Matrix Metalloproteinase-Responsive ... - ACS Publications

Article

Matrix Metalloproteinase-Responsive Multifunctional Peptide-Linked Amphiphilic Block Copolymers for Intelligent Systemic Anticancer Drug Delivery Wendong Ke, Zengshi Zha, Jean Felix Mukerabigwi, Weijian Chen, Yuheng Wang, Chuanxin He, and Zhishen Ge Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00330 • Publication Date (Web): 29 Jun 2017 Downloaded from http://pubs.acs.org on July 1, 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.

Bioconjugate Chemistry 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 33

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

Bioconjugate Chemistry

Matrix Metalloproteinase-Responsive

Multifunctional

Peptide-Linked

Amphiphilic

Block

Copolymers for Intelligent Systemic Anticancer Drug Delivery

Wendong Ke,† Zengshi Zha,† Jean Felix Mukerabigwi,† Weijian Chen,† Yuheng Wang,† Chuanxin He,*,‡

†

Zhishen Ge*,†

CAS Key Laboratory of Soft Matter Chemistry, Department of Polymer Science and Engineering,

University of Science and Technology of China, Hefei 230026, Anhui, China

‡

College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen 518060,

Guangdong, China.

*

Correspondence to:

Email address: [email protected]; [email protected]

1

ACS Paragon Plus Environment


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

Page 2 of 33

ABSTRACT The amphiphilic block copolymer anticancer drug nanocarriers clinically used or in the progress of clinical trials frequently suffer from the modest final therapeutic efficacy due to lack of intelligent features.

For

example,

the

biodegradable

amphiphilic

block

copolymer,

poly(ethylene

glycol)-b-poly(D,L-lactide) (PEG-PDLLA), has been approved for clinical applications as a paclitaxel (PTX) nanocarrier (Genexol-PM) due to the optimized pharmacokinetics and biodistribution, however, lack of intelligent features limits the intracellular delivery in tumor tissue. To endow the mediocre polymer with smart properties via a safe and facile method, we introduced a matrix metalloproteinase (MMP)-responsive peptide GPLGVRGDG into the block copolymer via efficient click chemistry and ring opening polymerization to prepare PEG-GPLGVRGDG-PDLLA (P1). P1 was further self-assembled into micellar nanoparticles (NPs) to load PTX, which show MMP-2-triggered dePEGylation due to cleavage of the peptide linkage. Moreover, the residual VRGDG sequences are retained on the surface of the NPs after dePEGylation, which can serve as ligands to facilitate the cellular uptake. The cytotoxicity of PTX loaded in P1 NPs against 4T1 cells is significantly enhanced as compared to free PTX or PTX-loaded PEG-GPLGVRG-PDLLA (P2) and PEG-PDLLA (P3) NPs. In vivo studies confirmed that PTX-loaded P1 NPs show prolonged blood circulation, which are similar to P2 and P3 NPs, but exhibit more efficient accumulation in the tumor site. Ultimately, PTX-loaded P1 NPs display statistically significant improvement of antitumor activity against tumor-bearing mice via systemic administration. Therefore, the strategy by facile incorporation of a responsive peptide linkage between PEG and PDLLA is a promising approach to improve the therapeutic efficacy of anticancer drug-loaded amphiphilic block copolymer micelles.

2


Page 3 of 33

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


INTRODUCTION Amphiphilic block copolymers can self-assemble into micelles in aqueous solution and have been extensively applied to encapsulate hydrophobic anticancer drugs.1 Compared with small-molecule anticancer drugs, polymeric micelle drug formulations improve the pharmacokinetics and accumulation of the drugs in tumor sites, which significantly improve bioavailability of drugs and reduce side effect of the drug toxicity to the body.2 The biocompatible and biodegradable polymer-based micelles have achieved great success and some systems have been translated into clinical applications or in the progress of clinical trials. Genexol-PM based on biocompatible poly(ethylene glycol)-b-poly(D,L-lactide) (PEG-PDLLA) copolymer as a typical example exhibited more efficacious and lower toxicity compared to solvent-based paclitaxel (PTX), which has been approved to treat metastatic breast cancer and non-small cell lung cancer in South Korea.3 Moreover, a series of other amphiphilic block copolymer micelle systems are under clinical trials, such as NK105, NK911, NK012, SP1049C, BIND014.4,5 On the other hand, the intrinsic characteristics of the polymeric micelles for long blood circulation and high tumor accumulation (e.g. PEG-covered surface) also compromise intracellular internalization at tumor sites whereas most anticancer drugs need take effect inside cells.6 To address this issue, various smart polymeric drug delivery systems with adaptive properties according to the delivery stages have been developed to further improve the therapeutic efficacy,7-12 yet few polymeric drug delivery systems have entered clinical stages so far. The complex polymer structure and undefined systemic toxicity restrict their further translation.13-15 The purpose to facilely engineer even smarter drug delivery systems while keeping the possibility to translate into clinical application provoked us to discreetly adjust the structure of clinically approved polymer carriers for better therapeutic efficacy while reserving the biosafety. To improve the delivery efficiency of PEG-PDLLA, various methods have been explored. Surface 3



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

modification with a ligand is a simple tool to endow such systems with some distinct features such as specific targeting, efficient cellular internalization, and high therapeutic efficiency.16-18 For example, cyclic RGD peptide c(RGDyk) functionalized PEG-PDLLA nanoparticles (NPs) realized the enhanced site-specific accumulation at the glioblastoma tumor site. Tumor-bearing mice treated with PTX-loaded c(RGDyk)-PEG-PDLLA micelles achieved improved antitumor efficacy and prolonged survival.19 However, the attachment of ligands on the surface of NPs not only changed the pharmacokinetics possibly but also limited the application range due to the differences among different cancers and varying persons.20 The roles of active targeting ligands for efficient tumor accumulation are still debatable.21 Thus, translation of the corresponding systems still remains a great challenge. Moreover, an alternative approach is incorporation of a stimuli-responsive linkage between PEG and PDLLA block to avoid contacting blood components and minimize the effect on the delivery systems. Disulfide bond has been embedded between the PEG and PDLLA blocks, redox-responsiveness of disulfide bond facilitated the control of payload release kinetics.22 However, detachment of PEG is difficult in tumor microenvironment because high reductive condition primarily exists inside tumor cells. Tumor pH-responsive linkage to PEG was developed successfully. Under the lowered pH at the tumor site, the linkage would be cleaved while the PEG detachment and retained amino group increased the zeta potentials, which induced enhanced cellular uptake and in vivo tumor inhibition.23 However, tiny pH difference between tumor microenvironment and normal tissues requires the responsive moieties to possess extremely high sensitivity for dePEGylation.24,25 It is difficult to ensure no release under normal tissue acidity.26 Therefore, improvement of the delivery efficiency of PEG-PDLLA via introduction of proper stimuli-responsive moieties is still urgent. Matrix metalloproteinases (MMPs) play critical roles in cancer cell motility and invasion, thereby 4


