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Open AccessCCS ChemistryRESEARCH ARTICLE1 Oct 2020

Robust and Tumor-Environment-Activated DNA Cross-Linker Driving Nanoparticle Accumulation for Enhanced Therapeutics

    https://doi.org/10.31635/ccschem.020.202000134
    Abstract
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    Agglomeration of therapeutic nanoparticles in response to tumor microenvironments is a promising approach to enhance drug accumulation and improve therapeutic efficacy. Cytosine-rich DNA sequences show potential as ideal cross-linkers to drive nanoparticle agglomeration because they can sensitively respond to weak acidity and form interchain folding. However, the in vivo application of DNA is generally limited by its poor biostability; as a consequence, modifications with unprotected DNA cross-linkers can enhance the accumulation of nanoparticles twofold at the tumor site. Facing this challenge, we have designed and developed a protection and tumor-environment activation strategy to enable the in vivo application of a DNA cross-linker. Specifically, reactive oxygen species (ROS)-responsive polyethylene glycol (PEG) was modified on the nanoparticle surface together with the DNA cross-linker, which protects DNA from degradation during the blood circulation; meanwhile, when arriving at the tumor site, the nanoparticles shed the PEG shell as a response to ROS to uncover and activate the DNA cross-linkers. Using this strategy, a sevenfold enhancement in tumor accumulation was achieved owing to both superior pH sensitivity and improved stability of DNA cross-linkers. Finally, significantly improved therapeutic efficacy in in vivo anticancer treatment was realized by using this agglomeration strategy driven by protected and stimuli-activated DNA cross-linkers.

    Introduction

    Malignant tumors pose a severe threat to human life and health. Achieving early diagnosis and treatment of tumors has always been a major topic of concern for the medical community and scientists.1 Nanotechnology has been widely used in the field of tumor theranostics.2 The nanoparticle sizes play an essential role in the enrichment of nanoparticles at the tumor sites.3 Compared with large-sized nanoparticles, small-sized nanoparticles show advantages of deep tissue penetration and long circulation lifetimes.4 However, when small nanoparticles reach the tumor site, they will return to the blood or metastasize to the tissues surrounding the tumor.5,6 This will greatly reduce the nanoparticle enrichment, thereby lowering the effectiveness in the diagnosis and treatment of tumors. Therefore, finding a simple and controllable strategy to enable small-sized nanoparticles to selectively aggregate at tumor sites has become a crucial issue in the field of nanomedicines.7,8 For instance, nanoparticles are designed to show impaired colloidal stability in the acidic environment at the tumor site to drive agglomeration.9 Alternatively, nanoparticles are modified by stimuli-responsive peptides10,11 or polymers,12,13 which can be cross-linked or cleaved (changing the solubility) by tumor-specific enzymes or molecules, and, as a result, noncovalent self-assembly or covalent cross-linking of nanoparticles was induced into the tumor site.11,12 These studies have proven that the agglomeration strategy is very promising in enhancing the tumor accumulation of nanoparticles, and further improvements are urgently needed.

    Functional DNA is a promising candidate as a nanoparticle cross-linker, due to its sensitive response to environmental variations, superior biocompatibility, and accessible chemistry for surface modification.14,15 Cytosine-rich DNA strands can sensitively recognize acidic pH and fold into a quadruplex structure (i-motif structure) through CH+·C interactions.16,17 The pH identification range of the i-motif forming strand (IFS) can be regulated by these sequences. Therefore, it is convenient to choose an IFS strand that can undergo cross-linking by forming an interchain quadruplex structure under weakly acidic pH, a typical physiological environment at the tumor site. However, DNA cross-linkers have been rarely used for in vivo therapy applications because DNA is easily degraded by nucleases during blood circulation.18 To enhance the availability of functional DNA molecules during in vivo uses, Mirkin and co-workers19–21 demonstrated that densely packed DNA molecules in spherical geometry exhibit substantially improved biostability compared with free DNA. The dense DNA arrangement hinders the approximation of nucleases and, therefore, protects DNA from degradation in the serum environment. However, such a close-packing strategy is not applicable to nanosystems utilizing DNA as a cross-linker. According to our research, for cross-linker applications, DNA modification at low density is necessary for inducing efficient particle–particle cross-linking; otherwise, higher densities will induce IFS cross-linking on the same nanoparticle. The loosely packed DNA can be more easily attacked by nuclease and degraded quickly during blood circulation. Thus, this observation prompted us to develop a new strategy to enhance the biostability of the loosely packed IFS DNA molecules on the nanoparticles.

