Reduction-Responsive Polypeptide Micelles for Intracellular Delivery

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Reduction-Responsive Polypeptide Micelles for Intracellular Delivery of Antineoplastic Agent Weiguo Xu, Jianxun Ding, and Xuesi Chen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00950 • Publication Date (Web): 06 Sep 2017 Downloaded from http://pubs.acs.org on September 7, 2017

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Reduction-Responsive Polypeptide Micelles for Intracellular Delivery of Antineoplastic Agent Weiguo Xu, Jianxun Ding*, Xuesi Chen Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China

KEYWORDS: polypeptide, micelle, reduction-responsiveness, intracellular drug delivery, chemotherapy

ABSTRACT:

Reduction-responsive

methoxy

poly(ethylene

glycol)-block-poly(S-tert-

butylmercapto-L-cysteine) copolymers (i.e., mPEG113-b-PBMLC4 and mPEG113-b-PBMLC9) were facilely synthesized through primary amino-initiated ring-opening polymerization (ROP) of disulfide-containing N-carboxyanhydride monomer. The reduction-responsive block copolymers were then investigated for intracellular delivery of antitumor drug after forming smart micelles in vitro and in vivo. The micelles were denoted as P4M and P9M, respectively. Doxorubicin (DOX) was selected as a model chemotherapeutic agent, which was loaded into micelles via hydrophobic interaction. The drug loading efficiency (DLE) were detected to be 55.4 and 61.7 wt.% for P4M and P9M, respectively. The loaded micelles, referred as P4M/DOX and

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P9M/DOX, exhibited spherical morphologies with hydrodynamic radii of 92.3 ± 2.3 and 80.2 ± 2.8 nm, respectively. Compared to P4M/DOX, P9M/DOX with a smaller size exhibited upregulated cell endocytosis and higher cytotoxicity to human breast cancer MCF-7 cells. Furthermore, the loading micelles, especially P9M/DOX, demonstrated improved antitumor efficacy toward MCF-7 breast tumor-bearing BALB/c nude mouse model compared with free doxorubicin hydrochloride (DOX·HCl). This was also confirmed by the histopathological and immunohistochemical results. The above results demonstrated that the facially prepared smart polypeptide micelles exhibited a potent prospect in intracellular drug delivery in vitro and in vivo.

1. INTRODUCTION Cancer is a common malignant disease, which threatens the health and even life of many people. Carcinogenesis is a multi-factor, multi-step, and multi-stage complex process, differentiating it from normal physiological processes.1, 2 As a result, tumor cells have certain specific features that enable researchers to target lesion site for more efficient treatment.3 For instance, glycolysis with lactate secretion, low pH level, high glutathione (GSH) concentration, and so forth have been investigated as characteristic microenvironments for targeted drug delivery to treat cancers.4, 5 Among them, the GSH-oriented studies are particularly popular recently owning to its significant concentration difference between extracellular and intracellular compartments of tumor cells, which endow the microenvironments with different reduction activities.6 Specifically, the concentration of intracellular GSH is 100 to 1,000 times higher than that of the extracellular one.2, 7 Moreover, with the help of low valence Fe(II) and high concentration of thiol (e.g., cysteine) in lysosomes, lysosomes maintain the reduction microenvironment.8 Based


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on the difference in the reduction potential, many GSH-responsive nanocarriers have been designed to deliver therapeutic agents into tumor cells.9,

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Because of their excellent

biocompatibility, responsiveness, and functionalization, polymer-based systems are in particularly being widely studied, and polypeptide is an important and promising one.

Scheme 1. Schematic illustration of polypeptide synthesis, drug loading, and in vivo metabolism. Synthesis pathway for mPEG-b-PBMLC, DOX loading by micelle, and its circulation, intratumoral accumulation, endocytosis, and targeted intracellular DOX release after intravenous injection. Polypeptides are amino acid-based biomaterials, exhibit good biocompatibility and biodegradability, and can be degraded into amino acids under the action of enzymes.11 Amino acids can rapidly metabolized without causing any side effects to the host tissue.12 In addition, the structures and properties of polypeptides can be adjusted according to their applications owning to abundant natural and synthetic amino acids. These features contributed to the fact that polypeptides are being widely investigated in biomedical researches and even clinical practices. For instance, various polypeptide-based systems have been developed for drug delivery.13 Recently, the stimuli-responsive polypeptide nanocarriers attract the wide attention of researchers as they can efficiently deliver therapeutic payloads to targeted sites.14, 15 However,