Page 4 of 33

Page 5 of 33

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


facilitating tumor progression and metastasis.27,28 MMPs are over-expressed at tumor sites, which have been considered as a tumor-specific stimuli for cancer imaging and drug delivery.29-35 Specific peptide sequences can be cleaved by highly active MMPs in tumor tissue. On the other hand, variability of peptide sequences makes the integration of MMP-responsiveness and specific targeting possible. Herein, we prepared a block copolymer PEG-GPLGVRGDG-PDLLA for PTX delivery, which can be regarded as an upgraded Genexol-PM. The incorporation of a MMP-responsive peptide linkage between the two blocks realized MMP-responsive dePEGylation in tumor tissue. Moreover, after cleavage of peptide sequence by MMP-2 at specific site between G and V, the remaining peptide sequence (VRGDG) has the ability to interact with tumor cells, hence enhances the cellular internalization (Scheme 1) 36-38. This design with simple and effective polymer preparation not only reserves high biosafety of PEG-PDLLA block copolymers but also endows them with responsive dePEGylation and targeting properties, which possesses great potentials for the valuable increment of therapeutic efficacy towards drug-loading PEG-PDLLA block copolymer micelles and can be considered as a general strategy to improve the efficiency of the amphiphilic block copolymer drug delivery systems.

5



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

Scheme 1. Schematic illustration of PTX-loaded PEG-GPLGVRGDG-PDLLA nanoparticle preparation. After intravenous injection, NPs achieved prolonged blood circulation, enhanced tumor accumulation, MMP-2-triggered dePEGylation, and residual RGD peptide-mediated high cellular internalization.

RESULTS AND DISCUSSION Polymer Synthesis and NP Preparation The GPLGVRGDG peptide-terminated PEG (PEG-GPLGVRGDG-NH2) was prepared according to the previous report via efficient click reaction between α-methoxy-ω-azido-poly(ethylene glycol) (PEG-N3) and peptide G(propargylglycine)-PLGVRGDG (alkynyl-GPLGVRGDG).31 The obtained PEG-GPLGVRGDG-NH2 was further used as an initiator to initiate ring-opening polymerization (ROP) 6


Page 6 of 33

Page 7 of 33

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


of D,L-lactide for the preparation of PEG-GPLGVRGDG-PDLLA (P1).39 Gel permeation chromatography (GPC) results showed the increased molecular weight of P1 with relative narrow molecular weight distribution (Mw/Mn = 1.23) as compared with the initiator, which indicated good control of the polymerization (Figure S1). DP of PDLLA block was calculated to be 100 by 1H NMR analysis of P1 via comparison of the integrals of PEG characteristic peak (a) and PDLLA peaks (b and c) (Figure S2). Moreover, the block copolymers PEG-GPLGVRG-PDLLA (P2) and PEG-PDLLA (P3) with comparable block lengths were also prepared successfully as controls. The characterizations of the block copolymers was summarized in Table 1. The amphiphilic block copolymers (P1, P2, and P3) were able to assemble into micelle NPs with well-defined core-shell structure in aqueous solution, which can be used to encapsulate hydrophobic PTX in the cores of the micelles. P1 NPs showed relatively uniform spherical morphology by transmission electron microscopy (TEM) measurements with the average size of 62.6 ± 5.1 nm (Figure 1D, before MMP-2 treatment) by using Image J software based on more than 50 NPs in TEM images. Dynamic laser light scattering (DLS) results indicated a diameter of 76 nm at a relatively narrow size distribution with polydispersity index (PDI) of 0.14 (Figure S3). The diameter from DLS was a little larger than that by TEM measurement which should be attributed to the hydrated state in aqueous solution. As controls, P2 and P3 amphiphilic block polymers were both assembled with PTX to prepare the core-shell micellar NPs. All the three NPs had comparable diameters of approximately 80 nm (Figure S3) which is believed to be favorable for long-term circulation, enhanced tumor accumulation based on enhanced permeability and retention (EPR) effect, and uniform distribution in tumor tissues.40 Moreover, the three NPs showed comparable critical micelle concentration (CMC) values, drug loading contents (DLCs), and drug loading efficiencies (DLEs) (Figure S4 and Table 1). The similar PTX 7



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 33

encapsulation efficiencies and particle properties indicated that the addition of a peptide linkage between PEG and PDLLA blocks don’t change the physicochemical properties of NPs significantly, which encouraged us to evaluate the influence of the peptide linkage on the therapeutic efficacy. Table 1. Characterization of the block copolymers and the corresponding PTX-Loaded NPs. Polymera

Mn(kDa)b

Mw/Mnb

sizec (nm)

PDIc

DLEd (%)

DLCe (wt%)

CMC (×10-3 mg/mL)

P3

PEG113-PDLLA110

11.6

1.13

82.4

0.15

57.2

5.2

3.3

P2

PEG113-GPLGVRG-PDLLA106

12.3

1.20

88.5

0.16

66

6.0

2.5

P1

PEG113-GPLGVRGDG-PDLLA100

12.0

1.23

76.2

0.14

51.7

4.7

2.1

a

The degrees of polymerization were calculated according to 1H NMR analysis. b Number average molecular weight and molecular weight distributions of the polymers were determined from DMF GPC. c The particle size and size distribution (PDI) were analyzed by DLS. d Drug loading efficiency (DLE) = w(drug in micelles)/ w(initial drug added). e Drug loading content ( DLC) = w(drug in micelle)/ w(total micelles).

MMP-2-Responsive Behavior The peptide linkage (GPLGVRG) has been verified to be cleaved at the site between G and V under treatment by activated MMP-2.37,41 In this work, to evaluate the MMP-responsive behavior of the GPLGVRGDG linkage, we first incubated alkynyl-GPLGVRGD with MMP-2 at 37 °C and subsequently the sample was applied to electrospray ionisation mass spectrometry (ESI-MS) analysis. The peaks of fragment peptides (alkynyl-GPLG and VRGDG) were both observed in the ESI-MS spectrum, which confirmed that the cleavage site is indeed between G and V (Figure S5). Then P1 NPs were examined to be treated by MMP-2 followed by GPC measurements. As shown in Figure 1A, GPC profiles show a new shoulder peak at 16.08 min appeared after treatment for 1 h, which corresponds to PEG peak. This result proved that MMP-2 treatment led to the cleavage of PEG from the NPs. With MMP-2 treatment time increased, the shoulder peak became more and more intensive with more than 70% 8


