Receptor and Microenvironment Dual-Recognizable Nanogel for

Subscriber access provided by EAST TENNESSEE STATE UNIV

Communication

Receptor and Microenvironment Dual-Recognizable Nanogel for Targeted Chemotherapy of Highly Metastatic Malignancy Jinjin Chen, Jianxun Ding, Weiguo Xu, Tianmeng Sun, Haihua Xiao, Xiuli Zhuang, and Xuesi Chen Nano Lett., Just Accepted Manuscript • Publication Date (Web): 23 Jun 2017 Downloaded from http://pubs.acs.org on June 24, 2017

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

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

Page 1 of 23

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

Nano Letters

Receptor and Microenvironment Dual-Recognizable Nanogel for Targeted Chemotherapy of Highly Metastatic Malignancy Jinjin Chen,†,§,⊥ Jianxun Ding,†,⊥ Weiguo Xu,† Tianmeng Sun,‡ Haihua Xiao,† Xiuli Zhuang,† and Xuesi Chen*,† †

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese

Academy of Sciences, Changchun 130022, People's Republic of China ‡

The First Hospital and Institute of Immunology, Jilin University, Changchun 130061, People's

Republic of China §

University of Chinese Academy of Sciences, Beijing 100039, People's Republic of China

ACS Paragon Plus Environment

1

Nano Letters

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

KEYWORDS:

nanogel,

receptor-mediated

targeting,

Page 2 of 23

environment-mediated

targeting,

intracellular drug delivery, metastatic malignancy therapy

ABSTRACT: Targeted delivery of chemotherapeutic drugs to the desired lesion sites is the main objective in malignancy treatment, especially in highly metastatic malignancies. However, extensive studies around the world on traditional targeting strategies of recognizing either overexpressed receptors or microenvironments in tumors show great limitations, owing to the offtarget effect and tumor homogeneity. Integration of both receptor-mediated targeting (RMT) and environment-mediated targeting (EMT) enhances the tumor accumulation and subsequent cell uptake at the same time, which may avoid these limitations. Herein, a dual targeting nanogel of PMNG engineered with both phenylboronic acid (PBA) and morpholine (MP) was reported for not only RMT via specific recognition of sialyl (SA) epitopes but also EMT toward extracellular acidity. Further engineering the nanoparticles via loading doxorubicin (DOX) brought a novel dual targeting system, i.e., PMNG/DOX. PMNG/DOX demonstrated a greater targeting effect to both primary and metastatic B16F10 melanoma than the single PBA-modified nanogel (PNG) with only RMT in vitro and in vivo. Moreover, PMNG/DOX was also proved to be highly potent on inhibiting primary tumor growth as well as tumor metastasis on B16F10 melanoma-grafted mouse model. The results demonstrated the dual targeting design as a translational approach for drug delivery to highly metastatic tumor.


2

Page 3 of 23

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

Nano Letters

Nowadays, malignancy has been a leading cause of human death in both developed and developing countries.1 As the improvement of clinical techniques and medical science, malignancies can be controlled temporarily through the combined treatment of surgery, radiotherapy, and chemotherapy.2 However, for the highly metastatic malignancies that account for more than 90% of human cancer deaths, the simple surgery and local irradiation cannot clear the metastatic tumor cells completely.3,

4

Chemotherapy may be an effective strategy for

treatment of highly metastatic malignancies. Nevertheless, the unspecific distribution of chemotherapy drugs results serious side effects, and also brings significant burdens to patients.5 Therefore, the selective accumulation of chemotherapy drugs to both primary and metastatic tumor sites is urgent for the improved therapy of highly metastatic malignancy.6 Nanocarriers have emerged as an effective platform for the targeted drug delivery through two major approaches based on either the over-expressed receptors (e.g., folate,7 glycosylations,8 and integrins9) or the abnormal microenvironments (e.g., irregular vessels,10 low pH,2 and high hypoxia11) in tumor sites, which are regarded as the receptor-mediated targeting (RMT) and the environment-mediated targeting (EMT), respectively. As sialyl (SA) epitopes are commonly over-expressed in highly metastatic tumors, it has been employed as an ideal receptor for RMT.12 Charge reversal groups, which are originally negative or neutral during circulation but transfer to be positively charged at tumor extracellular pH of about 6.5, become an efficient targeting ligand for EMT.13 However, the targeting efficiency of RMT or EMT alone is always hindered by their own limitations. Firstly, for RMT, the receptor SA is also present on red blood cells and vascular endothelium, and the nonspecific distribution of SA is the leading cause of the off-targeting of SA-mediated RMT.11, 14 Secondly, though the RMT increases the drug concentration at tumor


