Targeting and microenvironment-responsive lipid nanocarrier for the enhancement of tumor cell recognition and therapeutic efficiency.

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Targeting and Microenvironment-Responsive Lipid Nanocarrier for the Enhancement of Tumor Cell Recognition and Therapeutic Efficiency Wei Gao, Tingting Meng, Nianqiu Shi, Hongmeng Zhuang, Zhenzhen Yang, and Xianrong Qi* Although cell-penetrating peptides (CPPs) such as TAT and polyarginine have been shown to promote intracellular delivery of bioactive molecules with low membrane permeability, the majority of known CPPs are not cell or tissue specific; they come in contact with and are internalized by cells via heparan sulfates and other glycosaminoglycans in nearly all cell types in vitro and in vivo.[16,17] Macropinocytosis, receptor-mediated endocytosis and direct membrane penetration have all been determined to play a role for CPPs uptake. The nonspecific binding of CPPs can lead to a dramatic reduction in delivery to target cells. To overcome this dilemma, we introduced tumor microenvironment-sensitive polypeptide (TMSP)-decorated nanocarriers based on the sensitivity to locally overexpressed proteases[18–20] to generate an “on-off” switch of CPP activity. Several groups, including our own have experimented with the strategy in tumor targeted imaging and therapy.[21–25] The ability to modify and directly target nanocarriers has greatly increased their applicability in diagnostic and therapeutic studies. Folate receptor (FR) is overexpressed by many primary and metastatic cancers, while its expression is highly restricted in normal cells.[26,27] Folic acid is recognized by FR on specific types of cancer cell surfaces, which subsequently induce the cellular uptake of the ligand-decorated nanocarriers via receptor-mediated endocytosis.[28,29] So, the nanocarriers were simultaneous modified with folic acid to enhance the selectivity and internalization of the nanocarriers into tumor cells. We anticipate that the folic acid moiety binds quickly to the FR-positive tumors, and the TMSP moiety is cleaved by MMP-2 enriched in tumor tissues, and then the nanocarriers system turned to be modified with folic acid and CPP (Figure 1). Subsequent cellular uptake is triggered not only by the specific interaction between the ligands and target molecules but also by the uptake mediated by the CPP penetration. The specificity and efficacy of the folic acid and TMSP co-modified nanocarriers to tumor tissue and cancer cells were investigated in KB cells (high expression of FR), HT-1080 cells (oversecretion MMP-2) and A549 cells (negative FR and MMP-2 expression) in vitro, respectively. Furthermore, the pharmacokinetics,

Poor recognition and penetration of chemotherapeutic agents in solid tumors have been recognized as one of the major challenges limiting the efficacy of cancer therapies. Folic acid and tumor microenvironment-sensitive polypeptide (TMSP) co-modified lipid-nanocarrier (F/TMSP-NLC) are successfully formulated in response to the overexpression of folate receptor (FR) and the upregulation of matrix metalloproteinase-2 (MMP-2) in tumor microenvironment. The F/TMSP-NLC accumulates in tumor via the enhanced permeability and retention (EPR) effect, and folate moiety binds selectively to the FR once it reaches the tumor. In addition, cell-penetrating peptide (CPP)-penetrating activity is initiated by MMP-2 protease-oversecretion tumor. The specificity and efficacy of the co-modified nanocarriers to tumor are investigated in KB, HT-1080 and A549 cells in vitro. Multivalent interactions induce the enhancement of cancer cell recognition and internalization, which subsequently result in cancer cell apoptosis or death. The F/TMSP-NLC shows long-circulation effect, high accumulation in tumor, strong tumor inhibition, increased apoptotic indices, and negligible toxicity in vivo. In conclusion, the present nanocarrier modified with both TMSP and folic acid is a potential drug delivery system for tumor cell recognition and therapy, implying that using more than one target from the pool of tumor–stroma interactions is profoundly beneficial to therapeutic approaches.

1. Introduction Several conventional nanocarriers, e.g., liposomes,[1–4] drug–polymer conjugates,[5,6] polymeric nanoparticles,[7–9] and polymeric micelles[10–12] have drawn significant attention for the delivery of chemotherapeutic drugs to targeted sites. Stable, long-circulating nanocarriers can accumulate preferentially within a tumor via an enhanced permeability and retention (EPR) effect, resulting in tumor-specific delivery of the encapsulated drug.[13,14] However, poor penetration of chemotherapeutic agents in solid tumors has been recognized as one of the major challenges limiting the efficacy of nanocarrier cancer therapies.[15] Dr. W. Gao, T. Meng, Dr. N. Shi, H. Zhuang, Dr. Z. Yang, Prof. X. Qi State Key Laboratory of Natural and Biomimetic Drugs School of Pharmaceutical Sciences Peking University Beijing 100191, P. R China E-mail: [email protected]

DOI: 10.1002/adhm.201400675

Adv. Healthcare Mater. 2014, DOI: 10.1002/adhm.201400675

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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Figure 1. Schematic of targeting and microenvironment-responsive lipid nanocarrier and its multivalent interactions with cancer cells.

biodistribution, antitumor efficacy, and toxicity after systemic administration were also evaluated in KB and HT-1080 tumorbearing mice. The findings demonstrate that choosing more than one target from the pool of tumor–stroma interactions, which ensures tumor genesis, survival, growth and metastases, is profoundly beneficial to therapeutic approaches.

2. Results and Discussion 2.1. Development of Lipid Nanocarriers MMP-2 is cell-surface, Zn-dependent endoprotease associated with diverse processes throughout tumor formation and progression and play a critical role in tumor progression, angiogenesis, invasion, and metastasis.[30,31] The MMP-2-responsive peptide sequence (EGGEGGEGGEGG-PVGLIG-rrrrrrrrrC, where the lowercase “r” refers to D-arginine and the capital refers L-amino acid) includes three units: the cell-penetrating domain (CPP, oligoarginine), the MMP-2-sensitive cleavable peptide (PVGLIG) and the polyanionic shielding peptide (EGGEGGEGGEGG). Oligoarginine is a well-known CPP that enhances the delivery of molecules across biological barriers for increased intracellular access.[32] The MMP-2-responsive peptide is temporarily inert in circulation due to the attenuated CPP via the internal complex of the polyanionic shielding peptides, and its activation is triggered by overexpressed MMP-2 in tumors. Thus, TMSP exhibits target functions that are comparable to those of CPP due to the microenvironment-responsive cleavable peptides. We synthesized DSPE-PEG2000-TMSP and DSPE-PEG2000CPP via the thiol-ene “click” reaction of the maleimide group with the sulfhydryl group of MMP-2-responsive peptide or CPP peptide (Figure S1, Supporting Information). The major multipeaks were centered at mass-charge ratios of 5800–6400