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most of the platforms involve multi-step syntheses. There is a clear need for a system with facile synthesis procedure, high drug loading efficiency (DLE), and efficient targeted delivery. In the past several years, our group has synthesized and characterized a series of microenvironment-responsive polypeptide nanoplatforms for smart antitumor drug delivery.16-18 Basing on the previous researches, methoxy poly(ethylene glycol)-block-poly(S-tertbutylmercapto-L-cysteine) (mPEG-b-PBMLC) copolymers were synthesized through the ringopening polymerization (ROP) of S-tert-butylmercapto-L-cysteine N-carboxyanhydride (tBMLC NCA) with amino-terminated poly(ethylene glycol) (mPEG-NH2) as a macroinitiator in this study. The mPEG-b-PBMLC copolymers with PBMLC blocks of different degrees of polymerization (DPs) were prepared for the selectively intracellular delivery of antitumor drug in vitro and in vivo (Scheme 1). Specifically, a model antitumor drug doxorubicin (DOX) was loaded into the core of micelles (referred to as PM/DOX). The resulting micelles showed a GSHdependent release profile and an efficient inhibition of cell proliferation in vitro. Moreover, both PM/DOX micelles outperformed free doxorubicin hydrochloride (DOX·HCl) in terms of tumor growth suppression and safety for in vivo study. Regarding the length of PBMLC block, the higher DP of P9M/DOX exhibited better antitumor effect than the one with lower DP. 2. EXPERIMENTAL SECTION 2.1. Materials. mPEG was purchased from J&K Scientific Ltd. (Beijing, P. R. China). mPEGNH2 was synthesized according to the protocol described in Supporting Information.16 S-tertButylmercapto-L-cysteine (tBMLC) was bought from Tokyo Chemical Industry Co., Ltd. (Tianjin, P. R. China). tBMLC NCA was synthesized through the reaction between tBMLC and triphosgene according to the proposal reported in our previous works.18,

19

DOX·HCl was

obtained from Beijing Huafeng United Technology Co., Ltd. (Beijing, P. R. China). Dulbecco's


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modified Eagle's medium (DMEM) and new bovine serum (NBS) were provided by Gibco BRL Life Technology (Grand Island, NY, USA). Double antibiotics, i.e., penicillin and streptomycin, were obtained from Huabei Pharmaceutical Co., Ltd. (Shijiazhuang, P. R. China). GSH, 3-(4,5dimethyl-thiazol-2-yl)-2,5-diphenyl

tetrazolium

bromide

(MTT),

and

4′,6-diamidino-2-

phenylindole dihydrochloride (DAPI) were obtained from Sigma-Aldrich (Shanghai, P. R. China). Hematoxylin and eosin were purchased from Merck Company (Darmstadt, Germany). Cyanine 5.5 (Cy5.5) NHS ester was obtained from Lumiprobe Life Science Solutions (Hallandale Beach FL, USA). Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) kit was bought from Roche Company (Mannheim, Germany). Ki-67 antibody was purchased from Abcam Company (Cambridge, UK). The purified deionized water was produced by the Milli-Q plus system (Millipore Co., Billerica, MA, USA). 2.2. Syntheses of mPEG-b-PBMLC Copolymers. mPEG-b-PBMLC was synthesized through the ROP of tBMLC NCA with mPEG-NH2 as a macroinitiator. Typically, trace amount of water in mPEG-NH2 was first removed by azeotropic distillation with toluene at 120 °C for 2 h. Subsequently, tBMLC NCA and anhydrous N,N-dimethylformamide (DMF) were added. The polymerization was performed at room temperature for three days, and then the reaction solution was precipitated into a large excess of diethyl ether. The obtained solids (i.e., P4M and P9M) were dried overnight under vacuum at room temperature. The product yields of copolymers were decent (> 85%). 2.3. Characterizations. Proton nuclear magnetic resonance (1H NMR) spectra were recorded on a Bruker AV 600 NMR spectrometer (Rheinstetten, Germany) in deuterated trifluoroacetic acid (TFA-d). Fourier-transform infrared (FT-IR) spectra were recorded on a Bio-Rad Win-IR instrument (Bio-Rad, FTS-600; Cambridge, MA, USA) using potassium bromide (KBr) method.