Page 9 of 33

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


PEG detached within 8 h (Figure 1B). Finally, ~ 20% PEG still left on the NP surfaces. Presumably, a small portion of peptides were buried in the core of the micelles resulting in inaccessibility towards MMP-2. During the MMP-2 treatment, we also used DLS to investigate the NPs size change. Intriguingly, the nanoparticle size did not change significantly for at least 24 hours according to DLS results (Figure 1C). TEM measurement further confirmed that the NPs after dePEGylation can be still dispersed well in aqueous solution except very few relatively large particles can be observed (Figure 1D). After MMP-2 treatment, the average size of NPs is 65.2 ± 8.8 nm according to TEM images, which is slightly larger than that before MMP-2 treatment. This phenomenon is presumably attributed to ~ 20% PEG shells left on the surface of the NPs and VRGDG peptide residues, which can both stabilize the NPs to some extent.23 As controls, P2 also exhibited PEG detachment after incubation with MMP-2 while P3 did not respond to MMP-2 at all (Figure 1B). Similar to P1 NPs, the morphology of P2 didn’t change significantly in the presence of MMP-2 (Figure 1C and Figure S6). The above results showed that MMP-2 enzyme can efficiently cleave the peptide linkage between the two blocks of the drug-loading micellar NPs, but do not induce the remarkable aggregation. Considering VRGDG residuals on the surface of P1 NPs after dePEGylation, the small NPs size change is favorable for the interactions between NPs and cell membranes which may promote the cellular internalization. Moreover, to evaluate the effect of the presence of MMP-2 on the drug release, we examined the PTX release profiles from the NPs in the presence and absence of MMP-2. As shown in Figure 1E, the PTX formulations (P1, P2, and P3) exhibited similar release profiles in the absence of MMP-2 with approximately 80% PTX release within 72 hours. In the presence of MMP-2, the drug release profiles form P1, P2, and P3 NPs were not altered significantly as compared with those in the absence of 9



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

MMP-2 (Figure1F), which is likely owing to the small morphology change of the PTX-loaded NPs after treatment by MMP-2.

Figure 1. (A) GPC traces of P1 at different time points after treatment with 1 µg/mL MMP-2. (B) Quantified PEG release of P1, P2 and P3 block copolymers in the presence of MMP-2 (1 µg/mL). (C) Size changes of the NPs in the presence of MMP-2 (1 µg/mL). (D) Representative TEM images of P1 NPs before and after MMP-2 treatment. Scale bars represent 200 nm. The arrows indicate the representative spherical morphology of NPs. (E) and (F) PTX release profiles from the NPs in the absence (E) and presence (F) of MMP-2 (1 µg/mL) at pH 7.4, 37 °C. Data were expressed as mean ± s.d. (n = 3).

In Vitro Cellular Uptake and Cytotoxicity 10


Page 10 of 33

Page 11 of 33

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


The efficient MMP-responsive PEG detachment of P1 NPs and the small size change are expected to facilitate cellular uptake.42,43 Moreover, the remaining peptide VRGDG residuals on the surface of P1 NPs as one type of targeting RGD ligand are speculated to further promote the interactions between NPs and tumor cells.38 To investigate the cellular uptake behavior of the NPs in the presence of MMP-2, Nile red-loaded NPs were incubated with 4T1 cells upon addition of activated MMP-2 (1 µg/mL). The flow cytometric measurement was performed to investigate cellular internalization of NPs. As shown in Figure 2A, P1 NPs exhibited almost three times higher cellular uptake compared to P3 NPs, while P2 NPs exhibited only 1.2-fold increased uptake compared with P3 NPs. Moreover, as shown in the confocal laser scanning microscopy (CLSM) images, significantly stronger Nile red fluorescence intensity of P1 NPs was observed as compared with P2 and P3 also indicating that P1 NPs exhibited more efficient cellular internalization (Figure 2B). Excessive free RGD molecule was also added when incubating 4T1 cells with Nile red-loaded P1 NPs in the presence of MMP-2 (1 µg/mL). The cellular uptake was reduced remarkably as compared with that in the absence of free RGD, indicating that RGD-meditated cellular uptake played an important role for the enhanced cellular uptake of P1 NPs after MMP-2 triggered dePEGylation. In the presence of MMP-2, P2 NPs showed responsive dePEGylation while P1 NPs not only exhibited dePEGylation but also RGD residuals were left on the surface of NPs. Thus, P1 NPs showed highest efficiency of cellular uptake. Taken together, MMP-2-sensitive PEG detachment and the residual VRGDG peptide on the surface of P1 NPs synergistically facilitated the cellular uptake. Effective cellular internalization plays a critical role for the anticancer drugs acting inside cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were utilized to evaluate the cell viability against 4T1 cells after treatment by different drug formulations in the presence of 11



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

MMP-2. The blank NPs showed almost no toxicity even at quite high concentrations, which indicated the good biocompatibility of these block copolymers (Figure S7). As shown in Figure 2C, PTX-loaded P1 NPs exhibited significantly enhanced cytotoxicity compared to PTX-loaded P2 and P3 NPs as well as free PTX. Quantitatively, the half maximal inhibitory concentration (IC50) value of PTX loaded in P1 micelle was determined to be 79 ng/mL which is much lower as compared to free PTX (150 ng/mL), PTX-loaded P2 NPs (600 ng/mL), or P3 NPs (1130 ng/mL). As a control in the absence of MMP-2, PTX-loaded P1 NPs exhibited similar cytotoxicity to that of P3 NPs without dePEGylation (1070 ng/mL). Thus, the high cytotoxicity of PTX-loaded P1 NPs could be attributed to the increased cellular internalization in the presence of MMP-2.

Figure 2. (A) Flow cytometric results and (B) CLSM images of 4T1 cells after incubation with Nile red-loaded P1, P2, and P3 NPs in the presence of 1 µg/mL MMP-2. Scale bars represent 5 µm. (C) Cytotoxicity evaluation of different drug formulations at various PTX concentrations. Data were expressed as mean ± s.d. (n = 4). * p ˂ 0.05, versus PTX.

In Vivo Pharmacokinetics and Biodistribution The inspired in vitro results encouraged us to further study the in vivo performance of the 12


Page 12 of 33

Page 13 of 33

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


PTX-loaded NPs. First, the serum stability of the NPs was evaluated through incubation in DMEM containing 20% FBS. PTX-loaded P1, P2, and P3 NPs all showed high stability without obvious size and size distribution change after incubation for 48 h (Figure S8). Next, the pharmacokinetics of three drug formulations was evaluated in female 6-week-old CD-1 (ICR) mice. As shown in Figure 3A, free PTX was cleared rapidly form the blood circulation after intravenous injection. PTX-loaded P1, P2, and P3 NPs displayed similar pharmacokinetics profiles with relative long blood circulation times. The average elimination half-life t1/2 were determined to be 3.36 h for P1, 4.82 h for P2 and 3.92 h for P3, respectively, which did not show statistical significance. It is believed that the prolonged blood circulation enables high tumor accumulation of anticancer drugs. Considering the importance of MMP enzymes in the progression of various tumors,29 we chose H22 tumor models using female CD-1 (ICR) mice for in vivo biodistribution study due to the simple and efficient tumor model establishment with relatively uniform original tumor sizes. To quantify PTX amounts at the tumor site and major organs, the tissues were harvested after 12 or 24 h post intravenous injection of different drug formulations. Then PTX was extracted and analyzed with a high performance liquid chromatography (HPLC) system. It is clear that the drug accumulation of P1 NPs at tumor site was enhanced to some extent as compared to P2 NPs (1.47 times) and P3 (2.01 times) (*p < 0.05) while drug distribution in the organs showed no significant difference at 24 h (Figure 3B). To visualize the in vivo

fate

of

NPs,

hydrophobic

near

infrared

fluorescence-emission

dye,

1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR), was encapsulated into the NPs for real-time monitoring. As shown in Figure 3C, after intravenous injection of the three DiR-loaded NPs, the tumor sites showed obvious DiR fluorescence at 1 h and reached a maximum at 24 h with similar fluorescence intensities of P1, P2, and P3 NPs. However, it was found that DiR-loaded P1 NPs 13



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

exhibited slightly stronger fluorescence at 48 h and 72 h compared to P1 and P2 controls. DePEGylation and the residual peptide ligand of P1 NPs by over-expressed MMP-2 under tumor microenvironment were considered to be responsible for more efficient tumor accumulation of P1 NPs.