    In this study, [email protected]2-DOX was synthesized and proven to be an efficient therapeutic nanoparticle by combining photothermal therapy (PTT) with chemotherapy, in which doxorubicin hydrochloride (DOX) was loaded in the silica shell through physical adsorption.22 To enhance the accumulation of this nanoparticle at the tumor site, a DNA sequence was optimized to sensitively recognize weak acidic conditions up to pH 6.0 (approximately close to the interstitial pH value of the tumor)9 and undergo interchain cross-linking. However, barely using the DNA cross-linkers only produced a twofold enhancement in tumor accumulation of the therapeutic nanoparticles as a result of the poor stability. Therefore, the protection and tumor-environment activation strategy was developed. [email protected]2-DOX was modified with IFS DNA molecules and methoxy polyethylene glycol with labile thioketal linker (TK-mPEG)23,24 to produce [email protected]2-DOX/i-motif/TK-mPEG. In addition to extending the circulation time, the PEG layer was well shielded and protected the IFS from degradation during blood circulation.25 When the nanoparticle extravasated into the tumor through the enhanced permeability and retention (EPR) effect,26 sequential responses to multiple tumor microenvironments were triggered. Specifically, the nanoparticle shed the PEG shell in response to reactive oxygen species (ROS)27,28 and exposed IFS DNA, which subsequently formed interchain folding in response to the acidic environment at the tumor site, inducing nanoparticle agglomeration. Therefore, by this protection and stimuli-activation strategy, the very sensitive acid-responsiveness of the DNA cross-linker could be reserved for the in vivo application. As a consequence, a sevenfold enhancement in tumor accumulation, compared with the naked nanoparticle was observed, which is a superior result among recent, related studies ( Supporting Information Table S1), resulting in significantly improved therapeutic efficacy in in vivo anticancer experiments (Scheme 1).

    Scheme 1

    Scheme 1 | Schematic illustration of the self-assembly of [email protected]2-DOX/i-motif/TK-mPEG nanoparticles in response to tumor microenvironments.

    Experimental Methods

    Materials

    Chemicals and solvents were used as received without further purification unless specified otherwise. DNA sequences (HPLC-purified), thiazolyl blue tetrazolium bromide (MTT), and cetyltrimethylammonium bromide (CTAB) were obtained from Sangon Biotech Co., Ltd (Shanghai, China). Copper chloride (CuCl2), sodium sulfide (Na2S), acetic acid (HAc), sodium acetate (NaAc), DOX, 4-Morpholineethanesulfonic acid (MES), 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide (NHS), dimethyl sulfoxide (DMSO), 3-mercaptopropionic acid, trifluoroacetic acid (TFA), acetone, ether, and N,N-dimethylformamide (DMF) were obtained from Aladdin Chemistry Co., Ltd. (Shanghai, China). Polymethoxyethylene glycol modified with carboxyl groups (mPEG2k) (MW 2000) and PEG modified with both carboxyl groups and RhB (mPEG2k-RhB) (MW 2000) were purchased from ToYongBio (Shanghai, China). (3-aminopropyl) Triethoxysilane (APTES), tetraethylorthosilicate (TEOS), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were purchased from TCI Chemical Industry Co., Ltd. (Japan). Phosphate buffered saline (PBS) was prepared by dissolving tablets obtained from AMRESCO (Solon, OH, United States) in ultrapure water according to the manufacturer’s instructions (10.0 mM PBS, containing 137.0 mM Na,+ and 2.0 mM K+). Acetyl acetate solution (3.0%) and copper grid were purchased from Zhongjingkeyi Technology Co., Ltd. (Beijing, China). Ultrapure water (at 18.2 Mohm) was produced by a Millipore Synergy UV Ultrapure water purification system (MA, United States). The ultrafiltration tube (100,000-molecular-weight cutoff, 15.0 mL) was also purchased from the Millipore Co. Ltd. 4T1 cells were purchased from Oulu Biotechnology (Shanghai, China). RPMI 1640 medium, Dulbecco’s Modified Eagle’s Medium (DMEM), trypsin–EDTA solution, fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Gibco-BRL Co., Ltd. (Grand Island, NY, United States). Female BALB/c mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China).

    Instrumentation

    The 880 nm continuous-wave laser systems were purchased from Changchun Laser Technology Co., Ltd. (Changchun, China). The transmission electron microscopy (TEM) images were captured on Hitachi HT7700 (Hitachi, Japan). The hydrodynamic diameters and zeta potentials of the micelles were determined by a NanoBrook ZetaPALS potential analyzer (Brookhaven Instruments Corporation, United States) in ultrapure water. The surface area, pore size, and pore size distribution of the products were determined by Brunauer–Emmett–Teller (BET), nitrogen adsorption/desorption, and Barrett–Joyner–Halenda (BJH) methods (Micromeritics, ASAP2020). Fourier-transform infrared (FT-IR) spectra were recorded by using a Frontier spectrometer (PerkinElmer, United States). Copper content was obtained from an Agilent 7700× inductively coupled plasma mass spectrometry (ICP-MS). Powers X-ray diffraction (XRD) measurements were performed on a D8 Focus diffractometer (Rigaku SmartLab) using Cu-Kα radiation. Cellular fluorescence images were taken by a Leica TCS-SP8 confocal laser scanning microscope (CLSM, Leica, Germany). Circular dichroism (CD) spectra of the reporter and reporter-control sequences in buffers of pH 5.0 and 8.0 were measured on a Chirascan system (Applied Photophysics Ltd., United Kingdom). The 1H NMR spectra were recorded on a Bruker Advance 400 instrument. Native polyacrylamide gel electrophoresis (PAGE) experiments were carried out on 20% polyacrylamide gels run for 3 h under a field of 80 V at room temperature. TBE running buffer (0.5×) consisted of Tris-HAc (50 mM). Gels were stained with GelRed dye. The in vivo fluorescence images were obtained by an IVIS Spectrum Imaging System (PerkinElmer, United States), with an excitation wavelength at 480 nm and an emission wavelength at 580 nm.