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Transmission electron microscope (TEM) measurements were performed on a JEOL JEM-1011 TEM (Tokyo, Japan) with an accelerating voltage of 100 kV. The samples for TEM detection were prepared by putting a drop osample solution on a carbon-coated copper grid and then drying at room temperature. Dynamic laser scattering (DLS) measurements were performed with a vertically polarized He−Ne laser (DAWN EOS; Wyatt Technology, Santa Barbara, CA, USA). 2.4. Biodistribution and Pharmacokinetics. Female BALB/c nude mice (6 − 7 weeks, weighting ~ 21 g) were provided by Charles River laboratories (Beijing, P. R. China). All animals received care in compliance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals, and all procedures were approved by the Animal Care and Use Committee of Jilin University. The orthotopic breast cancer model was constructed by injecting human breast cancer MCF-7 cells at a dose of 2 × 106 cells per mouse into the second mammary fat pad of female BALB/c nude mouse. When tumors grew to about 250 mm3 in volume, DOX·HCl, P4M/DOX, or P9M/DOX was injected into human MCF-7 breast tumor-bearing mice at an equivalent DOX·HCl dose of 5.0 mg per kg body weight (mg (kg BW)−1), respectively. The mice were sacrificed at 3 or 6 h post-injection. The major organs (i.e., the heart, liver, spleen, lung, kidney, and sternum) and tumors were promptly excised, and the surface was washed with saline twice for biodistribution by measuring DOX fluorescence on a Maestro 500FL in vivo Imaging System (Cambridge Research & Instrumentation, Inc., USA). The collected fluorescence signals were quantitatively analyzed using a MaestroTM software (CRi, Inc., Woburn, MA). Sprague-Dawley rats were obtained from the Laboratory Animal Center of Jilin University, and used at seven weeks of age. For assessments of pharmacokinetics, the rats were randomly divided into three groups (n = 5 per group) after fasting (not water) for 12 h. DOX·HCl,


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P4M/DOX, or P9M/DOX was administered intravenously via tail vein at an equivalent DOX·HCl dose of 5.0 mg (kg BW)−1. The blood plasma samples were collected after 0.17, 0.5, 1, 3, 6, 12, and 24 h after injection. High-performance liquid chromatography (HPLC; Waters e2695 Separations Module, Waters Co., Milford, MA, USA) was used to detect the content of DOX in plasma. Specifically, 20.0 µL of daunorubicin hydrochloride (DAU·HCl) at a concentration of 10.0 µg mL−1 was added into 120.0 µL of plasma sample as an internal standard. Subsequently, the above mixture was mixed with 1.0 mL of methanol to deproteinize the plasma. Then the supernatant was collected and detected by HPLC after centrifugation at 8,000 rpm for 10 min. For HPLC analysis, C-18 Symmetry column (5 µm, 4.6 mm × 250 mm; Waters, Milford, MA, USA) and a fluorescence detector (Waters 2475 Multi λ Fluorescence Detector; Waters Co., Milford, MA, USA; λex = 480 nm, λem = 590 nm) were used. The mobile phase was consisted of methanol (80%) and phosphate buffer (20 mM). The flow rate was 1.0 mL min−1. All the data got from HPLC was analyzed by a PKSolver program.20 2.5. In Vivo Antitumor Efficacies. As mentioned above, the orthotopic breast cancer model was constructed. When tumor volume reached about 65 mm3, the mice bearing MCF-7 breast tumors were randomly divided into four groups with 6 mice in each group. The day was predetermined as day 0. Mice were treated with phosphate-buffered solution (PBS; as control), DOX·HCl, P4M/DOX, or P9M/DOX separately via intravenous injection on day 1, 6, 11, and 16. The equivalent DOX·HCl dose for all DOX formulations is 5.0 mg (kg BW)−1. The length (L) and width (W) of tumor were measured with vernier caliper (Guilin Guanglu Measuring Instrument Co., Ltd., Guilin, China) every two days, and the tumor volume (V) was calculated using Equation (1).


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Tumor volume (mm3 ) =

L ×W 2 2

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(1)

At the same time, the body weight was also monitored every two days. In addition, tumor inhibition rate was calculated using Equation (2).

Tumor inhibition rate (%) =

Vcontrol − Vtreatment × 100% Vcontrol

(2)

Figure 1. Chemical structure confirmation. (A) 1H NMR and (B) FT-IR spectra of mPEG113-bPBMLC4 and mPEG113-b-PBMLC9.


