560

Wavelength(nm)

580 540

560

580

600

620

Wavelength(nm)

Figure 3. Fluorescence emission spectra of (a) MSP@P(MAA-Cy)-SA-BT-FA; (b) MSP@P(MAA-Cy)-SA-BT-FITC; (c) MSP@P(MAA-Cy)-SA-BT-Rho and (d) MSP@P(MAACy)-SA mixed with BT-FITC, BT-FA and BT-Rho at same time and separated by using a magnet prior to measurement. In all cases, the nano-cargos exhibited a strong fluorescence emission and the characteristic emission bands appeared to be that of FA, FITC, and Rho-B, respectively, which indicated that a 15 ACS Paragon Plus Environment


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significant amount of the probe molecules was successfully immobilized, furnishing proof for the accessibility of the SA binding sites and the variability of the SA-BT assembly system. To further confirm the stability of the SA-BT binding, the fluorescence intensities of the nano-cargo dispersion after several purification steps as well as the supernatant solutions were additionally characterized as shown in Supporting Information Figure S1a. Comparison of the spectra revealed that no significant changes of the fluorescence intensities could be observed, and moreover, only after the first purification step a measurable fluorescence emission of the supernatant solution could be detected, providing further evidence for the remarkable stability of the SA-BT binding, which formed the basis for an efficient and quantitative on-site assembly. In order to prove the specificity of the SA-BT assembly and to validate that the fluorescent probe molecules were grafted onto the nano-cargos by the SA-BT interaction, control experiments were performed under identical conditions by mixing non-functionalized MSP@P(MAA-Cy) with BT-Rho and by mixing functionalized MSP@P(MAA-Cy)-SA with pristine Rho-B, respectively. In Supporting Information Figure S1b and c fluorescence emission spectra of the nano-cargos after the control experiments with five purification steps are given, showing that in both cases almost no fluorescence emission could be detected, which clearly demonstrated the high specificity of the SA-BT on-site assembly. To proof the efficiency of the SA-BT assembly and to quantify the amount of accessible SA binding sites, a series of solutions with molar ratios of BT-FITC with respect to theoretical immobilized SA (BT- /SA-) in the range of 0.15 to 1.00 mol/mol were added to MSP@P(MAA-Cy)-SA dispersion under identical reaction conditions. The fluorescence emission spectra of the dispersions after five purification steps are shown in Supporting Information Figure S1c. It was found that the fluorescence intensity increased with increasing BT-FITC/SA ratio, demonstrating that the SA-BT reaction 16 ACS Paragon Plus Environment

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proceeded efficiently and that the amount of biotinylated ligand can be precisely controlled by changing the BT/SA ratio. It is worth noting that at ratios above 1.00 mol/mol no further increase of the fluorescence intensity could be observed and, hence, a nearly full accessibility of the SA binding sites and an almost quantitative SA-BT reaction can be assumed. The utility and effectiveness of this on-site assembly strategy is predicated on the possibility of a parallelized conjugation with control over function and ligand composition, which is especially critical in creating personalized cancer theranostic agents. To demonstrate this facile parallelizable synthetic approach experimentally and to evaluate the degree of control, different combinations of BT-FA, BT-FITC and BT-Rho were simultaneously conjugated to the nanocargos in a one-step reaction. Supporting Information Figure S3 illustrates the fluorescence emission spectra of several representative samples of dual-modified nano-cargos. Again, with all combinations selected, the nano-cargos exhibited a strong fluorescence emission after 360 nm, 488 nm and 560 nm excitation, respectively, and the characteristic emission bands of FA, FITC, and Rho-B could be determined. Moreover, these three fluorescent probes showed distinct emission maximums, shared almost no overlapping and no fluorescence emission of the other probe molecules after respective excitation could be observed, so that no appreciable influence between the fluorescent molecules can be assumed. With this information, the ability of multi-modification based on the SA-BT on-site assembly strategy was evaluated by simply mixing all three kinds of fluorescent probes with an aqueous dispersion of MSP@P(MAA-Cy)-SA. The corresponding fluorescence emission spectra after respective excitation are shown in Figure 3d. It clearly turned out, that all three kinds of fluorescent probes could be detected, which proofed the success of the parallelized conjugation of the fluorescent probe molecules. This important evidence helped to validate our parallel on17 ACS Paragon Plus Environment