3

Nano Letters

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

Page 4 of 23

sites to some extent, subsequent cell uptake is still unsatisfied. In previous works, two kinds of RMT-related ligands were attempted to be integrated in one platform, but they were still burdened with the above limitations of RMT.15, 16 For EMT, though the cell internalization of these nanoparticles is enhanced, the complexities of tumor microenvironments, such as the regionally varied pH and hypoxia conditions in the same tumor, impede the accurate targeting of EMT without the co-recognition of RMT.17 Integration of RMT and EMT in one platform may overcome the above limitations of single targeting via either RMT or EMT, which probably lead to a broadly applicable targeting route to both primary and metastatic tumors. Herein, we constructed a first example of smart platform with both RMT and EMT for dual synergistic targeting. A phenylboronic acid (PBA) and morpholine (MP) dual-modified polypeptide nanogel (PMNG) was firstly fabricated for integration of RMT and EMT in one drug delivery system (Scheme 1). The design of this peculiar system is based on the following considerations: (1) PBA is applied for selective RMT toward highly metastatic cells with overexpressed SA on the membrane;18-20 (2) The charge-transformable MP, which is neutral at physiological pH but positively charged in tumor tissue, is employed for enhanced cell internalization at tumor microenvironment;21,

22

(3) The nanogel is core-cross-linked via a

disulfide bond, which can be broken down by the intracellular glutathione (GSH) to selectively release the loaded model drug doxorubicin (DOX). Owing to the integration of RMT and EMT mediated by PBA and MP, respectively, PMNG obtained excellent targeting property to the SAover-expressed primary and metastatic B16F10 tumors in vitro and in vivo. Moreover, PMNG/DOX exhibited great advantage in inhibiting the growth of both primary and metastatic tumors, implying the promising therapeutic of highly metastatic tumors in clinic. To the best of


4

Page 5 of 23

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

Nano Letters

our knowledge, this is the first example on the strategy of engineering the synergistic targeting of RMT and EMT, which may demonstrate the great promise on enhanced tumor targeting.12, 22

Scheme 1. Engineering PMNG with both EMT and RMT. (A) Synthesis route of PNG and PMNG. (B) Targeting mechanism of PMNG. On the one hand, the PBA ligand in PMNG selectively binds to the over-expressed SA on the highly metastatic tumor cells. On the other hand, the MP ligand targets to the extracellular pH condition (i.e., about 6.5) and further facilitates cell internalization. Finally, the intracellular GSH accelerates DOX release for superior antitumor therapy. To start with, PMNG with PBA or MP group in each terminal of polymer chain was synthesized through a two-step but one-pot ring-opening polymerization (ROP) of amino acid N-


5

Nano Letters

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

Page 6 of 23

carboxyanhydride monomers, as shown in Scheme 1. This is unlike the surface modifications of nanocarriers post-preparation with complicated steps and incomplete binding.23 In the same way, the PBA-modified nanogel noted as PNG was prepared using 4-(aminomethyl)phenylboronic acid as an initiator. The chemical structures of PNG and PMNG were subsequently well characterized by both proton nuclear magnetic resonance (1H NMR) and Fourier-transform infrared (FT IR) spectra, as depicted in Figures S1 and S2, Supporting Information (SI). Further, the weight percentages of boron (B) in PNG and PMNG were calculated to be about 0.21 and 0.17 wt.% by inductively coupled plasma optical emission spectroscopy (ICP-OES), respectively, corresponding to about 0.19 and 0.15 µmol mg−1 PBA groups in PNG and PMNG. Moreover, the numbers of MP groups were determined to be about 0.09 µmol mg−1 by the areas of peaks at 7.72 ppm (phenyl group in PBA) and 1.52 ppm (methylene group in MP). Then, the properties of PNG and PMNG were studied in vitro. Firstly, both PNG and PMNG exhibited the sizes of about 40 nm determined by dynamic light scattering (DLS) or about 30 nm by transmission electron microscope (TEM), as depicted in Figure 1A and Figure S3, SI. The size was beneficial for extravasation of nanoparticles from the vasculature to not only primary but also metastatic tumors.24 Moreover, owing to the disulfide cross-linker in the core, the nanogels exhibited reduction-responsiveness. As shown in Figure 1B and Figure S4, SI, the diameters of both PNG and PMNG increased from 40 to 700 nm, and the structures turned to be irregular after incubated with 10.0 mM GSH for 8 h. The dramatic increase in sizes was due to the cleavage of disulfide bond under reductive conditions and subsequent swelling or disintegration of the de-cross-linked core. Thirdly, the specific transition of zeta potential of PMNG from neutral to positive under extracellular condition was one key design for EMT. In Figure 1C, an increase in the zeta potential of PMNG from −1.3 to 7.8 mV was observed when


6

Page 7 of 23

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

Nano Letters

the pH value decreased from 7.4 to 6.0. In contrast, the zeta potential of PNG almost remained constant during the same experiment, suggesting its inability to charge reversal.

