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Core-Shell-Distinct Nanodrug Showing On-Demand Sequential Drug Release to Act on Multiple Cell Types for Synergistic Anticancer Therapy Jinsheng Huang, Yongmin Xu, Hong Xiao, Zecong Xiao, Yu Guo, Du Cheng, and Xintao Shuai ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b02149 • Publication Date (Web): 29 May 2019 Downloaded from http://pubs.acs.org on May 29, 2019

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Core-Shell-Distinct Nanodrug Showing On-Demand Sequential Drug Release to Act on Multiple Cell Types for Synergistic Anticancer Therapy

Jinsheng Huang,†,1,2 Yongmin Xu,†,1 Hong Xiao,1,2 Zecong Xiao,1 Yu Guo,3 Du Cheng,1 and Xintao Shuai*1

1PCFM

Lab of Ministry of Education, School of Materials Science and Engineering,

Sun Yat-sen University, Guangzhou 510275, China. 2College

of Chemistry and Materials Science, Jinan University, Guangzhou, 510632,

China. 3Department

of General Surgery, the First Affiliated Hospital of Sun Yat-Sen

University, Guangzhou 510275, China. †These

authors contributed equally.

1

ACS Paragon Plus Environment

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Abstract: Among various inflammatory factors/mediators, autocrine and paracrine prostaglandin 2 (PGE2), which is abundant in various tumors, promotes the proliferation and chemoresistance of cancer cells. Thus, eliminating the cytoprotective effect of PGE2 may strengthen the anti-tumor effect of chemotherapy. Chemo/anti-inflammatory combination therapy requires the programmed activities of two different kinds of drugs that critically depend on their spatiotemporal manipulation inside the tumor. Here, a micellar polymeric nanosphere encapsulating chemotherapeutic paclitaxel (PTX) in the core and conjugating anti-inflammatory celecoxib (CXB) to the shell through a peptide linker (PLGLAG) was developed. The PLGLAG linker was cleavable by the enzyme matrix metalloproteinase-2 (MMP-2) in tumor tissue, causing CXB release and turning the negatively charged nanosphere into a positively charged one to facilitate PTX delivery into cancer cells. The released CXB not only acted on cyclooxygenase-2 (COX-2) to suppress the production of pro-inflammatory PGE2 in multiple cell types but also suppressed the expression of the anti-apoptotic Bcl-2 gene to sensitize cancer cells to chemotherapy, thus resulting the in synergistic anti-cancer effect of PTX and CXB. This study represents an example of using surface charge-switchable nanosphere with on-demand drug release properties to act on multiple cell types for highly effective chemo/anti-inflammatory combination therapy of cancer.

Keywords: polymeric micelle, nanodrug, sequential drug release, multicell targeting, combination therapy

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Solid tumors possess heterogeneous structures and a microenvironment regulated by complex signaling pathway network, such that the combination therapy strategies using different drugs to act on multiple oncotargets have better chance to improve the therapeutic outcome.1, 2 It is well documented that chronic inflammation is one of the common

features

of

cancer,3

where

cyclooxygenase-2

(COX-2)

and

a

proinflammatory mediator, COX-2-derived prostaglandins 2 (PGE2) play critical roles in the maintenance of tumor viability, growth, metastasis and angiogenesis.4, 5 On the other hand, chemotherapy often induces the upregulation of COX-2, PGE2 and antiapoptotic genes (e.g., Bcl-2), upon which the malignant cells acquire resistance to chemotherapeutic agents.6,

7

Previous studies have shown that nonsteroidal

anti-inflammatory drugs (NSAIDs), e.g., celecoxib (CXB), could inhibit not only the inflammatory COX-2/PGE2 pathway to influence the hallmarks of cancer but also the anti-apoptosis genes to reduce chemoresistance.1, 8, 9 Therefore, a combination therapy using a chemotherapeutic drug and a NSAIDs could be a powerful strategy to alter the characteristic proinflammatory environment of tumor and sensitize cancer cells to chemotherapy.1, 10 Although the combination therapy strategy using COX-2 inhibitors and chemotherapeutic drugs has initiated several clinical trials,11-14 anti-inflammatory drugs as adjuvants for chemotherapy in clinical studies are usually administered as free form, making it hard to achieve a desirable synergistic anticancer effect and was even reported to likely lower the anticancer activity of chemotherapeutic drug due to two drug interactions.15 Additionally, the long-term administration of free NSAIDs may result in severe side effects.16 For combination drug therapy in vivo, two-in-one codelivery is advantageous over the separate delivery of two drugs in many aspects, including simplified pharmacokinetics, less carrier usage, and better synergistic effect,17-20 which has made 3


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the design of nanocarriers for codelivering two different kinds of drugs a hot research spot in recent years. Particularly, nanodrugs based on biodegradable polymers such as aliphatic polyesters and poly(amino acids) have drawn great attentions because of their superior performances in controlled drug release, improved biosafety, and easily achievable tumor targeting via convenient chemical modifications.21, 22 Because both cancer cells and other stromal cells in the tumor chronic inflammatory environment may be activated to secrete PGE2 to promote tumor growth and chemoresistance,9, 23-25 autocrine PGE2 of cancer cells and paracrine PGE2 of stromal cells should be both inhibited by CXB to sensitize cancer cells to paclitaxel (PTX) chemotherapy. In this context, the development of codelivery systems with on-demand sequential drug release properties inside the tumor is extremely important for cancer combination drug therapy. To date, although the well-programmed intracellular release of two drugs (e.g., siRNA and chemotherapeutic drug) targeting the same cancer cells has been intensively investigated,26,

27

research on the

intratumoral sequential release of two drugs targeting multiple cell types (e.g., both cancer cells and stromal cells) has been very rare.21,

22

Moreover, polymeric

nanocarriers for the intratumoral sequential release of one chemotherapeutic drug and another NSAIDs has not been reported yet. On the other hand, the surface charge-switching strategy has shown great potential to improve the tumor-targeting drug delivery efficiency of nanocarriers.28 Unlike the tumor-targeting ligand modification strategy that may have adverse effects on the pharmacokinetics of nanodrugs (e.g., shortened circulation time) by altering their size and surface chemistry,29,

30

this strategy could make the nanodrugs negatively charged in the

bloodstream to prolong circulation but positively charged inside the tumor to facilitate cancer cell uptake through a tumor microenvironment-responsive structural design.31, 4


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32

It may also be a feasible means to improve the drug delivery efficiency in cancer

combination therapy. Herein, we describe a polymer-based nanodrug showing on-demand sequential drug release and surface charge switching to act on multiple cell types for synergistic chemo/anti-inflammation therapy of cancer (Figure 1). An amphiphilic triblock copolymer

of

CXB-peptide/mPEG-grafted

(PPLG-g-(CXB-peptide&mPEG)), poly(-caprolactone)

polyethylene (PCL),

poly(L-glutamate)

glycol

(PEG)

abbreviated

and as

PPLG-g-(CXB-peptide&mPEG)-PEG-PCL (PCxbP), was synthesized and then self-assembled

into

a

nanosphere

with

distinct

core-shell

structure.