    Synthesis of [email protected]2 nanoparticles

    [email protected]2 core–shell nanoparticles were synthesized according to a previously reported method.22,29 CuCl2 (100 mM, 0.5 mL) was added to the aqueous solution of CTAB (49 mL), and aqueous Na2 S (0.5 mL,100 mM) was added dropwise to the mixture under magnetic stirring for 15 min. After that, the solution was heated to 90 °C under gentle nitrogen flow for 1 h. After CTAB-stabilized CuS nanoparticles were prepared, an extra 94 mg of CTAB (with a final concentration of 2 mg·mL−1) was added at 70 °C under stirring. After 20 min stirring, a NaOH solution (2 M, 350 μL) containing TEOS (180 μL) and ethyl acetate (1 mL) was added dropwise to the reaction solution. Then, APTES (80 μL) was added 1 h later, and the mixture was further stirred at 70 °C for 3 h. Then, the as-prepared [email protected]2 was collected by centrifugation and washed with water. Finally, the product was dispersed in an ethanol solution of NH4NO3 (50 mL, 10 mg·mL−1), and CTAB template was removed by ion exchange. The porous [email protected]2 nanoparticles were further purified by ethanol washing several times.

    Synthesis of the TK

    3-Mercaptopropionic acid (4.8 mL, 5.54 mol), anhydrous acetone (2.1 mL, 2.77 mol), and 1 mL of TFA were added into a round-bottom flask, and the mixture was stirred for 12 h under nitrogen flow. After the reaction, 20 mL of water was added into the mixture and chilled in ice until crystallization was complete. Then the product was collected by suction filtration, and the white crystals were washed with ice water to remove excess reagents and TFA. The product was further dried in a vacuum desiccator at 30 °C overnight.

    Synthesis of TK-mPEG

    mPEG2k-NH2 (400 mg, 0.2 mmol), TK (505 mg, 2 mmol), EDC (115 mg, 0.6 mmol), and NHS (69 mg, 0.6 mmol) were dissolved in 4 mL of anhydrous DMF. The reaction was stirred for 24 h under nitrogen flow. After the reaction, the solution was added dropwise to diethyl ether (20 mL) in an ice water bath to precipitate the TK-mPEG2k. Then the product was collected by centrifugation and further purified by extensively dialyzing against deionized water to remove EDC, NHS, and the remaining reagents. TK-mPEG2k was obtained as a white powder after lyophilization. The same method was used to synthesize TK-mPEG-RhB.

    ROS responsiveness of TK-mPEG

    The ROS responsiveness of TK-mPEG was investigated by 1H NMR spectrum. TK-mPEG2k (8 mg) was dissolved in 2 mL of the aqueous solution containing H2O2 (10 mM) and CuCl2 (3.2 μM) and then incubated at 37 °C for 24 h. Then the obtained solution was lyophilized and characterized by 1H NMR.

    Preparation of [email protected]2/i-motif

    After [email protected]2 (1 mg), EDC (5 mg, 26 μmol), NHS (5 mg, 44 μmol), and 100 μL of a random sequence (P1) modified with Cy5 (P1-Cy5, aqueous solution, 100 μM) were dispersed in 1 mL of HEPES buffer (10 mM, pH = 7.5). The solution was shaken for 2 h at 37 °C. After the reaction, [email protected]2/P1 was collected by centrifugation and washed with water three times. Then, we determined the content of the remaining sequence after the reaction and calculated the grafting efficiency of the nanoparticles. We used the same method to couple the [email protected]2 with i-motif sequences, and the grafting densities of the IFS sequences were controlled by adjusting the amount of added DNA.

    Preparation of [email protected]2/TK-mPEG

    1 mg of [email protected]2 was dispersed in 900 μL HEPES (10 mM, pH = 7.5), and EDC (5 mg, 26 μmol), NHS (5 mg, 44 μmol), and 100 μL of aqueous TK-mPEG-RhB (1 mg·mL−1) were added into the solution. The mixture was shaken at 37 °C for 2 h. The product was centrifuged and washed three times by water. Then we determined the content of the remaining sequence after the reaction and calculated the grafting efficiency of the nanoparticles. We used the same method to couple the [email protected]2/i-motif with TK-mPEG.

    Preparation of the DOX-loaded [email protected]2/i-motif/TK-mPEG

    500 μL of [email protected]2/i-motif/TK-mPEG nanoparticles (1.5 mg·mL−1) was added to 1.5 mL of PBS solution (pH = 7.5) containing DOX (0.75 mg·mL−1) and stirred overnight at room temperature. After DOX loading, [email protected]2-DOX/i-motif/TK-mPEG nanoparticles were centrifuged and washed with PBS solution to remove the free DOX.

    In vitro release of DOX

    The [email protected]2-DOX/i-motif/TK-mPEG nanoparticles were dispersed in PBS solution (pH 8.0) and MES buffer (pH 5.0), respectively. The DOX release by laser trigger was conducted by 10 min irradiation (880 nm, 0.8 W·cm−2). After that, the aqueous dispersions were centrifuged, and the supernatants were collected for UV–vis spectrophotometer analysis.