Figure 3. (A) Time-dependent PTX amount in the blood stream after intravenous injection of free PTX, PTX-loaded P1, P2 or P3 NPs at the PTX dose of 10 mg/kg mouse body. (B) Quantitative analysis of PTX amounts at 12 and 24 h in major organs and tumors post intravenous injection of varying PTX formulations into H22-bearing mice. Data were expressed as mean ± s.d. (n = 3). * p ˂ 0.05. (C) In vivo biodistribution post intravenous injection of DiR-loaded NPs.

Antitumor Efficacy The therapeutic performance of PTX-loaded NPs was evaluated using H22 tumor-bearing mice. Totally 25 mice were divided into five groups including phosphate buffer saline (PBS), free PTX, PTX-loaded P1, P2, and P3 micelles. As tumors grew to ~ 100 mm3, the different drug formulations were intravenously injected every two days for a total of three-time injection at PTX concentration of 10 mg/kg of mouse body weight. Tumor growth profiles as shown in Figure 4A,B revealed that tumors treated with PBS exhibited a nearly 40-fold volume increase after 24 days and 25-fold for free PTX, 14


Page 14 of 33

Page 15 of 33

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


16-fold for P3 NPs, and 10-fold for P2 NPs. In sharp contrast, PTX-loaded P1 NPs suppressed tumor growth efficiently with the tumor sizes just increased by a factor of 1.9. Considering H22 tumor model grows rapidly without treatment, this tumor growth suppression result showed potent therapeutic efficacy of PTX-loaded P1 NPs. At day 24, the excised tumors were weighted, and tumors treated with P1 NPs showed an average weight of 0.38 g, significantly lighter than P2 (0.75 g), P3 (1.50 g), free PTX (1.73 g) and PBS (2.11 g) (Figure S9). Tumor sections were stained with haematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining for evaluating the apoptosis cells in tumors. It was shown in Figure 4C that the tumor treated with PTX loaded P1 NPs exhibited the strongest green fluorescence which suggested the most abundant apoptotic cells. H&E staining also confirmed that the most enhanced therapeutic efficacy was realized through injection of PTX-loaded P1 NPs as evidenced by the large area of tissue necrosis. Considering that the only difference between P1, P2 and P3 is the peptide linkage between the polymer blocks, the enhanced therapeutic efficiency can be attributed to MMP-2-induced dePEGylation and residual peptide which enhanced accumulation and cellular internalization in tumor tissues. Moreover, the body weights of the mice from the five groups did not change significantly during treatment (Figure 4D). Moreover, H&E staining results of the major organs showed no obvious damage with the treatment of PTX-loaded NPs while remarkable liver damage was observed when the same dose of free PTX was injected intravenously (Figure S10). These results reveal high biosafety of P1, P2, and P3 NPs.

15



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

Figure 4. (A) In vivo antitumor performance after intravenous injection of different drug formulations at a PTX-equivalent dose of 10 mg/kg mouse body weight. Data were expressed as mean ± s.d. n = 5, *p ˂ 0.05, versus P2 and P3 NPs. (B) Typical tumor photographs collected from different treatment groups at the 24th day. (C) H&E staining results and representative fluorescence images of TUNEL assay of tumor sections after treatment with different drug formulations. (D) Mice body weight changes upon the treatment with different drug formulations.

CONCLUSION In summary, we demonstrated that the incorporation of a MMP-responsive peptide linkage GPLGVRGDG in the block copolymer (PEG-PDLLA) didn’t change the physicochemical properties of PEG-PDLLA remarkably, but significantly increased the in vivo performance of the amphiphilic block copolymer as drug delivery nanocarriers. In the presence of MMP-2, PEG surface of PTX-loaded P1 micelles was cleaved at the peptide site between G and V so that the residual short peptide VRGDG can serve as a ligand to enhance cellular internalization. In vivo biodistribution and pharmacokinetics evaluation confirmed that P1 micellar nanocarriers exhibited similar long blood circulation and enhanced tumor accumulation as compared with clinically used P3 NPs. Antitumor activity results

16


Page 16 of 33

Page 17 of 33

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


suggested that PTX-loaded P1 NPs significantly improved therapeutic efficacy as compared with P2 and P3 controls. Considering the high biosafety of the introduced peptide linkage, the intelligent amphiphilic block copolymer with significantly improved performance as anticancer nanocarriers possess great translational potentials. The incorporation of MMP-responsive peptides as a bridge represents a novel and efficient strategy to develop high-efficiency amphiphilic block copolymer drug nanocarriers.

EXPERIMENTAL PROCEDURES Materials Poly(ethylene glycol) monomethyl ether (PEG, Mn = 5,000), tin(II) 2-ethylhexanoate (Sn(Oct)2), Nile red (98%), and copper(I) bromide (CuBr, 98%) were purchased from Sigma-Aldrich and used as received. N,N,N',N'',N''-pentamethyldiethylenetriamine (PMDETA, 98%) and D,L-lactide (99%) were purchased from Energy Chemical. Peptide G(propargylglycine)-PLGVRG (alkynyl-GPLGVRG, purity 95.4% from HPLC) and G(propargylglycine)-PLGVRGDG (alkynyl-GPLGVRGDG, purity 97.9% from HPLC)

were

purchased

from

α-Methoxy-ω-azido-poly(ethylene PEG-GPLGVRG-NH2

China

glycol)

Peptides

(PEG113-N3,

32

, and PEG-GPLGVRGDG-NH2

Mn

31

Co., =

Ltd. 5,000,

(Shanghai, Mw/Mn

=

China).

1.07)

44,45

,

were synthesized according to the reported

methods. All the solvents such as toluene, benzene, N,N-dimethylformamide (DMF), and tetrahydrofuran (THF) were dried and distilled before use. Paclitaxel (PTX, 98%) was purchased from HVSF

United

Chemical

Materials

CO.,

Ltd.

(Beijing,

China).