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(Figure S2, Supporting Information) and 4500–5000 (Figure S3, Supporting Information), which was consistent with the calculated mean MWs of the corresponding conjugate (approximately 5936 of DSPE-PEG2000-TMSP and 4668 of DSPE-PEG2000-CPP, respectively). TMSP-specific cleavage by MMP-2 and unmasking of the polyanionic inhibitory peptide were confirmed by proteolysis experiments in vitro (Figure S4, Supporting Information). Cleavage of TMSP was correlated to the enzyme concentration and incubation time. Furthermore, the amount of TMSP did not decrease in the absence of MMPs after 4 h of incubation. It was demonstrated that TMSP was present and stable until it came into contact with MMP-2.[19] To obtain the folate-labeled conjugates, our approach involved carbodiimide-mediated coupling of folic acid to a readily attainable DSPE-PEG5000-NH2 (Figure S5, Supporting Information). The major multipeaks centered at a mass–charge ratio of 5200–6200, which indicated that the mean MW of DSPE-PEG5000-folate was consistent with the theoretical data (approximately 5965) (Figure S6, Supporting Information). Various payloads, including docetaxel (DTX) (a frontline chemotherapeutic agent), DiR (a classic near-infrared fluorescent lipophilic carbocyanine dye), and 6-coumarin (COU, a lipophilic fluorescent probe)-loaded lipid nanocarriers (NLC), including unmodified NLC and modified NLC (Figure 1), were prepared by a modified emulsification-ultrasonication method as previously described.[19] The threshold vesicle size for extravasation into a tumor's extracellular space was approximately 400 nm,[33] and a drug delivery system smaller than 200 nm is recommended.[34] All types of NLCs (Table 1) had a small size (hydrodynamic diameter 96%), which endowed the NLCs vectors with potential beneficial behavior in vivo. The melting behavior and inner phase status of docetaxel (DTX) itself, the physical mixed lipids, the blank NLCs (no




Preparations DTX-NLC

Diameter [nm]

Polydispersity index

Zeta potential [mV]

Entrapment efficiency [%]

33.16 ± 0.58

0.09 ± 0.01

−2.90 ± 0.86

98.48 ± 0.16

CPP-DTX-NLC

34.17 ± 0.04

0.10 ± 0.01

0.67 ± 1.03

97.47 ± 0.90

F/TMSP-DTX-NLC

35.37 ± 0.38

0.10 ± 0.01

−0.29 ± 0.21

98.49 ± 0.02

TMSP-DTX-NLC

58.03 ± 5.18

0.20 ± 0.03

1.25 ± 0.48

98.56

Folate-DTX-NLC

38.77 ± 1.15

0.11 ± 0.01

0.65 ± 1.13

99.20

COU-NLC

30.17 ± 0.51

0.09 ± 0.01

−2.11 ± 1.30

98.33 ± 0.73

CPP-COU-NLC

30.96 ± 0.67

0.12 ± 0.05

0.13 ± 0.24

98.39 ± 0.42

F/TMSP-COU-NLC

31.38 ± 0.85

0.13 ± 0.03

−0.57 ± 0.41

98.18 ± 0.33

DiR-NLC

35.57 ± 1.96

0.12 ± 0.01

−2.26 ± 0.87

97.30 ± 0.54

F/TMSP-DiR-NLC

36.90 ± 1.80

0.13 ± 0.03

−2.9 ± 0.95

96.89 ± 1.03

drug in nanocarriers), the physical mixtures of DTX and the blank NLCs, and the DTX-loaded NLCs were investigated by differential scanning calorimetry (DSC). As shown in Figure 2, the mixed lipids exhibited an endothermic peak occurring at 47.22 °C. All blank NLCs (NLC, CPP-NLC, F/TMSP-NLC) and all DTX-loaded NLCs (DTX-NLC, CPP-DTX-NLC, F/TMSP-DTXNLC) exhibited a lower melting point (approximately 44 °C). This melting depression was described in the Thomson equation (derived from the Kelvin equation), with the differences generally ascribed to the interaction of ingredients, or simply to the nanometric size of the particles.[35] Regarding the physical mixtures of DTX and all blank NLCs, the melting peak of DTX was determined. However, the melting event of DTX was no longer detected in all of the DTX-loaded NLCs. The absence of this melting event may be due to a super-cooled melted state, or an amorphous or molecular-dispersed state of DTX in the NLC matrix. For DTX, the passive-loading procedure was successful. The physical stabilities of DTX- and COU-loaded NLCs were evaluated at 4 °C over a period of one month. No significant changes in size, polydispersity index, zeta potential, and entrapment efficiency were found for the respective formulations during the storage time (Table S1, Supporting Information). The characteristics remained unchanged over the observation period, providing evidence that DTX or COU was enclosed in the lipid particle matrix. These results confirmed that the NLC could prevent drug expulsion during storage, and improve the physical and chemical long-term stability.

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Table 1. Characteristics of NLCs. The data are expressed as the mean ± SD value for at least three different preparations.