The

chemotherapeutic paclitaxel (PTX) was physically encapsulated into the core via hydrophobic interaction, while the anti-inflammatory celecoxib (CXB) was conjugated to the shell through a peptide (PLGLAG) linker sensitive to matrix metalloproteinase-2 (MMP-2) overexpressed in solid tumors. The PLGLAG chain could be cleaved inside tumor tissue, which was expected to result in the release of CXB acting on multiple cell types to regulate tumor-associated inflammation and meanwhile a surface charge switching of nanosphere to facilitate PTX delivery into cancer cells. The combinative chemo/anti-inflammatory anticancer potency of MMP-2-sensitive nanosphere was explored. RESULTS AND DISCUSSION Preparation of Polymers and Nanospheres. As peptides with the PLGLAG sequence are sensitive to the MMP-2 being enriched in solid tumors,33, 34 a triblock copolymer using the GGPLGLAGG peptide to conjugate CXB was synthesized to prepare

MMP-2-sensitive

nanocarriers.

A

triblock

copolymer

using

inert

GGLALGPGG peptide to conjugate CXB was also synthesized to prepare 5


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MMP-2-insensitive nanocarrier as a negative control. The details to synthesize prepolymers

and

final

amphiphilic

triblock

copolymers

[PPLG-g-(CXB-peptide&mPEG)]-PEG-PCL (PCxbP) via multistep reactions are described in the Supporting Information section. Characterizations using 1H NMR, FTIR, UV-Vis and GPC confirmed the successful syntheses of the designed polymers (Figure 2a,b and Figure S1-S11, Supporting Information). The amphiphilic triblock copolymer was composed of a hydrophobic PCL block with a molecular weight of 4 kDa and a hydrophilic [PPLG-g-(CXB-peptide&mPEG)]-PEG diblock with a molecular weight of 42.1 kDa (Table S1, Supporting Information). The CXB content of [PPLG-g-(CXB-peptide&mPEG)]-PEG-PCL was 10.2 wt.%, and the PEGylation extent of the PPLG-g-(CXB-peptide&mPEG) block was 66.7%, corresponding to a PEG content of 47.5 wt.% in the final copolymer (Table S2, Supporting Information). Instead of the CXB-terminated peptides, sodium succinate (SS)-terminated peptides with or without MMP-2 sensitivity were used to prepare the carrier PPLG-g-(SS-peptide&mPEG)-PEG-PCL containing no CXB (Scheme S10, Supporting Information). Next, the amphiphilic triblock copolymers were self-assembled into micelles in aqueous solution, and PTX was encapsulated in the micelle core via hydrophobic interaction

with

PCL.

Thus,

MMP-2-sensitive

nanospheres

(SN)

and

MMP-2-insensitive nanospheres (InN) carrying a single drug or two drugs were obtained. As shown in Table S2 (Supporting Information), the CXB loading contents of SN-CXB and InN-CXB were 10.2% and 10.5%, respectively. Meanwhile, the PTX loading contents of SN-PTX, InN-PTX, SN-CXB/PTX, and InN-CXB/PTX were 8.9%, 9.2%, 8.6% and 9.0%, respectively. Because both PCL and PTX possess high hydrophobicity,35,

36

a strong hydrophobic interaction might have facilitated a 6


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relatively high PTX loading content and efficiency. Their particle sizes and zeta potentials

are

summarized

in

Table

S3

(Supporting

Information).

The

MMP-2-sensitive and MMP-2-insensitive micelles showed approximately the same particle size and zeta potential, implying that the amino acid sequence of the peptide linker had no effect on the copolymer self-assembly. Moreover, the copolymer showed a low critical micellization concentration (CMC) of 28.2 μg/mL (Figure S12a, Supporting Information), indicating high stability of the nanospheres against dilution in the bloodstream, which may be due to crystallization,36 chain entanglement and the high hydrophobicity of PCL. Under transmission electron microscopy (TEM), the MMP-2-sensitive micelle appeared to be a highly uniform nanosphere—i.e., with a spherical morphology and a nanosize of approximately 130 ± 4 nm (Figure 2c). The nanosphere size, as determined by TEM, was slightly smaller than that measured by dynamic light scattering (DLS) (130 ± 4 nm vs 140 ± 5 nm), likely because drying the sample for TEM measurement caused particle shrinkage. Unlike most core-shell structural polymeric micelles whose hydrophilic shells (e.g., PEG) can hardly be seen under TEM,2, 17, 37 the hydrophilic shell of our micellar nanosphere was highly manifested due to the incorporation of CXB sodium salt with high electron density. The core-shell-distinct nanospheres has a shell thickness of 28 ± 2 nm and a core diameter of 75 ± 3 nm according to TEM analysis. Acid-base titration showed that the sulfonamide structure (-SO2NHCO-) conjugating CXB possesses a pKa of 4.5, indicating that it was completely ionized to form CXB sodium salt (-SO2N-Na+CO-) at pH 7.4 (Figure S12b, Supporting Information). Consequently, the nanospheres showed a negative surface charge (=-14.8 ± 1.1 mV), as determined by zeta potential measurement (Figure 2e). The 7