1,1'-Dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR) were purchased from Fanbo Chemicals (Beijing, China). Fetal bovine serum (FBS), trypsin, Dulbecco's modified Eagle's medium 17



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

(DMEM),

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

Page 18 of 33

bromide

(MTT),

and

4´,

6-diamidino-2-phenylindole (DAPI) were purchased from Beyotime Institute of Biotechnology (Shanghai, China) and used as received. Recombinant human MMP-2 (purity > 95%, the specific activity > 1000 pmoles/min/µg) was purchased from Sino Biological Inc. (Beijing, China). P-Aminophenyl mercuric acid (APMA) was purchased from Genmed Scientifics Inc. USA. TCNB buffer (50 mM Tris, pH 7.4, 10 mM calcium chloride, 150 mM sodium chloride, 0.05% BRIJ 35) and PBS (phosphate buffer saline, pH 7.4) were prepared freshly before use. Mouse breast cancer cell line 4T1 and mouse liver cancer cell line H22 were purchased from Shanghai Institute of Cell Biology (Shanghai, China). Female BALB/c mice of 5-6 weeks old were purchased from Vital River Laboratory Animal Technology Co. Ltd. in Beijing. The animal experiments were carried out according to the Regulations for the Administration of Affairs about Experimental Animals (Hefei, revised in June 2013). Characterizations All the 1H NMR spectra were obtained on a Bruker AV300 spectrometer. CDCl3 or D2O were used as the solvent. Gel permeation chromatography (GPC) equipped with G1316A PL gel columns, an Agilent G1362A differential refractive index detector that was set at 30 °C and an Agilent 1260 pump was used to analyze the molecular weights (Mn) and molecular weight distributions (Mw/Mn) of the polymers. Low-polydispersity PEG standards were utilized for molecular weight determination. DMF containing 1 g/L LiBr at a rate of 1.0 mL/min was utilized as the eluent. Particle sizes and particle size distributions were recorded with a zetasizer (Nano ZS) instrument containing a He-Ne ion laser (λ = 633 nm) at the scattering angle of 173°. Reversed-phase high performance liquid chromatography (RP-HPLC) analysis was conducted on a Shimadzu HPLC system equipped with a LC-20AP binary pump, a C18 column, and a SPD-20A UV-Vis detector. Transmission electron microscopy (TEM) 18


Page 19 of 33

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


samples were prepared by dropping 10 µL NPs solution on a copper TEM grid and dried overnight followed by observation using a Hitachi H-7650 electron microscope with an acceleration voltage of 100 kV. The confocal laser scanning microscopy (CLSM) (Leica TCS SP5 microscope) was used to perform the observation of CLSM images. Synthesis of PEG-GPLGVRGDG-PLA Block Copolymer (P1) The synthetic routes for synthesis of PEG-GPLGVRGDG-PDLLA are illustrated in Scheme S1. The synthesis of PEG-GPLGVRGDG-PDLLA was performed via ring opening polymerization (ROP) of D,L-lactide

using PEG-GPLGVRGDG-NH2 as the initiator according to reported procedure

39

. Briefly,

PEG-GPLGVRGDG-NH2 initiator was firstly lyophilized from benzene to remove trace water. Then, PEG-GPLGVRGDG-NH2 (0.117 g, 0.02 mmol), D,L-lactide (0.288 g, 2 mmol), Sn(Oct)2 (30 µL, 10 mg/mL in benzene), anhydrous THF (1 mL) and anhydrous benzene (5 mL) were charged into a 10-mL Schlenk flask. After lyophilization, the Schlenk flask was back-filled with N2 and another 4 mL anhydrous THF was added. The flask was sealed under vacuum and subsequently placed into an 80 °C oil bath. After stirring for 18 h, the reaction solution was precipitated into cold diethyl ether. The above dissociation-precipitation cycle was repeated twice and after drying in a vacuum oven, the final product was obtained as a white powder, denoted as P1 (0.26 g, yield: 64%; Mn,GPC = 12.0 kDa, Mw/Mn = 1.23, Figure S1). The degree of polymerization (DP) of PDLLA block was calculated to be 100 by 1H NMR analysis (Figure S2). PEG-GPLGVRG-PDLLA (P2) and PEG-PDLLA (P3) were also prepared based on similar procedures using PEG-GPLGVRG-NH2 and PEG-OH as the initiators, respectively. The characterizations of the samples were summarized in Table 1. Micelle Preparation and Drug Encapsulation First, the critical micelle concentration (CMC) values of P1, P2, and P3 block copolymers were 19



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 33

determined by using pyrene as the probe. Briefly, pyrene acetone solution (30 µL, 6 × 10−5 M) was added into nanoparticle solution (3 mL) with the polymer concentration fixed at 1 mg/mL followed by sonication for 2 h. After completely removing the organic solvent under reduced pressure, the fluorescence excitation spectra were recorded from 300 to 360 nm with emission wavelength fixed at 374 nm. CMC values were calculated from the intensity ratio at 339 and 332 nm (I339/I332) as a function of different polymer concentration. For the preparation of PTX-loaded NPs, a reported emulsion-solvent evaporation method was used 23

. Briefly, PEG-GPLGVRGDG-PDLLA (10 mg) and PTX (1 mg) were dissolved in ethyl acetate (0.2

mL), and PBS (1 mL) or TCNB (1 mL) was added. Next, the mixture was allowed to emulsify for 60 s by sonication at 90 W. Then, the emulsion was evaporated to remove ethyl acetate and centrifuged at 3000 g to remove the unencapsulated PTX. The drug loading efficiency (DLE) and drug loading content (DLC) were determined by RP-HPLC analysis after PTX-loaded NPs were lyophilized and dissolved in methanol. DiR or Nile red-loaded NPs were prepared according to the similar method mentioned above, just replacing PTX (1 mg) with DiR (0.1 mg) or Nile red (0.1 mg). Blank NPs were also prepared by the similar method mentioned as a control. MMP-2-Responsive Behavior First,

alkynyl-GPLGVRGDG

peptide

was

incubated

with

MMP-2

to

evaluate

the

MMP-responsiveness. Briefly, the peptide (0.1 µM) was dissolved in TCNB buffer which contain MMP-2 (1 µg/mL) activated by APMA and incubated at 37 °C for 12 h and then the mixture was lyophilized. The sample was dispersed in chloroform followed by ESI-MS analysis. Blank P1, P2, and P3 NPs were incubated with MMP-2 (1 µg/mL). Briefly, P1, P2, and P3 NPs prepared in TCNB buffer were treated with MMP-2 dissolved in TCNB buffer which was activated by 20


Page 21 of 33

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


APMA before use at 37 °C. The final concentration of MMP-2 was fixed at 1 µg/mL in the mixture. At different time intervals, the particle sizes and particle size distributions were measured, and aliquot samples were taken out, lyophilized, and measured by GPC to detect the changes of polymer structures. The GPC peak intensity was used to quantify the amount of detached PEG. Drug Release of PTX-Loaded NPs Freshly prepared PTX-loaded P1, P2, or P3 NPs in TCNB or PBS (pH 7.4, 1 mL) were added into a dialysis bag (MWCO: 3500 Da) and incubated with 4 mL released medium (TCNB or PBS, pH 7.4) with or without APMA-activated MMP-2 (1 µg/mL) at 37 °C. At different time intervals, released medium (1 mL) was taken out and another 1 mL fresh buffer solution was complemented. After lyophilization, the released amounts of PTX were quantified by RP-HPLC analysis (mobile phase: acetonitrile/H2O (3:7, v/v), 1 mL/min, UV-vis detection-wavelength fixed at 217 nm). Cytotoxicity 4T1 cells were cultured in 96-well plates at a density of 1 × 104 cells/well in 100 µL DMEM containing