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site assembly approach and highlighted the value of incorporating functional diversity through the parallelized SA-BT bioconjugation, which can be easily achieved by an exceptional simple but highly efficient one-step reaction. 3.3 Redox/pH-triggered drug release profile of DOX-MSP@P(MAA-Cy). In the next step, we used DOX as a model anti-cancer drug to test the controlled drug releasing properties of the as-prepared DOX-MSP@P(MAA-Cy).45 The use of herein investigated DOX-MSP@P(MAACy) nano-cargos as drug delivery system for a programmed release of DOX is particularly advantageous: first the P(MAA-Cy) shell is sensitive to reductive degradation of the disulfide crosslinks when it is exposed to glutathione (GSH)-rich environment inside tumor cells,46 second, the electrostatic interaction between the carboxylic acid groups in the polymer shell and the amine groups in DOX is sensitive to the acidic pH conditions (pH ~ 5.0-5.5) in the cytoplasm of tumor cells, and third, the MSP cores are completely acid-degradable under the same acidic conditions leaving no harm to other normal cells and organs.11 Thus, we assume an intracellular transport and metabolism of the nano-cargos following the cellular reduction of the polymer shell, the acid stimulated DOX release as well as the acidic degradation of the MSP in the cytoplasm of the tumor cells. In order to mimic the conditions of cancer cells experimentally and to distinguish between different intracellular environments of tumor cells and normal cells, we chose two pH conditions (pH 5.0 and 7.4) and two GSH concentrations (0 mM and 20 mM) to investigate the pH and redox dual stimuli-responsive DOX release of the nano-cargos.46 For this studies we have chosen GSH, because GSH/GSSH is the major redox pair found in animal cells that determines the antioxidative capacity, while in similar studies often dithiothreitol (DTT) was used, which does not occur in living cells.25 The release profiles were monitored by UV/vis spectroscopy and are shown in Figure 4. 18 ACS Paragon Plus Environment

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100 Cummulative release(%)

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Figure 4. The DOX release profile of the DOX-MSP@P(MAA-Cy) nano-cargos in different pH buffer solutions and with different concentrations of GSH (±SD, n=4) As can be seen from Figure 4, under physiological conditions similar to that of normal cells of pH 7.4 without GSH, the DOX release was about 20 wt% after 72 h, which confirmed the robustness of the polymer shell as well as the stability of the electrostatic interaction. Under acidic conditions of pH 5.0, however, almost 40 wt% DOX was released within 72 h, which can be attributed to the protonation of carboxylic acid groups accompanied with weakening the DOX-polymer interaction as well as the collapse of the polymer shell. In contrast, after addition of 20 mM GSH, the cumulative release of DOX increased to 54 wt% at pH 7.4, while under physiological conditions similar to that of tumor cells of pH 5.0 and 20 mM GSH the DOX drug release was much faster with almost 50 wt% DOX release in the first 10 h and continued rapid release to reach almost 80 wt% within 72 h., which can be ascribed to the complete dissolution of the polymer shell. From the release experiments, it can be concluded that herein investigated DOX-MSP@P(MAA-Cy) nano-cargos revealed very low premature drug release and minimized the undesired drug leaking to normal cells, while they showed a very fast response accompanied 19 ACS Paragon Plus Environment