    Photothermal performance

    [email protected]2 nanoparticles at different concentrations (25, 50, 100, and 200 µg mL−1) were irradiated by an 880 nm laser (power density of ∼ 0.8 W cm−2), and the temperatures of the dispersions were measured every 30 s.

    Cytotoxicity tests

    The in vitro cytotoxicity was determined by the MTT assay in the mouse breast cancer cell line (4T1). 4T1 cells were seeded in 96-well plates and precultured for 24 h. Then the old medium was replaced by the fresh medium with the [email protected]2, [email protected]2-DOX, [email protected]2/i-motif/TK-mPEG, and [email protected]2-DOX/i-motif/TK-mPEG nanoparticles at a series of concentrations at 37 °C with 5% CO2 for an additional 4 h. Finally, the standard MTT assay determined the relative cell viabilities.

    CLSM imaging

    The 4T1 cells were seeded in a 96-well plate and cultured for 12 h and then incubated with the medium concentration of (100 µL) [email protected]2-DOX/i-motif/TK-mPEG nanocomposites (with a DOX concentration of 5 μg mL−1) for 1 h. Thereafter, the cells were washed with PBS three times and fixed with 4% paraformaldehyde for 15 min at room temperature. The cells were then rinsed with PBS and stained with the DAPI solution for another 10 min. Finally, the collected cells were imaged on a CLSM at excitation wavelengths of 405  (DAPI) and 480 nm (DOX), respectively.

    Chemo-PTT

    The 4T1 cells were seeded in 96-well plates and precultured for 24 h. Then the old medium was replaced by the fresh medium with [email protected]2, [email protected]2-DOX, [email protected]2/i-motif/TK-mPEG, and [email protected]2-DOX/i-motif/TK-mPEG at various concentrations. For different samples, the concentrations of [email protected]2 were equivalent to 50, 100, 150, and 200 µg mL−1. After 4 h of incubation, the cells were washed with PBS and replaced with fresh media. Then the cells were treated with 5 min irradiation (880 nm laser, 0.8 W cm−2). Finally, the cell viabilities were determined by the MTT assay.

    Animal experiments

    All animal experiments were in accordance with Institutional Animal Use and Care Regulations protocol no. SUSTC-JY2017078, approved by the Laboratory Animal Ethics Committee of the Southern University of Science and Technology. After being acclimated and tested for infectious diseases for 1 week, female BALB/c mice (aged 6–8 weeks) were subcutaneously injected with 4T1 cells (1×107 cells each mouse) at the flank region. When the tumor size reached about 200 mm3, the mice were randomly divided into five groups and administered with the following treatments: saline only (1); [email protected]2-DOX only (2); [email protected]2-DOX and exposed to the laser (3); [email protected]2-DOX/i-motif/TK-mPEG only (4); [email protected]2-DOX/i-motif/TK-mPEG and exposed to the laser (5). The tumor size was calculated using the following formula: volume = (length × width2)/2, and the tumor volumes and body weights were measured every three days for 22 days.

    For in vivo fluorescence studies, tumor-bearing mice were intravenously injected with [email protected]2-DOX, [email protected]2-DOX/i-motif, [email protected]2-DOX/TK-mPEG, and [email protected]2-DOX/i-motif/TK-mPEG (the concentrations of [email protected]2 were equivalent to 25 mg kg−1), respectively. Then the mice were sacrificed at designed time points (12, 24, 36, and 48 h), and ex vivo fluorescence of the major organs was recorded. Then all the organs and tumors were managed by vacuum freeze-drying, weighed, and dissolved in nitric acid for Cu quantification by ICP-MS.

    Pharmacokinetics studies

    The pharmacokinetics of [email protected]2/i-motif/TK-mPEG and [email protected]2/i-motif nanoparticles were systematically investigated using ICP-MS for the quantified amount of Cu in the blood samples. Specifically, the SD mice were intravenously injected with the nanoparticles (25 mg kg−1 according to [email protected]2), and the blood samples were collected at the designed time points (0.25, 0.5, 1, 3, 4, 8, 12, and 24 h), which were further dissolved in nitric acid for Cu quantification by ICP-MS.

    Cy5 dye-labeled i-motif sequence (i-motif-Cy5) was utilized to fabricate the nanoparticles to monitor the stability of DNA during blood circulation. Consequently, [email protected]2/i-motif-Cy5/TK-mPEG and [email protected]2/i-motif-Cy5 nanoparticles (25 mg kg−1, according to [email protected]2) were intravenously injected into the healthy mice, and the blood samples were collected 12 h later. The fluorescence intensity of Cy5 in the blood was measured by an ex vivo IVIS Lumina Imaging System (PerkinElmer, United States) at excitation and emission wavelengths of 650 and 670 nm, respectively.