Figure 1. Characterizations of PMNG. (A) Typical TEM image and DLS intensity (Insert) of PMNG. (B) Changes of sizes for PMNG and PNG in the presence of 10.0 mM GSH at predetermined time interval. (C) Zeta potential of PNG and PMNG under different pH values from 6.0 to 7.4. (D) DOX release behavior of PMNG/DOX incubated in PBS at pH 7.4, 6.5, or 7.4 in the present of 10.0 mM GSH. For further assessment of therapeutic efficacy, DOX was loaded into the nanogels through diffusion with drug loading contents (DLCs) of 7.2 and 7.3 wt.% for PNG/DOX and PMNG/DOX, respectively. Targeting and triggered release of drug right at the tumor site is necessary for effective chemotherapy. As shown in Figure 1D and Figure S5, SI, both


7

Nano Letters

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

Page 8 of 23

PMNG/DOX and PNG/DOX showed minimal drug release (< 25%) at pH 7.4 due to the diffusion, which was also happened in other non-responsive systems.25 However, rapidly triggered release (> 90%) in the reductive environment was observed, resulting from the disassembly of nanogels after break of disulfide bond. The release rate was also slightly accelerated at pH 6.5 due to the increased hydrophilicity of DOX under acidic condition. It seemed that the different modifications showed no obvious influence on drug loading and release behaviors, which ensured the high accuracy of the further comparative studies of PNG/DOX and PMNG/DOX. To reveal the targeting abilities of PNG/DOX and PMNG/DOX in vitro, the cell uptake against B16F10 cells was measured by flow cytometer (FCM) and confocal laser scanning microscope (CLSM). B16F10 cells are highly metastatic and over-express SA on the cell membrane.12 By monitoring the fluorescence intensity of DOX via FCM, PNG/DOX and PMNG/DOX exhibited similar cell uptakes after incubation at pH 7.4 for 3 h (Figure 2A). Interestingly, the cells treated with PMNG/DOX at pH 6.5 displayed 1.38 (P < 0.01) and 1.17 times (P < 0.05) fluorescence intensity of those incubated with PMNG/DOX at pH 7.4 and with PNG/DOX at pH 6.5, respectively. A higher fluorescence intensity in the PMNG/DOX-treated cells than that in the PNG/DOX-treated cells should be attributed to (i) a greater binding affinity between PBA and SA at pH 6.5 and (ii) enhanced cell uptake induced by positive charged MP group at pH 6.5. To further address the combined efficiency, free PBA was added to the cell culture media as a competing ligand. As expected, compared with that incubated at pH 6.5 without free PBA, both the cell uptakes of PNG/DOX and PMNG/DOX decreased to 46.9% (P < 0.001) and 61.4% (P < 0.001), respectively. The above finding confirmed that the enhanced targeting abilities of PNG and PMNG were related with the interaction of PBA moiety with the


8

Page 9 of 23

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

Nano Letters

cells. However, the decreased cell internalization in the PMNG/DOX-treated cells after treatment of free PBA was much slighter than that in the PNG/DOX-treated cells owing to the presence of MP. This was similar by pretreating the cells with sialidase, which cleaved SA epitopes from cells and resulted in an obvious decrease in cell internalization of PNG/DOX. Moreover, the CLSM images of cell internalization in pH 7.4 or 6.8 or after pretreatment of PBA or sialidase in Figures S6 and S7, SI, also demonstrated the same tendency. Taken together, all these results indicated that the cell uptake of PMNG/DOX was not only based on the recognition of PBA but also the interaction of MP with the cell membrane. To reveal whether this dual targeting strategy exhibited advantage in cell proliferation inhibition, the viabilities of B16F10 cells treated with various formulations of DOX were determined. In Figure 2B, the half-maximal inhibitory concentration (IC50) of PNG/DOX at pH 6.5 (i.e., 3.1 ± 0.3 µg mL−1) was approximately equal to that at pH 7.4 (i.e., 3.2 ± 0.1 µg mL−1). However, the IC50 of PMNG/DOX at pH 6.5 decreased to 2.2 ± 0.3 µg mL−1 with respect to that at pH 7.4 (i.e., 3.2 ± 0.1 µg mL−1) (P < 0.01). The decreased IC50 further suggested the superiority of the dual targeting approach. To confirm the reduction-triggered drug release, B16F10 cells were pre-incubated with 10.0 mM GSH for 2 h before addition of PNG/DOX or PMNG/DOX. The increase of DOX fluorescence was observed in both the PNG/DOX and PMNG/DOX groups revealed by FCM (Figure 2A) and CLSM (Figures S6 and S7, SI), which resulted from the intracellular degradation of disulfide bonds in the nanogels and subsequent burst release of the self-quenched DOX. Moreover, the triggered releases of DOX from both PNG/DOX and PMNG/DOX were further supported by dramatic decrease of IC50 from 3.2 ± 0.1 to 1.5 ± 0.2 µg mL−1 toward the cells pretreated with 10.0 mM GSH (P < 0.01; Figure 2B).