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with a controlled drug release in the environment of tumor cells, which is the basic prerequisite for an efficient targeting cancer therapy. 3.4 Cellular uptake behavior and cytotoxicity of the DOX-loaded multi-targeting nanocargos to different types of cells. As a proof of concept of herein investigated parallelized onsite

assembly

strategy

towards

personalized

cancer

targeting

therapy,

biotinylated

arginylglycylaspartic acid (BT-RGD) and biotinylated folic acid (BT-FA) that are known to specifically target to integrin or folate receptors in tumor cells, respectively, were use as targeting ligands and were simultaneously immobilized on DOX-MSP@P(MAA-Cy)-SA with the molar ratio of 1 (BT-/SA-) at 25 oC. In order to test the targeting efficiency and the controlled drug releasing capabilities of the dual-ligand modified DOX-MSP@P(MAA-Cy)-FA/RGD to specific tumor sites, the cellular uptake behavior as well as the cytotoxicity were investigated in three typical cell lines, including SK-OV-3 cells, HeLa cells and normal HEK-293T cells and the results were compared to respective single-ligand (DOX-MSP@P(MAA-Cy)-FA, DOXMSP@P(MAA-Cy)-RGD) and non-ligand (DOX-MSP@P(MAA-Cy)-SA) versions. The SKOV-3 human ovarian cancer cell line and HeLa cells were selected as model systems for cancer cells, considering similar GSH-rich and acidic intracellular environment accompanied with different receptor expression to confirm the reliability of the experiments. The HeLa cells represented model cells which express folate receptors,49 while SK-OV-3 are known to express both, folate as well as integrin receptors such as αvβ3.48 HEK-293T cells were selected for control experiments as model system for normal cells without folate and αvβ3 integrin receptor expression, the lack of GSH and with neutral cellular environment. To evaluate the cellular uptake, the cells were incubated with non-targeting (DOXMSP@P(MAA-Cy)-SA), single-targeting (DOX-MSP@P(MAA-Cy)-FA, DOX-MSP@P(MAA20 ACS Paragon Plus Environment

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Cy)-RGD) and double-targeting (DOX-MSP@P(MAA-Cy)-FA/RGD) nano-cargos and flow cytometric analyses of SK-OV-3, HeLa and HEK-293T was carried out using the DOX fluorescence emission. The results of the cytometric analysis as well as the comparison of the extent of cellular uptake for the non-, single- and dual-ligand systems using SK-OV-3 and HeLa cell lines are shown in Figure 5. The cellular uptake of DMEM was set as a control. The results for HEK-293T cells are given in Supporting Information Figure S4.

Figure 5. The flow cytometer graphs of SK-OV-3 cells that were incubated in (a) DMEM; (b) DOX-MSP@P(MAA-Cy) in DMEM; (c) DOX-MSP@P(MAA-Cy)-RGD in DMEM; (d) DOXMSP@P(MAA-Cy)-FA in DMEM; (e) DOX-MSP@P(MAA-Cy)-FA/RGD in DMEM for 2 h, 24 h and 36 h (all DMEM are without folic acid). The concentration of DOX is 2µg/mL and the long strings above align at the peak of (b) DOX-MSP@P(MAA-Cy); (h)-(l) was the same sample order incubated with HeLa cells for 2 h, 12 h and 24 h; The mean DOX fluorescence