FBS (10%) at 37 °C under humidified CO2 atmosphere (5%) for 24 h. Then the medium

was replaced with 100 µL fresh DMEM containing 10% FBS. Next, blank or PTX-loaded NPs were added at different concentrations and incubated for 24 h in the presence of APMA-activated MMP-2 (1 µg/mL). The medium was then aspirated completely, and 100 µL fresh medium was added followed by another 48 h incubation. The cell viability was measured by MTT assays. MTT solutions (20 µL, 5 mg/mL in PBS) was added to each well and incubated at 37 °C in the dark for 4 h. The medium was aspirated completely and DMSO (200 µL) was added to dissolve the formed purple formazan crystals. After all the crystals were dissolved in about 30 minutes, the absorbance at 490 nm was measured by a microplate reader. The half maximal inhibitory concentrations (IC50) values were calculated by 21



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

GraphPad Prism software according to MTT results. Cellular Uptake For CLSM observation, 4T1 cells (5 × 104) were cultured in each well of a 4-well glass-bottom Petri dish in DMEM (500 µL) with 10% FBS at 37 °C under humidified 5% CO2 atmosphere for 24 h. Then the medium was replaced with fresh DMEM (500 µL) with 10% FBS followed by addition of Nile red-loaded NPs with the Nile red concentration of 0.1 µg/mL. As one control, RGD peptide (20 molar × concentration of peptide linkage) was added in Nile red-loaded P1 NPs solution. After 24 h incubation in the absence or presence of APMA-activated MMP-2 (1 µg/mL), the medium was removed and the cells were washed with cold PBS for three times and fixed with 4% paraformaldehyde for 15 min. Then the cell nuclei were stained with DAPI for CLSM observation. For flow cytometric measurements, 4T1 cells (1 × 105) were cultured in each well of a 24-well plate in DMEM (500 µL) with 10% FBS at 37 °C under humidified 5% CO2 atmosphere for 24 h. Then the medium was replaced with fresh DMEM (500 µL) with 10% FBS followed by addition of Nile red-loaded NPs with the Nile red concentration of 0.1 µg/mL. As one control, RGD peptide (20 molar × concentration of peptide linkage) was added in Nile red-loaded P1 NPs solution. After 24 h incubation in the absence or presence of APMA-activated MMP-2 (1 µg/mL), the medium was removed and the cells were detached by trypsin treatment carefully and collected by centrifugation. The cells were resuspended in cold PBS (1 mL) and measured by flow cytometry (EasyCyte2, 484 nm/591 nm). The results were analyzed by Flow Jo software. Pharmacokinetic Studies Female 6-week-old CD-1 (ICR) mice were randomly divided into three groups (n = 3 for each group). Free PTX, PTX-loaded P1, P2, or P3 NPs in PBS were intravenously injected into the tail vein 22


Page 22 of 33

Page 23 of 33

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


at the PTX concentration of 10 mg/kg mouse body weight. At different time intervals, 100 µL plasma samples collected from mouse orbital were charged into heparinized tubes and mixed with 10 mL ethyl acetate. Followed by centrifugation at 10000 × g for 5 min, the organic solvent was separated and dried under N2. The amount of paclitaxel was determined by RP-HPLC (mobile phase: acetonitrile/H2O (3:7, v/v), 1 mL/min, UV-vis detection-wavelength fixed at 217 nm). H22 Tumor Model H22 cells (3 × 106) suspended in 200 µL PBS were subcutaneously injected into right limb armpits of female 6-week-old CD-1 (ICR) mice. Tumor size was monitored using a digital vernier caliper. And the tumor volume (V) was determined by the equation V = L × W2/2, where L and W represent longest and shortest diameters of the tumors, respectively. Biodistribution H22 tumor-bearing mice were used to investigate the biodistribution of NPs. When tumor volume reached 100 mm3, DiR-loaded P1, P2, or P3 NPs in PBS were intravenously injected into the tail vein with the DiR concentration of 1 mg/kg mouse body weight. At different time intervals (1, 12, 24, 48 and 72 h), the mice were anesthetized and an IVIS small-animal imaging system was used to obtain the images. To quantify the amount of PTX accumulation at organs and tumors, major organs and tumors were harvested at 12 and 24 hours after intravenous injection of PTX loaded drug formulations at the dose of 10 mg/kg. Then the samples were washed with PBS, weighed after drying, homogenized in 0.5 mL acetonitrile, and centrifuged at 10000 × g for 10 min, then, the supernatant was further extracted with 1 mL CHCl3. The organic phase was dried under N2 flow and the residue was dissolved in 100 µL methanol.

The amount of PTX was determined by RP-HPLC (mobile phase: acetonitrile/H2O (3:7, 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

v/v), 1 mL/min, UV-vis detection-wavelength fixed at 217 nm).

In Vivo Antitumor Efficacy H22 tumor-bearing mice were divided randomly into 5 groups (n = 5). When the tumor volume reached to ~ 100 mm3, free PTX (10 mg/kg), PTX-loaded P1, P2, and P3 NPs in PBS were intravenously injected into the tail vein at PTX concentration of 10 mg/kg every two days for a total of three-time injection. Tumor volume was monitored by measuring the tumor size using a digital Vernier caliper. Meanwhile, the body weight of each mouse was recorded every two days. At the end of treatment (24th day), all the mice were sacrificed and main organs (heart, liver, spleen, lung, kidney) and tumors were collected. The weights of tumors were recorded. Then the tumors and organs were embedded in paraffin and sectioned into 5 µm slices. The sections were stained with haematoxylin and eosin (H&E) according to manufacturer’s protocols. Moreover, the tumor sections were further stained with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay kit to identify the apoptotic cells. Statistical Analysis All the data were expressed as mean ± standard deviation (s.d.). P-value assessed by Student’s t-test lower than 0.05 was considered statistically significant.