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Figure 2. Morphology and size. (A, B) Typical TEM morphologies and (C, D) Dhs of (A, C) P4M/DOX and (B, D) P9M/DOX. 2.6. Histological and Immunofluorescence Analyses. At the end of all treatments, the tumors and main organs (i.e., the heart, liver, spleen, lung, kidney, and sternum) were collected, fixed in 4% (W/V) PBS-buffered paraformaldehyde over 48 h, and then embedded in paraffin. The paraffin-embedded organs or tissues were cut into 5 µm slices that were stained with hematoxylin and eosin (H&E) and into ~ 3 µm sheets for immunofluorescence analyses (i.e., TUNEL and Ki-67). TUNEL assay was performed with a commercial kit following the manufacturer's instructions. The bone marrow cell micronucleus rates of sternum were counted


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in five different areas of H&E images of sternums. The histological and immunofluorescence images were obtained by using a microscope (Nikon Eclipse Ti, Optical Apparatus Co., Ardmore, PA, USA) and confocal laser scanning microscope (CLSM; LSM 780, Carl Zeiss, Oberkochen, Germany), respectively. In order to further analyses of the data, all the images were analyzed with Image J software (National Institutes of Health, Bethesda, Maryland, USA). 2.7. Evaluations of Maximum Tolerated Doses (MTDs). Kunming mice were purchased from the Laboratory Animal Center of Jilin University, and used at seven weeks of age. Equal amount of male and female Kunming mice weighting 18 − 21 g were used to evaluate the MTDs of DOX·HCl, P4M/DOX, and P9M/DOX. All group received a single-injection by tail vein with four different doses. In particular, mice received an equivalent DOX·HCl dose of 5.0, 10.0, 20.0, or 30.0 mg (kg BW)−1. After injection, the body weight, survival, and average daily diet were recorded every day.

Figure 3. Drug release. Reduction-dependent DOX release from PM/DOX in PBS without or with 10.0 mM GSH. Each set of data was represented as mean ± SD (n = 3). 2.8. Statistical Analyses. All experiments were performed at least thrice and expressed as means ± standard deviation (SD). Data were analyzed for statistical significance using SPSS 14.0


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(SPSS Inc., Chicago, IL, USA). *P < 0.05 was considered statistically significant, and #P < 0.01 and &P < 0.001 were considered highly significant. 3. RESULTS AND DISCUSSION 3.1. Preparation and Characterization of Loading mPEG-b-PBMLC Micelles. The ROP of amino acid NCA offers a greatest potential strategy for the syntheses of polypeptides.21 Moreover, polypeptides with biocompatibility and biodegradability play an essential role in drug delivery system.22 As illustrated in Figure 1, mPEG-NH2 was used as a macroinitiator to synthesize mPEG-b-PBMLC copolymers through the ROP of tBMLC NCA in our work. The chemical structures were confirmed by both 1H NMR and FT-IR spectra. The DPs of tBMLC segment in copolymers were evaluated based on the integrated area of signal at ~1.2 ppm (e) assigned to the side methyl protons (−CH(CH3)3) and that of peak at 3.8 ppm (b) attributed to the methylene proton in PEG (Figure 1A). As shown in Figure 1B, the results of FT-IR spectra also confirmed the successful syntheses of tBMLC blocks from the appearance of typical amide bands at 1657 cm−1 (νC=O) and 1547 cm−1 (vC(O)−NH), respectively. All these findings demonstrated the successful syntheses of mPEG-b-PBMLC copolymers. Nanoprecipitation is a common but efficient strategy for drug loading by polymer micelles.23, 24

In this work, DOX was loaded into the cores of mPEG-b-PBMLC micelles through

nanoprecipitation, and the loading micelles were marked as PM/DOX. As indicated by the TEM results shown in Figure 2A and 2B, both P4M/DOX and P9M/DOX exhibited spherical morphologies with mean diameters of 91.5 ± 6.8 and 77.3 ± 5.3 nm, respectively. Similar results were obtained by the detection of DLS. As depicted in Figure 2C and 2D, the hydrodynamic diameters (Dhs) of P4M/DOX and P9M/DOX were 92.3 ± 2.3 and 80.2 ± 2.8 nm, respectively.


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Both of them had suitable size distributions for passive intratumoral targeting through the enhanced permeability and retention (EPR) effect.25-27

Figure 4. Cell internalization and proliferation inhibition. (A) Typical CLSM and (B) FCM determinations of intracellular DOX release from PM/DOX, and (C) in vitro cytotoxicities of PM/DOX against MCF-7 cells. For B and C, each set of data was represented as mean ± SD (n = 3). Scale bar = 10 µm.