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intensity of (f) sample (a)-(e) and (g) sample (h)-(l) were analysed according to the processing software FlowJo_v10. The excitation wavelength is 520 nm. As can be extracted from Figure 5a-f, in SK-OV-3, about 1.8-fold enhancement in cellular uptake could be observed for both single-ligand modified nano-cargos (DOX-MSP@P(MAACy)-FA, DOX-MSP@P(MAA-Cy)-RGD) after 2 h, 24 h and 36 h, as compared to the nonmodified version (DOX-MSP@P(MAA-Cy), indicating that both, the SA as well as the RGD ligands recognize the target receptors. In comparison, the dual-ligand system (DOXMSP@P(MAA-Cy)-FA/RGD) showed similar uptake behavior after 2h and 24h, which can be attributed to the efficient recognition of both target receptors by both targeting ligands, as a result of the specific interaction of FA and RGD with target folate and integrin αvβ3 receptor expressed from SK-OV-3. However, after 36 h about 2.2-fold enhancement in cellular uptake compared to the non-modified version could be observed, indicating that the use of the dual-ligand system led to a continuous and significantly enhanced uptake in SK-OV-3 due to a synergistic effect on the cellular uptake in folate and integrin αvβ3 expressing cells. To the contrary, in HeLa, no significant difference in cellular uptake was observed between single-ligand and double-ligand modified nano-cargos even after 24 h (Figure 5g-l). In contrast to DOX-MSP@P(MAA-Cy)-RGD, that showed only about 1.4-fold enhancement compared to non-modified version after 24 h, MSP@P(MAA-Cy)-FA exhibited about 1.7-fold enhancement in cellular uptake after 24 h, which can be attributed to the efficient recognition of targeted folate receptors expressed from HeLa. However, no significant difference in cellular uptake was observed for double-ligand modified DOX-MSP@P(MAA-Cy)-FA/RGD, as a result of the absence of specific interactions between RGD and corresponding receptors on the cell surface. It therefore turned out that, in the case of HeLa, the enhancement in cellular uptake of the dual22 ACS Paragon Plus Environment

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ligand system compared to non-modified version was only derived from the interaction of FA with folate receptors. It should be noted that all herein investigated nano-cargos showed similar uptake behavior to HEK-293T cells due to absence of receptor expression (Supporting Information Figure S4).

Figure 6. The CLSM images of SK-OV-3 cells incubated with (a, f) DOX; (b, g) DOXMSP@P(MAA-Cy); (c, h) DOX-MSP@P(MAA-Cy)-FA; (d, i) DOX-MSP@P(MAA-Cy)-RGD; (e, j) DOX-MSP@P(MAA-Cy)-FA/RGD for 2 h (left panel) and 24 h (right panel), respectively (all DMEM recipes are without folic acid). The concentration of DOX is 2 µg/mL. All the scale bars are 20 µm. To further confirm the cellular uptake of the differently modified nano-cargos and to accurately examine the targeting drug release in SK-OV-3, the distribution of released DOX was



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investigated by confocal laser scanning microscopy (CLSM). The cellular uptake of free DOX was set as a control. Figure 6 displays the localization of DOX, originally loaded to the nanocargos, 2 h (Figure 6a-d) and 24 h (Figure 6f-j) after cell incubation followed by fluorescent staining with Hoechst 33342. For free DOX (Figure 6a) and non-targeting DOX-MSP@P(MAACy)-SA (Figure 6b) only a slight DOX signal was detectable after 2 h of incubation, while after 24 h of incubation (Figure 6f, g) the DOX signal was slightly enhanced, which could be attributed to a passive diffusion pathway for entry into the cells. For the single-targeting DOXMSP@P(MAA-Cy)-FA (Figure 6c) and DOX-MSP@P(MAA-Cy)-RGD) (Figure 6d), as well as double-targeting DOX-MSP@P(MAA-Cy)-FA/RGD, a nuclear localization of DOX after 2 h of incubation could clearly be observed by the overlap of the DOX and Hoechst 33342 signal. However, after 24 h incubation the ligand modified nano-cargos showed a strong DOX signal, confirming that the ligand modified nano-cargos are efficiently internalized and accumulated into the target cells accompanied with a programmed drug release inside the SK-OV-3 cells. To elucidate the anti-tumor efficacy and the harm to normal cells, the CCK-8 method was further applied to investigate the cytotoxicity of the nano-cargos. For quantitative cytotoxicity evaluation, the SK-OV-3, HeLa and HEK-293T were incubated with non-targeting (DOXMSP@P(MAA-Cy)-SA), single-targeting (DOX-MSP@P(MAA-Cy)-FA, DOX-MSP@P(MAACy)-RGD) and double-targeting (DOX-MSP@P(MAA-Cy)-FA/RGD) nano-cargos containing different concentrations of DOX (0.25-2.00 µg/mL) and the cytotoxicity was quantified by determining the viability of treated cells relative to untreated controls. The results of the cytometric analysis for the non-, single- and dual-ligand systems at different DOX concentrations and incubation time using SK-OV-3 and HeLa cell lines are shown in Figure 7. The results for HEK-293T cells are given in Supporting Information Figure S5. 24 ACS Paragon Plus Environment