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introduction of sodium succinate (SS) allowed the formation of CXB-free nanoparticles with shell properties (zeta potential and MMP-2 sensitivity) comparable to that of CXB-bearing nanoparticles. The pKa values of CXB sodium of CXB-bearing nanoparticles (SN-CXB) and sodium carboxylate of the CXB-free nanoparticles (SN) were very close (pKa 4.5 vs 4.8, Figure S12b, Supporting Information). Additionally, the same grafting densities of CXB sodium and carboxylate sodium endowed the SN-CXB/PTX and SN-PTX with similar zeta potentials (Table S3, Supporting Information). Nanoparticles with negative surface charges were found to show a long circulation time enhancing tumor accumulation in previous studies.31, 32, 38, 39 Indeed, the size of MMP-2-sensitive nanospheres in PBS containing 10% fetal bovine serum (FBS) or in mouse plasma remained almost unchanged over 48 h at 37 C, implying a high stability and no protein binding in the bloodstream (Figure S13a-d, Supporting Information). Moreover, Coomassie blue staining directly showed the anti-protein-fouling capacity of both SN-CXB/PTX and InN-CXB/PTX nanospheres grafted with PEG, while protein adsorption was clearly shown for positively charged PEI-PCL nanoparticles without PEG modification (Figure S13f, Supporting Information). According

to

TEM

measurement,

the

MMP-2-sensitive

nanospheres

(SN-CXB/PTX) no longer displayed the distinct core-shell structure under TEM because of CXB release (Figure 2d). Instead, uniform spheres with approximately the same core diameter (75 nm in diameter) prior to enzymatic digestion were observed. Although the enzymatically treated nanospheres should still possess a hydrophilic shell comprising the PPLG backbone, PEG graft chains and residual LAG peptide pendants, the shell of the nanospheres became unobservable under TEM owing to the

8


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loss of CXB sodium salts. Meanwhile, the zeta potential of nanospheres was switched to +11.5 mV from -14.8 mV (Figure 2f). HPLC eluograms analyzed using a UV-Vis detector at 254 nm further confirmed that the CXB-conjugated MMP-2-sensitive linker was cleavable by MMP-2. As shown in Figure 2g, the enzyme cleaved the CXB-conjugated GGPLGLAGG peptide in a time-dependent manner, causing a gradual decrease in peak intensity for the intact peptide and a gradual increase in the peak intensity for the CXB moiety cleaved-off peptide. By contrast, the peak intensity for CXB-conjugated GGLALGPGG peptide (negative control) showed not change at all against time, indicating that MMP-2 could not break the insensitive peptide linker. Consequently, both the particle size and zeta potential of the MMP-2-insensitive nanospheres (InN-CXB/PTX) in PBS (pH 7.4) were not affected by treatment with 10 nM MMP-2 (Table S3, Supporting Information). Drug Release in Vitro. Fluorescence resonance energy transfer (FRET) using fluorescein isothiocyanate (FITC) as a donor and rhodamine B (RhoB) as an acceptor was employed to verify the MMP-2-triggered release of CXB. The nanospheres for the FRET study in solution were assembled from the FITC and RhoB decorated copolymer shown in Scheme S9 (Supporting Information). MMP-2 cleavage of the peptide linking FITC/CXB will cause FITC release, thus terminating the FRET between FITC and RhoB.40 As shown in Figure 3a,c, after the nanospheres were excited at the maximum FITC absorption of 480 nm, not only FITC fluorescence emission at 520 nm but also strong fluorescence emission at 590 nm for RhoB was shown due to energy transfer between the closely spaced FRET molecular pair. After 10 nM MMP-2 was added, the SN-CXB/FITC/RhoB (MMP-2-sensitive) and InN-CXB/FITC/RhoB (MMP-2-insensitive) solutions excited at 480 nm showed very 9


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different behaviors in FITC and RhoB fluorescence emissions. Thus, the SN-CXB/FITC/RhoB solution showed a gradual increase in FITC fluorescence and a gradual decrease in RhoB fluorescence against the MMP-2 digestion time (Figure 3a), likely due to the loss of FRET caused by FITC release via peptide cleavage. At 4 h, the 480-nm light excitation only induced FITC fluorescence emission (also the strongest in this case) due to the complete loss of FRET from FITC to RhoB. Furthermore, along with the increase in the MMP-2 digestion time to 4 h from 0 h, DLS detected a gradual increase in the nanospheres zeta potential to +12.0 mV from -15.2 mV (Figure 3b), apparently because the FITC/CXB release increased the primary amino groups in the shell of the nanospheres. By contrast, both the FITC/RhoB fluorescence and zeta potential of InN-CXB/FITC/RhoB solution were not affected by MMP-2 digestion (Figure 3c,d) because MMP-2 was unable to cleave the insensitive peptide linker for FITC/CXB release; thus, FRET between FITC and RhoB exists all the time. Quantitative determination of CXB release from nanospheres in solution (pH 7.4) containing 10 nM MMP-2 showed consistent results. CXB release from InN-CXB/PTX was not detected regardless of whether the solution contained 10 nM MMP-2. By contrast, CXB release from SN-CXB/PTX and SN-CXB appeared much different (Figure 3e and Figure S14a, Supporting Information). CXB release from SN-CXB/PTX and SN-CXB was not detected when MMP-2 was absent in the solution. However, CXB release was rapid in solution containing 10 nM MMP-2, reaching a plateau of approximately 88% cumulative release for SN-CXB/PTX within just 8 h (Figure 3e). Thus, 8 h was regarded as the time for the nanospheres to complete CXB release. It was noted that the time to complete CXB release seemed longer than that needed to complete surface charge switching upon MMP-2 digestion 10


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(Figure 3b), likely because it took extra time for CXB to diffuse out of the dialysis bag. The MMP-2-triggered CXB release from SN-CXB/PTX was in line with the results of DLS, TEM and HPLC analyses (Figure 2c-g). Because the MMP-2 concentration is below 0.5 nM in serum,41, 42 the CXB release from nanosphere was evaluated in serum and buffered solution containing 0.5 nM MMP-2. As shown in Figure 3e, CXB release was less than 10% even over a prolonged experimental time of 48 h, in both serum and solution containing 0.5 nM MMP-2, suggesting negligible early release of CXB into the bloodstream. In addition, only approximately 22% of CXB was released from SN-CXB/PTX after incubating with liver homogenate over 48 h, indicating low levels of nonspecific cleavage and unwanted drug release in normal organs as compared to the significantly accelerated release at 10 nM MMP-2. The release of PTX from nanospheres with prereleased CXB was then investigated (Figure 3f). At pH 5.0, mimicking the pH value of lysosomal compartments where PTX was expected to be released, 43% of PTX that were encapsulated into the PCL core of nanospheres were released within 48 h. Considering that lipase that may degrade PCL is abundant inside lysosomes,43 we further examined the PTX release behavior in nanospheres solutions containing lipase. Excitingly, PTX release was indeed accelerated obviously in this case, reaching the cumulative release of 87% at 0.1 mg/mL of lipase or 96% at 0.5 mg/mL of lipase (Figure 3f). The PTX release profile from nanospheres without prereleasing CXB was very similar to that from the CXB-released one (Figure S14b, Supporting Information), indicating that CXB in the shell had little effect on PTX release. Additionally, PTX release was accelerated as the pH decreased, likely due to easier hydrolysis of PCL at lower pH.36 However, at pH 6.5 of the tumor extracellular microenvironment,44, 45 the cumulative release of PTX at 10 nM MMP-2 only reached 11