NLC, and modified DTX-loaded NLC was evaluated in KB cell (high-level FR expression),[36] HT-1080 cell (overexpression of MMP-2),[37,38] and A549 cell (negative FR and MMP-2 expression)[39,40] using the MTT assay after 48, 72, and 96 h of incubation, individually. Blank NLCs showed no cytotoxicity to each of the cells during the indicated experimental period, indicating the safety of the nanocarriers (Figure S7, Supporting Information). Obviously, all cell viability was decreased with an increase in DTX concentration and incubation period (Figure S7, Supporting Information and Figure 3). Consistent with our expectations, folic acid and TMSP double-modified NLC promoted cytotoxic effects after 48, 72, and 96 h of incubation compared to the nonmodified NLC, both in KB cells which expressed a high level of FR (Figure 3A) and in HT-1080 cells which oversecreted MMP-2 (Figure 3B). To confirm that high cytotoxicity of KB and HT-1080 cells was related to a high-level expression of FR and oversecretion of MMP-2, we performed the following three tests: 1) competition block the FR with free folic acid in the KB cell

2.2. Avidity and Specificity of Targeted Nanocarriers Toward HT-1080, KB and A549 Cells The specificity and efficacy of the folic acid and TMSP co-modified nanocarriers to tumor tissue and cancer cells were investigated in various cell lines in vitro. The in vitro cytotoxicity of Taxotere (a commercial product of DTX), blank NLC, DTX-loaded


Figure 2. DSC thermograms of DTX, mixed lipids, various blank nanoparticles (NLC, CPPNLC, F/TMSP-NLC), mixture of DTX with various blank nanocarriers, and various DTX-loaded nanoparticles (DTX-NLC, CPP-DTX-NLC, F/TMSP-DTX-NLC). The melting peaks of DTX disappeared in all DTX-loaded nanocarriers.



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Figure 3. A) IC50 of KB cell, B) HT-1080 cell, and C) A549 cells cultured with various DTX-loaded NLCs compared with Taxotere at the same DTX dose and the same amount of blank NLC for 48, 72, and 96 h, respectively. Blank NLC showed no cytotoxicity to each of the cells during the indicated experimental period. The data is presented as the mean ± SD (n = 6).

(Figure 3A). The results showed that the cytotoxicity of F/ TMSP-DTX-NLC was returned to the level of nonmodified NLC after being blocked with free folic acid in the KB cell for 2 h indicating that internalization of the F/TMSP-DTX-NLC was blocked by free folate. 2) use of negative FR and MMP-2 expressed A549 cells (Figure 3C). The results showed that F/ TMSP-DTX-NLC exhibited a similar cytotoxicity compared with nonmodified NLC in A549 cells indicating that the cytotoxicity of F/TMSP-DTX-NLC was associated with the FR or

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MMP-2 expression. 3) use of a scissor CPP-DTX-NLC as the positive control of TMSP-DTX-NLC to evaluate cytotoxicity (Figure 3). CPP-DTX-NLC exhibited a comparable cytotoxicity with F/TMSP-DTX-NLC in KB and HT-1080 cells but a dramatically higher cytotoxicity than F/TMSP-DTX-NLC in A549 cell, which cleared the actions of the FR and MMP-2. All of these factors confirmed that the FR-mediated endocytosis and MMP-2-triggered cleavage on TMSP could enhance NLC internalization.




FULL PAPER Figure 4. Intracellular distribution of 6-coumarin (COU) with different formulations on A) KB cells and B) HT-1080 cellsusing confocal laser scanning microscopy (scale bar, 50 µm). a) Blue color indicates the nucleus of KB or HT-1080 cells stained with Hoechst 33342. b) Green color, and c) overlapping images of a and b shows the COU distribution in KB or HT-1080 cells after application of 1) free COU, 2) COU-NLC, and 3) F/TMSP-COU-NLC.

The confocal laser scanning microscope (CLSM) was applied to qualitative determination of the cellular uptake and intracellular distribution of COU-loaded NLCs. With the high hydrophobic nature, free COU readily partitioned into the cell membranes and then diffused into both HT-1080 cells and KB cells, resulting in a greater extent of cellular accumulation, which served as the positive control group. CLSM images also confirmed that COU was accumulated in the cytoplasm for COU-loaded NLCs. Double-modified F/TMSP-COU-NLC demonstrated much stronger fluorescent intensities in the KB cell compared to COU-NLC (Figure 4A), demonstrating that folic acid modification on NLC could enhance NLC internalization. F/TMSP-COU-NLC-treated HT-1080 cells exhibited stronger intracellular fluorescence than COU-NLC (Figure 4B), which revealed the contribution of MMP-2-triggered cleavage of TMPS in F/TMSP-COU-NLC to cellular uptake. As shown in Figure 3, the IC50 of Taxotere displayed a large difference among the three cancer cell lines (e.g., approximately 24 ng mL−1 for KB cells, 580 ng mL−1 for HT-1080 cells, and 19 944 ng mL−1 for A549 cells after exposure for 48 h), indicating that the KB cells, the HT-1080 cells, and the A549 cells have different sensitivities for DTX. In other words, KB cells demonstrate high sensitivity, HT-1080 cells show moderate sensitivity, and A549 cells exhibit low sensitivity for DTX. However, all of the NLCs, including the nonmodified and modified NLCs, significantly decreased the IC50 in the three cell lines compared to Taxotere (e.g., approximately 11 ng mL−1 and 3 ng mL−1 for KB cells, 338 ng mL−1 and 212 ng mL−1 for HT-1080 cells, 2938 ng mL−1 and 3298 ng mL−1 for A549 cells after exposure of DTX-NLC and F/TMSP-DTX-NLC for 48 h, respectively). These phenomena revealed a change in viability not only as a function of the ligand number but also in the nature of the cells.


2.3. Apoptosis and Cell Cycle Analysis An apoptosis study was performed to elucidate the mode of cell death after cell treatment with Taxotere and DTX-loaded NLC. Annexin V-FITC staining combined with PI could distinguish early apoptosis from late apoptosis as well as living cells from dead cells. The cell apoptosis ratio increased significantly in a concentration-dependent manner, while the control treatment group showed insignificant presence of apoptosis and necrotic cells (less than 10%) in KB and HT-1080 cells (Figure 5). F/ TMSP-DTX-NLC induced more apoptosis than DTX-NLC and Taxotere, although this was not statistically significant in KB cell (Figure 5A,B). In the apoptosis study using the HT-1080 cell line, similar results were obtained. However, as a moderately sensitive cell line for DTX, the between-group differences in the HT-1080 cells were significant (P < 0.01) at a dose of 10 ng mL−1 (Figure 5C,D). Here, our results signified the effectiveness of F/TMSP-DTX-NLC as an effective DTX carrier. DTX has been shown to target tubulin, which causes a stabilization of microtubules, thereby resulting in cell-cycle arrest, mitosis damage, and apoptosis. Cell cycle arrest was found to be concentration and time dependent (Figure 6). After treatment with F/TMSP-DTX-NLC, DTX-NLC, and Taxotere, the majority of KB and HT-1080 cells were predominantly accumulated in G2/M phases and fewer cells in G0/G1 phases. Cell cycle analysis of both KB and HT-1080 cells revealed that F/TMSP-DTXNLC demonstrated superior antitumor activity compared to DTX-NLC and Taxotere. In the case of NLC-mediated activity by the tumor microenvironment and FR, more drug is available for a longer period of time compared to Taxotere, resulting in a greater efficacy of DTX in arresting cell growth, which is consistent with the cytotoxicity study. Overall, the cytotoxicity, intracellular distribution, cell apoptosis, and cell-cycle arrest results