    Results and Discussion

    Synthesis and characterization of dual-modified nanoparticles for tumor accumulation

    [email protected]2 core–shell nanoparticles were functionalized with amine groups using APTES together with TEOS. The crystalline structure of [email protected]2 was characterized by powder XRD ( Supporting Information Figure S1a), for which the diffraction peaks of the CuS core matched well with a hexagonal-phase structure (JCPDS card no. 79-2321); after silica coating, the structure of CuS was maintained, and an additional amorphous peak at 2θ = 22.6° was observed due to the silica formation. According to the TEM image of the as-prepared [email protected]2 (Figure 1a and b), the CuS core exhibited an average diameter of 15 nm with a silica shell averaging 20 nm thick, and the CuS core showed a fringe spacing of 0.282 nm, in accordance with the (103) crystal planes of the hexagonal-phase CuS crystal.22 The porous structure of the silica shell was produced by removing the CTAB template, which was monitored by FT-IR analysis ( Supporting Information Figure S1b). The disappearance of the peak at 2926 cm−1 (from the stretching vibration of the alkyl chain of CTAB) indicated the removal of CTAB. After that, the nanoparticles exhibited a typical IV isotherm during BET analysis, which proved the porous nature of the silica shell. A surface area of 516 m2 g−1 and a pore size of 2.6 nm were determined, and the BJH pore volume of the [email protected]2 nanoparticles was calculated to be 1.16 m3 g−1, which provide the high DOX loading efficiency in the next step ( Supporting Information Figure S1c and d).

    Figure 1

    Figure 1 | TEM image (a) and high-resolution TEM image (b) of [email protected]2 core–shell nanoparticles. Size distribution (c) and zeta potentials (d) of [email protected]2, [email protected]2/i-motif, and [email protected]2/i-motif/TK-mPEG nanoparticles.

    Next, [email protected]2 was successively modified by carboxylic-acid-functionalized DNA and TK-mPEG by EDC/NHS amidation coupling. A 15-mer random DNA strand with carboxyl functionalization at the 5’ end (P1) was selected to study the conjugation yield ( Supporting Information Figure S2a). The carboxyl-functionalized TK-mPEG ( Supporting Information Figure S3a) was synthesized according to the literature method28 and characterized by 1H NMR ( Supporting Information Figure S3b). The resonance frequency at 1.62 ppm belongs to the –CH3 protons of the TK linker, which would be eliminated by the addition of H2O2 ( Supporting Information Figure S3c), indicating that TK is a labile linker and TK-PEG will be a sheddable layer. The maximum conjugation yields for DNA and TK-PEG were determined to be 5.5 nmol DNA ( Supporting Information Figure S2b and c) per mg [email protected]2 and 4.1 nmol TK-mPEG ( Supporting Information Figure S4a–c) per mg [email protected]2, respectively. The successful functionalizations were certified by DLS and zeta potential tests to reveal the changes in surface properties. As shown in Figure 1c and d, the hydrodynamic diameter of [email protected]2 was about 80 nm with a zeta potential of 17.4 mV due to positively charged amine groups on the surface. After conjugation with the negatively charged DNA, the hydrodynamic diameter increased to about 100 nm accompanied by a negative zeta potential of –11.2 mV. Finally, the hydrodynamic diameter of [email protected]2/i-motif/TK-mPEG increased to 160 nm, and an almost neutral zeta potential was observed, implying that the PEG layer can shield the DNA layer from the surface and overwhelmingly govern the surface properties of the final nanoparticles. Subsequently, due to the good porous properties of the silica shell, a high DOX loading efficiency was achieved, that is, ∼ 1.9 g DOX on 1.0 g of [email protected]2, resulting in the final dual-modification therapeutic nanoparticle, [email protected]2-DOX/i-motif/TK-mPEG.

    Stimuli-responsive self-assembly of nanoparticles

    The self-assembly capability of the nanoparticles under acidic conditions was first optimized. An IFS sequence (5′-TTTTTCCCCCTCCCCC-3′) was selected from the literature, which prefers the formation of interchain i-motif structures, according to literature.16,30 Analyzed by CD spectrum ( Supporting Information Figure S2d), this IFS strand showed a positive band at ∼ 270 nm at pH 8.0; while at pH 5.0, the main band shifted to 290 nm and a new negative band appeared at ∼ 255 nm, consistent with the spectral characteristics of an i-motif structure. We also revealed that the conjugated IFS strand on the nanoparticle surface exhibited similar CD spectral changes ( Supporting Information Figure S2e), indicating that the chemical conjugation will not affect the pH-responsiveness of the IFS sequence. Further, the specific folding structure of this IFS strand was studied by native polyacrylamide gel electrophoresis. If the IFS forms an interchain i-motif folding during pH variation, a lower electrophoretic migration rate will be observed. In contrast, if an intramolecular i-motif is the dominant structure, the molecular size of the sequence will be reduced, resulting in a higher electrophoretic migration rate. As shown in Supporting Information Figure S2f, this IFS strand did prefer an interchain folding structure at pH of 5.0, noticeably which could also sensitively respond to a weakly acidic pH of 6.0 and fold up. The grafting density of the IFS strand on the nanoparticle was found to be very critical for inducing particles’ aggregation. According to the DLS analysis, under a high graft density, the IFS strands on the same particle become very close and prefer cross-linking with each other; therefore, the possibility to form particle–particle aggregation was low (Figure 2a). When the density decreased to 0.5 nmol IFS per mg nanoparticle, the distance between the IFS strands on the same particle was sufficiently attenuated, and consequently significant aggregation among particles was observed. The interstitial pH value of the tumor is around 6.0–6.2,9 which is a weakly acidic condition. As shown in Figure 2b, although the most prominent aggregate was formed at pH 5.0, due to the very sensitive acid-responsiveness of IFS, distinct aggregations were also formed at pH 6.0. Therefore, the sensitive response of IFS to weak acid will greatly benefit the tumor retention of nanoparticles.