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well as RGD, to the respective receptors in SK-OV-3. However, after 72 h of incubation the synergistic effect of both ligands reinforcing the inhibiting ability got more obvious, which demonstrated the great potential for the dual-ligand modified nano-cargos for targeting drugdelivery to SK-OV-3 cells. As can be extracted from Figure 7c and d, also for HeLa a significant enhancement of the cytotoxicity of dual-ligand modified DOX-MSP@P(MAA-Cy)-FA/RGD compared to nonmodified and single-modified nano-cargos after 24h and 48 h of incubation could be detected. Again, for the non-modified nano-cargos a much lower cytotoxicity was determined in HeLa at all concentrations and any incubation time. Moreover, single-modified MSP@P(MAA-Cy)-FA showed higher cytotoxicity than MSP@P(MAA-Cy)-RGD, which could be attributed to the targeting of FA to the folate receptor in HeLa. The high specificity MSP@P(MAA-Cy)-FA can be observed especially at very high DOX concentrations after 48 h incubation time, in which the inhibiting ability of the dual-modified nano-cargos was even exceeded by single-modified MSP@P(MAA-Cy)-FA. Again, it is worth mentioning that all herein investigated nano-cargos showed very low cytotoxicity to HEK-293T even after 48 h of incubation, which could be attributed to the neutral cellular environment and lack of GSH in HEK-293T (Supporting Information figure S5). 3.5 Validation of the on-site assembly strategy. The utility and effectiveness of herein developed on-site assembly strategy is predicated on the ability to assemble these multifunctional nano-cargos through highly specific and parallelizable bioconjugation with control over nanocargo/ligand ratio, activity and functionality, but also by the simplicity and efficiency of this approach, which can be carried out without additional purification and post-treatment. These factors offer an additional degree of control that is often more difficult to achieve with large 26 ACS Paragon Plus Environment

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biomolecular ligands, which is critical in creating personalized cancer theranostic agents. To demonstrate this outstanding possibility experimentally, we mixed an aqueous dispersion of DOX-MSP@P(MAA-Cy)-SA and biotinylated targeting molecules (BT-FA, BT-RGD) and evaluated the cell uptake and cytotoxicity already after 10 min of assembly time without further purification or post-treatment. The results for these so-called “on-site nano-cargos” were directly compared to the results of so-called “purified nano-cargos”, that were purified by magnetic separation for five times after 12 h incubation. The results of the cytometric analysis for the non-, single- and dual-ligand systems using SK-OV-3 are shown in Figure 8.

Figure 8. The flow cytometer graphs of SK-OV-3 cells incubated with purified nano-cargos and onsite nano-cargos for (a) 12 h and (b) 24 h, the concentration of DOX is 2µg/mL. The DOXMSP denoted the DOX-MSP@P(MAA-Cy) sample. 27 ACS Paragon Plus Environment


As can be concluded from Figure 8, the on-site nano-cargos showed completely identical uptake behavior in SK-OV-3 after 2h and 24h of incubation, furnishing proof for the exceptional efficiency of our on-site assembly approach. Furthermore, a comparison of the fluorescence intensity of the on-site nano-cargos and the purified nano-cargos revealed an identical enhancement of the fluorescence intensity with increasing incubation time, which additional demonstrated the identical and efficient uptake behavior in SK-OV-3. Finally, the cytotoxicity for both the on-site nano-cargos as well as the purified nano-cargos were evaluated and directly compared. For quantitative cytotoxicity analysis, again the CCK-8 method was applied and the SK-OV-3 were incubated with the non-, single- and dual-ligand systems containing different concentrations of DOX (1-10 µg/mL). The results of the cytometric analysis are shown in Figure 9.