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7.8% at 12 h and 12.5% at 48 h (Figure S14b, Supporting Information). Because the nanospheres could accomplish surface charge switching within 4 h in the presence of 10 nM MMP-2 (Figure 3a,b) and may be easily taken up thereafter by HT-1080 cells as will be discussed, it implied that significant extracellular PTX leakage would not occur in the tumor microenvironment. Inhibition of PGE2 Secretion in Multiple Cell Types via MMP-2-Triggered CXB Release. The PGE2 was reported to induce chemo-resistance and promote proliferation through antiapoptotic protein Bcl-2, EP2 and EP4 signaling cascade.9, 46 As PGE2 could be secreted not only by cancer cells (autocrine) but also by other cell types (paracrine) inside the inflammatory microenvironment of tumors, the effect of adscititious PGE2 on the proliferation of HT-1080 cancer cells was evaluated. As shown in Figure S15a (Supporting Information), the viabilities of HT-1080 cancer cells were increased in a PGE2 concentration-dependent manner until saturation at a PGE2 concentration of 44 ng/mL. Additionally, adding PGE2 into the cell culture medium elevated the expression levels of cell proliferation marker Ki-67 and antiapoptotic protein Bcl-2 in cancer cells (Figure S15b,c, Supporting Information). These results demonstrated that PGE2 was closely associated with HT-1080 cell proliferation, and it may exert a cytoprotective effect on cancer cells to counteract the chemotherapeutic effect of PTX. Obviously, PGE2 secretion, whether autocrine or paracrine, should be inhibited to make HT-1080 cancer cells sensitive to chemotherapy. Free CXB has been reported to inhibit PGE2 production by binding to the catalytic site of COX-2.8 To visualize the CXB binding to COX-2 and distribution inside cells under CLSM observation, the distal end of the peptide moiety-tailed CXB (CXB-GGPLG) as the exact molecular structure after MMP-2 cleavage was 12


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functionalized with the fluorescent dye 7-nitrobenzofurazan (NBD) (Scheme S7, Supporting Information). As shown in Figure S16a (Supporting Information), Coomassie blue staining of COX-2 and fluorescence imaging of the CXB-peptide moiety indicated the binding of released CXB to COX-2 protein. The released CXB-containing entity showed a dose-dependent inhibitory effect on the COX-2 catalytic activity, with a IC50 value of 30.1 ng/mL (calculated based on the CXB pharmacophore; Figure S16b, Supporting Information) that was only slightly higher than that of free CXB (15.2 ng/mL).47 Additionally, as shown in Figure 4a, the NBD-labeled CXB effectively entered not only the HT-1080 cancer cells but also the LPS-stimulated RAW 264.7 and LPS-stimulated L-929 cells as analogs of major stromal cells involved in the COX-2/PGE2 pathway in the inflammatory tumor microenvironment.20,

48

Moreover, the fluorescence distribution pattern of CXB

seemed identical to that of COX-2, indicating that the released CXB may effectively bind COX-2 inside cells to block its catalytic activity in PGE2 synthesis. Next, PGE2 secretion was analyzed in multiple cell types incubated with different nanospheres. As shown in Figure S17c (Supporting Information), SN-PTX treatment was further elevated whereas SN-CXB and SN-CXB/PTX treatments significantly decreased the PGE2 secretion in HT-1080 cells. By contrast, neither the InN-CXB and InN-CXB/PTX treatments suppressed nor the InN-PTX treatment upregulated the COX-2 protein expression and PGE2 secretion effectively, a finding that was reasonable because the MMP-2-insensitive nanospheres could not release CXB for a surface charge switching to enhance their cell uptake. Using SN-CXB nanospheres as an example, we further confirmed that PGE2 secretion in all three cell types was effectively inhibited in a CXB concentration-dependent manner. At CXB concentrations above 1.9 g/mL, the PGE2 secretions of three cell types were all 13


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decreased down to or below their basal levels (Figure 4b). Besides binding to the COX-2, high dose celecoxib was reported to downregulate the mRNA and protein levels of COX-2 via the inhibition of the NF- κ B, a master transcriptional regulator involving in the regulation of COX-2 gene expression.49,

50

Therefore, the COX-2

protein expression of cells was analyzed to gain more comprehensive insight into the nanosphere-inhibited PGE2 secretion. The SN-PTX incubation upregulated, whereas the SN-CXB or SN-CXB/PTX incubation obviously downregulated the expression of COX-2 protein in HT-1080 cells (Figure S17c, Supporting Information). In line with PGE2 secretion, the COX-2 protein levels in the three cell types treated with SN-CXB were effectively suppressed, especially at high CXB concentrations (Figure 4c). Based on the above results, CXB released from the MMP-2-sensitive nanospheres may effectively inhibit the PGE2 synthesis of multiple cell types in the tumor inflammatory microenvironment not only through blocking the COX-2 catalytic activity but also via downregulating COX-2 expression. These results highlighted the significance of MMP-2-sensitive CXB release to act on multiple cell types in the tumor inflammatory microenvironment. Synergistic Anticancer Effect of CXB and PTX in Vitro. Enhanced PTX delivery via surface charge switching of the nanosphere was confirmed in HT-1080 cancer cells overexpressing MMP-2.41,

51

To visualize the cellular uptake and

intracellular distribution of nanospheres under CLSM, the fluorescent dye Nile red (NR) replacing PTX was loaded into the nanospheres. As shown in Figure S18 (Supporting Information), the intracellular trafficking results using CLSM showed that the CXB-released and charge-switched SN-CXB/NR nanosphere was internalized into lysosomes of cancer cells. Moreover, the cells incubated with SN-CXB/NR showed strong NR fluorescence, indicating efficient cellular uptake of the 14