    Figure 2

    Figure 2 | Dynamic light scattering analysis of (a) the size distribution of [email protected]2/i-motif with different IFS modification density at pH 5.0; (b) the size distribution of [email protected]2/i-motif at different pH; (c) the size distribution of [email protected]2/i-motif incubated with 10% FBS at 35 °C over different times; (d) the size distribution of [email protected]2/i-motif/TK-mPEG incubated with 10% FBS at 35 °Cover different times; (e) the size distribution of [email protected]2/i-motif/TK-mPEG at different pH with and without H2O2; (f) the Zeta potentials of [email protected]2/i-motif and [email protected]2/i-motif/TK-mPEG incubated with 10% FBS at 35 °Cover different times. (g) TEM images of [email protected]2/i-motif at pH 5.0, 6.0, 7.0, and 8.0; (h) and (i) the aggregation properties of nanoparticles under different situations.

    However, the loosely packed IFS DNA molecules will face problems of poor biostability, and the additional PEG layer is introduced to shield and protect the IFS strands during blood circulation. When incubated in 10% FBS, the [email protected]2/i-motif lost its aggregation capability in 2 h (Figure 2c). According to zeta potential analysis (Figure 2f), its surface charge had changed from negative to positive during the incubation process, indicating complete degradation of IFS strands. On the contrary, [email protected]2/i-motif/TK-mPEG showed different properties. After a 4 h incubation in 10% FBS, the aggregation properties of [email protected]2/i-motif/TK-mPEG were still well preserved. Further proven by zeta potential analysis, [email protected]2/i-motif/TK-mPEG could go through a prolonged incubation in FBS environment and still showed negative charge after shedding the PEG layer, due to the preservation of IFS strands during this process (Figure 2d), which showed that the PEG layer could protect DNA from degradation. As shown in Figure 2e, only when PEG was shed from the particle surface, the particles would aggregate at pH 5.0; no pronounced aggregation formed in the absence of ROS or acidic environment, which implies that the stealth PEG layer will shield the particle before the delivery to the tumor site and reduce the uptake by the reticuloendothelial system.26 The aggregation process was also imaged by TEM, as shown in Figure 2g. In neutral and basic buffers, the particles showed good monodispersity, while in acidic buffers (pH 5.0 and 6.0), significant particle aggregation was observed. According to the kinetic aggregation curve ( Supporting Information Figure S5), we revealed that [email protected]2/i-motif/TK-mPEG could sensitively respond to ROS and low pH and quickly complete the aggregation process within 30 min.

    In vivo tumor accumulation of the dual-modified nanoparticle

    From the aforementioned studies, we demonstrated that IFS could very sensitively respond to very weak acids (Figure 2h), which helps to enhance the accumulation of particles at tumor interstitium. The protection and tumor-microenvironment activation strategy can well protect IFS from degradation in the biological environment (Figure 2i). Before the in vivo application, the Cy5 dye-labeled IFS sequence (i-motif-Cy5) was utilized to monitor the pharmacokinetics of IFS sequences during blood circulation. [email protected]2/i-motif-Cy5/TK-mPEG and [email protected]2/i-motif-Cy5 nanoparticles were intravenously injected into the healthy mice, and the blood samples were collected 12 h later. As shown in Supporting Information Figure S6, the fluorescence intensity of [email protected]2/i-motif-Cy5/TK-mPEG in the blood sample was much higher than that of [email protected]2/i-motif-Cy5. The result demonstrated that the stability of the IFS sequence was notably enhanced by the protection of TK-mPEG, which makes the IFS DNA applicable in in vivo experiments. Therefore, four groups were compared to investigate the in vivo tumor accumulation of the nanoparticles: (i) the naked nanoparticle [email protected]2-DOX; (ii) single modification with IFS, [email protected]2-DOX/i-motif; (iii) single modification with PEG, [email protected]2-DOX/TK-mPEG; and (iv) dual-modification/response system, [email protected]2-DOX/i-motif/TK-mPEG. The 4T1 subcutaneous tumor-bearing mice were administered with the four groups of nanoparticles (all the samples had equivalent doses of [email protected]2 core at 200 µg mL−1) via intravenous tail injection. The mice were sacrificed at certain time points post injection, and the organs (heart, lung, liver, spleen, and kidney) and tumors were collected for further studies. First, the tumor retention was monitored by ex vivo DOX fluorescence (Figure 3a; Supporting Information Figure S7). For group (i), DOX accumulation reached a peak level at 12 h postadministration, since then, which quickly decayed. Also, the naked nanoparticles were more captured by the liver and kidney. Single modification with PEG (group iii) showed no significant difference from group (i). The single modification with IFS (group ii) and dual-modification/response (group iv) groups exhibited enhanced DOX delivery due to the cross-linking capability of IFS on the particle surface; moreover, group iv showed much higher signals compared with group ii, because the stability of the IFS strands was enhanced in the dual-modification system. Besides, due to the presence of PEG, the blood half-life (t1/2) of group iv was determined to be ∼ 2.1 h, which became longer than that of group ii (∼ 1.3 h) ( Supporting Information Figure S8a). For more accurate quantification, the Cu content in the collected tissues was studied and quantified by ICP-MS (Figure 3b; Supporting Information Figure S8b–f). The results were consistent with that monitored by DOX fluorescence. Single modification with IFS strands showed around a twofold increase in particle retention, which was not significant enough because unprotected IFS strands suffered from the instability during blood circulation. Benefiting from the protection by the PEG shield, the dual-modification/response system showed a sevenfold enhancement in nanoparticle accumulation compared with the naked system (Figure 3b; Supporting Information Figure S8). As shown in Figure 3c, 24 h after injection of [email protected]2-DOX/i-motif/TK-mPEG to mice bearing 4T1 tumor, it was found that large clusters of nanoparticle aggregates were retained in the tumor. Accordingly, the IR thermography disclosed that the surface temperatures of the tumors from the mice treated with the dual-modification/response system rapidly increased to 56.5 °C after 5-min irradiation, which was much higher than 50.2 °C of the naked nanoparticles. It has been reported that hyperthermia to ∼57 °C can result in irreversible tumor cell damage and the best PTT effect (Figure 3d).31,32 This declaration was proven by the histopathological tissue analysis following hematoxylin and eosin (H&E) staining of tumor sections from mice after the treatments. As shown in Figure 3e, compared with [email protected]2-DOX, [email protected]2-DOX/i-motif/TK-mPEG resulted in more necrotic areas in tumor tissues, indicating more severe tumor damage by both enhanced PTT and chemotherapy. All these results prove that the protection and tumor-microenvironment activation strategy can deal well with the whole delivery process and, therefore, drive nanoparticles to reach an enhanced accumulation at the tumor site, which will greatly affect the therapy results of the nanoparticles.