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Figure 5. In vitro cell apoptosis evaluation of various DTX formulations with different DTX concentration against A) KB cells, B) HT-1080 cells, C) apoptotic indices for KB cells and D) HT-1080 cells in each group. LL: necrotic cells; LR: late apoptotic cells; UL: live cells; UR: early apoptotic cells. The data are presented as the mean ± SD (n = 3).

strongly supported our hypothesis that multivalent interactions can play a key role in the enhancement of cancer cell recognition and the uptake and killing of cancer cells.

2.4. In Vivo Pharmacokinetics and Distribution of Nanocarriers One of the drawbacks of the use of nanoparticles is the fast elimination from blood and the capture of nanoparticles by the reticuloendothelial system (RES), which is primarily in the liver and spleen. To increase the likelihood of success in drug targeting by nanocarriers, it is necessary to prolong the circulation of nanocarriers in vivo. This can be achieved by the surface coating of nanocarriers with hydrophilic biocompatible polyethylene glycol (PEG), which forms a protective layer over the NLC surface and delays NLC recognition by opsonins and subsequent clearance. In addition, the size of the nanoparticle is paramount for effective delivery to tumor tissues. Particles ranging from 10 to 100 nm have the optimal size for a drug vector[41–43] because they are sufficiently large to be retained by the body yet sufficiently small to access the cell surface receptors and pass through the cell membrane via endocytosis.[42] The DTX concentrations in plasma versus time curves after intravenous administration of Taxotere, DTX-NLC, and F/TMSP-DTX-NLC (Figure 7) showed that NLC prolonged DTX retention in blood circulation compared to Taxotere. The pharmacokinetic parameters that are best described by the two-compartment model according to the Akaike Information Criterion are summarized in Table S2 (Supporting Information). Pharmacokinetic studies

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revealed that NLCs were capable of preventing drug molecules from being easily eliminated from the physiological environment. These results were consistent with the small size and biocompatibility matrix of these nanocarriers.[44,45] For effective drug-based cancer treatment, the drug must be accumulated within tumors. After the establishment of a KB and HT-1080 xenograft tumor model, physiological saline, free DiR, DiR-NLC, or F/TMSP-DiR-NLC samples were administered, and the tissue distribution was recorded using non-invasive live animal imaging technology with a DiR for near-infrared fluorescence imaging. Analysis of whole body imaging (Figure 8A) revealed no fluorescence signals detected in mice treated with physiological saline during the experimental period. Free DiR only accumulated in the liver and spleen, and tumor accumulation did not occur. In contrast, significant tumor accumulation was observed for DiR-NLC and F/TMSP-DiR-NLC, with the tumor fluorescence intensity at a high level over the entire period (2 d). Enhanced distribution of all NLCs at the tumor sites is thought to be mediated by EPR effects in solid tumors, the vasculature and endothelial cell junction become leaky.[46] Moreover, the fluorescence was intensified gradually in the tumor regions after 6 h. The most intense distribution in tumors was found in the F/TMSP-DiRNLC-treated mice and was further confirmed by the strongest fluorescence identified in isolated tumors (Figure 8B,C) both in KB and HT-1080 xenograft tumor-bearing mice, providing decisive evidence of the activity of tumor targeting, the function of TMSP for tumor tissue permeability and cell penetration, and enhancement of cellular uptake.




FULL PAPER Figure 6. Effects of treatment with Taxotere, DTX-NLC and F/ACPP-DTX-NLC for 4, 8, 12, 24 h on the cell cycle of A) KB and B) HT-1080 cells. Treatment with equivalent DTX concentrations of a) 1 ng mL−1, b) 10 ng mL−1, and c) 100 ng mL−1. The data is expressed as the mean ± SD (n = 3).

2.5. In Vivo Antitumor Efficacy and Toxicity Evaluation

Figure 7. Concentration–time curves of DTX based on plasma levels after intravenous administration of Taxotere, DTX-NLC and F/TMSP-DTX-NLC to Sprague–Dawley rats at a single dose of 10 mg kg−1, respectively. The results are presented as the mean ± SD (n = 6).


To further evaluate the biocompatibility of NLC on intravenous injection, hemolysis assays were performed using goat red blood cells. The hemolytic percentages of DTX-NLC and F/TMSP-DTX-NLC exposed were less than 5%. Thus, NLC systems exhibited tolerable hemolysis of erythrocytes ( Folate-DTX-NLC > DTX-NLC >> blank NLC and saline. There was greater than 25-fold shrinkage in tumor volume for three treatments of F/ TMSP-DTX-NLC (8 mg kg−1) compared to the saline group at the end of the test. For KB cell-derived tumors in nude mice following systemic administration of various DTX formulations (DTX dosage of 10 mg kg−1 and an excess dosage of 15 mg kg−1 for F/TMSP-DTXNLC), the tumor volume ranked as follows: F/TMSP-DTX-NLC (15 mg kg−1) > F/TMSP-DTX-NLC (10 mg kg−1) ≈ TMSP-DTXNLC ≈ CPP-DTX-NLC ≈ folate-DTX-NLC > DTX-NLC > Taxotere >> blank NLC and saline. There was greater than 85-fold shrink in the tumor volume for F/TMSP-DTX-NLC (10 mg kg−1 of DTX) treatment three times compared to the saline group at the end of the test.[19] By analyzing DNA strand breaks, the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay can be extensively employed to assess tumor cell apoptosis. As observed in Figure 9D, neither saline nor blank NLC resulted in a pronounced induction of cell apoptosis, as demonstrated by the absence of detectable TUNEL-positive tumor cells (red). In contrast, exposure to DTX, particularly F/TMSP-DTX-NLC (8 and 10 mg kg−1), exhibited significant