ASSOCIATED CONTENT Supporting Information Description Scheme S1: Synthetic routes for the preparation of PEG-GPLGVRGDG-PDLLA. Figure S1: DMF GPC traces of PEG-GPLGVRGDG-NH2 and PEG-GPLGVRGDG-PDLLA. Figure S2: 1H NMR spectra of PEG-GPLGVRGDG-NH2 and PEG-GPLGVRGDG-PDLLA. Figure S3: DLS results of PTX-loaded P1, 24


Page 24 of 33

Page 25 of 33

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


P2 and P3 NPs. Figure S4: Intensity ratio (I339/I332) as a function of concentration of P1, P2 and P3 by using pyrene as the probe. Figure S5: ESI-MS spectrum of peptide alkynyl-GPLGVRGDG after incubation with MMP-2. Figure S6: Representative TEM images of P2 and P3 NPs before (a) and after (b) treatment with MMP-2. Figure S7: MTT results of blank NPs. Figure S8: Serum stability of NPs. Figure S9: The tumor weights of H22 tumor at the end of treatment by different drug formulations. Figure S10: H&E-stained heart, liver, spleen, lung and kidney sections after treatment with PBS, PTX, P1, P2 and P3. This information is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGEMENTS We gratefully acknowledge financial support from National Natural Scientific Foundation of China (NNSFC) Project (21674104) and the Fundamental Research Funds for the Central Universities (WK3450000002).

Notes The authors declare no competing financial interest.

ABBREVIATIONS PEG: poly(ethylene glycol) PDLLA: poly(D,L-lactide) ROP: ring-opening polymerization NP: nanoparticle TEM: transmission electron microscopy 25



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

DLS: dynamic laser light scattering GPC: gel permeation chromatography MMP: matrix metalloproteinase ESI-MS: electrospray ionisation mass spectrometry PTX: paclitaxel CMC: critical micelle concentration DLE: drug loading efficiency DLC: drug loading content MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide H&E: haematoxylin and eosin TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling IC50: half maximal inhibitory concentrations CLSM: confocal laser scanning microscopy 4T1: mouse breast cancer cell line H22: mouse liver cancer cell line

REFERENCES (1)

Kataoka, K., Harada, A., and Nagasaki, Y. (2001) Block copolymer micelles for drug delivery: design, characterization and biological significance. Adv. Drug Delivery Rev. 47, 113-131.

(2)

Maeda, H., Nakamura, H., and Fang, J. (2013) The EPR effect for macromolecular drug delivery to solid tumors: Improvement of tumor uptake, lowering of systemic toxicity, and distinct tumor imaging in vivo. Adv. Drug Delivery Rev. 65, 71-79. 26


Page 26 of 33

Page 27 of 33

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

(3)


Ahn, H. K., Jung, M., Sym, S. J., Shin, D. B., Kang, S. M., Kyung, S. Y., Park, J.-W., Jeong, S. H., and Cho, E. K. (2014) A phase II trial of Cremorphor EL-free paclitaxel (Genexol-PM) and gemcitabine in patients with advanced non-small cell lung cancer. Cancer Chemoth. Pharm. 74, 277-282.

(4)

Min, Y., Caster, J. M., Eblan, M. J., and Wang, A. Z. (2015) Clinical translation of nanomedicine. Chem. Rev. 115, 11147-11190.

(5)

Cabral, H., and Kataoka, K. (2014) Progress of drug-loaded polymeric micelles into clinical studies. J. Controlled Release 190, 465-476.

(6)

Sun, Q., Zhou, Z., Qiu, N., and Shen, Y. (2017) Rational Design of Cancer Nanomedicine: Nanoproperty Integration and Synchronization. Adv. Mater. DOI: 10.1002/adma.201606628.

(7)

Ge, Z. S., and Liu, S. Y. (2013) Functional block copolymer assemblies responsive to tumor and intracellular microenvironments for site-specific drug delivery and enhanced imaging performance. Chem. Soc. Rev. 42, 7289-7325.

(8)

Mura, S., Nicolas, J., and Couvreur, P. (2013) Stimuli-responsive nanocarriers for drug delivery. Nat. Mater. 12, 991-1003.

(9)

Ganta, S., Devalapally, H., Shahiwala, A., and Amiji, M. (2008) A review of stimuli-responsive nanocarriers for drug and gene delivery. J. Controlled Release 126, 187-204.

(10)

Fleige, E., Quadir, M. A., and Haag, R. (2012) Stimuli-responsive polymeric nanocarriers for the controlled transport of active compounds: Concepts and applications. Adv. Drug Delivery Rev. 64, 866-884.

(11)

Cheng, R., Meng, F. H., Deng, C., Klok, H. A., and Zhong, Z. Y. (2013) Dual and multi-stimuli responsive polymeric nanoparticles for programmed site-specific drug delivery. Biomaterials 34, 27



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

3647-3657. (12)

Sawant, R. M., Hurley, J. P., Salmaso, S., Kale, A., Tolcheva, E., Levchenko, T. S., and Torchilin, V. P. (2006) "SMART" drug delivery systems: Double-targeted pH-responsive pharmaceutical nanocarriers. Bioconjugate Chem. 17, 943-949.

(13)

Hare, J. I., Lammers, T., Ashford, M. B., Puri, S., Storm, G., and Barry, S. T. (2016) Challenges and strategies in anti-cancer nanomedicine development: an industry perspective. Adv. Drug Delivery Rev.

(14)

Sanhai, W. R., Sakamoto, J. H., Canady, R., and Ferrari, M. (2008) Seven challenges for nanomedicine. Nat. Nanotechnol. 3, 242-244.

(15)

Sakai-Kato, K., Nishiyama, N., Kozaki, M., Nakanishi, T., Matsuda, Y., Hirano, M., Hanada, H., Hisada, S., Onodera, H., Harashima, H., et al. (2015) General considerations regarding the in vitro and in vivo properties of block copolymer micelle products and their evaluation. J. Controlled Release 210, 76-83.

(16)

Hu, Q., Gao, X., Gu, G., Kang, T., Tu, Y., Liu, Z., Song, Q., Yao, L., Pang, Z., and Jiang, X. (2013) Glioma therapy using tumor homing and penetrating peptide-functionalized PEG–PLA nanoparticles loaded with paclitaxel. Biomaterials 34, 5640-5650.

(17)

Gu, G., Hu, Q., Feng, X., Gao, X., Menglin, J., Kang, T., Jiang, D., Song, Q., Chen, H., and Chen, J. (2014) PEG-PLA nanoparticles modified with APT EDB peptide for enhanced anti-angiogenic and anti-glioma therapy. Biomaterials 35, 8215-8226.

(18)

Hu, Q., Gu, G., Liu, Z., Jiang, M., Kang, T., Miao, D., Tu, Y., Pang, Z., Song, Q., and Yao, L. (2013) F3 peptide-functionalized PEG-PLA nanoparticles co-administrated with tLyp-1 peptide for anti-glioma drug delivery. Biomaterials 34, 1135-1145. 28


Page 28 of 33

Page 29 of 33

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

(19)


Zhan, C., Gu, B., Xie, C., Li, J., Liu, Y., and Lu, W. (2010) Cyclic RGD conjugated poly (ethylene glycol)-co-poly (lactic acid) micelle enhances paclitaxel anti-glioblastoma effect. J. Controlled Release 143, 136-142.

(20)

Zhang, X. Q., Xu, X., Bertrand, N., Pridgen, E., Swami, A., and Farokhzad, O. C. (2012) Interactions of nanomaterials and biological systems: Implications to personalized nanomedicine. Adv. Drug Delivery Rev. 64, 1363-1384.