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nanospheres (Figure 5a). In comparison, cells incubated with InN-CXB/NR showed very weak NR fluorescence due to much lower cellular uptake of nanospheres. Furthermore, quantitative flow cytometric assay verified that almost all HT-1080 cells became NR fluorescence-positive upon incubation with SN-CXB/NR, whereas only 62.9% of the cells incubated with InN-CXB/NR became NR fluorescence positive. It is well-known that a positively charged surface may facilitate the endocytosis of nanoparticles via electrostatic interaction with negatively charged cell membranes.31, 38

Obviously, cleavage of the peptide linker by MMP-2 inside tumor caused not only

the extracellular release of CXB but also a negative-to-positive surface charge switching to promote the cellular uptake of nanospheres encapsulating PTX (here Nile red as an analogue). By contrast, because the surface charge switching did not occur for InN-CXB/NR bearing MMP-2-insensitive peptide as the CXB linker, CXB could not be released; thus, the always-negative nanospheres could hardly deliver PTX into cancer cells. These results further rationalized the structural design of our MMP-2-sensitive nanospheres for the sequential release of two drugs on demand—i.e., the 1st-stage extracellular CXB release to act on multiple cell types involved in the COX-2/PGE2 pathway and 2nd-stage PTX release inside cancer cells to exert a chemotherapeutic effect. The joint effect of CXB and PTX on cancer cell growth was then evaluated. The HT-1080 cells incubated with drug-free nanospheres at a high concentration of 500 g/mL still showed a viability above 95%, indicating low cytotoxicity of the carrier itself (Figure S19, Supporting Information). Owing to the effective CXB release triggered by MMP-2, SN-CXB behaved more cytotoxic than InN-CXB especially at high CXB concentrations (e.g., 35 μg/mL) (Figure S20, Supporting Information), a finding that is consistent with previous report that CXB at a high concentration could 15


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inhibit cell cycle stagnation and induce apoptosis.52 At low CXB concentrations such as 1.9 μg/mL, even SN-CXB exhibited little effect on the viability of HT-1080 cells, a finding that is also in line with previous report that CXB at low concentrations inhibited PGE2 production but had a minor inhibitory effect on cancer cell growth.53 SN-PTX appeared much more cytotoxic than InN-PTX because of more effective PTX delivery into HT-1080 cells via MMP-2-triggered surface charge switching (Figure 5b). The IC50 values of PTX were 83.6 and 152.3 ng/mL for SN-PTX and SN-PTX+PGE2, respectively. The decrease in cytotoxicity of PTX was attributed to the PGE2-induced drug resistance. However, the IC50 value of PTX was further decreased to 33.8 ng/mL for SN-CXB/PTX—i.e., SN-CXB/PTX behaved more efficiently in inhibiting HT-1080 cell growth than SN-PTX at low PTX concentrations below 250 ng/mL. Considering that the CXB concentration in this case was low and could not affect cell growth as mentioned above, the higher cytotoxicity of SN-CXB/PTX at low PTX concentrations implied that the released CXB acted on the COX-2/PGE2 pathway to inhibit PGE2 synthesis and thus suppressing PGE2-induced drug resistance to sensitize the HT-1080 cells to PTX treatment. The values of combination index (CI) were less than 1 at CXB concentration of 1.9 μg/mL and PTX concentrations below 300 ng/mL (Figure S21, Supporting Information), clearly demonstrating the synergistic anticancer effect.54 By contrast, InN-CXB/PTX without surface charge switching ability showed cytotoxicity even lower than that of SN-PTX due to its poor cell uptake. Flow cytometric analysis in cell apoptosis obtained consistent results (Figure 5c). The HT-1080 cells incubated with various nanospheres at the same CXB concentration of 1.9 g/mL displayed different apoptosis levels. Due to the low CXB concentration, SN-CXB only induced 6.7% cell apoptosis that was slightly higher 16


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than that induced by InN-CXB (2.1%). Moreover, MMP-2 sensitivity was critical for the PTX-loaded nanospheres to induce cell apoptosis. Due to the poor cell uptake, InN-PTX and InN-CXB/PTX only induced 9.7% and 6.4% cell apoptosis, respectively. By comparison, SN-PTX and SN-CXB/PTX appeared more potent in inducing cell apoptosis, owing to the effective PTX delivery into HT-1080 cells upon the MMP-2-triggered surface charge switching of nanospheres. Moreover, the cell apoptosis level (71.4%) induced by SN-CXB/PTX was even significantly higher than the sum (45.8%) induced by SN-CXB (6.7%) and SN-PTX (39.1%), clearly indicating a synergistic anticancer effect of PTX and CXB codelivered by the MMP-2-sensitive nanospheres. Western blotting assay is highly supportive of a synergistic anticancer effect of PTX and CXB (Figure 5d). When the MMP-2-sensitive nanospheres were employed to mediate therapy, PTX treatment not only induced cell apoptosis according to the upregulated expression of cleaved Caspase-3 but also activated the COX-2/PGE2 pathway and anti-apoptosis Bcl-2 gene to counteract the therapeutic effect (Figure 5d and Figure S17, Supporting Information). However, the protein expression of Bcl-2 was suppressed by CXB treatment because CXB acted on the COX-2/PGE2 pathway to inhibit PGE2 synthesis of HT-1080 cells while PGE2 was found to upregulate Bcl-2 protein expression (Figure 4b and Figure S15c, Supporting Information). Furthermore, HT-1080 cells were incubated with SN-CXB/PTX at various concentrations of exogenous PGE2 to mimic the treatment in the tumor inflammatory microenvironments. As shown in Figure S22 (Supporting Information), the apoptotic rate of HT-1080 cells showed a negligible decrease in cell apoptosis at a low concentration of exogenous PGE2 (e.g., 4.4 ng/mL). However, it was decreased obviously at high concentration of exogenous PGE2. For example, the addition of 44 17


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ng/mL of PGE2 significantly decreased the cell apoptotic rate to 48.7% from 71.4%. These results provided direct evidence that PGE2 synthesis inside tumors should be suppressed to achieve a synergistic anticancer effect of CXB and PTX, requiring the carrier systems to provide on-demand sequential drug release to act on multiple cell types. Excitingly, the in vitro results underlined the great potential of our core-shell-distinct nanospheres (SN-CXB/PTX) to do so. In Vivo Drug Delivery. Fluorescence imaging was performed to monitor the distribution and tumor accumulation of nanospheres after intravenous (IV) injection into nude mice bearing subcutaneous HT-1080 tumor xenografts. The near-infrared fluorescent dye DIR instead of PTX was loaded into the nanospheres to enable fluorescence imaging. As shown in Figure 6a, the surface charge-switchable nanospheres