9

Nano Letters

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

Page 10 of 23

Figure 2. In vitro targeting and anti-metastatic behaviors of PNG and PMNG. (A) Cell uptake of DOX in B16F10 cells incubated with PNG/DOX or PMNG/DOX at an equivalent DOX concentration of 1.0 mg L−1 for 3 h and measured by FCM. Cells are incubated in PBS at pH 7.4, 6.5, or 7.4 with 10.0 mM GSH. To cleave SA specifically, the cells are pretreated with PBA (4.0 mM) or sialidase (40.0 mU mL−1) before incubation with loading nanogels. (B) IC50 values of PNG/DOX and PMNG/DOX to B16F10 cells for 48 h. Cells are incubated in PBS at pH 7.4, 6.5, or preincubated with 10.0 mM GSH for 2 h. (C) Cell migration of B16F10 cells after incubation with DOX, PNG/DOX, or PMNG/DOX at an equivalent DOX concentration of 1.0


10

Page 11 of 23

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

Nano Letters

mg L−1 for 0 or 12 h by wound-healing assay. (D) Cell invasion of B16F10 cells after incubation with DOX, PNG/DOX, or PMNG/DOX at an equivalent DOX concentration of 1.0 mg L−1 for 12 h by transwell assay. Data are represented as mean ± standard deviation (SD; n = 3, t-test; *P < 0.05, **P < 0.01, ***P < 0.001). The metastatic tumor cells show great capability of migration and invasion.26, 27 Moreover, the migration and invasion allowed the tumor cells to enter the blood vessels and lymphatic system, and then undergo the metastatic growth in other organs. A well-established wound-healing assay was applied to evaluate the ability of migration of a certain cell. The cell migration distance was determined by subtracting the final width from the initial width of the wound. As shown in Figure 2C, the relative cell migration in cultures was much smaller in the PNG/DOX and PMNG/DOX groups, namely, 9.2% and 6.6%, respectively, than those in the control (52.2%) and DOX (30.1%) groups after 48 h. For evaluation of cell invasion, a transwell assay was carried out on B16F10 cells.28 As shown in Figure 2D, fewer B16F10 cells could invade through the Matrigel® in the PNG/DOX and PMNG/DOX groups compared with those in the control and DOX groups. Furthermore, the purple areas of each image in Figure 2D were semi-quantitatively analyzed and shown in Figure S8, SI. Results showed that the invasion ability of B16F10 cells treated with PNG/DOX and PMNG/DOX was significantly reduced compared with that of the DOX group (P < 0.001). Taken together, the inhibited migration and invasion behaviors of B16F10 cells in the PMNG/DOX group than that of other groups suggested the enhanced antimetastatic ability of nanogel with the dual target modification. The in vivo targeting efficiency of PMNG/DOX was further evaluated against both primary tumor and lung metastases, which were constructed after subcutaneous or intravenous injection of green fluorescent protein (GFP) expressing B16F10 cells (B16F10-GFP). The mice were