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apoptosis compared to other treatments with formulated DTXs. The trend observed for apoptotic analysis was consistent with the results of antitumor efficacy in vivo (Figure 9A,C). DTX could promote microtubule polymerization and stabilization via bundle formation, thereby inhibiting mitotic spindle formation.[47] Acetylation of α-tubulin at lysine 40 is an established marker of microtubule stability.[48] Thus, the amount of acetylated tubulin is thought to be proportional to the stability of the microtubule to evaluate the inhibition of tumor caused by various DTX formulation treatments. Nearly all of the untreated control cancer cells in the physiological saline and blank NLC groups demonstrated a fine and organized microtubule network. In contrast, treatment with DTX-loaded NLCs that were modified with folate, CPP, or TMSP resulted in an increase in microtubule bundling compared with identical doses of the non-targeted DTX-NLC and Taxotere (Figure 9E). As expected, the most extensive microtubule bundles were observed in the F/TMSP-DTXNLC group. Histopathologic imaging exhibited more necrosis after F/TMSP-DTX-NLC exposure in excised KB and HT-1080 tumors (Figure S8 and S9, Supporting Information). In addition, despite multiple injections of various DTXcontaining formulations over the duration of the treatment for HT-1080 and KB tumor-bearing mice, no pronounced changes in mouse body weight (Figure 9B) were observed compared




FULL PAPER Figure 9. A) Tumor volume–time profile and B) body weight changes of HT-1080 tumor-bearing mice after docetaxel-loaded NLC treatment. Data is presented as the mean ± SD (n = 10). C) Photograph of the solid tumors removed from different treatment groups at the termination of the study. Apoptosis of the dissected HT-1080 tumor (D, scale bar: 75 µm). Acetyl-α-tubulin of the dissected KB tumor (E, scale bar: 100 µm).

with the control, suggesting that there was negligible acute or severe toxicity related to the indicated treatment at the test dose. In addition, the relatively large weight loss of the mice in the DTX-NLC and CPP-DTX-NLC groups was speculated to be due to the unspecific distribution of formulation. For KB tumor cell-derived mice, more than 15% weight loss was observed in the Taxotere (10 mg kg−1) groups at the end of the experimental period, likely due to the nontargeted characteristics and toxicity of the solvent system of the formulations (ethanol and nonionic surfactant Tween-80).[11,49–51] A high drug dose was associated with a higher risk of normal tissue toxicity for cytotoxic drugs. To confirm whether NLC


impaired major organ function, microscopic changes in organ tissues were evaluated. There were no obvious pathological changes observed in either type of DTX-loaded NLC (Figure 10). Although DTX-NLC accumulated in the liver (Figure 8) during circulation, it did not have a visible detrimental effect on the histology of the liver and other organs. The organ coefficient of all groups (Figure S10 and S11, Supporting Information) showed no significant difference, which indicated little damage to the organs. Thus, F/TMSP-DTX-NLC achieved a better antitumor efficacy with low systemic toxicity. The white blood cell (WBC), red blood cell (RBC), hemoglobin (HGB), platelet (PLT), granulocyte (GRN), and



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Figure 10. Histological analysis of KB tumor-bearing mouse tissue sections obtained from different treatment groups. The photomicrographs are at a magnification of 200×.

lymphocyte (LYM) counts varied during antitumor treatments. The Taxotere group showed a marked and comparable decrease in WBC counts on day 7 for KB tumor-bearing mice (Figure S12, Supporting Information), which was likely due to myelosuppression effects. For HT-1080-bearing mice, similar results were obtained. The WBC showed a little decrease in the group of Taxotere and F/TMSP-DTX-NLC (10 mg kg−1) compared to the saline-treated group, while there was no different between F/TMSP-DTX-NLC (8 mg kg−1) and the saline-treated group (Figure S13, Supporting Information). Then, the BMC cycle was analyzed to evaluate the myelosuppression effects. There was no significant difference in the BMC counts (Figure S14, Supporting Information) and cell numbers in S phase among various groups (Table S3, Table S4, Supporting Information). The overall results of the blood study indicated that treatment with various DTX-loaded NLCs showed no obvious myelosuppression effects and were less toxic, demonstrating the potential of this treatment for therapeutic applications. During the antitumor experiment, a large number of the mice in Taxotere had a phenomenon of psoriasis, low skin temperatures, and lack of vigor, while few mice in F/TMSP-DTX-NLC had psoriasis with normal temperatures. The toxicity evaluation showed that the modified DTX-NLCs were safe with better antitumor efficacy compared with Taxotere.

3. Conclusion All data revealed that folic acid and TMSP co-modified lipid nanocarrier (F/TMSP-DTX-NLC) were successfully formulated in response to the simultaneous overexpression of FR and the upregulation of MMP-2 in the tumor microenvironment. The F/TMSP-DTX-NLC first accumulated in the tumor tissue via the EPR effect, and the folate moiety binds selectively to the FR once it reaches the tumor. Simultaneously, CPP penetrating activity was initiated by MMP-2 protease-oversecretion tumor tissue. Multivalent interactions induced the enhancement of cancer cell recognition and internalization, which subsequently

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resulted in cancer cell apoptosis or death. In conclusion, the present DTX-NLC modified with both TMSP and folic acid is a potential drug delivery system for tumor cell recognition and therapy.