    Figure 3

    Figure 3 | (a) Ex vivo images of organs and tumors excised from 4T1 tumor-bearing mice at different time points after the injection of different samples. (b) The copper contents in tumors at different time points after injection of different samples. (c) Representative TEM images of the sections of 4T1 tumor tissue after injection with [email protected]2-DOX/i-motif/TK-mGEG for 24 h. (d) In vivo IR thermography of tumors from 4T1 tumor-bearing mice intravenously injected with various samples at different time points after laser irradiation (880 nm, 1.0 W cm−2). (e) H&E stained slices of tumors collected from 4T1 tumor-bearing mice 12 h after receiving treatments. (i) [email protected]2-DOX, (ii) [email protected]2-DOX/i-motif, (iii) [email protected]2-DOX/TK-mPEG, and (iv) [email protected]2-DOX/i-motif/TK-mPEG.

    Antitumor properties of the dual-modified and multimodual-therapy nanoparticle

    By both in vitro and in vivo tests, the protection and tumor-environment activation strategies have been proven to be effective in improving the biostability of IFS DNA. Therefore, the antitumor properties of [email protected]2/i-motif/TK-mPEG were investigated. As shown in Supporting Information Figure S9a, the aqueous dispersion of [email protected]2 showed a broad and intense absorption from 700 to 1200 nm. As the absorption of hemoglobin and water is mainly located at 980 nm, an 880 nm laser was selected for the photothermal conversion and PTT experiments.33,34 First, the in vitro photothermal conversion capability of [email protected]2 was studied. As shown in Supporting Information Figure S9b, after 10 min irradiation at 880 nm with a power density of 0.8 W cm−2, only negligible temperature changes were observed for pure water; but, in the presence of [email protected]2, a significant elevation in temperature can be realized. For instance, when 200 μg mL−1 of [email protected]2 was used, a temperature increment of 40 °C was realized. Moreover, [email protected]2 exhibited a good photo-/thermal stability. After five cycles of the irradiation on/off process ( Supporting Information Figure S9c), it showed a consistent photothermal conversion capability, without visible decay. Second, further studies revealed that DOX release from [email protected]2-DOX exhibited a sustained and pH-responsive manner ( Supporting Information Figure S9d). At pH 8.0, the cumulative DOX release during an 8 h period was only 5.1%, which significantly increased to 32.3% for the condition of pH 5.0. We also found that the hyperthermia induced by [email protected]2 under photoirradiation (10 min irradiation at 880 nm of 0.8 W cm−2) could accelerate DOX release, resulting in a 69.9% accumulative release (at pH 5.0). Besides, even with photoirradiation, the total release amount of DOX in 8 h was less than 10% at pH 8.0. These results demonstrated that [email protected]2-DOX showed a tumor-specific drug release property, and the PTT treatment will facilitate DOX release and subsequently enhance chemotherapy. Therefore, increasing the accumulation of nanoparticles at the tumor site will promote not only the efficacy of PTT but also that of chemotherapy. Third, the in vitro therapeutic effects of the nanoparticles were evaluated on the 4T1 cell line. The dark cytotoxicities of [email protected]2, [email protected]2-DOX, [email protected]2/i-motif/TK-mPEG, and [email protected]2-DOX/i-motif/TK-mPEG were tested by MTT assay ( Supporting Information Figure S10a). The nanoparticles without DOX loading showed negligible cytotoxicity to 4T1 cells, and not surprisingly, among which [email protected]2/i-motif/TK-mPEG with PEGylation showed better biocompatibility, while the nanoparticles with DOX loading showed obvious cytotoxicity. According to the CLSM investigations ( Supporting Information Figure S11), the uptake of [email protected]2-DOX/i-motif/TK-mPEG by 4T1 cells was very efficient, and DOX could be released and enter the cell nucleus. The nanoparticles were probably delivered through the endosome and lysosome pathway; therefore, efficient DOX release was triggered in the acidic environment. Further, the cytotoxicity of the nanoparticles under laser irradiation was compared ( Supporting Information Figure S10b). After irradiation, the nanoparticles exhibited obvious PTT toxicity. Benefiting from the synergistic interaction between chemotherapy and PTT, the cell-killing capability of the nanoparticles was remarkably enhanced. Accordingly, the final agent [email protected]2/i-motif/TK-mPEG was demonstrated to be capable of killing 97.8% cancer cells at a concentration of 200 μg mL−1.