11

Nano Letters

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

Page 12 of 23

treated with different formulations at an equivalent DOX concentration of 5.0 mg per kg body weight (mg (kg BW)−1). After 12 h, the tumors were homogenized and then characterized by FCM in Figure S10, SI. B16F10-GFP cells exhibited obvious green fluorescence, and the tumor cell-targeted uptake of DOX or DOX-loaded formulations showed red fluorescence. The targeting abilities of various DOX formulations were evaluated by the percentage of DOXpositive tumor cells, as presented in Figure 3A. PMNG/DOX showed the best ability of tumor targeting with DOX-positive proportion of 15.6% compared with those of DOX (1.7%) and PNG/DOX (10.1%). As depicted in Figure S11, SI, the targeting ability of PMNG/DOX to metastatic tumor was also well evaluated in the similar proposal. B16F10-GFP cells were firstly distinguished from the mixture of lung cells and tumor cells, and further analyzed in Figure 3B. In the GFP-positive tumor cells, PMNG/DOX exhibited the best targeting efficiency with a DOX-positive proportion of about 23.9%. Such impressive targeting ability to metastatic tumor was further confirmed by the results of CLSM. As shown in Figure 3C, the tumor cells in the metastatic nodules of the lungs expressed GFP and were marked by green fluorescence. The DOX intensity distributed with no difference between the lung and tumor in the DOX group, suggesting there was no selectivity of free DOX. However, in the PNG/DOX and PMNG/DOX groups, the DOX intensities in tumor sites were stronger than those in the lung sites, especially in the PMNG/DOX group. Moreover, the DOX accumulated in the metastatic nodules in the PMNG/DOX group was also much stronger than those in both the DOX and PNG/DOX groups. All the results significantly proved the superior targeting efficiency of PMNG/DOX to both primary and metastatic tumors owing to the reasonable dual-modification. Lastly, the blood cells showed no significant uptakes of DOX formulations (Figure S12, SI), and there was also no obvious


12

Page 13 of 23

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

Nano Letters

adhesion of DOX formulations in normal blood vessels after the treatments (Figure S13, SI), which demonstrated the minimized off-targeting of PMNG/DOX during the blood circulation.

Figure 3. In vivo targeting efficiency of PMNG/DOX to both primary and metastatic melanomas. (A) FCM analyses of primary tumor cells after treatments with different formulations for 12 h. (B) FCM analyses of metastatic tumor cells after treatments with different formulations for 12 h. (C) CLSM images of metastatic lung sections at 12 h post-treatment. Nucleus is stained blue by 4′,6-diamidino-2-phenylindole (DAPI). B16F10-GFP cells stably express GFP, which are marked with green. DOX fluorescence is red. Data are represented as mean ± SD (n = 3, t-test; *P < 0.05, **P < 0.01). The prolonged blood circulation and improved tumor accumulation are the foundation for upregulated antitumor efficacy in vivo. In Figure 4A and Table S2, SI, both PNG/DOX and PMNG/DOX showed prolonged blood circulation (t1/2 > 13 h) compared with that of free DOX (t1/2 = 8.2 h). Moreover, PNG and PMNG significantly increased the area under the curve (AUC)


13

Nano Letters

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

Page 14 of 23

of DOX in blood, which were 3.0 and 3.5 times greater than that of free DOX, respectively. The extended blood circulation time and high blood drug concentration of nanoformulations might be due to the interference by the poly(γ-(methoxy triethylene glycol)-L-glutamate) (PmTEGLG) shell that proved to be resistant to protein adsorption and cell adhesion.

Figure 4. Pharmacokinetics and tumor accumulation of PMNG/DOX as compared to free DOX and PNG/DOX. (A) DOX concentrations of DOX-, PNG/DOX-, and PMNG/DOXtreated mice in plasma compared with initial concentration. (B) Tumor accumulation of DOX, PNG/DOX, and PMNG/DOX in C57/BL mice bearing B16F10 tumors. (C) Biodistribution of DOX in major organs after treatments with different DOX formulations. (D) Quantization of DOX distribution in C. Data are represented as mean ± SD (n = 3, t-test; *P < 0.05, ***P < 0.001).