(SN-CXB/DIR)

showed

better

tumor

accumulation

than

the

always-negative one (InN-CXB/DIR) after tail vein injection. At 14 h after injection, the animals receiving SN-CXB/DIR and animals receiving InN-CXB/DIR both showed strong DIR fluorescence at tumor sites, indicating the efficient anti-biofouling capacity of nanospheres could facilitate good tumor accumulation via the enhanced permeability and retention (EPR) effect.55 Nevertheless, animals receiving SN-CXB/DIR showed much slower attenuation of tumor fluorescence than animals receiving

InN-CXB/DIR,

indicating

much

better

tumor

retention

of

the

MMP-2-sensitive nanospheres via surface charge switching to enhance the interaction with cancer cells. The ex vivo fluorescence imaging for major organs and tumors from mice sacrificed at 48 h after nanospheres injection showed consistent results (Figure 6b). The tumor fluorescence intensities (FI) of mice receiving SN-CXB/DIR was 3.0 times higher than that of mice receiving InN-CXB/DIR. Additionally, the biodistribution of PTX in tumor were 24.5 ± 4.3% and 7.1 ± 2.5% ID/g for 18


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SN-CXB/PTX and InN-CXB/PTX, respectively, once again demonstrating much better tumor-targeting effect of the MMP-2-sensitive nanosphere (Figure S23, Supporting Information). CLSM observation of the distribution of drug analogs on frozen sections further showed the MMP-2 responsiveness of nanospheres inside the tumor. Mice bearing subcutaneous HT-1080 tumor were tail vein injected with nanospheres that conjugated CXB-mimicking FITC through peptide linker and Rhodamine B (RhoB) through stable 1,2,3-triazole bond (Figure 6c and Scheme S9, Supporting Information). As shown in Figure 6d, because MMP-2-insensitive nanospheres without charge switching may not be effectively endocytosed to enhance tumor retention, RhoB fluorescence and FITC fluorescence were both weak on tumor sections of animals receiving InN-CXB/FITC/RhoB. By contrast, both the RhoB fluorescence and FITC fluorescence were obviously intensified on tumor sections of animals receiving SN-CXB/FITC/RhoB. Furthermore, when InN-CXB/FITC/RhoB was injected, the fluorescence distribution of FITC seemed identical to that of RhoB on tumor sections, implying that FITC was not released from nanospheres due to the MMP-2-insensitive peptide linkage. However, when SN-CXB/FITC/RhoB was injected, the fluorescence distribution of FITC appeared obviously different from that of RhoB on tumor sections. In this event, the MMP-2-sensitive peptide was cleaved, allowing FITC to be released and diffuse away from the nanospheres. Furthermore, the intratumoral fluorescence distribution of Nile red (NR) mimicking PTX released from SN-CXB/FITC/NR/AF647 after tail vein injection was studied. CLSM observation showed obvious separation of fluorescence between FITC mimicking CXB and Alexa Fluor® 647 (AF647) labeling nanospheres at 3 h (Figure S24, Supporting Information), indicating a large amount of CXB release. Notablely, at 3 h, 19


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the fluorescece of NR mimicking PTX still overlaped mostly with that of AF647, implying rare leakage of PTX from the nanospheres. At 6 h, partial release of NR was shown. Moreover, an intracellular NR release might be the primary cause in this case because positively charged nanospheres (i.e., nanospheres after CXB release) entered cancer cells easily and lysosomal release of NR would follow. It is worth noting that, according to the results introduced in the previous sections, the earlier released CXB may act on multiple cell types to enhance the chemotherapeutic effect of PTX delivered into cancer cells. In Vivo Synergistic Anti-cancer Activity. Animal study in nude mice bearing HT-1080 tumor was performed to explore whether a synergistic anticancer effect could be achieved in vivo. As shown in Figure 7a,b, measurements on the tumor size and survival rate indicated that SN-CXB/PTX exhibited the best therapeutic effect among the five experimental animal groups. Although all treatments with nanospheres loading one or two drugs showed clear therapeutic effects to inhibit tumor growth, only the treatment with SN-CXB/PTX completely inhibited the tumor growth of animals. At 35 d after the first treatment, the tumor volume in this group was 69.4 ± 28.9 mm3, representing even a 54.5% shrinkage upon the treatment. In comparison, the tumors dramatically grew to 1595.1 ± 110.4 mm3, 1161.1 ± 102.5 mm3, and 863.6 ± 30.8 mm3 for the SN-CXB, InN-CXB/PTX and SN-PTX treatment groups, respectively. In line with the data of tumor growth inhibition, SN-CXB/PTX treatment also showed the best outcome in prolonging animal survival (Figure 7b). In this treatment group, 50% of animals survived longer than 66 d and 10% animals survived for 87 d. However, no animal survived longer than 60 d in other treatment groups. Particularly, no animal survived longer than 44 d in the animal group receiving PBS. Moreover, mice receiving SN-CXB/PTX exhibited a stable body 20


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weight, whereas animals groups receiving other treatments all showed an obvious decrease in body weight at 35 d after the first treatment (Figure 7c). Histological and immunohistochemical analyses of tumor sections further confirmed the synergistic action of PTX and CXB delivered by the MMP-2-sensitive nanospheres. As shown in Figure 7d, animals receiving PBS showed the most hypercellular tumor tissue with obvious nuclear polymorphism and negligible apoptosis. By contrast, animals receiving SN-CXB, SN-PTX or InN-CXB/PTX showed medium levels of cancer cell density and apoptosis. Moreover, animals receiving SN-CXB/PTX showed the fewest cancer cells and highest apoptosis level. Free CXB for anti-inflammation was orally administered every day at a dose of 200 mg (roughly 3 mg/kg). Although the per IV injection dose (5 mg/kg) in our study was higher than the routine dose of orally administered CXB, the interval between two consecutive injections in our study was longer (i.e., 4 d), which was supposed to reduce stress to mice. The drug-loaded nanospheres neither altered the liver/renal serum function markers nor caused structural damage to major organs, indicating their low side effects to major organs (Figure 7e, f). Additionally, compared with the PBS group, the haematological parameters in the four treated groups were not statistically significant (P value > 0.05), indicating low myelotoxicity (Table S5, Supporting Information). Quantitative analyses of the PGE2, COX-2, Bcl-2, and cleaved Caspase-3 levels in the tumor tissues of mice receiving treatments of various nanospheres were performed to further explore the synergistic action of PTX and CXB in vivo (Figure 8a-c and Figure S25, Supporting Information). According to the expression levels of cleaved Caspase-3 indicating apoptosis, SN-PTX treatment could induce considerable tumor apoptosis, whereas such an effect could be detracted by the upregulated expression of anti-apoptotic Bcl-2 protein accompanying the chemotherapy-activated 21