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Figure 5. Biodistribution. Biodistribution and semiquantitative analyze after injection of DOX·HCl or PM/DOX to MCF-7 breast tumor-xenografted mice for 3 or 6 h. Data were presented as a mean ± SD (n = 3; *P < 0.05, #P < 0.01, &P < 0.001). The in vitro DOX release behaviors of P4M/DOX and P9M/DOX were carried out using the dialysis method in PBS without or with 10.0 mM GSH. As shown in Figure 3, both P4M/DOX and P9M/DOX exhibited decent stability in PBS at pH 7.4 without GSH with less than 27.8% of DOX release after 72 h. The stability could provide the advantage of long-term maintenance of


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drug concentrations in the blood, which was the key properties of nanomedicines.28 In addition, P9M/DOX had a lower DOX release than P4M/DOX, which resulted from the dense microstructure of P9M/DOX. On the contrary, more than 58.3% of DOX were released from PM/DOX in 24 h with GSH. The cumulative DOX release at 72 h increased to as high as 73.6%. Likewise, P4M/DOX showed a slightly quicker cumulative release of DOX than that of P9M/DOX in the presence of GSH due to a bigger size and looser structure. All the characteristics mentioned above demonstrate the fact that PM/DOX had a long circulation time in plasma and an efficient intracellular GSH-triggered release of DOX. 3.2. Cell Internalization and Cell Proliferation Inhibition. The internalization of tumor cells is a key factor for the efficiencies of therapeutic drugs.29 The intracellular and extracellular GSH concentrations were detected using the GSH-GloTM Glutathione Assay Kit (Promega; San Luis Obispo, CA, USA; Figure S1). The intracellular GSH concentration of MCF-7 cells was 6.1 times higher than that of extracellular one. The cell internalization of Cy5.5-marked PM was verified by flow cytometry (FCM). As shown in Figure S2, the amount of Cy5.5-PM endocytosis increased along with the extension of incubation time from 1 to 6 h. The order of red Cy5.5 fluorescence was as follow: P9M/DOX (6 h) > P4M/DOX (6 h) > P9M/DOX (2 h) > P4M/DOX (2 h) > P9M/DOX (1 h) > P4M/DOX (1 h). The cell internalization of PM/DOX was detected by CLSM and FCM against MCF-7 cells without or with GSH pretreatment. As shown in Figure 4A, the red DOX fluorescence was found in the nuclei of MCF-7 cells after incubation for 2 h, indicating a colocalization with a blue fluorescence of DAPI. The cells cultured with free DOX·HCl presented a stronger DOX fluorescence intensity than that incubated with P4M/DOX or P9M/DOX. It manifested that DOX·HCl exhibited higher intracellular distribution than that of PM/DOX. The reason is that the


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cell uptake of DOX·HCl is through simple diffusion, which is faster than endocytosis of nanomedicines.30, 31 For PM/DOX micelles, the S−S bond in polypeptides could be restored by high concentration of GSH in cells, then the micelles disintegrated and the loaded DOX was released quickly. The sequence of red DOX fluorescence was as follows: P9M/DOX (with GSH pretreatment) > P4M/DOX (with GSH pretreatment) > P9M/DOX > P4M/DOX. Obviously, the above results demonstrated the efficient endocytosis of PM/DOX and intracellular GSHresponsive drug release. For PM/DOX, it had an opposite trend as compared to the in vitro DOX release. This may correlate to the smaller size of P9M/DOX, which contributes to a higher endocytosis. As show in Figure 4B, FCM analysis was carried out to further confirm the cell uptake of PM/DOX. The DOX fluorescence signal intensity of PM/DOX and DOX·HCl exhibited a similar result with CLSM. The cells cultured with free DOX·HCl had the highest fluorescence intensity, and there was no difference in signal intensity between the cells pretreated with 10.0 mM GSH and nonpretreated ones. The GSH-responsive DOX release was found in PM/DOX. P9M/DOX had a stronger signal intensity than P4M/DOX, indicating that a higher endocytosis of P9M/DOX than that of P4M/DOX. For the cytotoxicity evaluation of PM/DOX toward MCF-7 cells, MTT colorimetric assay was conducted as shown in Figure 4C. After 48 h incubation, as the increase of DOX concentration, the improved anti-proliferative activities were found in the DOX·HCl, P4M/DOX, and P9M/DOX groups. In detail, free DOX·HCl exhibited the highest cytotoxicity in the three test formulations. When pretreated with GSH, the cytotoxicity of DOX·HCl had not changed much as compared to no GSH pretreatment. After quantitative calculation, the half-maximal inhibitory concentration (IC50) values of free DOX·HCl without and with GSH pretreatment were 1.09 and