4. Experimental Section Materials: DTX was purchased from Norzer Pharmaceutical Co. Ltd. Peptides were custom synthesized via a standard Fmoc solid-phase peptide synthesis method by ChinaPeptides Co. Ltd. Folate was obtained from Sinopharm Group Co., Ltd. 1,2-distearoylphosphoethanolaminepolyethyleneglycol-amine (DSPE-PEG5000-NH2) and DSPE-PEG2000maleimide (DSPE-PEG2000-Mal) were purchased from NOF Corporation. Fluorescent probe DiR was supplied from Biotium Inc. (Hayward, CA, USA). 6-Coumarin (COU) were purchased from Sigma–Aldrich. Propidium iodide (PI), RNase A, and Annexin V-FITC were the products of KeyGen bio-technology (Nanjing, China). 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma– Aldrich. Hoechst 33258 was from Molecular Probes Inc. (Oregon, USA). Taxotere was provided by the National Cancer Center. Folate-free RPMI-1640 medium and fetal bovine serum (FBS) were obtained from Gibco. RPMI-1640 medium, modified eagle's medium (MEM), F-12K medium, nonessential amino acids, penicillin, and streptomycin were provided by Macgene Co. Ltd. All other chemicals were analytical or high-performance liquid chromatography (HPLC) grade. Three cell lines, human mouth epidermoid carcinoma KB cells, human fibrosarcoma HT-1080 cells, and human pulmonary adenocarcinoma A549 cells, were cultured in folate-free RPMI-1640, MEM, and F-12K medium, respectively. Each cell culture medium was supplemented with 10% FBS, 100 units mL−1 penicillin, and 100 µg mL−1 streptomycin. The cultures were maintained at 37 °C, 95% relative humidity and 5% CO2. Male Sprague– Dawley rats (180–220 g) and female BALB/c nude mice (18–20 g) were purchased from Vital Laboratory Animal Center (Beijing, China). The animals were raised at 25 °C and 55% humidity under natural light/ dark conditions. All care and handling of animals were performed with the approval of the Institutional Animal Care and Use Committee at Peking University Health Science Center. Preparation of Nanocarriers: DTX-loaded lipid nanocarrier (DTX-NLC) was prepared using a modified emulsification ultrasonication method as previously described.[19] For the preparation of various modified DTX-NLC, such as folate-DTX-NLC, CPP-DTX-NLC, and F/TMSP-DTX-NLC, an





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identical procedure was followed except that either only 3% DSPEPEG-CPP or both 3% molar DSPE-PEG-TMSP and 1% DSPE-PEG-folate were used to replace an equivalent quantity of soya lecithin. Differential Scanning Calorimetry: To evaluate the interaction of DTX with lipids and the lipid crystallinity, the thermal behavior was determined using a DSC TA-60 apparatus (Shimadzu, Japan). The NLCs were freeze-dried prior to the DSC measurements. Lyophilized nanocarriers (5 mg) were weighed into aluminum pans that were sealed hermetically. The thermal analysis profiles were obtained as the temperature was increased from 30 °C to 280 °C at a rate of 10 °C min−1 under nitrogen. Baselines were determined using an empty pan, and all thermograms were baseline corrected. The DSC measurements were performed on the following samples: DTX, mixed lipids, blank NLC, mixture of DTX and blank NLC, DTX-NLC, blank CPP-NLC, mixture of DTX and blank CPP-NLC, CPP-DTX-NLC, blank F/TMSP-NLC, mixture of DTX and blank F/TMSP-NLC, and F/TMSP-DTX-NLC, respectively. Cytotoxicity Assay: The cytotoxicity of DTX formulations including Taxotere, DTX-NLC, CPP-DTX-NLC, and F/TMSP-DTX-NLC against three tumor cell lines (HT-1080, KB and A549) was evaluated using MTT-based cytotoxicity assay. Briefly, cell suspension diluted with the corresponding growth medium was added to each well (4000 cells well−1) of a 96-well plate and allowed to attach for 24 h. Then, the cells were treated with various DTX formulations at a range of concentrations. After incubation for 48, 72, and 96 h, 20 µL MTT (5 mg mL−1 in PBS) was added to each well for 4 h at 37 °C. The medium containing MTT was removed from the wells and the remaining MTT-formazan crystals were dissolved by adding 200 µL DMSO. The absorbance of each well was measured by an iMark microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) at a wavelength of 570 nm. Percent of cell survival was defined as the relative absorbance of treated cells versus respective controls. Intracellular Distribution: COU was used as a hydrophobic fluorescent probe to study cellular uptake and intracellular distribution of NLCs. Following 24 h incubation of KB or HT-1080 cells on glass bottom dishes containing growth media at 37 °C, COU loaded NLCs (150 ng mL−1 of COU) were added to each dish and were incubated at 37 °C for 2 h. The medium was removed and cells were washed with cold PBS followed by fixing with 4% paraformaldehyde in PBS. Subsequently, the cells were washed with PBS and incubated with Hoechst 33258 for staining the cell nuclei. After washing with PBS, the fluorescent images of cells were analyzed using a CLSM (Leica, TCS SP5, Germany). Apoptosis Assay: The transversion of phosphatidyl serine (PS) from the inner to outer plasma membrane leaflet, as an initial event in the apoptotic pathway, was able to bind by Annexin V-FITC. Furthermore, PI could conjugate to necrotic cells. So the Annexin V-FITC/PI kit was used to detect apoptotic and necrotic cells induced by DTX formulations. KB cells or HT-1080 cells with various DTX formulations at dosages of 1, 10, and 100 ng mL−1 incubated for 24 h. After that, the medium was collected and treated cells were detached using trypsin (without EDTA) solution and suspended in fresh medium. The cells were centrifuged at 2000 rpm for 5 min. Then, the cells were washed twice with PBS and resuspended in 500 µL Annexin V binding buffer at a density of 1 × 106 per mL. Annexin V-FITC and PI (5 µL) were added and the cells were incubated in the dark for 15 min at room temperature. Finally, the cells were analyzed within 1 h by flow cytometry (Becton Dickinson, San Jose, CA, USA). Cell Cycle Analysis: Cell cycle analysis was assessed by flow cytometry. Briefly, HT-1080 and KB cells were seeded in six-well plates (Corning, NY, USA). After incubating for 24 h, the medium in each well was replaced with fresh cell medium containing DTX formulations at dosage of 1, 10, and 50 ng mL−1 for KB cells, and 1, 10, or 100 ng mL−1 for HT-1080 cells, respectively. After the treatment for 4, 8, 12, and 24 h, the cells were harvested, washed with cold PBS, and fixed with 70% precooled alcohol overnight at 4 °C, respectively. Before measurements, the cells were washed to eliminate alcohol and then incubated with 500 µL RNase A (100 mg mL−1) containing Triton X-100 (2 mg mL−1) for 30 min at 37 °C, and then stained with 5 µL PI solution (1 mg mL−1) for 15 min in the dark. The DNA content was determined using flow cytometry (Merck Millipore, USA) and the cell cycle profile was analyzed by Guava CytoSoft 5.3 software (Guava Technologies, Hayward, CA).