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COX-2/PGE2 pathway. These results were in line with the aforementioned results that adscititious PGE2 inhibited cancer cell apoptosis and upregulated the expression of anti-apoptotic Bcl-2 protein, thus leading to chemotherapy resistance of the tumor. Excitingly, SN-CXB treatment behaved highly effectively in suppressing the COX-2/PGE2 pathway and Bcl-2 expression in vivo. Consequently, when CXB and PTX were codelivered by the MMP-2-sensitive nanospheres, both the COX-2/PGE2 pathway and Bcl-2 expression were effectively suppressed, leading to the highest level of tumor apoptosis. By contrast, when the two drugs were codelivered with the MMP-2-insensitive nanospheres, a much lower level of tumor apoptosis was detected because CXB was not effectively released to exert its effect. Obviously, the good anticancer efficacy of SN-CXB/PTX in vivo was owing to the synergistic effect of PTX and CXB, which was achieved based on the bistaged sequential drug release enabling multiple cells targeting inside tumor. Furthermore, the surface charge switching of nanosphere was also important because it enhanced PTX delivery into cancer cells. Anti-inflammatory drugs can treat cancer-related complications such as fever and pain, and may reduce the dosage as well as enhance the anticancer efficacy of chemotherapy.10,

12, 56

In this study, inhibition of COX-2/PGE2 pathway by the

celecoxib, a NSAIDs, enhanced the anticancer effect of chemotherapeutic PTX in xenograft HT-1080 tumor. However, caution is needed when using different types of anti-inflammatory

drugs

which

may

decrease

the

anticancer

effect

of

chemotherapeutic agents. For example, Obradović et al found that dexamethasone and triamcinolone acetonide as glucocorticoid anti-inflammatory drugs promoted breast cancer metastasis, reduced the overall survival rates of the mice and decreased xenograft cancer response to PTX.57 22


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CONCLUSION A serum-stable and MMP-2-sensitive nanosphere possessing a distinct core-shell structure was developed for the tumor-targeted codelivery of anti-inflammatory CXB and chemotherapeutic PTX. This nanodrug demonstrated great potential to provide on-demand sequential drug release allowing the targeting of multiple cell types inside the tumor, which is essential to achieve a synergistic anticancer effect of the two drugs. After the nanospheres entered the tumor tissue, the enriched MMP-2 therein triggered CXB release to turn the negatively charged nanosphere into a positively charged one. Consequently, the released CXB acted on multiple cell types to suppress their secretion of the inflammatory mediator PGE2, which promotes tumor growth, and then the residual positive nanosphere carrying PTX was readily taken up by cancer cells to exert chemotherapeutic effect. In addition, the expression of anti-apoptotic Bcl-2 protein in cancer cells was suppressed by CXB therapy as well, which sensitized the cancer cells to chemotherapy. Consequently, the core-shell distinct nanodrug exhibited a good therapeutic outcome in an animal study—i.e., a 54.5% shrinkage of HT-1080 tumor being achieved. Theoretically, this surface charge-switchable drug delivery system may be applied in most types of solid tumors overexpressing MMP-2. Our study provides a proof of concept to design nanocarrier with on-demand sequential drug release and surface charge switching inside tumor to realize highly effective synergistic anticancer therapy.

EXPERIMENTAL SECTION Synthesis and Characterization of Triblock Copolymers. CXB-bearing and CXB-free triblock copolymers with or without MMP-2 sensitivity were synthesized via multiple reactions (Scheme S1-S10, Supporting Information). The structure of 23


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[PPLG-g-(FITC/CXB-peptide&mPEG&RhoB)]-PEG-PCL

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carrying

FITC

and

Rhodamine B is shown in Scheme S9 (Supporting Information). Polymer compositions were characterized by 1H NMR, FTIR and GPC analyses. The critical micellization concentrations (CMCs) of the final copolymer and pKa values of the nanospheres were detected using pyrene as a fluorescent probe and acid-base titration, respectively. Details for these experiments were provided in Supporting Information. Preparation of Drug-Loaded Nanospheres. The MMP-2-sensitive nanospheres (SN) and MMP-2-insensitive nanospheres (InN) were prepared using four triblock copolymers containing MMP-2 sensitive and insensitive peptides, respectively. Briefly, 15 mg of copolymer and 1.7 mg of paclitaxel were dissolved in a 6 mL mixture of DMF and THF (1/5, V/V). The solution was added into deionized water under sonication (60 Sonic Dismembrator; Fisher Scientific), dialyzed (MWCO: 14 kDa) against deionic water for 1 d, concentrated to 4 mL by ultrafiltration, filtered through a 220 nm syringe filter to remove aggregates of nonencapsulated paclitaxel, and stored at 4 oC for further experiments. The prepared nanospheres with PTX loading include SN-PTX, SN-CXB/PTX, InN-PTX and InN-CXB/PTX, respectively. The PTX-free nanosphere was prepared via the same procedure except that PTX was not used. The prepared PTX-free nanospheres included SN-CXB and InN-CXB. To study cellular uptake, the Nile red (NR) fluorescent molecule as a model drug replacing paclitaxel was loaded into the nanospheres using the same method described above. SN-CXB/NR and InN-CXB/NR were prepared and the loading content of NR was determined by fluorescence spectroscopy. The emission spectrum of Nile red was measured at the excitation wavelength of 570 nm in DMSO. Similarly, the nanospheres self-assemblied from triblock copolymer with FITC and Rhodamine B