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1.10 µg mL−1, respectively. As expected, with the assistance of S−S, PM/DOX exhibited a higher cytotoxicity toward the cells pretreated with GSH. The IC50 sequence of cells was as follows: P4M/DOX (i.e., 2.15 µg mL−1) > P9M/DOX (i.e., 2.03 µg mL−1) > P4M/DOX (i.e., 1.68 µg mL−1; with GSH pretreatment) > P9M/DOX (i.e., 1.55 µg mL−1; with GSH pretreatment). Obviously, with the GSH-triggered drug release and high cell proliferation inhibition against MCF-7 cells, PM/DOX offered a high potential for treatment of tumors.

Figure 6. Pharmacokinetics. In vivo pharmacokinetic profiles after injection of DOX·HCl or PM/DOX in rats. Each set of data was represented as mean ± SD (n = 3). * represented the interruption of HPLC signal. 3.3. Biodistribution and Pharmacokinetics. The biodistribution is closely related to the drug efficacy and side effects. For biodistribution determinations, the ex vivo DOX fluorescence imaging of tumors and main organs (i.e., the heart, liver, spleen, lung, kidney, and sternum) at 3 and 6 h post-injection were performed in MCF-7 breast tumor-xenografted mice as shown in Figure 5. As depicted in Figure 5A, the heart and spleen showed little and even no DOX fluorescence for all the formulations. For all the groups, the livers had the highest fluorescence intensities ascribed to the phagocytosis by macrophages of liver.32 The DOX fluorescence intensity of tumor in the DOX·HCl group was higher than that in the PM/DOX groups at 3 h. As the time reached to 6 h post-injection, the DOX fluorescence intensities of tumors in the


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PM/DOX groups, increased, especially in the P9M/DOX group. The results should be ascribed to the selectively intratumoral accumulation by the EPR effect of tumor tissue and the responsively intracellular release of DOX from PM/DOX.

Figure 7. Antitumor efficacy and body weight changes. (A, C) Tumor volumes, (B) tumor inhibition rate, and (D) body weights of MCF-7 breast tumor-xenografted BALB/c nude mice after treatment with PBS, DOX·HCl or PM/DOX at an equivalent DOX·HCl dose of 5.0 mg (kg BW)−1. Each set of data was represented as mean ± SD (n = 6; *P < 0.05, #P < 0.01, &P < 0.001). Furthermore, the fluorescence intensity was semi-quantitatively analyzed and shown in Figure 5B. The average signals of the liver, lung, kidney, and tumor were higher than those of the heart and spleen. Generally, for the tumor, the average signal intensity of DOX·HCl group was higher than those of P4M/DOX and P9M/DOX groups at 3 h. However, the 2.43 and 1.28 times higher


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intratumoral average signal of P9M/DOX group was observed than those of the DOX·HCl and P4M/DOX groups at 6 h, respectively. All the above data indicated that PM/DOX, especially P9M/DOX, exhibited selectively intratumoral accumulation.

Figure 8. Histopathology and immunohistochemistry. Histopathological (i.e., H&E) and immunohistochemical (i.e., TUNEL and Ki-67) analyses of tumor tissue sections after treatment with PBS, DOX·HCl, or PM/DOX at an equivalent DOX·HCl dose of 5.0 mg (kg BW)−1. Scale bar = 50 µm.


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Figure 9. Histopathologies of organs. Ex vivo histopathology analyses of visceral organ sections (i.e., the heart, liver, spleen, lung, kidney, and marrow) from MCF-7 breast tumor-xenografted BALB/c nude mouse model after treatment with PBS or with DOX·HCl or PM/DOX at an equivalent DOX·HCl dose of 5.0 mg (kg BW)−1. Scale bar = 50 µm.