Pharmacokinetic Study: In vivo pharmacokinetic study was carried out using Sprague-Dawley rats (female, 180–220 g). These rats were fasted overnight with free access to water before drug administration. The rats were randomly divided into three groups and injected intravenously through the tail vein with DTX formulations at a dosage of 10 mg DTX kg−1 body weight. The blood samples were taken from the retroorbital plexus at predetermined time points with heparinized tubes and centrifuged for 5 min at 10 000 rpm. 0.2 mL plasma was obtained and stored at −20 °C until further analysis. DTX concentration in plasma was determined by an established and validated bioanalytical method using HPLC. The HPLC system consisted of a Shimadzu LC-20AT Pump, a Shimadzu SPD-20A UV detector, and a SIL-20A autosampler (Shimadzu, Japan). The mobile phase consisted of acetonitrile: double-distilled water (53:47, v/v) introduced at a flow rate of 1.0 mL min−1. The detection wavelength was 230 nm. An RP-18 column (4.6 mm × 250 mm, pore size 5 µm, Diamonsil) was used. Briefly, plasma samples (200 µL) were spiked with PTX solution (internal standard) and 2 mL of ether, vortexed, and filtrated. After filtration, the organic phase was collected. Subsequently, the organic phase was dried under a nitrogen stream at 40 °C, the residue was reconstituted in 100 µL methanol. Following further centrifugation, 20 µL of clear supernatant was injected into the HPLC system and the DTX concentration in plasma was calculated by standard curve. The main pharmacokinetic parameters were calculated using the DAS 2.0 software (Shanghai, China). The area under the plasma concentration time profiles (AUC), the distribution half-life (t1/2α), and elimination half-life (t1/2β), and total plasma clearance (CL) were calculated. Animal Xenograft Tumor Model: To establish the tumor model, BALB/c nude mice (female, 18–20 g) were subcutaneously injected in the right axilla with 200 µL of cell suspension containing 3 × 106 KB cells or 2 × 106 HT-1080 cells. The mice were used in in vivo imaging when the tumors reached a size of approximately 600 mm3, or antitumor efficacy and toxicity evaluation when the tumors reached a size of approximately 100–200 mm3. In Vivo Biodistribution Study: DiR as a near-infrared fluorophore encapsulated in NLC was used to evaluate the tumor-targeting capability in the KB cells and HT-1080 cells bearing BALB/c nude mice model, respectively. Mice were injected with 0.2 mL of physiological saline, free DiR, DiR-NLC, or F/TMSP-DiR-NLC (80 µg DiR kg−1 body weight) via the tail vein, respectively, and then anesthetized by 2% isoflurane delivered via a nose cone system. There were three mice for each group. Near-infrared fluorescence imaging experiments using a Maestro in vivo imaging system (CRI, Woburn, MA, USA) were performed at 1, 3, 6, 12, 24, 36, and 48 h post-injection. After 48 h, mice were sacrificed. Tumors and organs including heart, liver, spleen, lung, and kidney were excised and imaged. All images were analyzed using the imaging station IS2000MM software. Antitumor Efficacy in HT-1080 and KB Tumor-Bearing Mice: The antitumor efficacy in vivo was evaluated in HT-1080 tumor-bearing mice. When the tumor volume reached approximately 100 mm3, mice were randomly divided into 9 groups (10 mice per group). Then, each group of mice was treated twice every 3 d by tail vein injection with physiological saline; blank NLC; a 8 mg kg−1 dose of Taxotere, DTX-NLC, FolateDTX-NLC, TMSP-DTX-NLC, CPP-DTX-NLC, F/TMSP-DTX-NLC, and a 10 mg kg−1 dose of F/TMSP-DTX-NLC. The tumor volume was measured every day and calculated based on the equation (a × b2)/2, where a and b equaled the length and the width of the tumor, respectively. The animals were also weighed every day during the experimental period. After 20 d, the mice were sacrificed, and the tumor tissues were removed, weighed, and photographed. For KB tumor-bearing mice, each group of mice was treated three times every 3 d by tail vein injection with physiological saline; blank NLC; a 10 mg kg−1 dose of Taxotere, DTX-NLC, F-DTX-NLC, TMSP-DTX-NLC, CPP-DTX-NLC, F/TMSP-DTX-NLC, and a 15 mg kg−1 dose of F/TMSP-DTX-NLC, respectively. Statistical Analysis: All data were expressed as the mean ± standard deviation (SD) of independent measurements unless specifically outlined. Student's t-test or one-way analyses of variance (ANOVA) were performed in the statistical evaluation. A p value less than 0.05 was considered to be statistically significant, and a p value less than 0.01 was considered to be highly significant.



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Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements The authors acknowledge the 973 Program (No. 2013CB932501), National Nature Science Foundation (No.81273454, No.81473156), Beijing Science Foundation (No.7132113), Doctoral Foundation of the Ministry of Education (No. 20130001110055), and Innovation Team of Ministry of Education (No. BMU20110263) for funding of this work. Received: November 3, 2014 Revised: November 30, 2014 Published online:

[1] M. L. Immordino, P. Brusa, S. Arpicco, B. Stella, F. Dosio, L. Cattel, J. Controlled Release 2006, 91, 417. [2] X. R. Qi, Y. Maitani, T. Nagai, S. L. Wei, Int. J. Pharm. 1997, 146, 31. [3] R. Mo, Q. Sun, J. Xue, N. Li, W. Li, C. Zhang, Q. Ping, Adv. Mater. 2012, 24, 3659. [4] Z. Z. Yang, J. Q. Li, Z. Z. Wang, D. W. Dong, X. R. Qi, Biomaterials 2014, 35, 5226. [5] E. Lee, H. Kim, I. H. Lee, S. Jon, J. Controlled Release 2009, 140, 79. [6] D. Zhou, H. Xiao, F. Meng, X. Li, Y. Li, X. Jing, Y. Huang, Adv. Healthcare Mater. 2013, 2, 822. [7] A. Kumari, S. K. Yadav, S. C. Yadav, Colloid. Surf. B, Biointerfaces 2010, 75, 1. [8] H. J. Cho, H. Y. Yoon, H. Koo, S. H. Ko, J. S. Shim, J. H. Lee, K. Kim, I. C. Kwon, D. D. Kim, Biomaterials 2012, 32, 7181. [9] D. W. Dong, B. Xiang, W. Gao, Z. Z. Yang, J. Q. Li, X. R. Qi, Biomaterials 2013, 34, 4849. [10] S. W. Lee, M. H. Yun, S. W. Jeong, C. H. In, J. Y. Kim, M. H. Seo, C. M. Pai, S. O. Kim, J. Controlled Release 2011, 155, 262. [11] S. W. Tong, B. Xiang, D. W. Dong, X. R. Qi, Int. J. Pharm. 2012, 434, 413. [12] T. Chen, X. Guo, X. Liu, S. Shi, J. Wang, C. L. Shi, Z. Y. Qian, S. B. Zhou, Adv. Healthcare Mater. 2012, 1, 214. [13] J. Fang, H. Nakamura, H. Maeda, Adv. Drug Delivery Rev. 2011, 63, 136. [14] A. K. Iyer, G. Khaled, J. Fang, H. Maeda, Drug Discovery Today 2006, 11, 812. [15] A. I. Minchinton, I. F. Tannock, Nat. Rev. Cancer 2006, 6, 583. [16] Y. Ren, S. Hauert, J. H. Lo, S. N. Bhatia, ACS Nano 2012, 6, 8620. [17] D. Ma, N. Shi, X. R. Qi, Int. J. Pharm. 2011, 419, 200. [18] N. Shi, W. Gao, B. Xiang, X. R. Qi, Int. J. Nanomed. 2012, 7, 1613. [19] W. Gao, B. Xiang, T. Meng, F. Liu, X. R. Qi, Biomaterials 2013, 34, 4137. [20] B. Xiang, D. Dong, N. Shi, W. Gao, Z. Yang, Y. Cui, D. Cao, X. R. Qi, Biomaterials 2013, 34, 6976. [21] T. Jiang, E. S. Olson, Q. T. Nguyen, M. Roy, P. A. Jennings, R. Y. Tsien, Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 17867.