(21)

Bae, Y. H., and Park, K. (2011) Targeted drug delivery to tumors: Myths, reality and possibility. J. Controlled Release 153, 198-205.

(22)

Li, Y., Tong, R., Xia, H., Zhang, H., and Xuan, J. (2010) High intensity focused ultrasound and redox dual responsive polymer micelles. Chem. Commun. 46, 7739-7741.

(23)

Sun, C. Y., Liu, Y., Du, J. Z., Cao, Z. T., Xu, C. F., and Wang, J. (2016) Facile Generation of Tumor-pH-Labile Linkage-Bridged Block Copolymers for Chemotherapeutic Delivery. Angew. Chem. Int. Ed. 55, 1010-1014.

(24)

Gao, W. W., Chan, J. M., and Farokhzad, O. C. (2010) pH-Responsive Nanoparticles for Drug Delivery. Mol. Pharm. 7, 1913-1920.

(25)

Lee, E. S., Gao, Z. G., and Bae, Y. H. (2008) Recent progress in tumor pH targeting nanotechnology. J. Controlled Release 132, 164-170.

(26)

Zhou, Z. X., Shen, Y. Q., Tang, J. B., Fan, M. H., Van Kirk, E. A., Murdoch, W. J., and Radosz, M. (2009) Charge-Reversal Drug Conjugate for Targeted Cancer Cell Nuclear Drug Delivery. Adv. Funct. Mater. 19, 3580-3589.

(27)

Dufour, A., and Overall, C. M. (2013) Missing the target: matrix metalloproteinase antitargets in inflammation and cancer. Trends Pharmacol. Sci. 34, 233-242. 29



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

(28)

Overall, C. M., and Kleifeld, O. (2006) Tumour microenvironment - Opinion - Validating matrix metalloproteinases as drug targets and anti-targets for cancer therapy. Nat. Rev. Cancer 6, 227-239.

(29)

Kessenbrock, K., Plaks, V., and Werb, Z. (2010) Matrix metalloproteinases: regulators of the tumor microenvironment. Cell 141, 52-67.

(30)

Zhu, L., Wang, T., Perche, F., Taigind, A., and Torchilin, V. P. (2013) Enhanced anticancer activity of nanopreparation containing an MMP2-sensitive PEG-drug conjugate and cell-penetrating moiety. Proc. Natl. Acad. Sci. USA 110, 17047-17052.

(31)

Ke, W., Li, J., Zhao, K., Zha, Z., Han, Y., Wang, Y., Yin, W., Zhang, P., and Ge, Z. (2016) Modular design and facile synthesis of enzyme-responsive peptide-linked block copolymers for efficient delivery of doxorubicin. Biomacromolecules 17, 3268-3276.

(32)

Li, J., Ge, Z., and Liu, S. (2013) PEG-sheddable polyplex micelles as smart gene carriers based on MMP-cleavable peptide-linked block copolymers. Chem. Commun. 49, 6974-6976.

(33)

He, H. N., Sun, L., Ye, J. X., Liu, E. G., Chen, S. H., Liang, Q. L., Shin, M. C., and Yang, V. C. (2016) Enzyme-triggered, cell penetrating peptide-mediated delivery of anti-tumor agents. J. Controlled Release 240, 67-76.

(34)

Shi, L. L., Hu, Y., Lin, A., Ma, C., Zhang, C., Su, Y., Zhou, L. Z., Niu, Y. M., and Zhu, X. Y. (2016) Matrix Metalloproteinase Responsive Nanoparticles for Synergistic Treatment of Colorectal Cancer via Simultaneous Anti-Angiogenesis and Chemotherapy. Bioconjugate Chem. 27, 2943-2953.

(35)

Yamamoto, N., Bryce, N. S., Metzler-Nolte, N., and Hambley, T. W. (2012) Effects of Enzymatic Activation on the Distribution of Fluorescently Tagged MMP-2 Cleavable Peptides in Cancer 30


Page 30 of 33

Page 31 of 33

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


Cells and Spheroids. Bioconjugate Chem. 23, 1110-1118. (36)

Zhang, J., Yuan, Z.-F., Wang, Y., Chen, W.-H., Luo, G.-F., Cheng, S.-X., Zhuo, R.-X., and Zhang, X.-Z. (2013) Multifunctional envelope-type mesoporous silica nanoparticles for tumor-triggered targeting drug delivery. J. Am. Chem. Soc. 135, 5068-5073.

(37)

Bremer, C., Tung, C.-H., and Weissleder, R. (2001) In vivo molecular target assessment of matrix metalloproteinase inhibition. Nat. Med. 7, 743-748.

(38)

Danhier, F., Le Breton, A., and Preat, V. (2012) RGD-Based Strategies To Target Alpha(v) Beta(3) Integrin in Cancer Therapy and Diagnosis. Mol. Pharm. 9, 2961-2973.

(39)

Kowalski, A., Libiszowski, J., Biela, T., Cypryk, M., Duda, A., and Penczek, S. (2005) Kinetics and mechanism of cyclic esters polymerization initiated with tin (II) octoate. Polymerization of ε-caprolactone and L, L-Lactide co-initiated with primary amines. Macromolecules 38, 8170-8176.

(40)

Cabral, H., Matsumoto, Y., Mizuno, K., Chen, Q., Murakami, M., Kimura, M., Terada, Y., Kano, M. R., Miyazono, K., Uesaka, M., et al. (2011) Accumulation of sub-100 nm polymeric micelles in poorly permeable tumours depends on size. Nat. Nanotechnol. 6, 815-823.

(41)

Lee, S., Cha, E. J., Park, K., Lee, S. Y., Hong, J. K., Sun, I. C., Kim, S. Y., Choi, K., Kwon, I. C., Kim, K., et al. (2008) A near-infrared-fluorescence-quenched gold-nanoparticle imaging probe for in vivo drug screening and protease activity determination. Angew. Chem. Int. Ed. 47, 2804-2807.

(42)

Mok, H., Bae, K. H., Ahn, C.-H., and Park, T. G. (2008) PEGylated and MMP-2 specifically dePEGylated quantum dots: comparative evaluation of cellular uptake. Langmuir 25, 1645-1650.

(43)

Li, S. D., and Huang, L. (2010) Stealth nanoparticles: high density but sheddable PEG is a key 31



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

for tumor targeting. J. Controlled Release 145, 178. (44)

Gao, H. F., and Matyjaszewski, K. (2007) Synthesis of molecular brushes by "grafting onto" method: Combination of ATRP and click reactions. J. Am. Chem. Soc. 129, 6633-6639.

(45)

Cernovska, K., and König, B. (2003) Safe and convenient preparation of azido-methoxy polyethylenglycole on an azide-anion exchange resin. Synth. Commun. 33, 3789-3794.

32


Page 32 of 33

Page 33 of 33

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


ToC

33


Matrix Metalloproteinase-Responsive ... - ACS Publications

Recommend Documents