    Finally, 4T1 tumor-bearing mice were randomly divided into five groups to comprehensively evaluate the in vivo therapy efficacies of the nanoparticles over a 22-day follow-up period. Figure 4a showed tumor growth curves of mice treated with saline (group 1), [email protected]2-DOX (group 2), [email protected]2-DOX + NIR (group 3), [email protected]2-DOX/i-motif/TK-mPEG (group 4), and [email protected]2-DOX/i-motif/TK-mPEG + NIR (group 5). The groups with photoirradiation (1.0 W cm−2, 5 min) (groups 3 and 5) showed better therapeutic effects compared with the corresponding groups without photoirradiation (groups 2 and 4), attributable to the PTT effect of the nanoparticles. For mice injected with saline (group 1), the tumors eventually increased approximately sixfold in volume, while for mice administrated with naked nanoparticles, the tumor growth rate was greatly inhibited to ∼2.5 fold. Finally, due to the significantly enhanced tumor accumulation by virtue of the protection and tumor-microenvironment activation strategy, group 5 showed the best therapeutic effect in that the tumor was reduced to 0.3-fold of the original size. The comparison between group 5 and group 3 showed that the enhancement in tumor accumulation by the protection and tumor-microenvironment activation strategy did improve the in vivo therapeutic efficacy of the nanoparticles (Figure 4b). The mice in all the treatment groups did not show weight loss (Figure 4a insert) or other abnormalities. A histopathological analysis was conducted on a treated mouse in group 5, which was compared with a healthy mouse. We found that, after the treatment, the mice showed no visible signs of damage in the main organs (Figure 4c). These results exhibited that in addition to the superior therapeutic performance, [email protected]SiO2-DOX/i-motif/TK-mPEG is also a safe and reliable nanoparticle without long-term toxicity and side effects.

    Figure 4

    Figure 4 | (a) Tumor growth curves in different groups with various treatments and the inset is body-weight monitoring over 22 days. (b) Photographs of tumor tissues from different groups after treatment: (group 1) Saline; (group 2) [email protected]2-DOX; (group 3) [email protected]2-DOX+NIR; (group 4) [email protected]2-DOX/i-motif/TK-mPEG; (group 5) [email protected]2-DOX/i-motif/TK-mPEG+NIR, (c) Histological H&E staining for different organs collected from mice in group 5, and the organs in control group were collected from healthy mice, the scale bar is 100 μm.

    Conclusion

    In this study, [email protected]2-DOX/i-motif/TK-mPEG based on protection and tumor-microenvironment activation strategies was synthesized, for which IFS DNA and TK-mPEG polymer were successively modified on the surface of [email protected]2-DOX. We found that DNA can sensitively recognize weakly acidic conditions up to pH 6.0. Also, the grafting density of DNA on the particle surface was optimized to realize efficient cross-linking among particles. Notably, PEG protection was proven to be very important for the effectiveness of the system. For the single modification with IFS DNA, [email protected]2-DOX/i-motif only exhibited a twofold enhancement in tumor accumulation compared with naked [email protected]2-DOX, while, for the dual-modification system of [email protected]2-DOX/i-motif/TK-mPEG, a sevenfold enhancement was observed. Due to the significantly enhanced tumor accumulation of the therapeutic nanoparticles, hyperthermia of 56.5 °C in 5 min irradiation was realized at the tumor site. Therefore, more drugs remained in the tumor zone, which finally resulted in an improved in vivo therapeutic efficacy compared with the naked nanoparticles.

    Supporting Information

    Supporting Information is available.

    Conflicts of Interest Statement

    The authors declare no conflict of interest.

    Acknowledgments

    The authors acknowledge financial support by grants from the National Natural Science Foundation of China (no. 51973089), Shenzhen Fundamental Research Programs (nos. JCYJ20160226193029593 and JCYJ20170817105645935), Shenzhen Science and Technology Innovation Commission (grant no. KQTD20170810111314625), Guangdong Innovative and Entrepreneurial Research Team Program (no. 2016ZT06G587).

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