24


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labeling were abbreviated as SN-CXB/FITC/RhoB and InN-CXB/FITC/RhoB, respectively. Characterization of Nanospheres. High-performance liquid chromatography (HPLC) was performed to determine the drug-loading contents as well as efficiency, in vitro drug release and inhibitory effect of the released CXB on COX-2 catalytic activity. The stabilities and anti-biofouling capacities of nanospheres were detected by DLS and SDS-PAGE, respectively. Fluorescence resonance energy transfer (FRET) was used to characterize MMP-2 sensitivity. Details for these experiments were provided in Supporting Information. Cell Assays. Cellular uptake, cell viability, apoptosis, Ki-67 immunofluorescence, and Bcl-2 and Caspase-3 protein expressions were determined in HT-1080 cells. Analyses of the PGE2 level, COX-2 binding, and COX-2 mRNA and protein expression were performed on three cell types, HT-1080 cells, RAW 264.7 cells and L-929 cells. Details for these experiments were provided in Supporting Information. Animal Assays. In vivo and ex vivo fluorescence imaging, the anticancer effect, the biochemistry index and histology analyses were determined in mice bearing HT-1080 tumors. Drug distributions, in situ TUNEL assay, and gene and protein expression analyses were performed on excised tumor tissue. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of the Sun Yat-sen University. Details for these experiments were provided in Supporting Information. Statistical Analysis. Data are expressed as the means ± standard deviation (SD), and statistical analysis of two-sided data was performed using ANOVA with a post-hoc test. P < 0.05 was considered to be of statistical significance.

25


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Syntheses and characterizations of compounds and polymers, biological experiments; 1H

supporting Figure S1-S25:

NMR spectra, FTIR spectra, GPC curves,

determination of CMC, acid-base titration curves, stabilities and anti-protein-fouling capacities

of

nanospheres,

in

vitro

drug

release,

cell

viabilities,

Ki-67

immunostaining, COX-2 binding and inhibitory effect of the released CXB, expressions of Bcl-2 and COX-2, intracellular distribution of nanospheres, combination index (CI), cell apoptosis, biodistribution of PTX, intratumoral drug distribution and relative protein expression level; supporting Table S1-S5: characteristics of the synthesized polymers and the as-prepared nanospheres, particle sizes and zeta potentials of nanospheres, primer sequences for RT-PCR and haematological data of mice.

AUTHOR INFORMATION Corresponding Author * Prof. Xintao Shuai, Email: [email protected]. Tel.: +86-20-84110365 ORCID Jinsheng Huang: 0000-0002-6371-4532; Du Cheng: 0000-0001-5105-7777; Yu Guo：0000-0002-8886-0106; Xintao Shuai: 0000-0003-4271-0310 ACKNOWLEDGEMENTS This research was supported by the National Natural Science Foundation of China 26


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(U1401242), the National Basic Research Program of China (2015CB755500), the Natural Science Foundation of Guangdong Province (2014A0303120182), the Guangdong Innovative and Entrepreneurial Research Team Program (2013S086), the Fundamental Research Funds for the Central Universities (17lgjc01).

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Figure Legends

Figure 1. Preparation and in vivo performance of MMP-2-sensitive nanospheres carrying anti-inflammatory CXB and chemotherapeutic PTX (SN-CXB/PTX). Sequential drug release and surface charge-switching to act on multiple cell types inside the tumor was expected.

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Figure 2. Characterizations of the polymer and nanosphere. (a) Prepolymers and CXB-conjugated peptide for the click reaction. (b) Final copolymer for nanosphere preparation. (c) Transmission electron microscopy (TEM) imaging showing the distinct core-shell structure of nanosphere before MMP-2-triggered CXB release, because the sodium salt of CXB increased the electron density of shell for high TEM visibility. (d) TEM only observed the core of nanosphere after CXB release, due to the loss of sodium salt in the shell. (e) Nanosphere were negatively charged before MMP-2-triggered CXB release but (f) positively charged after CXB release according to zeta potential measurement. (g) HPLC traces of the CXB-conjugated peptide with 10 nM MMP-2 treatment at different times in TCNB solution (pH 7.4). The MMP-2-sensitive peptide (peak I) was cleaved gradually by MMP-2 to release CXB-GGPLG (peak II), whereas the MMP-2-insensitive peptide (peak III) remained intact to show one peak was unaffected by MMP-2. Scale bars in TEM images represent 100 nm.

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Figure 3. FRET, zeta potential and drug release analyses of nanospheres in vitro. (a) Fluorescence spectra and (b) surface charges of MMP-2-sensitive nanospheres (SN-CXB/FITC/RhoB) in solution containing 10 nM MMP-2. The MMP-2 was set at 10 nM to mimic the tumor microenvironment. (c) Fluorescence spectra and (d) surface charges of MMP-2-insensitive nanospheres (InN-CXB/FITC/RhoB) in solution containing 10 nM MMP-2. (e) CXB release of nanospheres in mouse serum, liver homogenate, and pH 7.4 solutions with or without MMP-2 (means ± SD, n = 3). (f) PTX release of MMP-2-sensitive nanospheres (SN-CXB/PTX) after CXB release at pH 5.0 and different lipase concentrations mimicking lysosomal conditions (means ± SD, n = 3).

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Figure 4. CXB released from MMP-2-sensitive nanospheres (SN-CXB) acts on multiple cells to inhibit PGE2 secretion. (a) Confocal images showed the colocalization of CXB and COX-2 inside HT-1080 cancer cells, LPS-stimulated RAW 264.7 macrophages and LPS-stimulated L-929 fibroblasts, indicating the binding of released CXB to COX-2 protein. The CXB-peptide moiety (CXB-GGPLG) cleaved off nanosphere were labeled with the 7-nitrobenzofurazan (NBD) fluorophore (green fluorescence), and COX-2 was labeled with Cy-3 (red fluorescence) by immunostaining. Scale bars represent 25 μm. (b) ELISA indicated that the PGE2 secretion of HT-1080 cancer cells, LPS-stimulated RAW 264.7 macrophages and L-929 fibroblasts were decreased by the treatment with MMP-2-sensitive nanospheres in a dose-dependent manner. A decrease in PGE2 secretion in the three cell types was owing to the inhibition of COX-2 activity and COX-2 expression by CXB released from MMP-2-sensitive nanospheres (SN-CXB). The Data are expressed as the means ± SD (n=3). *P

Core-Shell-Distinct Nanodrug Showing On ... - ACS Publications

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