14

Page 15 of 23

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

Nano Letters

The intratumoral accumulation and biodistribution of DOX in the mice bearing both primary and metastatic tumors were also measured for assessment of the targeting efficiency in vivo. After 12 h post-injection, the amount of DOX in primary tumors of the PNG/DOX and PMNG/DOX groups were around 1.8 and 3.1 times higher than that of the DOX group (P < 0.01, Figure 4B). Moreover, as shown in Figure 4C, the increased DOX intensity in the lungs of PNG/DOX and PMNG/DOX groups also proved their enhanced targeting to lung metastatic tumors owing to the surface modification. More importantly, the lung in the PMNG/DOX group exhibited the highest DOX accumulation among all the formulations in Figure 4D. Overall, the promoted DOX distribution to both primary and metastatic tumors further confirmed the upregulated targeting efficiency after dual-modification. The antitumor activities of DOX-loaded nanogels against primary tumors were evaluated in C57/BL mice bearing subcutaneous melanomas. When the tumors grew to about 50 mm3, the mice were treated with phosphate-buffered saline (PBS), DOX, PNG/DOX, or PMNG/DOX by tail vein injection. As shown in Figure 5A, though the initial inhibition of tumor growth was found in the DOX group, the tumor volume increased rapidly after the last seven-day treatment. It was probably because that the low accumulation of free DOX in tumor tissues could not inhibit the rapidly growing tumors. However, both PNG/DOX and PMNG/DOX significantly reduced the tumor growth rate with the tumor volumes only 7.2% and 2.4% compared with that in the PBS group, respectively (P < 0.001). Notably, PMNG/DOX exhibited the greatest antitumor efficacy among all experiment groups owing to the enhanced synergistic targeting effect by integration of RMT and EMT. Moreover, the apoptosis areas were calculated to be 1.3%, 45.7%, 68.6%, and 89.5% of the whole tumor section in the PBS, DOX, PNG/DOX, and PMNG/DOX groups by hematoxylin and


15

Nano Letters

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

Page 16 of 23

eosin (H&E) staining (Figures S14 and S15, SI), respectively, which were consistent with their antitumor efficacies. The antitumor effects were further confirmed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) and Ki67 proliferation antigen staining in Figure S14, SI. Both in the PNG/DOX and PMNG/DOX groups, the tumor sections were TUNEL-positive and Ki67-negative. The strongest apoptotic signal was found in the PMNG/DOX group, consisting with the best antitumor efficacy of PMNG/DOX. The nanoformulations also reduced the systemic toxicity of free DOX. As shown in Figure 5B, the body weight decreased seriously in the DOX group, while the body weight remained almost unchanged in the PNG/DOX and PMNG/DOX groups. As depicted in Figure S16, SI, there was also no obvious damage to major organs after treatments with PNG/DOX and PMNG/DOX, further suggesting the increased systemic safety of nanoformulations. There are plenty of nanomedicines exhibiting excellent treatment efficacies to primary tumors, but the anti-metastatic effect is more important for the survival of patients. Fortunately, the PBAmodified nanocarriers had been proved to be efficient for inhibition of tumor metastasis due to the excellent targeting property to the SA-over-expressed tumor cells with high metastatic potentials. Based on the above background, the efficacies of DOX-loaded nanogels against lung metastasis were also evaluated to C57/BL mice bearing lung metastases. In detail, the mice were treated with PBS, DOX, PNG/DOX, or PMNG/DOX by tail vein injection. The mice without treatment died quickly with no survivors after 16 days due to the severe lung collapse as shown in Figure 5C. Although free DOX could extend the survival time of some mice with the half-time of 16 days, it failed to obtain the desirable long-term efficacy. The survival rates were significantly increased both in the PNG/DOX (P < 0.01) and PMNG/DOX (P < 0.001) groups


16

Page 17 of 23

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

Nano Letters

compared with that in the DOX group. No death was observed even at 21 days in the PMNG/DOX group, and the half-time of survivor was extended to 30 days.

Figure 5. In vivo antitumor efficacy of PMNG/DOX on primary and metastatic melanomas. (A) Tumor volumes of primary B16F10 tumors after treatment with PBS, free DOX, PNG/DOX, or PMNG/DOX at an equivalent DOX dose of 5.0 mg (kg BW)−1 on day 1, 4, 7, and 10. (B) Body weight loss during treatment with PBS, DOX, PNG/DOX, or PMNG/DOX. (C) Survival rate during treatment of metastatic B16F10 melanoma. (D) Images of the lungs after treatments with different formulations. (E) Numbers of metastatic nodules with the diameters above 5 mm in D. (F) H&E, TUNEL, and Ki67 staining of lung sections at the end of experiments. In H&E staining, cell nucleus is stained blue, and extracellular matrix and cytoplasm are stained red. In TUNEL assay, the broken DNA is stained by FITC-labeled dUTP (green). In Ki67 staining, the