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Figure 9. The cell survival of SK-OV-3 cells incubated with purified nano-cargos and onsiteassembly nano-cargos of (i) DOX-MSP@P(MAA-Cy), (ii) DOX-MSP@P(MAA-Cy)-RGD, (iii) 28 ACS Paragon Plus Environment

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DOX-MSP@P(MAA-Cy)-FA, (iv) DOX-MSP@P(MAA-Cy)-FA/RGD at different DOX concentrations of (a, d) 1 µg/mL; (b, e) 5 µg/mL; (c, f) 10 µg/mL for 24 h (top) and 48 h (bottom), ±SD, n=3. As can be concluded from Figure 9, the onsite-assembly nano-cargos showed almost similar cytotoxicity to SK-OV-3 cells compared to the purified nano-cargos at DOX concentration of 1 µg/mL, 5 µg/mL and 10 µg/mL after 48-h and 72-h incubation (Figure 9), providing further evidence for the simplicity and efficiency of this approach, which opens access to highly efficient targeting drug delivery systems without the use of any purification and post-treatment. 4. CONCLUSION In summary, a convenient and highly variable on-site assembly strategy for the flexible conjugation of specific targeting ligands to binding sites of magnetic nano-cargos based on highly efficient and parallelizable streptavidin-biotin (SA-BT) bioconjugation was reported. Exceptional uniform and water-dispersible magnetic nano-cargos composed of magnetic supraparticle (MSP) cores and redox-degradable poly(methylacrylic acid-co-N,N-bis(acryloyl) cystamine)

(P(MAA-co-Cy)

shells

were

synthesized

through

precipitation-distillation

polymerization, that were additionally modified with streptavidin moieties. Furthermore, the cancer drug doxorubicin (DOX) was loaded as a guest molecule into the polymer shell and could be selectively released under glutathione (GSH)-rich and acidic conditions, similar to the environment in the cytoplasm of tumor cells. Prepared DOX-loaded magnetic nano-cargos could be simultaneously modified with multiple biotinylated ligands through parallelized SA-BT bioconjugation. The stability, specificity and efficiency of the SA-BT assembly was verified through the conjugation of means of biotinylated probe molecules and it was found that it could provide a remarkable high degree of control over nano-cargo/ligand ratio, activity and 29 ACS Paragon Plus Environment


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functionality in an almost quantitative one-step reaction without the need of any purification and post-treatment. As a proof of concept of herein investigated parallelized on-site assembly strategy towards cancer targeting therapy, biotinylated arginylglycylaspartic acid (BT-RGD) and biotinylated folic acid (BT-FA) were use as targeting ligands and were simultaneously immobilized. Corresponding dual-ligand modified DOX-loaded nano-cargos showed remarkable proliferation inhibition for the HeLa cells and the SK-OV-3 cells, giving rise that herein described on-site assembly strategy opens access to highly efficient targeting drug delivery systems by incorporating functional diversity, which can be easily achieved through highly efficient and parallelized one-step bioconjugation. Therefore, we consider that this on-site assembly approach can be used as a modular synthetic toolkit to tailor targeting properties of cancer theranostic agents towards personalized cancer targeting therapy.

ASSOCIATED CONTENT Supporting Information. Photographs of fluorescence spectra, flow cytometer graphs and the cell survival.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]; Fax: +86-21-65640293. Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. Funding Sources This work was supported by State Key Project of Research and Development (Grant No. 2016YFC1100300) and National Science Foundation of China (Grant No 21474017, 51633001).

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SYNOPSIS

Specific On-site Assembly of Multifunctional Magnetic Nano-Cargos Based on Highly Efficient and Parallelized Bioconjugation: Towards Personalized Cancer Targeting Therapy Luyan Sun, † Jiaxun Wan, † Christian G. Schaefer,† Zihao Zhang,† Jing Tan, † Jia Guo, † Limin Wu,‡ Changchun Wang* † ,


Specific On-site Assembly of Multifunctional ... - ACS Publications

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