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The long blood circulation and clearance period of nanoparticles is a critical factor for efficient drug delivery to the tumor.33 In the present study, as shown in Figure 6, pharmacokinetics of the DOX·HCl, P4M/DOX, and P9M/DOX formulations were evaluated with HPLC. The area under concentration versus time curve from 0 to last time t (AUC0-t) of P4M/DOX and P9M/DOX were 0.52 and 1.10 µg (mL h)−1, respectively, which were 1.8 and 3.7 times higher than that of DOX·HCl. The calculated mean half-life of the elimination phase (t1/2) of DOX·HCl was 1.02 h. After nanostructure formation, the t1/2 of P4M/DOX and P9M/DOX increased up to 3.83 and 4.91 h, respectively. The blood circulation time of PM/DOX was significantly prolonged compared to DOX·HCl, indicating that great potential of the facilely prepared polypeptide micelles in antitumor drug delivery. 3.4. In Vivo Antitumor Efficacy. For a chemotherapy drug, the antitumor efficacy is a key factor to consider before entering clinical trial stage. The antitumor efficacies of DOX·HCl and PM/DOX were investigated toward the BALB/c nude mouse models bearing MCF-7 breast tumors. When tumor volumes reached to ~ 65 mm3, the mice were injected with free DOX·HCl, P4M/DOX, or P9M/DOX at an equivalent DOX·HCl dose of 5.0 mg (kg BW)−1 via tail vein. As shown in Figure 7A, the tumor volume was real-time measured every two days. In particular, the average tumor volume of Control group reached to ~ 3600 mm3 in 19 days (Figure 7C). After chemotherapy of DOX·HCl, the tumor volume was controlled by an inhibition rate of 72.0% (Figure 7B). Nevertheless, the tumor was still growing in the whole treatment period. On the other hand, the tumor growth was controlled by the treatment of P4M/DOX or P9M/DOX as shown in Figure 7B. The tumor inhibition rates were 87.5% and 92.2%, respectively. Moreover, the tumor inhibition rates of P4M/DOX and P9M/DOX were 1.3 and 1.2 times higher than that


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of DOX·HCl after four treatments, respectively. The suitable particle size and intracellular GSHresponse contributed to a remarkable antitumor efficacy of PM/DOX. The antitumor efficacy of DOX formulations in vivo were further verified by both histopathological and immunohistochemical analyses. As shown in Figure 8 (upper), growth of tumor cells was observed in the H&E-stained tumor tissue section in the Control group. However, the different degrees of apoptosis appeared in the DOX·HCl and PM/DOX groups. Furthermore, the tumor necrosis areas were counted thrice on different observation fields and calculated by the NIS-Elements D 4.20.00 Image Analysis software (Nikon Instruments Inc., Ardmore, PA, USA). The necrosis area of tumor section in the P4M/DOX group was (77.4 ± 4.4)%, which was 1.5 times higher than that of the DOX·HCl group (50.3 ± 5.3)%. For the P9M/DOX group, the ratio became higher up to 1.9 times. One main reason was that P9M/DOX with smaller particle size had better tumor penetration, which induced larger necrosis area in tumor section than that of P4M/DOX.34 Moreover, the condition of tumor cell apoptosis was evaluated by TUNEL assays. As shown in Figure 8 (middle), the green fluorescence of FITC-dyed DNA fragmentation was observed in tumor sections of all groups. More fluorescence expression indicated more necrosis or apoptosis induced by various DOX formulations. The amount of fluorescence expression areas (i.e., tumor necrosis areas) were counted thrice on different observation fields and calculated by ImageJ software (National Institute of Health, Bethesda, MD, USA). The amount of fluorescence in four groups was significantly different, the order of fluorescence density was calculated as follow: P9M/DOX (i.e., 86.2 ± 3.2) > P4M/DOX (i.e., 67.2 ± 4.9) > DOX·HCl (i.e., 25.9 ± 6.5) > Control (i.e., 8.6 ± 1.4). It was worth mentioning that the Control group showed a little apoptosis due to the spontaneous necrosis changes of tumors in the process of rapid growth.35 Ki-67 stain


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was used to assess the tumor cell proliferation ability. The amount of fluorescence expression areas (i.e., proliferation areas) were obtained according to the above method. As shown in Figure 8 (lower), the expression of Ki-67 showed a contrary tendency with that of TUNEL sections, and the same experiment result was proved once again. To summarize, for the in vivo antitumor efficacy, both P4M/DOX and P9M/DOX showed dramatic antitumor effect, which was definitely higher than DOX·HCl. Furthermore, it was important that PM/DOX had less toxicity compared to free DOX·HCl, and the advantage pushed forward the clinical pharmacotherapy of PM/DOX.