17

Nano Letters

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

Page 18 of 23

Ki67 proliferation antigen is stained with FITC-labeled secondary antibody (green). Data are represented as mean ± SD (n = 8 for A and B, t-test; n = 10 for C, log-rank test; n = 3 for D, ttest; *P < 0.05, **P < 0.01, ***P < 0.001). After 15 days of the first treatment, the lungs of treated mice in each groups were collected for further evaluation of anti-metastatic efficiency (n = 3). As shown in Figure 5D, the B16F10 cells metastasized to the lung and formed visible black nodules, making it possible to track the metastatic nodules. The anti-metastatic efficacy was firstly assessed by calculating the number of metastatic nodule with the diameter larger than 5 mm in Figure 5E. The number of metastatic nodule in the PMNG/DOX group decreased dramatically compared with those in the control, DOX, and PNG/DOX groups, revealing predominant anti-metastatic efficacy of the DOX-loaded nanogels than that of free DOX. The excellent inhibition of metastatic nodule after treatment with PMNG/DOX should be attributed to the great dual targeting efficiency of PMNG/DOX. To further reveal the specificity of PMNG/DOX to the metastatic site in the lung, the histopathology analyses of the lung were performed. The H&E staining of whole lung sections and typical metastatic nodules were displayed in Figure 5F and Figure S17, SI, respectively. In the control and DOX groups, the metastatic areas were high to 59.4% and 54.3% of the whole lung sections, respectively. However, the metastatic area reduced to 15.5% after treatment with PNG/DOX, and the metastatic area was greatly suppressed to 6.4% in the PMNG/DOX group. Further immunofluorescence staining of TUNEL and Ki67 was also carried out in Figure 5F. In TUNEL staining, the fluorescence intensity around the metastatic foci in the PMNG/DOX group increased significantly. Additionally, much weaker fluorescence intensity of stained Ki67 could be also observed in the lung of PMNG/DOX group, which confirmed the desirable antimetastatic efficacy of PMNG/DOX.


18

Page 19 of 23

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

Nano Letters

The targeted treatments of highly metastatic malignancies have been becoming the most urgent challenge for chemotherapy. In this work, PMNG was integrated with RMT and EMT as the first attempt for targeting highly metastatic malignancies was explored. The increased targeting behavior of PMNG/DOX was demonstrated in both primary and metastatic B16F10 tumors in vitro and in vivo owing to the integration of dual targeting strategy. As predicted, the superior antitumor efficacy of PMNG/DOX was also observed in not only primary but also metastatic tumor models. All the results confirmed the superiority of the dual targeting strategy in targeted therapy of highly metastatic malignancy. Notably, the facilely smart surfaceengineered nanogels might be a promising platform for integration of multi-targeting properties by adjusting the targeted ligands for broad recognition of various tumors. ASSOCIATED CONTENT Supporting Information Characterizations of PNG and PMNG; TEM evaluations of PNG, disassembled PMNG, and PNG under 10.0 mM GSH; CLSM images and detailed viabilities of B16F10 cells after treatment with PNG/DOX or PMNG/DOX under different conditions; FCM analyses of red blood cells, primary tumor cells, and metastatic tumor cells after treatments with different formulations for 12 h; CLSM images of normal tissues after treatments with different formulations for 12 h; CLSM images of tumor sections after TUNEL or Ki67 staining, H&E images of major organs; detailed H&E images of tumors and the lungs after treatments with various formulations. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author


19

Nano Letters

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

Page 20 of 23

* E-mail: [email protected] (X. Chen). Author Contributions ⊥

J. Chen and J. Ding contributed equally to this work.

ACKNOWLEDGMENTS This study was financially supported by National Natural Science Foundation of China (Grant Nos. 51673190, 51603204, 51390484, 51233004, 51473165, 51520105004, and 81422026). REFERENCES 1.

Torre, L. A.; Bray, F.; Siegel, R. L.; Ferlay, J.; Lortet‐ Tieulent, J.; Jemal, A. CA: Cancer

J. Clin. 2015, 65, (2), 87−108. 2.

Donker, M.; van Tienhoven, G.; Straver, M. E.; Meijnen, P.; van de Velde, C. J.; Mansel,

R. E.; Cataliotti, L.; Westenberg, A. H.; Klinkenbijl, J. H.; Orzalesi, L. Lancet Oncol. 2014, 15, (12), 1303−1310. 3.

Wan, L.; Pantel, K.; Kang, Y. Nat. Med. 2013, 19, (11), 1450−1464.

4.

Mehlen, P.; Puisieux, A. Nat. Rev. Cancer 2006, 6, (6), 449−458.

5.

Blanco, E.; Shen, H.; Ferrari, M. Nat. Biotechnol. 2015, 33, (9), 941−951.

6.

Cabral, H.; Makino, J.; Matsumoto, Y.; Mi, P.; Wu, H.; Nomoto, T.; Toh, K.; Yamada,

N.; Higuchi, Y.; Konishi, S.; Kano, M. R.; Nishihara, H.; Miura, Y.; Nishiyama, N.; Kataoka, K. ACS Nano 2015, 9, (5), 4957−4967. 7.