12


[22] J. Levi, S. R. Kothapalli, T. J. Ma, K. Hartman, B. T. Khuri-Yakub, S. S. Gambhir, J. Am. Chem. Soc. 2010, 132, 11264. [23] Q. T. Nguyen, E. S. Olson, T. A. Aguilera, T. Jiang, M. Scadeng, L. G. Ellies, R. Y. Tsien, Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 4317. [24] J. L. Crisp, E. N. Savariar, H. L. Glasgow, L. G. Ellies, M. A. Whitney, R. Y. Tsien, Mol. Cancer Ther. 2014, 13, 1514. [25] S. Huang, K. Shao, Y. Kuang, Y. Liu, J. Li, S. An, Y. Guo, H. Ma, X. He, C. Jiang, Biomaterials 2013, 34, 5294. [26] S. D. Weitman, R. H. Lark, L. R. Coney, D. W. Fort, V. Frasca, V. R. Zurawski, B. A. Kamen, Cancer Res. 1992, 52, 3396. [27] J. F. Ross, P. K. Chaudhuri, M. Ratnam, Cancer 1994, 73, 2432. [28] a) Y. Lu, P. S. Low, Adv. Drug Delivery Rev. 2002, 54, 675; b) S. Sabharanjak, S. Mayor, Adv. Drug Delivery Rev. 2004, 56, 1099. [29] a) X. Guo, C. Shi, J. Wang, S. Di, S. Zhou, Biomaterials 2013, 34, 4544; b) Z. Tang, D. Li, H. Sun, X. Guo, Y. Chen, S. Zhou, Biomaterials 2014, 35, 8015. [30] T. Kline, M. Y. Torgov, B. A. Mendelsohn, C. G. Cerveny, P. D. Senter, Mol. Pharm. 2004, 1, 9. [31] J. D. Vassalli, M. S. Pepper, Nature 1994, 370, 14. [32] P. A. Wender, D. J. Mitchell, K. Pattabiraman, E. T. Pelkey, L. Steinman, J. B. Rothbard, Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 13003. [33] F. Yuan, M. Dellian, D. Fukumura, M. Leunig, D. A. Berk, V. P. Torchilin, R. K. Jain, Cancer Res. 1995, 55, 3752. [34] D. Peer, J. M. Karp, S. Hong, O. C. FaroKHzad, R. Margalit, R. Langer, Nat. Nanotechnol. 2007, 2, 751. [35] H. Bunjes, T. Unruh, Adv. Drug Delivery Rev. 2007, 59, 379. [36] J. M. Saul, A. V. Annapragada, R. V. Bellamkonda, J. Controlled Release 2006, 114, 277. [37] T. A. Giambernardi, G. M. Grant, G. P. Taylor, R. J. Hay, V. M. Maher, J. J. McCormick, R. J. Klebe, Matrix Biol. 1998, 16, 483. [38] Y. Chau, R. F. Padera, N. M. Dang, R. Langer, Int. J. Cancer 2006, 118, 1519. [39] F. Canal, M. J. Vicent, G. Pasut, O. Schiavon, J. Controlled Release 2010, 146, 388. [40] K. Watanabe, M. Kaneko, Y. Maitani, Int. J. Nanomed. 2012, 7, 3679. [41] K. K. Jain, Drug Discovery Today 2005, 10, 1435. [42] W. Li, F. C. Szoka Jr, Pharm. Res. 2007, 24, 438. [43] H. Gao, W. Shi, L. B. Freund, Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 9469. [44] M. J. Ernsting, M. Murakami, A. Roy, S. D. Li, J. Controlled Release 2013, 172, 782. [45] H. S. Choi, W. Liu, P. Misra, E. Tanaka, J. P. Zimmer, B. I. Ipe, M. G. Bawendi, J. V. Frangioni, Nat. Biotechnol. 2007, 25, 1165. [46] R. K. Jain, T. Stylianopoulos, Nat. Rev. Clin. Oncol. 2010, 7, 653. [47] L. G. Wang, X. M. Liu, W. Kreis, D. R. Budman, Cancer Chemother. Pharmacol. 1999, 44, 355. [48] G. Piperno, M. Ledizet, X. J. Chang, J. Cell Biol. 1987, 104, 289. [49] A. Eschalier, J. Lavarenne, C. Burtin, M. Renoux, E. Chapuy, M. Rodriguez, Cancer Chemother. Pharmacol. 1988, 21, 246. [50] Z. Weiszhar, J. Czucz, C. Revesz, L. Rosivall, J. Szebeni, Z. Rozsnyay, Eur. J. Pharm. Sci. 2012, 45, 492. [51] M. Elsabahy, M. E. Perron, N. Bertrand, G. E. Yu, J. C. Leroux, Biomacromolecules 2007, 8, 2250.



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