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Figure 10. MTDs. Body weight, survival rate, and diet of Kunming mice after injection of DOX·HCl or PM/DOX at the equivalent DOX·HCl doses of 5.0, 10.0, 20.0, and 30.0 mg (kg BW)−1. Each set of data was represented as mean ± SD (initial value; n = 10). 3.5. Upregulated Safety of PM/DOX In Vivo. The security is necessary for the successful application of nanoscale drug delivery system. For in vivo antitumor verification experiment, three DOX formulations were used four times by intravenous injection during the 19-day treatment course. The body weight of mouse was recorded every two days. As shown in Figure 7C, the body weights of both P4M/DOX and P9M/DOX groups after the treatments increased by 16.7% and 19.1% during the period of treatment, respectively. However, 20.4% loss of body weight was observed for the DOX·HCl group because of the side effects of DOX·HCl. The side effects of drug were greatly reduced by loaded into polypeptide micelles. To further evaluate the safety of PM/DOX, the main organs or tissues (i.e., the heart, liver, spleen, lung, kidney, and marrow) were harvested, sliced, and stained with H&E at the end of all treatments. As shown in Figure 9, the H&E-imaged sections were analyzed. For the DOX·HCl group, the most obvious change was that the renal cavity shrank appeared in the H&E-stained kidney tissue section, which was pointed out with black arrows. Nevertheless, normal glomeruli were distributed relatively equally in the kidney tissue sections of PM/DOX groups. To further confirm the security of P4M/DOX and P9M/DOX, the MTD assay was performed by a single intravenous injection to Kunming mice (Figure 10). In detail, the survival rate, body weight, and diet were continuously monitored for 10 days after the injection of DOX·HCl or PM/DOX. The higher survival rates and less weight loss of PM/DOX group were found compared with that of the DOX·HCl group, which indicated that PM/DOX reduced the side effect of free DOX·HCl. For three groups, the body weight become smaller with the


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concentration of DOX·HCl increasing from 5.0 to 30.0 mg (kg BW)−1. The smaller change of body weight of PM/DOX group was found than that of the DOX·HCl group. The MTD of DOX·HCl was calculated to be between 5.0 – 10.0 mg (kg BW)−1, being consistent with the results of previous studies.18 The MTD values of P4M/DOX and P9M/DOX were both between 10.0 and 20.0 mg (kg BW)−1, which benefited from that the toxicity of DOX was reduced by being loaded in polypeptide micelle. Under living condition, a significant death rate was observed at the equivalent DOX·HCl doses of 10.0 – 30.0 mg (kg BW)−1 from the third day after injection in the DOX·HCl group (Figure 10 (middle)). However, only when the dose of DOX·HCl increased up to 20.0 mg (kg BW)−1, the mice death in the PM/DOX group was observed. For the dose of 30.0 mg (kg BW)−1, the 5-day survival rates of DOX·HCl, P4M/DOX, and P9M/DOX were 10%, 80%, and 100%, respectively. The average diet of every group mice was recorded every day to surmise the physical conditions of mice. As shown in Figure 10 (below), the average diet of every group decreased with the doses of DOX·HCl increased in general. In the DOX·HCl group, the average diet of one mouse was less than 1.2 g day−1 at an equivalent DOX·HCl dose of 30.0 mg (kg BW)−1. It was encouraging that the encapsulation of polypeptide micelle improved the diet level. In summary, the result of MTD assays showed that both P4M/DOX and P9M/DOX micelles decreased the systemic toxicity of DOX·HCl, which showed a prolongation of survival time. One step further, the low toxicity of PM/DOX indicates that PM/DOX can be applied in clinical practice. 4. CONCLUSION In the present study, the reduction-responsive polypeptide micelles were prepared with facile preparation strategy, high drug loading capacity, and the ability of stimuli-response to reductive


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environment. PM/DOX with prolonged blood circulation enhanced the drug distribution in tumor. Furthermore, PM/DOX exhibited stronger tumor inhibition effect and lower side effects. This delivery platform also has the potential to be used for delivery of various hydrophobic antitumor drugs besides DOX. In addition, this convenient and efficient synthesis method makes mass production of this micelle platform possible for clinical transformation.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author E-mail: [email protected].

ACKNOWLEDGMENT This research was financially supported by the National Natural Science Foundation of China (Grant Nos. 51673190, 51603204, 51303174, 51473165, 51390484, and 51520105004) and the Science and Technology Development Program of Jilin Province (Grant Nos. 20160204015SF and 20160204018SF).

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Reduction-Responsive Polypeptide Micelles for Intracellular Delivery

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