Lu, Y.; Low, P. S. Adv. Drug Deliv. Rev. 2012, 64, 342−352.

8.

Kawakami, S.; Hashida, M. J. Control. Release 2014, 190, 542−555.

9.

Li, C. Nat. Mater. 2014, 13, (2), 110−115.

10.

Fang, J.; Nakamura, H.; Maeda, H. Adv. Drug Deliv. Rev. 2011, 63, (3), 136−151.


20

Page 21 of 23

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

Nano Letters

11.

Perche, F.; Biswas, S.; Wang, T.; Zhu, L.; Torchilin, V. Angew. Chem., Int. Ed. 2014, 53,

(13), 3362−3366. 12.

Deshayes, S.; Cabral, H.; Ishii, T.; Miura, Y.; Kobayashi, S.; Yamashita, T.; Matsumoto,

A.; Miyahara, Y.; Nishiyama, N.; Kataoka, K. J. Am. Chem. Soc. 2013, 135, (41), 15501−15507. 13.

Chen, J.; Ding, J.; Xiao, C.; Zhuang, X.; Chen, X. Biomater. Sci. 2015, 3, (7), 988−1001.

14.

Gao, W.; Hu, C. M. J.; Fang, R. H.; Luk, B. T.; Su, J.; Zhang, L. Adv. Mater. 2013, 25,

(26), 3549−3553. 15.

Lee, J. Y.; Termsarasab, U.; Park, J. H.; Lee, S. Y.; Ko, S. H.; Shim, J. S.; Chung, S. J.;

Cho, H. J.; Kim, D. D. J. Control. Release 2016, 236, 38−46. 16.

Shi, S.; Zhou, M.; Li, X.; Hu, M.; Li, C.; Li, M.; Sheng, F.; Li, Z.; Wu, G.; Luo, M.; Cui,

H.; Li, Z.; Fu, R.; Xiang, M.; Xu, J.; Zhang, Q.; Lu, L. J. Control. Release 2016, 235, 1−13. 17.

Helmlinger, G.; Yuan, F.; Dellian, M.; Jain, R. K. Nat. Med. 1997, 3, (2), 177−182.

18.

Wu, X.; Li, Z.; Chen, X. X.; Fossey, J. S.; James, T. D.; Jiang, Y. B. Chem. Soc. Rev.

2013, 42, (20), 8032−8048. 19.

Matsumoto, A.; Cabral, H.; Sato, N.; Kataoka, K.; Miyahara, Y. Angew. Chem., Int. Ed.

2010, 49, (32), 5494−5497. 20.

Sanjoh, M.; Miyahara, Y.; Kataoka, K.; Matsumoto, A. Anal. Sci. 2014, 30, (1), 111−117.

21.

Guo, X.; Wei, X.; Jing, Y.; Zhou, S. Adv. Mater. 2015, 27, (41), 6450−6456.

22.

Zhang, Y.; Wang, C.; Xu, C.; Yang, C.; Zhang, Z.; Yan, H.; Liu, K. Chem. Commun.

2013, 49, (66), 7286−7288. 23.

Nicolas, J.; Mura, S.; Brambilla, D.; Mackiewicz, N.; Couvreur, P. Chem. Soc. Rev. 2013,

42, (3), 1147−1235.


21

Nano Letters

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

24.

Page 22 of 23

Cabral, H.; Makino, J.; Matsumoto, Y.; Mi, P.; Wu, H.; Nomoto, T.; Toh, K.; Yamada,

N.; Higuchi, Y.; Konishi, S. ACS Nano 2015, 9, (5), 4957−4967. 25.

Feng, X.; Ding, J.; Gref, R.; Chen, X.. Chinese J. Polym. Sci. 2017, 35, (6), 693−699.

26.

Sadok, A.; McCarthy, A.; Caldwell, J.; Collins, I.; Garrett, M. D.; Yeo, M.; Hooper, S.;

Sahai, E.; Kuemper, S.; Mardakheh, F. K.; Marshall, C. J. Cancer Res. 2015, 75, (11), 2272−2284. 27.

Friedl, P.; Wolf, K. Nat. Rev. Cancer 2003, 3, (5), 362−374.

28.

Albini, A.; Iwamoto, Y.; Kleinman, H. K.; Martin, G. R.; Aaronson, S. A.; Kozlowski, J.

M.; McEwan, R. N. Cancer Res. 1987, 47, (12), 3239−3245.


22

Page 23 of 23

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

Nano Letters

ToC Graphic


23

Receptor and Microenvironment Dual-Recognizable Nanogel for

Recommend Documents